TAK1 kinase inhibitors

Abstract

Disclosed are compounds of Formula I, which can be used to inhibit TAK1 kinase.

Description

TAK1 KINASE INHIBITORS by Mark A. Lovell Thomas E. Prisinzano Assignee: University of Kentucky Research Foundation Attorney Docket No.: 13177N / 2778WO RELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional Application Serial No.63 / 612,217 filed December 19, 2023, the entire disclosure of which is incorporated herein by this reference. TECHNICAL FIELD

[0002] The presently-disclosed subject matter generally relates to compounds that areTransforming Growth Factor-β (TGF-β)-Activated Kinase 1 (“TAK1” or “TAK1 kinase”) inhibitors and methods for use thereof. In particular, certain embodiments of the presently- disclosed subject matter relate to TAK1 kinase inhibitors that are soluble, orally bioavailable, and brain penetrant. INTRODUCTION

[0003] TAK1 is a mitogen-activated protein kinase (MAP3K) that plays an importantrole in various cellular signaling pathways. For example, TAK1 is a key mediator in the activation of several signaling pathways, including the nuclear factor kappa B (NF-κB) pathway and the mitogen-activated protein kinase (MAPK) pathways. These pathways are important for regulating various cellular processes such as inflammation, immune response, cell survival, and differentiation.

[0004] TAK1 is also a regulator of inflammatory responses. It is involved in the signalingcascades triggered by pro-inflammatory cytokines, Toll-like receptors (TLRs), and other immune receptors. Activation of TAK1 leads to the activation of NF-κB and otherdownstream effectors, resulting in the expression of genes involved in inflammation and immune responses.

[0005] TAK1 also plays a role in determining cell fate decisions, including cell survival,apoptosis, and differentiation. The activation of TAK1 can influence these processes by regulating the activity of various downstream kinases and transcription factors.

[0006] TAK1 is also a key component in the transforming growth factor-beta (TGF-β)signaling pathway. TGF-β is a multifunctional cytokine that regulates cell growth, differentiation, and various other cellular processes. TAK1 activation is important for transmitting signals downstream in response to TGF-β ligands.

[0007] TAK1 also contributes to the maintenance of cellular homeostasis by integratingsignals from various stimuli and coordinating appropriate cellular responses. It acts as a central player in the cellular response to environmental stress, growth factors, and cytokines.

[0008] Dysregulation of TAK1 signaling has been implicated in various diseases,including inflammatory disorders, autoimmune diseases, cancer, and neurodegenerative diseases. Consequently, TAK1 has become a target for drug development, and inhibitors of TAK1 are being explored for their therapeutic potential in these conditions.

[0009] Indeed, TAK1 kinase inhibitors have attracted attention in the field of drugdiscovery and development due to the role of TAK1 in signaling pathways associated with inflammation, immunity, and cancer. Inhibition of TAK1 activity has potential therapeutic applications in conditions where dysregulated TAK1 signaling is implicated.

[0010] While there is promise for TAK1 kinase inhibitors, currently available TAK1kinase inhibitors have a number of drawbacks. For example, Takinib is not brain penetrant, and 5Z-7-oxozeaenol has been shown to have substantial off target effects.

[0011] For therapeutic compounds targeting neurodegeneration or other central nervoussystem (CNS) conditions, the blood-brain barrier (BBB) presents a formidable obstacle. The BBB is a highly selective and protective interface that restricts the passage of most substances from the bloodstream into the brain. Many potentially effective neurotherapeutic compounds either fail to cross the BBB or exhibit poor brain penetration, which severely limits their ability to reach therapeutic concentrations within neural tissue. This barrierfunction is particularly problematic for addressing neurodegenerative diseases, where the ability to target degenerating neurons directly is essential for treatment efficacy. Without effective brain penetration, even promising compounds may fail to deliver therapeutic benefits or mitigate disease progression, necessitating innovative strategies to overcome this limitation.

[0012] Many compounds with promising therapeutic potential suffer from poor solubilityin aqueous environments. Solubility is an important factor in drug development because it directly affects the compound’s dissolution rate, bioavailability, and overall therapeutic efficacy. A poorly soluble compound often leads to insufficient drug concentrations in systemic circulation, hindering its ability to achieve therapeutic levels at the target site. This problem is particularly pronounced for oral formulations, where the compound must dissolve in gastrointestinal fluids before absorption. Inadequate solubility can result in inconsistent pharmacokinetics, reduced efficacy, and challenges in formulating stable and effective drug products. Addressing solubility issues is therefore an important hurdle in translating a therapeutic compound from discovery to clinical use.

[0013] Oral bioavailability, the fraction of an orally administered drug that reachessystemic circulation in an active form, is another significant challenge for many therapeutic compounds. Poor oral bioavailability can arise from several factors, including low solubility, poor permeability across the intestinal epithelium, extensive first-pass metabolism, or degradation in the gastrointestinal tract. When bioavailability is low, achieving therapeutic drug concentrations often requires higher or more frequent dosing, which can increase the risk of adverse effects and reduce patient compliance. Furthermore, compounds with poor bioavailability often exhibit variable pharmacokinetics, complicating dose optimization and limiting their therapeutic potential.

