N-desmethylruboxistaurin as a kinase inhibitor

Abstract

Aspects of the invention relate to the use of N-desmethylruboxistaurin and its pharma- ceutically acceptable formulations to modulate GSK3β signaling. Some aspects of the invention relate to the use of N-desmethylruboxistaurin to inhibit protein kinase C. Some aspects of the invention provide a method of using N-desmethylruboxistaurin in treating subjects with neurological and / or psychiatric disorders, including Alzheimer's disease, frontotemporal dementia, behavioral complications of dementia, bipolar disorder, depression, schizophrenia, Parkinson's disease, or neuroinflammation. Some aspects of the invention provide a method of using N-desmethylruboxistaurin in treating diabetes or its complications, or conditions associated with ischemia, inflammation, pulmonary hypertension, congestive heart failure, cardiovascular disease, skin disease, or cancer. Some aspects of the invention relate to the use of N-desmethylruboxistaurin as an alternative to ruboxistaurin, with a more favorable pharmacokinetic profile, less potential for electrocardiogram QT interval prolongation, and / or less potential for adverse drug interactions. In some embodiments, N-desmethylruboxistaurin is administered in combination with lithium or another treatment for bipolar disorder.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 362,293, filed March 31, 2022, the disclosure of which is incorporated herein by reference.

[0002] Aspects of the invention relate to methods of treating neurological or psychiatric disorders including Alzheimer's disease, frontotemporal dementia, behavioral complications of dementia, bipolar disorder, depression, schizophrenia, Parkinson's disease or neuroinflammation, and methods of treating diabetes and its complications, or ischemia, inflammation, pulmonary hypertension, congestive heart failure, cardiovascular disease, skin diseases, cancer, or GM2 gangliosidosis, or other conditions in which ruboxistaurin is clinically useful. [Background technology]

[0003] Ruboxistaurin has been shown to modulate GSK3 signaling and inhibit protein kinase C.

[0004] As a GSK3 inhibitor, ruboxistaurin has been proposed as a treatment for subjects with neurological and / or psychiatric disorders, including Alzheimer's disease, frontotemporal dementia, behavioral complications of dementia, bipolar disorder, depression, schizophrenia, Parkinson's disease, or neuroinflammation. Inhibitors of GSK3 are known to increase the expression of WNT proteins, thereby enhancing regenerative medicine pathways that have been widely proposed for the treatment of neurological and psychiatric disorders and for reducing neuroinflammation. Inhibition of GSK3 or enhancement of WNT signaling has been implicated as a potential treatment for type 2 diabetes and kidney disorders including diabetic nephropathy, chronic kidney disease, polycystic kidney disease, focal segmental glomerulosclerosis, atherosclerosis, alopecia, bone and joint disorders including osteoarthritis and osteoporosis, inflammatory disorders including alcoholic hepatitis, inflammatory bowel disease and septic shock, and eye disorders including exudative age-related macular degeneration, dry age-related macular degeneration, diabetic macular edema, Fuchs' corneal dystrophy, corneal epithelial cell defect, dry eye, glaucoma, familial exudative vitreoretinopathy (FEVR), Norrie's disease, Coats' disease, retinopathy of prematurity, macular telangiectasia, retinal vein occlusion and Sjogren's syndrome. Inhibition of GSK3 or enhancement of WNT signaling has been implicated as a potential treatment for ear disorders including sensorineural and conductive hearing loss, lung disorders including chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis, short bowel syndrome, and cancers including melanoma, pancreatic cancer, prostate cancer, colon cancer, and leukemia. As a GSK3 inhibitor, the use of ruboxistaurin has been proposed as a monotherapy for treating bipolar disorder, or in combination with lithium, or in combination with other bipolar disorder medications.

[0005] As a protein kinase C inhibitor, ruboxistaurin has been proposed to treat conditions associated with diabetes, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, ischemia, inflammation, pulmonary hypertension, congestive heart failure, cardiovascular disease, skin diseases, cancer, and GM2 gangliosidosis. Protein kinase C inhibition has also been suggested to be useful in the treatment of bipolar disorder and Alzheimer's disease.

[0006] However, ruboxistaurin's pharmacokinetics include a high peak-to-trough ratio, and ruboxistaurin has been shown to prolong the QT interval in human subjects. Additionally, ruboxistaurin levels can be elevated by drugs that inhibit CYP3A4 metabolism.

[0007] N-desmethylruboxistaurin is a metabolite of ruboxistaurin. Summary of the Invention

[0008] Aspects of the present invention relate to the use of N-desmethylruboxistaurin as a therapeutic agent in situations where ruboxistaurin is clinically useful, and as a potentially safer alternative to the use of ruboxistaurin.

[0009] One aspect of the present invention relates to a method of treating a disorder involving aberrant signaling of GSK3β or protein kinase C by administering a therapeutically effective dose of N-desmethylruboxistaurin or a pharma- ceutically acceptable salt thereof or a pharmaceutical composition thereof to a subject in need thereof. In other words, the present invention provides N-desmethylruboxistaurin or a pharma- ceutically acceptable salt thereof for use in the treatment of a disorder characterized by aberrant signaling of GSK3β or protein kinase C by administering a therapeutically effective dose of N-desmethylruboxistaurin or a pharma- ceutically acceptable salt thereof to a subject in need thereof. The method may be applicable when N-desmethylruboxistaurin is administered to a subject who: 1) has never taken ruboxistaurin; 2) is taking ruboxistaurin and experiencing side effects; 3) has demonstrated a prolonged QT interval; 4) has demonstrated high plasma concentrations of ruboxistaurin; 5) may be receiving drugs that may interfere with the metabolism of ruboxistaurin; or 6) may require high doses of ruboxistaurin and for which there is a concern about side effects, QT prolongation, or adverse drug interactions.

[0010] The subject may have a neurological disease and / or a psychiatric disorder. The disease / disorder may be selected from Alzheimer's disease, frontotemporal dementia, behavioral complications of dementia, bipolar disorder, depression, schizophrenia, Parkinson's disease and / or neuroinflammation.

[0011] The subject is a patient suffering from diabetes, diabetic neuropathy, diabetic retinopathy, diabetic nephropathy, chronic kidney disease, atherosclerosis, alopecia, osteoarthritis, osteoporosis, alcoholic hepatitis, inflammatory bowel disease, exudative age-related macular degeneration, atrophic age-related macular degeneration, diabetic macular edema, Fuchs' corneal dystrophy, corneal epithelial cell defect, dry eye, glaucoma, familial exudative vitreoretinopathy (FEVR), Norrie's disease, Coats' disease, or retinopathy of prematurity. , macular telangiectasia, retinal vein occlusion, Sjogren's syndrome, sensorineural hearing loss, conductive hearing loss, schizophrenia, Parkinson's disease, polycystic kidney disease, focal segmental glomerulosclerosis, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, short bowel syndrome, melanoma, pancreatic cancer, prostate cancer, colon cancer, leukemia, septic shock, ischemia, inflammation, pulmonary hypertension, congestive heart failure, cardiovascular disease, skin disease, cancer, or GM2 gangliosidosis.

[0012] N-desmethylruboxistaurin or a pharma- ceutically acceptable salt thereof or a pharmaceutical composition thereof may be administered in combination with lithium for bipolar disorder or other conditions in which inhibition of GSK3, protein kinase C, or both is beneficial. Concomitant treatments for bipolar disorder other than lithium may include valproic acid, lamotrigine, quetiapine, olanzapine, risperidone, aripiprazole, lurasidone, lumateperone, cariprazine, asenapine, and carbamazepine.

[0013] Lithium may be administered at a non-effective dose as a monotherapy and N-desmethylruboxistaurin may be administered at a non-effective dose as a monotherapy, the non-effective dose being a dose that reduces kidney damage or does not cause kidney damage.

[0014] The subject may be lithium non-responsive or lithium responsive.

[0015] Another aspect of the invention relates to a method for establishing a diagnosis of bipolar disorder or other conditions in which GSK3 inhibition is clinically useful by administering to a subject to be evaluated a therapeutically effective dose of N-desmethylruboxistaurin or a pharma- ceutically acceptable salt thereof or a pharmaceutical composition thereof and assessing the clinical response of said subject.

[0016] A further aspect of the invention relates to a method of establishing an appropriate therapeutic dose of N-desmethylruboxistaurin in a subject by administering increasing doses of N-desmethylruboxistaurin to said subject and assessing the response using GSK3 imaging or GSK3 serology.

[0017] Yet another aspect of the present invention relates to a method of treating a subject having Alzheimer's disease, bipolar disorder or depression who shows evidence of elevated GSK3 by administering to the subject a therapeutically effective dose of N-desmethylruboxistaurin or a pharma- ceutically acceptable salt thereof or a pharmaceutical composition thereof and evaluating and monitoring the subject using positron emission tomography (PET) or serology.

[0018] Yet another aspect of the invention relates to a method for establishing a diagnosis of bipolar disorder or other conditions in which GSK3 inhibition is clinically useful by administering to a subject to be evaluated a therapeutically effective dose of N-desmethylruboxistaurin or a pharma- ceutically acceptable salt thereof or a pharmaceutical composition thereof together with a therapeutically effective dose of lithium, and assessing the clinical response of said subject, wherein the doses of both N-desmethylruboxistaurin and lithium may be non-effective doses for monotherapy.

[0019] A further aspect of the invention relates to a method of treating a subject with Alzheimer's disease having evidence of elevated GSK3 activity by administering to the subject a therapeutically effective dose of N-desmethylruboxistaurin or a pharma- ceutically acceptable salt thereof or a pharmaceutical composition thereof and a therapeutically effective dose of lithium, and monitoring the subject using positron emission tomography (PET).The doses of both N-desmethylruboxistaurin and lithium may be non-effective doses for monotherapy.

[0020] Another aspect of the invention relates to a method of establishing an appropriate therapeutic dose of N-desmethylruboxistaurin in a subject by administering to the subject increasing doses of N-desmethylruboxistaurin and lithium and assessing the response using positron emission tomography (PET). [Brief description of the drawings]

[0021] [Figure 1] Illustrates the QT interval of an electrocardiogram (ECG) tracing, which shows the portion of the cardiac contraction cycle that begins with contraction of the left ventricle and ends with expansion of the left ventricle.

[0022] [Diagram 2] 1 shows a diagram of the therapeutic window of a drug on a graph of % of maximum effect versus drug concentration.

[0023] [Diagram 3] 1 shows the chemical structure of ruboxistaurin.

[0024] [Figure 4] The chemical structure of N-desmethylruboxistaurin is shown.

[0025] [Diagram 5] 1 shows a graph of the mean plasma concentration versus time profile following a single 32 mg dose of ruboxistaurin in healthy subjects, with the results in the inset being in the fed state.

[0026] [Figure 6] 1 shows a synthesis scheme of N-desmethylruboxistaurin.

[0027] [Figure 7] 1 shows the ability of ruboxistaurin and N-desmethylruboxistaurin to inhibit GSK3β and GSK3α at various concentrations.