[0014] Accordingly, there remains a need in the art for improved TAK1 kinase inhibitorsand new methods of using TAK1 kinase inhibitors, including TAK1 kinase inhibitors that are capable of crossing the blood brain barrier, have improved solubility, and are orally bioavailable.SUMMARY

[0015] The presently-disclosed subject matter meets some or all of the above-identifiedneeds, as will become evident to those of ordinary skill in the art after a study of information provided in this document.

[0016] This Summary describes several embodiments of the presently-disclosed subjectmatter, and in many cases lists variations and permutations of these embodiments. This Summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently-disclosed subject matter, whether listed in this Summary or not. To avoid excessive repetition, this Summary does not list or suggest all possible combinations of such features.

[0017] The presently-disclosed subject matter includes compounds that are TAK1 kinaseinhibitors and methods for use thereof. In some embodiments, the TAK1 kinase inhibitors are water soluble. In some embodiments, the TAK1 kinase inhibitors are orally bioavailable. In some embodiments, the TAK1 kinase inhibitors are brain penetrant, such that they can cross the blood-brain barrier (BBB) and enter the central nervous system (CNS).

[0018] In some embodiments, the TAK1 kinase inhibitors are formulated in apharmaceutical composition. In some embodiments, the compounds disclosed herein can be used to inhibit TAK1 kinase in a cell. In some embodiments, the compounds disclosed herein can be used to inhibit TAK1 kinase in a subject. In some embodiments, the subject has or is at risk of developing neurodegenerative condition or a condition characterized by degenerating neurons. In some embodiments, the subject has or is at risk of Alzheimer’s disease. In some embodiments, the subject has or is at risk of head injury.

[0019] The presently-disclosed subject matter includes compounds that are TAK1 kinaseinhibitors and methods for use thereof. In some embodiments, the TAK1 kinase inhibitors are water soluble. In some embodiments, the TAK1 kinase inhibitors are orally bioavailable. In some embodiments, the TAK1 kinase inhibitors are brain penetrant, such that they can cross the blood-brain barrier (BBB) and enter the central nervous system (CNS).

[0020] In some embodiments, the TAK1 kinase inhibitors are formulated in apharmaceutical composition. In some embodiments, the compounds disclosed herein can be used to inhibit TAK1 kinase in a cell. In some embodiments, the compounds disclosed herein can be used to inhibit TAK1 kinase in a subject. In some embodiments, the subject has or is at an increased risk of developing neurodegenerative condition or a condition characterized by degenerating neurons. In some embodiments, the subject has or is at an increased risk of Alzheimer’s disease. In some embodiments, the subject has or is at an increased risk of head injury.

[0021] The presently-disclosed subject matter includes a compound of formula (I):O R2,II) N N or a pharmaceutically-acceptable salt thereof, wherein R1 ,NO R2N N N or a pharmaceutically-acceptable salt thereof, , N N N HHNNN N N O O ,

[0024] The presently-disclosed subject matter includes a compound of formula (IV):or a pharmaceutically-acceptable salt thereof.

[0025] The presently-disclosed subject matter includes a compound of formula (V):or a

[0026] The presently-disclosed subject matter further includes a composition comprisinga compound as disclosed herein and a pharmaceutically-acceptable carrier.

[0027] The presently-disclosed subject matter further includes a method of inhibitingTAK1 in a cell, which comprises administering a compound as disclosed herein to the cell. In some embodiments, the cell is in a subject. In some embodiments, the subject has or is at an increased risk of developing neurodegeneration in Alzheimer’s disease. In some embodiments, the subject has or is at an increased risk of head injury. In some embodiments, the compound is orally administered.

[0028] The presently-disclosed subject matter further includes a method of treating acondition characterized by degenerating neurons, which comprises administering a compound as disclosed herein to a subject in need thereof. In some embodiments, thecondition is Alzheimer’s disease. In some embodiments, the condition is head injury, concussions, traumatic brain injury, or traumatic chronic encephalopathy. DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0029] The details of one or more embodiments of the presently-disclosed subject matterare set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document. The information provided in this document, and particularly the specific details of the described exemplary embodiments, is provided primarily for clearness of understanding and no unnecessary limitations are to be understood therefrom. In case of conflict, the specification of this document, including definitions, will control.

[0030] The presently-disclosed subject matter includes compounds that are TAK1 kinaseinhibitors and methods for use thereof. In some embodiments, the TAK1 kinase inhibitors are water soluble. In some embodiments, the TAK1 kinase inhibitors are orally bioavailable. In some embodiments, the TAK1 kinase inhibitors are brain penetrant, such that they can cross the blood-brain barrier (BBB) and enter the central nervous system (CNS).

[0031] In some embodiments, the TAK1 kinase inhibitors are formulated in apharmaceutical composition. In some embodiments, the compounds disclosed herein can be used to inhibit TAK1 kinase in a cell. In some embodiments, the compounds disclosed herein can be used to inhibit TAK1 kinase in a subject. In some embodiments, the subject has or is at an increased risk of developing neurodegenerative condition or a condition characterized by degenerating neurons. In some embodiments, the subject has or is at an increased risk of Alzheimer’s disease. In some embodiments, the subject has or is at an increased risk of head injury.