[0028] [Figure 8] 1 shows the stability of ruboxistaurin and N-desmethylruboxistaurin in human liver microsomes.

[0029] [Figure 9] We demonstrate the ability of N-desmethylruboxistaurin to provide pharmacological effects similar to lithium in rats and to reduce dextroamphetamine-induced positive ultrasonic vocalizations. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Unless otherwise defined, all terms used in disclosing the present invention, including technical and scientific terms, have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0031] As disclosed herein, several ranges of values ​​are provided. Unless the context dictates otherwise, it is understood that each intervening value between the upper and lower limits of that range is also specifically disclosed to one tenth of the unit of the lower limit. Each smaller range between any stated value or intervening value within a stated range and any other stated value or intervening value within that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the range, and each range with either or neither limit or both limits included is also encompassed within the invention, subject to the specifically excluded limit within the stated range. If one or both limits are included in a stated range, then ranges excluding either or both of those included limits are also encompassed within the invention. The term "about" generally includes ±10% of the stated value. For example, "about 10%" may indicate a range of 9% to 11%, and "about 20" may mean 18 to 22. Preferably, "about" includes ±6% of the stated value. Alternatively, "about" includes ±5% of the stated value. Other meanings of "about" may be apparent from the context, such as rounding, so that, for example, "about 1" may mean from 0.5 to 1.4.

[0032] The term "pharmaceutical acceptable salt" of a compound refers to a salt that is pharmaceutically acceptable and possesses the desired pharmacological activity of the parent compound. Pharmaceutically acceptable salts are understood to be non-toxic. Such salts include acid addition salts formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, or with acids such as formic acid, acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanes ... These include acid addition salts formed with organic acids such as sulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, glucoheptonic acid, 4,4'-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like. Additional information regarding suitable pharma- ceutically acceptable salts is found in Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Company, Easton, PA, 1985, which is incorporated herein by reference.

[0033] As used herein, the term "therapeutically effective dose" refers to the amount of the compound of the present invention that can alleviate the symptoms of various pathological conditions described herein. Of course, the specific dose of the compound administered according to the present invention is determined by the particular circumstances surrounding the case, including, for example, the compound administered, the route of administration, the condition of the patient, and the pathological condition being treated. Administration can be once a day or multiple times a day (e.g., two, three or more times a day).

[0034] The effective dose of N-desmethylruboxistaurin or a pharma- ceutically acceptable salt thereof in monotherapy is about 32 to about 320 mg once a day, or about 16 to about 160 mg twice a day. Pharmaceutical compositions of N-desmethylruboxistaurin or a pharma- ceutically acceptable salt thereof further comprise at least one pharma- ceutically acceptable adjuvant or excipient.

[0035] In the case of combination therapy, the non-effective dose of N-desmethylruboxistaurin or its pharma- ceutically acceptable salt is about 8 to about 32 mg once a day, or about 4 to about 16 mg twice a day. When N-desmethylruboxistaurin is combined with lithium, the non-effective dose of lithium is about 60 mg to about 600 mg once a day, or about 30 mg to about 300 mg twice a day. This non-effective dose of lithium can prevent kidney damage typically caused by lithium therapy. The effective dose of N-desmethylruboxistaurin or its pharma-ceutically acceptable salt once a day may be about 32, about 64, about 96, about 128, about 160, about 192, about 224, about 256, about 288, or about 320 mg. An effective dose twice daily of N-desmethylruboxistaurin or a pharmaceutically acceptable salt thereof may be about 16, about 32, about 48, about 64, about 80, about 96, about 112, about 128, about 144 or about 160 mg. An ineffective dose once daily of N-desmethylruboxistaurin or a pharmaceutically acceptable salt thereof may be about 8, about 16, about 24 or about 32 mg. An ineffective dose twice daily of N-desmethylruboxistaurin or a pharmaceutically acceptable salt thereof may be about 4, about 8, about 12 or about 16 mg.

[0036] Ruboxistaurin has been studied in several clinical trials for the treatment of diabetes and its complications, including diabetic retinopathy, diabetic neuropathy, and diabetic nephropathy. See U.S. Patent Application Publication No. 2008 / 0096923 to A. Girach, incorporated herein by reference in its entirety. It has been described as having good safety profile and a lower incidence of serious adverse events than placebo. The clinical safety profile of ruboxistaurin and the growing interest in the use of therapeutic agents capable of inhibiting GSK3 encourage further development of ruboxistaurin. In addition, ruboxistaurin is currently proposed to enter clinical trials soon as a treatment for GM2 gangliosidosis.

[0037] However, careful review of the clinical and preclinical data for ruboxistaurin has determined that ruboxistaurin has the potential to prolong the QT interval in human subjects, potentially increasing the risk of dangerous cardiac arrhythmias, particularly when ruboxistaurin is administered in the setting of accidental or intentional overdose, when used in combination with other drugs that prolong the QT interval, or when extremely high ruboxistaurin levels are induced by concomitant administration of drugs that inhibit CYP3A4.

[0038] Evidence of these potential risks is documented in a Withdrawal Assessment Report prepared by the European Medicines Agency (EMA). The report comes as a Marketing Authorization Application for ruboxistaurin was submitted to the European Medicines Agency but was withdrawn. The Withdrawal Assessment Report showed that ruboxistaurin inhibits hERG, a potassium ion channel known to contribute to the electrical activity of the heart. This preclinical assessment is commonly used to identify compounds with a potential risk of QT prolongation.

[0039] The QT interval is part of the electrocardiogram (ECG) tracing and represents the portion of the cardiac contraction cycle that begins with left ventricular contraction (the letter "Q" represents the beginning of the "QRS" complex in the waveform) and ends with left ventricular expansion (the end of the "T" wave). The QT interval should generally be 425 milliseconds or less. If the QT interval is too long, the heart's expansion will not be complete before the next electrical signal is initiated, which can cause dangerous arrhythmias. See Figure 1.

[0040] In the hERG assay, ruboxistaurin had an IC of 35.6 nM. 50 Ruboxistaurin blocks the hERG channel at 100-200 Hz, resulting in a predicted increase in the QT interval of 5 milliseconds. Further evaluation of the drug's cardiac safety in dogs revealed no issues, but there were concerns over the results of a "through QT" study conducted in human subjects administered ruboxistaurin. This type of study is performed by taking multiple ECG tracings at various time intervals after exposure to the test drug.

[0041] Ruboxistaurin prolonged the QT interval by 6 ms in a through human QT study. Furthermore, several patients in the study were identified who experienced a prolongation of the QT interval of 10 ms or more. This potential safety concern was the basis for major opposition by the European Medicines Agency.

[0042] A positive hERG assay predicts QT prolongation, which is associated with life-threatening cardiac arrhythmias, including torsades de pointes. These tests alone are not perfect predictors of cardiac risk. However, avoidance of QT prolongation is considered clinically desirable. Pharmaceutical companies have sometimes discontinued development of products that prolong the QT interval by more than 5 milliseconds.

[0043] Even when a drug with QT prolongation is approved, it may carry a safety warning on the prescription, such as ziprasidone, whose FDA label states, "When selecting alternative available treatments for schizophrenia, prescribers should consider the finding that ziprasidone has a greater ability to prolong the QT / QTc interval compared with some other antipsychotics (see WARNINGS)." Although torsades de pointes are not listed on ziprasidone's FDA label, cardiologists prefer other agents in this class because of concerns about QT prolongation.

[0044] QT prolongation poses great concern for potential cardiac arrhythmias when high doses are administered, when used concomitantly with other drugs that prolong the QT interval, in the setting of accidental or intentional overdose, and when other drug interactions inhibit the metabolism of the drug resulting in high plasma exposure to the drug. All of these situations apply to ruboxistaurin.

[0045] Higher doses of ruboxistaurin are currently being studied for the treatment of congestive heart failure. Although 32 mg of ruboxistaurin was widely used in the development of ruboxistaurin for the treatment of diabetic retinopathy, doses up to 256 mg have been studied for the treatment of heart failure. One of the primary outcome measures in heart failure trials is the proportion of patients with QT interval prolongation.

[0046] The pharmacokinetics of a compound are also important when considering the potential for QT prolongation. The arrhythmia potential is related to the maximum plasma concentration of the compound. This has been observed in ECG studies that showed maximum prolongation of the QT interval at the highest concentration of the test drug.

[0047] High peak concentrations that cause serious prolongation of the QT interval may not occur in most patients in most cases, but may be a problem if there is an accidental or intentional overdose. This principle is clearly stated in the guidelines for the use of tricyclic antidepressants. Physicians are instructed to monitor patients' ECGs during treatment with tricyclic antidepressants that prolong the QT interval. Importantly, physicians are further instructed to reduce the possibility of dangerous overdoses and cardiac arrhythmias by prescribing only small amounts at a time. As a general rule, an overdose of 30 tablets is safer than an overdose of 100 tablets.

[0048] In typical use, the peak concentration of a drug can be reduced (compared to the trough concentration of the drug) when the half-life is longer. For example, a drug with a half-life of 24 hours can be administered once daily with a peak drug concentration that is only about twice the trough concentration. In contrast, a drug with a half-life of 6 hours, when administered once daily, can have a peak concentration that is about 16 times the trough concentration.

[0049] A principle of drug development is that low peak concentrations (compared to trough concentrations) are usually desirable to keep drug levels within the therapeutic window. There is a dose response for efficacy and a dose response for toxicity. A low peak / trough ratio helps keep drug concentrations at levels that are effective without being so high that they cause toxicity. See Figure 2.

[0050] Ruboxistaurin has a high peak concentration of approximately 90 nmol / L compared to its trough concentration of approximately 5 nmol / L (peak / trough ratio of approximately 18, consistent with a half-life of less than 6 hours), which increases the risk of QT prolongation as well as other toxicities, where worsening glycemic control and elevated creatine kinase are of concern to the European Medicines Agency.

[0051] A third consideration affecting the peak concentration of a drug is potential interactions with other drugs. Ruboxistaurin is metabolized by CYP3A4, which converts ruboxistaurin to N-desmethylruboxistaurin (see Figures 3 and 4). Thus, the peak concentration of ruboxistaurin may be elevated in the presence of CYP3A4 inhibitors.

[0052] These considerations are particularly important in situations where a patient may be taking one or more medications that prolong the QT interval. Medications that prolong the QT interval include antipsychotics (haloperidol, ziprasidone, quetiapine, thioridazine, olanzapine, risperidone), antiarrhythmics (amiodarone, sotalol, dofetilide, procainamide, quinidine, flecainide), antibiotics (macrolides, fluoroquinolones), antidepressants (amitriptyline, imipramine, citalopram), and others (methadone, sumatriptan, ondansetron, cisapride).

[0053] Drugs that prolong the QT interval are often prescribed for bipolar disorder, depression, and / or schizophrenia. Patients with these conditions may be prescribed antidepressants or atypical antipsychotics, which are indicated for the treatment of bipolar disorder, depression, and schizophrenia.