[0032] The presently-disclosed subject matter includes a compound of formula (I):O R2F N N CF3(I)N or a pharmaceutically-acceptable salt thereof, wherein R1 N ,NN N N N N N or a pharmaceutically-acceptable salt thereof, wherein R1, ,N,N O S HN N O S N N N HNII) or a pharmaceutically-acceptable salt thereof, ,O O ,:N or a pharmaceutically-

[0036] The presently-disclosed subject matter includes a compound of formula (V):V) or a pharmaceutically-acceptable salt thereof.

[0037] The presently-disclosed subject matter further includes a composition comprisinga compound as disclosed herein and a pharmaceutically-acceptable carrier. Suitable formulations include aqueous and non-aqueous sterile injection solutions that can contain antioxidants, buffers, bacteriostats, bactericidal antibiotics and solutes that render the formulation isotonic with the bodily fluids of the intended recipient; and aqueous and non- aqueous sterile suspensions, which can include suspending agents and thickening agents.

[0038] The compositions can take such forms as suspensions, solutions or emulsions inoily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0039] The formulations can be presented in unit-dose or multi-dose containers, forexample sealed ampoules and vials, and can be stored in a frozen or freeze-dried (lyophilized) condition requiring only the addition of sterile liquid carrier immediately prior to use.

[0040] The presently-disclosed subject matter further includes a method of inhibitingTAK1 in a cell, which comprises administering a compound as disclosed herein to the cell.

[0041] As will be recognized by one of ordinary skill in the art, the term “inhibiting” or“inhibition” does not refer to the ability to completely inactivate all target biological activity in all cases. Rather, the skilled artisan will understand that the term “inhibiting” refers to decreasing biological activity of a target, such as TAK1 kinase. Such decrease in biological activity can be determined relative to a control, wherein an inhibitor is not administered and / or placed in contact with the target. For example, in some embodiments, a decrease inactivity relative to a control can be about a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% decrease.

[0042] As used herein, the terms “administering” and “administration,” when used inconnection with providing a pharmaceutical preparation to a subject, refer to any of the methods that are well known to those skilled in the art. For example, such methods include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. A preparation can be administered therapeutically; that is, administered to treat an existing condition of interest. A preparation can be administered prophylactically; that is, administered for prevention of a condition of interest.

[0043] In some embodiments of the methods disclosed herein, the compound is orallyadministered. For oral administration, the compositions can take the form of, for example, tablets or capsules prepared by a conventional technique with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycollate); or wetting agents (e.g., sodium lauryl sulphate). The tablets can be coated by methods known in the art. For example, an ACE inhibitor can be formulated in combination with hydrochlorothiazide, and as a pH stabilized core having an enteric or delayed release coating which protects the ACE inhibitor until it reaches the colon.

[0044] Liquid preparations for oral administration can take the form of, for example,solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional techniques with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifyingagents (e.g. lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethylalcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p- hydroxybenzoates or sorbic acid). The preparations can also contain buffer salts, flavoring, coloring and sweetening agents as appropriate. Preparations for oral administration can be suitably formulated to give controlled release of the active compound. For buccal administration the compositions can take the form of tablets or lozenges formulated in conventional manner.

[0045] The compounds can also be formulated as a preparation for implantation orinjection. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).

[0046] The compounds can also be formulated in rectal compositions (e.g., suppositoriesor retention enemas containing conventional suppository bases such as cocoa butter or other glycerides), creams or lotions, or transdermal patches.

[0047] In some embodiments of the method of inhibiting TAK1, the cell is in a subjectand the method involves administering the compound to the subject. In some embodiments, the subject has or is at an increased risk of developing neurodegeneration in Alzheimer’s disease. In some embodiments, the subject has or is at an increased risk of head injury, concussions, traumatic brain injury, or traumatic chronic encephalopathy.

[0048] The presently-disclosed subject matter further includes a method of treating acondition characterized by degenerating neurons, which comprises administering a compound as disclosed herein to a subject in need thereof. In some embodiments, the condition is Alzheimer’s disease. In some embodiments, the condition is head injury, concussions, traumatic brain injury, or traumatic chronic encephalopathy.

[0049] As used herein, the term “subject” refers to an animal, including humans and non-human animals, including mammals.

[0050] Alzheimer’s disease is a progressive brain disorder that gradually destroysmemory, thinking skills, and the ability to carry out simple tasks. It is the most common cause of dementia among older adults. The disease is characterized by the buildup of amyloid plaques and neurofibrillary tangles in the brain, which lead to the death of brain cells and theshrinkage of brain tissue. Symptoms typically begin with mild memory loss and confusion but eventually progress to severe cognitive and functional impairments.

[0051] A head injury refers to any damage to the head, including the scalp, skull, orbrain, caused by trauma. This broad term encompasses a variety of injuries, ranging from minor bumps and bruises to severe conditions like skull fractures, concussions, and traumatic brain injuries (TBI). Head injuries can result from falls, vehicle accidents, sports activities, or violent incidents. Symptoms can vary widely depending on the severity and type of injury, and may include headaches, dizziness, confusion, bleeding, and loss of consciousness.

[0052] A concussion is a type of mild traumatic brain injury (mTBI) that occurs when ablow to the head or a sudden jolt causes the brain to move rapidly back and forth within the skull. This movement can stretch and damage brain cells, leading to temporary changes in brain function. Symptoms of a concussion can include headaches, confusion, dizziness, nausea, and memory problems. While most people recover fully, repeated concussions can lead to more serious long-term effects.

[0053] Traumatic brain injury (TBI) is a serious condition caused by an external force,such as a blow, jolt, or object penetrating the skull, that disrupts normal brain function. TBIs can range from mild, such as a brief change in mental status or consciousness, to severe, involving extended periods of unconsciousness or amnesia after the injury. Common causes include falls, vehicle accidents, sports injuries, and explosive blasts.

[0054] Chronic traumatic encephalopathy (CTE) is a degenerative brain disorder causedby repeated head injuries, such as concussions. It is typically found in athletes who participate in contact sports like football, boxing, and hockey, as well as military personnel exposed to blast injuries. CTE leads to the progressive degeneration of brain tissue, including the buildup of an abnormal protein called tau. Symptoms can include memory loss, confusion, impaired judgment, aggression, depression, and eventually progressive dementia. The condition typically develops over many years, often appearing long after the initial injuries.

[0055] “Degenerating neurons” or “neurodegeneration” refer to the progressive loss ofstructure or function in neurons, the cells that make up the nervous system. This process can lead to the death of neurons and is a hallmark of various neurodegenerative diseases. Neurodegeneration often involves mechanisms like protein misfolding, oxidative stress,mitochondrial dysfunction, and inflammation. As neurons degenerate, they lose their ability to communicate effectively, leading to the gradual decline in cognitive and motor functions associated with these diseases.

[0056] Examples of some neurodegenerative diseases include: Alzheimer’s disease,characterized by memory loss, confusion, and cognitive decline; Parkinson’s disease, known for causing tremors, stiffness, and difficulty with balance and coordination; amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease, which affects motor neurons, leading to muscle weakness and atrophy; Huntington’s disease, which is a genetic disorder that causes the progressive breakdown of nerve cells in the brain, affecting movement, cognition, and behavior; multiple sclerosis (MS), which is an autoimmune disease that damages the myelin sheath, leading to communication problems between the brain and the rest of the body; frontotemporal dementia (FTD), which refers to a group of disorders caused by progressive nerve cell loss in the brain’s frontal and temporal lobes; chronic traumatic encephalopathy (CTE), which is a condition associated with repeated head injuries, often seen in athletes and military personnel.

[0057] The phrase “increased risk” is used herein to refer to those subjects whoselikelihood of developing a condition in their lifetime is increased, as compared to a normal subject. As will be appreciated by one skilled in the art of diagnosis, prognosis, and / or identification of risk, such subjects may be identified by factors that include, but are not limited to, a genetic pre-disposition to certain conditions, environmental factors impacting risk, and life style choices.

[0058] For example, several factors can be considered when determining whether apatient is at an increased risk for Alzheimer’s disease. Age is the most significant risk factor, with the likelihood of developing Alzheimer’s doubling every five years after age 65. Family history also plays a crucial role; individuals with a parent, sibling, or child with Alzheimer’s are more likely to develop the disease themselves. Genetic predisposition, particularly the presence of the APOE-e4 gene, can increase risk. Additionally, certain medical conditions such as cardiovascular disease, diabetes, and hypertension are linked to a higher risk of Alzheimer’s disease. Lifestyle factors, including smoking, lack of physical activity, and poor diet, can further elevate the risk. Head injuries and traumatic brain injuries are also associated with an increased risk of developing Alzheimer’s later in life.

[0059] For another example, several factors are considered when determining whether apatient is at an increased risk for head injury, including concussions. Participation in contact sports, such as football, hockey, and soccer, significantly increases the risk due to the high likelihood of collisions and falls. Previous head injuries or concussions also elevate the risk, as individuals who have had one concussion are more susceptible to subsequent ones. Certain medical conditions, such as Attention Deficit Hyperactivity Disorder (ADHD), have been linked to a higher incidence of concussions. Additionally, risky behaviors like not wearing helmets while biking or engaging in activities with a high potential for falls, such as skateboarding, can increase the likelihood of head injuries. Age and sex may also play a role, with younger individuals and males generally being at higher risk.

[0060] As used herein, the terms “treatment” or “treating” relate to any treatment of acondition of interest, such as a condition characterized by degeneration of neurons. Such treatment includes, but is not limited to, prophylactic treatment to prevent development or reduce severity of a condition. The terms “treatment” or “treating” include: preventing a condition associated with degeneration of neurons from occurring in a subject who may be predisposed to these diseases but who has not yet been diagnosed as having them; inhibiting these conditions, i.e., arresting their development; or ameliorating or relieving the symptoms of these conditions, i.e., causing regression of one or more of the symptoms associated with the conditions.

[0061] While the terms used herein are believed to be well understood by those ofordinary skill in the art, certain definitions are set forth to facilitate explanation of the presently-disclosed subject matter.

[0062] Unless defined otherwise, all technical and scientific terms used herein have thesame meaning as is commonly understood by one of skill in the art to which the invention(s) belong.