[0054] QT prolongation is also a concern because patients with bipolar disorder and depression are at increased risk of attempting suicide, including drug overdose. Additionally, confusion or delusions in patients with psychiatric illness may lead to accidental drug overdose.

[0055] Alzheimer's disease also results in frequent prescription of drugs that prolong the QT interval. Depression or agitation may be present (behavioral complications of dementia), and these symptoms may be treated with antidepressants or atypical antipsychotics, or both at the same time. Patients with Alzheimer's disease are generally older, which increases the risk of being prescribed antibiotics and other medications. In addition, confusion or delusions in patients with Alzheimer's disease may lead to accidental drug overdose.

[0056] Ruboxistaurin is metabolized by CYP3A4, and inhibitors of CYP3A4 include grapefruit juice, itraconazole, voriconazole, ketoconazole, ritonavir, boceprevir, danoprevir, telaprevir, saquinavir, azamulin, erythromycin, troleandomycin, telithromycin, verapamil, diltiazem, ciprofloxacin, cyclosporine, imatinib, cimetidine, ranitidine, and the antidepressants nefazodone and fluvoxamine. Use of these medications may increase peak concentrations of ruboxistaurin and exacerbate QT interval prolongation.

[0057] For these reasons, the use of ruboxistaurin is of particular concern in the treatment of neuropsychiatric disorders, including bipolar disorder, depression, and Alzheimer's disease, all conditions for which new medications are needed, and research into the use of GSK3 inhibitors and ruboxistaurin has been encouraged. The use of ruboxistaurin is also of concern in the treatment of elderly subjects, as older adults are more likely to take multiple medications and are at higher risk for adverse drug interactions.

[0058] Furthermore, the half-life of ruboxistaurin is suboptimal when used in combination with lithium; the elimination half-life of lithium is approximately 18 hours. In a combination product of two drugs, the levels of both products must be within the therapeutic window throughout the dosing interval. Combining two products, one with a short half-life and one with a long half-life, can result in unnecessarily high levels of the short-acting drug immediately after administration and potentially low levels of the same component later in the dosing interval.

[0059] New drugs may be important alternatives to ruboxistaurin if they have similar or greater potency against protein kinase C or GSK3 while at the same time possessing at least one or more characteristics such as similar or less hERG inhibition, longer half-life allowing for lower peak concentrations during the dosing interval (better pharmacokinetics), and lower potential for drug interactions. One aspect of the invention relates to the use of N-desmethylruboxistaurin, which has these characteristics, in situations where ruboxistaurin is clinically useful.

[0060] In the hERG assay, the ability of N-desmethylruboxistaurin to block the hERG channel was slightly lower than that of ruboxistaurin, requiring higher concentrations and an IC 50 However, N-desmethylruboxistaurin was reported to have a t of 62.6 nM compared to 35.6 nM for ruboxistaurin. The half-life (t1 / 2) of N-desmethylruboxistaurin was also reported to be longer than that of ruboxistaurin, 23.9 hours compared to 5.25 hours (Figure 5). CYP3A4 inhibitors can increase the plasma concentration of ruboxistaurin, but have not been reported to increase the plasma concentration of N-desmethylruboxistaurin to the same extent, and the long half-life of N-desmethylruboxistaurin suggests an alternative metabolic and elimination pathway, i.e., hydroxylation. Thus, N-desmethylruboxistaurin is slightly less likely to inhibit hERG, has a better pharmacokinetic profile, can reduce interactions with CYP3A4 inhibitors, and has a more suitable half-life for use with lithium.

[0061] IC for hERG 50 Because the difference in QT interval between ruboxistaurin and N-desmethylruboxistaurin has been reported to be approximately twice that of ruboxistaurin, and the peak plasma concentrations have been reported to be only half that of ruboxistaurin, these features combine to reduce the potential for N-desmethylruboxistaurin to prolong the QT interval or cause other toxicity to approximately one-quarter the level of ruboxistaurin. The difference is even greater in the presence of drugs that inhibit CYP3A4.

[0062] The preparation of N-desmethylruboxistaurin (compound 1) generally follows the method depicted in Figure 6. As shown, starting material 1 is reacted with vinyl Grignard in the presence of copper iodide to produce alcohol intermediate 2. One of skill in the art will recognize that alternatives to the vinyl Grignard are useful in effecting similar transformations. Such alternatives include, but are not limited to, vinyl zinc, vinyl cuprate and vinyl lithium reagents. One of skill in the art will recognize that alternatives to copper iodide are useful in facilitating the conversion of intermediate 1 to intermediate 2. Such alternatives include, but are not limited to, alternative Lewis acid reagents and chelating agents such as crown ethers.

[0063] Following isolation of intermediate 2, Figure 6 shows its conversion to intermediate 3 upon reaction with allyl bromide. One of skill in the art will recognize that alternative allylation agents are useful for allylation of intermediate 2 to intermediate 3. Such allylation agents generally employ alternatives to the bromide leaving group and include, but are not limited to, allyl chloride, allyl iodide, and allyl mesylate. One of skill in the art will also recognize that alternatives to the potassium tert-butoxide base shown are useful in effecting the reaction of intermediate 2 with an allylation agent. Such bases include, but are not limited to, hydride reagents, carbonate reagents, bicarbonate reagents, lithium diisopropylamide, sodium hexamethyldisilazide, and the like.