[0063] Although any methods, devices, and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presently-disclosed subject matter, representative methods, devices, and materials are described herein.

[0064] The present application can “comprise” (open ended) or “consist essentially of”the components of the present invention as well as other ingredients or elements described herein. As used herein, “comprising” is open ended and means the elements recited, or theirequivalent in structure or function, plus any other element or elements which are not recited. The terms “having” and “including” are also to be construed as open ended unless the context suggests otherwise.

[0065] Following long-standing patent law convention, the terms “a”, “an”, and “the”refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a cell” includes a plurality of such cells, and so forth.

[0066] Unless otherwise indicated, all numbers expressing quantities of ingredients,properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.

[0067] As used herein, the term “about,” when referring to a value or to an amount ofmass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, in some embodiments ±0.1%, in some embodiments ±0.01%, and in some embodiments ±0.001% from the specified amount, as such variations are appropriate to perform the disclosed method.

[0068] As used herein, ranges can be expressed as from “about” one particular value,and / or to “about” another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0069] As used herein, the term “pharmaceutically acceptable carrier” refers to sterileaqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example,by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.

[0070] The term “pharmaceutically acceptable salts” refers to salts prepared frompharmaceutically acceptable non-toxic bases or acids. When the compound of the present invention is acidic, its corresponding salt can be conveniently prepared from pharmaceutically acceptable non-toxic bases, including inorganic bases and organic bases. Salts derived from such inorganic bases include aluminum, ammonium, calcium, copper (-ic and -ous), ferric, ferrous, lithium, magnesium, manganese (-ic and -ous), potassium, sodium, zinc and the like salts. Particularly preferred are the ammonium, calcium, magnesium, potassium and sodium salts. Salts derived from pharmaceutically acceptable organic non- toxic bases include salts of primary, secondary, and tertiary amines, as well as cyclic amines and substituted amines such as naturally occurring and synthesized substituted amines.

[0071] The presently-disclosed subject matter is further illustrated by the followingspecific but non-limiting examples. The following examples may include compilations ofdata that are representative of data gathered at various times during the course of development and experimentation related to the present invention. EXAMPLES

[0072] Example 1: Identification of Initial Compounds for Testing

[0073] NFD-L1 (found in U.S. Patent No. 9,968,574) is brain penetrant and wasdiscovered to be a mild TAK1 kinase inhibitor. It was further tested in mouse models of AD pathology, but studies indicated that it has limited bioavailability in this context.

[0074] Work was undertaken to develop compounds that are potent TAK1 kinaseinhibitors, brain penetrant, orally bioavailable, and that have improved solubility. Infinisee (BioSolveIT) software was used to identify theoretical structures of compounds that might serve as TAK1 inhibitors. Approximately 500 theoretical structures were identified.

[0075] Each of the 500 compounds was subjected to in silico ADMET to identifycompounds predicted to pass the blood brain barrier. Based on this screen several theoretical structures were identified, including Compounds 1-4 in Table 1. After identification, Compounds 1-4 were synthesized.

[0076] The synthesized compounds were tested for TAK1 kinase inhibition activity(Table 1). Three of the compounds did not demonstrate TAK1 kinase inhibition. However,

[0077] 1-{[3-fluoro-4-(1H-imidazol-1-yl)phenyl]methyl}-3-{4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl}urea dihydrochloride[HCl] (referred to herein as “UK5141”) was found to be of particular interest. UK5141 had potent TAK1 kinase inhibition (Kd = 87 nM) and is predicted to have beneficial water solubility and oral bioavailability.n n on . n

[0078] Data that has been obtained clearly show TAK1 kinase is strongly associated withdegenerating neurons in AD brain and that overexpression of the kinase can drive neurodegeneration. Data using NFD-L1 show that TAK1 inhibition in a mouse model of neurodegeneration decreases tau pathology and improves performance in behavioral tests. It is contemplated that UK5141 will have improved water solubility relative to NFD-L1, will be brain penetrant as predicted in silico, and that it will be a potent compound against tau pathology in Alzheimer’s disease.

[0079] Data also show that active TAK1 kinase is strongly associated with degeneratingneurons in traumatic chronic encephalopathy (TCE) associated with repeat head injuries in athletes. Therefore, it is contemplated that UK5141 would also have utility in connection with head injury.

[0080] Example 2: Synthesis of Additional Compounds

[0081] Derivatives of UK5141 were considered and identified. Synthetic schemes weredeveloped. Scheme 1aa

[0082] Reaction and Conditions: (a) K2CO3 (1 equiv), 1- (bromomethyl)-4-nitro-2- (trifluoromethyl)benzene (1 equiv), DMF, rt, overnight; (b) Pd / C (0.04 equiv), H2, rt, 2 h; (c) CDI (1 equiv), Et3N (2 equiv), (3-fluoro-4-(1H-imidazol-1-yl)phenyl)methanamine (1 equiv), DMF, rt, overnight. Scheme 2aBoc, - (trifluoromethyl)benzene (1 equiv), DMF, rt, overnight; (b) TFA (7.5 equiv), DCM, rt, 2 h; (c) NaH (2 equiv), MeI (1 equiv), THF, 0 ⁰C – rt, 15 mins; (d) Pd / C (0.04 equiv), H2, rt, 2 h; (e) CDI (1 equiv), Et3N (2 equiv), (3-fluoro-4-(1H-imidazol-1-yl)phenyl)methanamine (1 equiv), THF, 0 ⁰C – rt, overnight.