[0064] FIG. 6 further illustrates a two-step conversion of intermediate 3 to intermediate 4. As shown, the first step is an ozonolysis reaction, resulting in the cleavage of the bis-olefin to a bis-aldehyde, and the second step is a sodium borohydride reduction of the bis-aldehyde to a bis-alcohol. Those skilled in the art will recognize that ozonolysis is only one of several reactions or combinations of reactions suitable for cleaving an olefin to an aldehyde. Such conversions include, but are not limited to, dihydroxylation of the olefin followed by cleavage of the resulting diol to an aldehyde. Reagents suitable for dihydroxylation of the olefin include, but are not limited to, osmium tetroxide, and the like. Reagents suitable for cleaving the diol to an aldehyde include, but are not limited to, sodium periodate, lead tetraacetate, and the like. Those skilled in the art will also recognize that alternatives to the sodium borohydride reducing agent are suitable for the reduction of the aldehyde to an alcohol. Such reagents include, but are not limited to, lithium aluminum hydride, diisopropylaluminum hydride, lithium borohydride, borane, etc. Those skilled in the art will also recognize that alternatives to boron and aluminum based reducing agents are also useful in the reduction of aldehydes to alcohols. Such alternatives include, but are not limited to, samarium iodide and triethylsilane.

[0065] As shown in Figure 6, the diol of intermediate 4 is converted to the bis-mesylate intermediate 5 upon reaction with methanesulfonyl chloride and triethylamine. One of skill in the art will recognize that mesylate as a leaving group is generally useful, as are common alternative leaving groups including, but not limited to, chloride, bromide, iodide, tosylate, and the like. One of skill in the art will also recognize that alternatives to triethylamine are useful in the conversion of alcohols to mesylates. Such alternatives include, but are not limited to, diisopropylethylamine, pyridine, carbonate reagents, bicarbonate reagents, and the like.

[0066] The reaction of bismesylate intermediate 5 with bisindolylmaleimide intermediate 6 to form intermediate 7 is shown in Figure 6 using cesium carbonate as the base. One of skill in the art will recognize that alternative bases are useful in effecting the reaction to the depicted intermediate 7. Such bases include, but are not limited to, hydrides, alkoxides, carbonates, bicarbonates, and the like.

[0067] The process of converting methylmaleimide to its demethylated version involves the initial hydrolysis of intermediate 7 to maleic anhydride intermediate 8. As shown in Figure 6, this conversion is carried out using potassium hydroxide in ethanol. One of ordinary skill in the art will recognize that the conversion of intermediate 7 to intermediate 8 can use alternatives to potassium hydroxide, including, but not limited to, caustic soda and lithium hydroxide. Additionally, one of ordinary skill in the art will recognize that ethanol can be replaced with any protic solvent, including, but not limited to, methanol and water.

[0068] 6, conversion of maleic anhydride intermediate 8 to the corresponding maleimide intermediate 9 is completed upon reaction of intermediate 8 with hexamethyldisilazane. One of skill in the art will recognize that maleic anhydride can be converted to a maleimide using alternative reagents including, but not limited to, ammonia, sodium amide, and the like.

[0069] With the maleimide established, Figure 6 illustrates the cleavage of the trityl-based protecting group from intermediate 9 to the alcohol intermediate 10. While Figure 6 highlights hydrochloric acid as the reagent that effects the trityl cleavage, one of skill in the art will recognize that alternative acids can be used, including but not limited to hydrobromic acid, trifluoroacetic acid, acetic acid, and the like.

[0070] As shown in Figure 6, the alcohol of intermediate 10 is converted to the mesylate intermediate 11 upon reaction with methanesulfonyl chloride and pyridine. One of skill in the art will recognize that mesylate as a leaving group is generally useful, as are common alternative leaving groups including, but not limited to, chloride, bromide, iodide, tosylate, and the like. One of skill in the art will also recognize that alternatives to triethylamine are useful in the conversion of the alcohol to the mesylate. Such alternatives include, but are not limited to, diisopropylethylamine, pyridine, carbonate reagents, bicarbonate reagents, and the like.

[0071] In the final step of the synthesis, Figure 6 shows the conversion of intermediate 11 to compound 1 upon reaction with methylamine. Although not shown in Figure 6, the methylamine is further converted to its corresponding hydrochloride salt. Those skilled in the art will appreciate that there are alternative strategies for converting compounds such as intermediate 11 to structures such as compound 1. Such strategies are generally recognizable by those skilled in the art, and the above strategies generally include the steps of: Comprehensive Organic Transformations (Larock, Wiley) and other resources.

[0072] Those of skill in the art will recognize that there are many additional reactions useful for the preparation of N-desmethylruboxistaurin (Compound 1), in addition to those described above and illustrated in Figure 6. Suitable reactions can be readily identified by those of skill in the art, Comprehensive Organic Transformations (Larock, Wiley). Strategies for the introduction and cleavage of protecting groups can be readily identified by one of skill in the art. Protective Groups in Organic Synthesis( 6 and other resources such as Greene and Wutz, Wiley. Those skilled in the art will also recognize that all alternative and variant routes to those depicted in FIG. 6 are generally applicable, and alternative combinations of reagents, solvents, temperature conditions and reaction times will result in similar chemical outcomes that allow for the preparation of compound 1.

[0073] The ability of N-desmethylruboxistaurin to inhibit GSK3β (a specific form of GSK associated with bipolar disorder and other neuropsychiatric disorders) was previously unknown. Surprisingly, N-desmethylruboxistaurin was found to be approximately twice as potent as ruboxistaurin in inhibiting GSK3β (FIG. 7). This higher efficacy provides the ability to use N-desmethylruboxistaurin at lower doses than ruboxistaurin.