[0084] 1H and 13C NMR spectra were recorded on a Bruker 400 MHz and 600 MHz. Thechemical shifts were reported in parts per million (ppm) using the δ 7.26 signal of CDCl3, δ 3.31 signal of CD3OD and δ 2.50 signal of DMSO-d6 (1H NMR), the δ 77.16 signal of CDCl3, δ 49.00 signal of CD3OD and δ 39.52 signal of DMSO- d6(13C NMR) as internal standards. The following abbreviations explained the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet. HR-ESI-MS experiments were acquired with an Agilent 6230 TOF system. Flash column chromatography was performed using Biotage Isolera Foyrand Biotage KP-SIL SNAP cartridges. Analytical thin-layer chromatography with fluorescence F254 indicator (TLC; EMD Chemicals Inc., Darmstadt, Germany) was used for routine reaction progress assessment. Purity was assessed using an analytical HPLC on an Agilent 1260 infinity II with diode array detection at 214 or 280 nm. A Poroshell 120 EC- C18 column (4.6 x 100 mm, 2.7 mm) with a gradient mobile phase of 5-100% acetonitrile / 0.1% TFA in water was utilized. All compounds were confirmed to be ≥ 95% pure before testing. All commercially available reagents were used without further purification, and purchased from Sigma-Aldrich, Ambeed, and Enamine.

[0085] Example 3: 1-methyl-4-(4-nitro-2-(trifluoromethyl)benzyl)piperazine (2)

[0086] To a solution of 1- (bromomethyl)-4-nitro-2-(trifluoromethyl)benzene (200 mg, 1equiv.) in DMF (2 mL), was added N-methylpiperazine (155 μL, 2 equiv.) and potassium carbonate (97 mg, 1 equiv.). The reaction mixture was stirred overnight at room temperature. Upon completion, the resulting mixture was poured into water (15 mL) and extracted with DCM (3 x 15 mL). Combined organic extracts were washed with brine water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography on silica gel using ethyl acetate / hexanes to produce 2 as a light yellow solid (192 mg, 90% yield).1H NMR (400 MHz, CDCl3) δ 8.49 (d, J = 2.2 Hz, 1H), 8.36 (dd, J = 8.6, 2.3 Hz, 1H), 8.07 (d, J = 8.6 Hz, 1H), 3.75 (s, 2H), 2.58 (s, 8H), 2.36 (s, 3H).

[0087] Example 4: 4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)aniline (3)

[0088] To a solution of 2 (192 mg, 1 equiv.) in ethanol (2 mL), Pd / C (40 mg, 10% wt / wt,0.04 equiv.) was added and stirred under H2(balloon) for 2 hours at room temperature. The solid was filtered off, and the filtrate was concentrated. The filtrate was purified by flash column chromatography on silica gel using methanol / dichloromethane to produce 3 as a white solid (164 mg, 95% yield).1H NMR (400 MHz, CDCl3) δ 7.42 (d, J = 8.3 Hz, 1H), 6.89 (d, J = 1.8 Hz, 1H), 6.76 (d, J = 8.3 Hz, 1H), 3.88 (s, 2H), 3.51 (s, 2H), 2.49 (s, 8H), 2.30 (s, 3H).

[0089] Example 5: 1-(3-fluoro-4-(1H-imidazol-1-yl)benzyl)-3-(4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)urea (4)

[0090] To a solution of 3 (30 mg, 1 equiv.) in DMF (1 mL), 1,1’-Carbonyldiimidazole(52 mg, 1 equiv.) and triethylamine (31μL, 2 equiv.) were added. The reaction mixture was stirred at room temperature for 3 hours then (3-fluoro-4-(1H-imidazol-1- yl)phenyl)methanamine (21 mg, 1 equiv.) was added. The reaction was stirred at room temperature overnight. Upon completion, the resulting mixture was poured into water (15 mL) and extracted with DCM (3 x 15 mL). Combined organic extracts were washed with brine water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography on silica gel using methanol / dichloromethane to produce 4 as a white solid (38 mg, 70% yield).1H NMR (400 MHz, DMSO- d6) δ 9.04 (s, 1H), 7.98 (s, 1H), 7.90 (d, J = 1.6 Hz, 1H), 7.58 (t, J = 8.1 Hz, 1H), 7.55 – 7.49 (m, 3H), 7.37 (dd, J = 12.0, 1.8 Hz, 1H), 7.27 (dd, J = 8.2, 1.5 Hz, 1H), 7.12 – 7.07 (m, 1H), 6.89 (t, J = 6.1 Hz, 1H), 4.35 (d, J = 6.1 Hz, 2H), 3.49 (s, 2H), 2.36 (s, 8H), 2.18 (s, 3H).13C NMR (151 MHz, DMSO- d6) δ 155.28 (s, 1C), 154.14 (d, J = 247.8 Hz, 1C), 142.58 (d, J = 6.6 Hz, 1C), 139.69 (s, 1C), 137.28 (d, J = 3.5 Hz, 1C), 131.32 (s, 1C), 129.12 (s, 1C), 128.83 (s, 1C), 127.46 (q, J = 29.4 Hz, 1C), 124.40 (q, J = 274.5 Hz, 1C), 125.60 (s, 1C), 123.71 (d, J = 2.8 Hz, 1C), 123.33 (d, J = 11.7 Hz, 1C), 120.86 (s, 1C), 120.13 (s, 1C), 115.23 (d, J = 20.5 Hz, 1C), 114.40 (q, J = 5.6 Hz, 1C), 57.20 (s, 1C), 54.20 (s, 2C), 51.81 (s, 2C), 44.82 (s, 1C), 41.86 (s, 1C). HR-ESI-MS (m / z): calcd for C24H27F4N6O, 491.2177 [M + H]+; found 491.2171. HPLC Rt = 2.203 min; purity = 98%.