[0074] N-desmethyl ruboxistaurin was found to have a higher stability than ruboxistaurin in human liver microsomes. After 15 minutes, 76.38% of N-desmethyl ruboxistaurin was found to remain in the liver compared to 3.51% of ruboxistaurin remaining (Figure 8). In this study, the CYP3A4 inhibitor troleandomycin increased the level of ruboxistaurin by 23-fold at 15 minutes, but only increased the level of N-desmethyl ruboxistaurin by 1.2-fold. This indicates that the effect of CYP3A4 on the metabolism of N-desmethyl ruboxistaurin is less than that of ruboxistaurin (Table 1). [Table 1]

[0075] The ability of N-desmethylruboxistaurin to cross the blood-brain barrier was previously unknown. Pharmacokinetic studies in rats demonstrated brain penetration of N-desmethylruboxistaurin with a brain / plasma ratio of 1.18 after 4 hours, supporting the potential therapeutic use of N-desmethylruboxistaurin to treat central nervous system conditions (Table 2). [Table 2]

[0076] The ability of N-desmethylruboxistaurin to have pharmacological effects similar to lithium was previously unknown. N-desmethylruboxistaurin was able to reduce ultrasonic appetitive vocalizations in rats administered dextroamphetamine, and the magnitude of the effect was similar to that of lithium (Figure 9). This supports the possibility that N-desmethylruboxistaurin may provide similar clinical benefits as lithium.

[0077] As an alternative to ruboxistaurin, N-desmethylruboxistaurin can be administered to subjects who have never used ruboxistaurin. As an alternative to ruboxistaurin, N-desmethylruboxistaurin can be administered to subjects who have experienced side effects from ruboxistaurin, to subjects with a prolonged QT interval, to subjects who have demonstrated high drug levels of ruboxistaurin, to subjects who may be receiving drugs that may interfere with the metabolism of ruboxistaurin, or to subjects who may require high doses of ruboxistaurin and are concerned about side effects, QT prolongation, or adverse drug interactions.

[0078] The risk of QT prolongation and other potential toxicities can be further mitigated by administering N-desmethylruboxistaurin with lithium. Both N-desmethylruboxistaurin and lithium inhibit GSK3, and combining lithium with another GSK3 inhibitor has been shown to have synergistic effects in the treatment of bipolar disorder in animal models. When administered in combination with lithium, N-desmethylruboxistaurin can achieve the desired amount of GSK3 inhibition at a lower concentration. Thus, N-desmethylruboxistaurin or a pharmacologic acceptable salt thereof can be administered in combination with lithium for bipolar disorder or other conditions in which inhibition of GSK3, protein kinase C, or both is useful.

[0079] This combination allows for a reduction in the dose of N-desmethylruboxistaurin while simultaneously reducing the dose of lithium required to treat bipolar disorder, Alzheimer's disease, and other conditions where GSK3 inhibition by lithium is clinically desirable, improving safety. The dose of N-desmethylruboxistaurin in the combination may be lower than that required as monotherapy (non-effective dose), and the dose of lithium in the combination may be lower than that required as monotherapy (non-effective dose). This combination can be used to provide efficacy to subjects who do not respond to lithium or who cannot tolerate lithium at standard doses. N-desmethylruboxistaurin can be used to provide additional efficacy as an alternative to higher lithium doses in subjects who have only a partial response to lithium.

[0080] Additionally, ruboxistaurin has been proposed for use in combination with valproic acid, lamotrigine, carbamazepine, gabapentin, and topiramate for the treatment of neurological and / or psychiatric disorders. N-desmethylruboxistaurin has better pharmacokinetics and a lower potential for QT prolongation, and can therefore be used as an alternative to ruboxistaurin in combination with valproic acid, lamotrigine, quetiapine, olanzapine, risperidone, aripiprazole, lurasidone, lumateperone, cariprazine, asenapine, and carbamazepine.

[0081] Antipsychotics that are not known to prolong the QT interval, including olanzapine, risperidone, aripiprazole, lumateperone, xanomeline-trospium, iloperidone, and lurasidone, are also used to treat neurological and / or psychiatric disorders, including bipolar disorder, depression, Parkinson's disease, and schizophrenia. N-desmethylruboxistaurin can be used in combination with these antipsychotics to treat these conditions.

[0082] Furthermore, the response to N-desmethylruboxistaurin or the combination of N-desmethylruboxistaurin and lithium can help establish the diagnosis of bipolar disorder and other conditions in which GSK3 inhibition is clinically useful. In Alzheimer's disease, positron emission tomography (PET) of GSK3β activity has been developed as a diagnostic method. N-desmethylruboxistaurin can be administered to subjects who show excessive GSK3β activity on PET, either alone or in combination with lithium, to treat Alzheimer's disease, and the reduction in GSK3β activity on PET after administration of N-desmethylruboxistaurin can support the use of N-desmethylruboxistaurin (alone or in combination with lithium) as a suitable treatment administered at an appropriate dose.

[0083] The presently disclosed compositions and methods of treatment are appropriate for any instance in which ruboxistaurin may be clinically useful, including psychiatric and neurological disorders such as bipolar disorder, depression, Alzheimer's disease, frontotemporal dementia, behavioral complications of dementia, autism spectrum disorder, Fragile X syndrome, Pitt-Hopkins syndrome, Rett syndrome, traumatic brain injury, stroke, acute spinal cord injury, schizophrenia, Parkinson's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), neurofibromatosis type 1, neuronal ceroid lipofuscinosis, chronic pain, neuropathic pain, chemotherapy-induced neuropathy, and chemotherapy-induced cognitive impairment.