[0091] Example 6: General procedure for oxalate salt formation. To a stirred clearsolution of the appropriate free base (1 equiv.) in DCM, a solution of oxalic acid (1 equiv.) in ether was added resulting in white precipitation. The solvent was removed under vacuum and washed with ether. The compound was dried completely using a lyophilizer to get the corresponding oxalate salt. Oxalate salt of 1-(3-fluoro-4-(1H-imidazol-1-yl)benzyl)-3-(4-((4-methylpiperazin-1- yl)methyl)-3-(trifluoromethyl)phenyl)urea (4) A white solid. HPLC Rt = 2.202 min; purity = 100%. Anal. Calcd for C26H28F4N6O5·0.5H2O: C, 53.06; H, 4.95; N, 14.06%. Found: C, 52.97; H, 4.96; N, 14.26%. Oxalate salt of 1-(3-fluoro-4-(1H-imidazol-1-yl)benzyl)-3-(4-((-3-methyl-3,8- diazabicyclo[3.2.1]octan-8-yl)methyl)-3-(trifluoromethyl)phenyl)urea (12)A white solid. HPLC Rt = 2.257 min; purity = 100%. Anal. Calcd for C28H30F4N6O5·1H2O: C, 53.84; H, 5.16; N, 13.46%. Found: C, 53.77; H, 5.16; N, 13.46%.

[0092] Example 7: Toxicity Studies, Solution Properties

[0093] ADME-Toxicity Studies to determine solution properties, in vitro absorption, invitro metabolism and in vitro toxicity were conducted for UK5141.

[0094] Aqueous solubility was measured in PBS (pH 7.4) using the shake flask methodwith an incubation time of 24 hours at room temperature. Soluble drug concentrations were measured using HPLC with UV / Vis detection. The mean partition coefficient (log D) was measured using the shake-flask method with an incubation time of 60 minutes at RT using n- octanol / PBS (pH 7.4). Drug concentrations were quantified using HPLC with UV / Vis detection with a mean partition coefficient = 2.79.

[0095] Example 8: Membrane Permeability

[0096] To assess membrane permeability of UK5141, Caco-2 and MDR1-MDCKII cellswere cultured on semipermeable membranes and treated with UK514 added to either side of the cell monolayer. The plate was incubated at 37° C for 60 minutes, and UK5141 concentrations quantified in each chamber using HPLC / MS / MS to measure the permeability of the compound. Drug concentrations were measured at time 0 and at 60 minutes for A-B transport and time 0 and 40 minutes for B-A transport. To assess the role of p-glycoprotein (P-gp) on transport experiments were carried out in the presence or absence of verapamil to block P-gp transport. Mean permeability and percent recovery are shown in Table 2. Collectively, the data show that UK5141 is membrane permeable and not a potential substrate for efflux transporters. Table 2. Mean permeability and percent recovery of Caco-2 and MDR1-MDCKII cells treated with UK5141 in the presence or absence of verapamil. Mean Mean yy

[0097] Intrinsic clearance of UK5141 was measured in a mixture of microsomes andcytosol (S9 fraction 0.3 mg / ml) prepared from human, mouse and Sprague-Dawley rat livers as well as human liver microsomes (0.1 mg / ml). UK5141 was incubated with S9 fractions and appropriate cofactors at 37°C and aliquots collected and enzyme activity stopped at T = 0,15,30,45 and 60 minutes. Drug concentrations in samples at each time point were quantified using HPLC / MS / MS with results reported as % compound remaining, the clearance half-life (minutes) and the Clint (µl / min / mg protein) (Table 3). Intrinsic clearance by human liver microsomes was measured using microsomes at 0.1 mg / ml incubated at 37°C with samples collected for drug quantification at 0,15,30,45 and 60 min using HPLC / MS / MS. Collectively, the data are consistent with UK5141 having a relatively long half-life and being slowly cleared. Table 3. Clearance of UK5141 by human, rat and mouse liver microsomal fraction S9 andhuman liver microsomes. )maximal inhibitory concentration (IC50) of UK5141 was measured for CYP1A (phenacetin substrate), CYP2C9 (diclofenac substrate), CYP2C19 (omeprazole substrate) CYP2D6 (dextromethorphan substrate), and CYP3A (midazolam substrate). Results of the assays showed IC50values of 1.6 X 10-6M for CYP1A, 6.8 X 10-7M for CYP2C9, < 3.0 X 10-8M for CYP2C19, 5.8 X 10-8M for CYP2D6 and <3.0 X 10-8M for CYP3A.

[0099] Example 9: In vitro toxicity

[0100] To test in vitro toxicity of UK5141 Eurofins carried out an image-based highcontent assays that measured sensitive cellular parameters including mitochondrial membrane potential, intracellular free calcium, membrane permeability, nuclear size and cell proliferation in live HepG2 cells. Effects of UK5141 tested at a concentration of 10 µM for 72 hours were compared to those observed for a standard drug (cerivastatin). Results of the assays showed 10 µM UK5141 led to 80% reduction in cell number compared to 100% reduction with 0.3 µM cerivastatin. Nuclear size showed a 19% reduction with UK5141 treatment compared to 100% reduction with 5.7 µM cerivastatin. Intracellular free calcium levels showed a 58% increase with 10 µM UK5141 compared to a 100% increase with 0.046 µM cerivastatin. Membrane permeability increased 110% with 10 µM UK5141 compared to a 100% increase with 0.4 µM cerivastatin. Mitochondrial membrane potential was reduced 58% with 10 µM UK5141 treatment compared to 100% decrease with 0.6 µM cerivastatin.

[00101] Example 10: Preclinical Pharmacokinetics

[0102] To verify brain penetrance of UK5141 and to determine single dosepharmacokinetics groups of male C57Bl / 6J mice were provided 9.2 mg / kg UK5141 by oral gavage and plasma and brain concentrations of active measured using HPLC-MS / MS at 1,2,4,8 and 24 hours post gavage. No adverse clinical observations were recorded. Pharmacokinetic calculations were carried out using WinNolin 8.3.5 IP & Tissue- Noncompartmental model 200 and a linear / log trapezoidal / nominal dose method. Pharmacokinetic parameters for brain and plasma are shown in Table 4. Collectively, the data show that UK5141 is able to cross the blood brain barrier. Table 4. PK Parameter Mean Brain Mean Plasma l

[00103] Example 11: In vivo efficacy in mouse model of tau pathology.

[0104] To test the efficacy of UK5141 in a mouse model of tau pathology UK5141(~200 mg / kg / d in diet) or control diet was provided to male mice containing the P301S mutation in the microtubule associated protein tau associated with frontotemporal dementia from 6.5 to 7.5 months of age (7 in each group). P301S mice were chosen for study because they demonstrate progressive accumulation of phosphorylated tau beginning at ~3 months of age and generally demonstrate prominent phosphorylated tau by 8 months of age. The mice can also begin to demonstrate hind limb weakness by the same age.

[00105] To determine if a high dose of UK5141 provided therapeutically late inprogression of tau pathology dosing for 1 month from 6.5 – 7.5 months of age was conducted. Following dosing animals were sacrificed and brains collected for pTau and activated (phosphorylated) TAK1 (pTAK1) quantification by immunofluorescence microscopy. Blinded analysis of immunostained samples showed UK5141 treatment led to a significant (p < 0.05) 75% decrease in TAK1 phosphorylation that corresponded to a marginally significant (P = 0.08) 43% decrease in tau pathology. The immunostaining also suggested that the TAK1 phosphorylation and activation preceded tau phosphorylation.

[00106] It will be understood that various details of the presently disclosed subjectmatter can be changed without departing from the scope of the subject matter disclosed herein. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.

Claims

CLAIMS What is claimed is:

1. A compound of the formula:N , ,N a pharmaceutically-acceptable salt thereof,, 3. The compound of claim 1, of the formula: O R2H ,N a pharmaceutically-acceptable salt thereof.of the formula:a pharmaceutically-acceptable salt thereof.

6. A composition comprising a compound of any one of claims 1-5 and a pharmaceutically-acceptable carrier.

7. A method of inhibiting TAK1 in a cell, comprising: administering the compound of any one of claims 1-5 to the cell.

8. The method of claim 7, wherein the cell is in a subject.

9. The method of claim 8, wherein the subject has or is at an increased risk of developing neurodegeneration in Alzheimer’s disease.

10. The method of claim 8, wherein the subject has or is at an increased risk of head injury.

11. The method of claim 8, wherein the compound is orally administered.

12. A method of treating a condition characterized by degenerating neurons, comprising: administering the compound of any one of claims 1-5 to a subject in need thereof.

13. The method of claim 12, wherein the condition is Alzheimer’s disease.

14. The method of claim 12, wherein the condition is head injury, concussions, traumatic brain injury, or traumatic chronic encephalopathy.

15. The use of the compound of any one of claims 1-5 in the manufacture of a medicament for the treatment of a condition characterized by degenerating neurons.

16. The use of claim 15, wherein the condition is Alzheimer’s disease.

17. The use of claim 15, wherein the condition is head injury, concussions, traumatic brain injury, or traumatic chronic encephalopathy.

18. The use of the compound of any one of claims 1-5 in the manufacture of a medicament for the treatment of a subject having or at an increased risk of developing neurodegeneration in Alzheimer’s disease.

19. The use of the compound of any one of claims 1-5 in the manufacture of a medicament for the treatment of a subject having or at an increased risk of having head injury.

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