[0084] Ruboxistaurin and N-desmethylruboxistaurin have been reported to be equally effective at inhibiting protein kinase C, and therefore these compositions and methods of treatment are suitable for conditions in which ruboxistaurin is indicated as a protein kinase C inhibitor, including diabetes, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, ischemia, inflammation, cardiovascular disease, pulmonary hypertension, congestive heart failure, skin diseases, cancer, and GM2 gangliosidosis.

[0085] These compositions and methods of treatment are also appropriate for conditions where inhibition of GSK3 and / or enhancement of WNT signaling has been proposed, including alopecia, osteoarthritis, osteoporosis, alcoholic hepatitis, inflammatory bowel disease, wet age-related macular degeneration, dry age-related macular degeneration, diabetic macular edema, Fuchs' corneal dystrophy, corneal epithelial cell deficiency, dry eye, glaucoma, familial exudative vitreoretinopathy (FEVR), Norrie's disease, Coats' disease, retinopathy of prematurity, macular telangiectasia, retinal vein occlusion, Sjogren's syndrome, sensorineural hearing loss, conductive hearing loss, schizophrenia, Parkinson's disease, polycystic kidney disease, focal segmental glomerulosclerosis, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, short bowel syndrome, melanoma, pancreatic cancer, prostate cancer, colon cancer, leukemia, septic shock, and ischemia / reperfusion injury. Such compositions and methods are also suitable for GM2 gangliosidosis, where the use of ruboxistaurin is suggested.

[0086] The presently disclosed compositions and methods of treatment may also be used in veterinary applications to improve the health and well-being of farm animals and pets by treating any of the aforementioned conditions occurring in animals. <Example> Materials and Methods Example 1 Regarding compound synthesis, N-desmethylruboxistaurin was synthesized according to the following procedure. Step 1: Synthesis of (S)-1-(trityloxy)penten-4-en-2-ol (2) [ka]

[0087] To a solution of vinylmagnesium bromide (1M in THF, 840 mL, 0.84 mol) was added copper iodide (4.5 g, 23.62 mmol) at −40° C. under nitrogen atmosphere. After stirring at −40° C. for 20 min, compound 1 (150 g, 0.46 mmol) dissolved in dry THF (750 mL) was added dropwise to the reaction mixture, and the resulting reaction mixture was stirred at −40° C. for 2 h. After the reaction was complete (monitored by TLC), saturated ammonium chloride (1000 mL) was added. The reaction was allowed to warm to room temperature and extracted with ethyl acetate (1000 mL) with stirring. The organic layer was separated and washed with aqueous ammonia (250 mL). The organic layer was separated, dried over sodium sulfate, filtered, and evaporated under vacuum to give compound 2 (166 grams, 100% crude yield) as a dark brown sticky material. 1 H NMR (400MHz, CDCl3): δ7.45-7.42(m,6H),7.32-7.28(m,6H),7.26-7.22(m,3H),5.74-5.71(m,1H),5.09-5.02( m,2H),3.85-3.82(m,1H),3.18(dd,J=9.6Hz,J=4.0Hz,1H),3.09(dd,J=9.2Hz,J=6.8Hz,1H),2.27-2.22(m,3H). Step 2: Synthesis of (S)-(((2-(allyloxy)penten-4-en-1-yl)oxy)methanetrityl)tribenzene (3) [ka]

[0088] To a stirred solution of compound 2 (165 g, 0.48 mol) in dry THF (1500 mL) was added potassium tert-butoxide (70.0 g, 0.62 mmol) portionwise under nitrogen atmosphere. The resulting reaction contents were heated to 45° C. and stirred for 2 h, then cooled to room temperature and allyl bromide (145.5 g, 1.22 mol) was added at room temperature and stirring was continued for 1 h at room temperature. After the reaction was completed (monitored by TLC), saturated ammonium chloride (1500 mL) was added to the reaction and extracted with ethyl acetate (1500 mL). The organic layer was separated, dried over sodium sulfate, filtered and evaporated under vacuum to give crude compound 3, which was further purified by silica gel column chromatography (100-200 mesh) eluted with 0.5-1% ethyl acetate in hexane. Pure fractions were collected and evaporated under reduced pressure to give the desired compound 3 (106 grams, 58% yield) as a pale yellow semi-solid. 1 H NMR (400MHz, CDCl3): δ7.48-7.44(m,6H),7.31-7.26(m,6H),7.25-7.20(m,3H),5.95-5 .88(m,1H),5.75-5.70(m,1H),5.27(dd,J=17.2Hz,J=2.0Hz,1H),5.15(dd,J=10.4Hz,J= 2.0Hz,1H),5.03(dd,J=17.2Hz,J=2.0Hz,1H),4.96(dt,J=10.4Hz,J=1.2Hz,1H),4.12- 4.10(m,1H),4.04-4.02(m,1H),3.52-3.49(m,1H),3.17-3.09(m,2H),2.35-2.31(m,2H) Step 3: Synthesis of (S)-3-(2-hydroxyethoxy)-4-(trityloxy)butan-1-ol (4) [ka]

[0089] Ozone gas was bubbled into a stirred solution of compound 3 (100 g, 0.26 mol) in MeOH:DCM (1:1) (800 mL) at -45 °C for 18 h. After the reaction was complete (monitored by TLC), it was poured into a solution of sodium borohydride (21.5 g, 0.57 mol) in 0.5 N NaOH solution (370 mL) at 0 °C. The resulting reaction mixture was allowed to stir at room temperature for 16 h. After the reaction was complete (monitored by TLC), it was quenched with 1 N HCl solution until pH was 6-7. The resulting solution was then extracted with ethyl acetate (750 mL). The organic layer was separated, dried over sodium sulfate, filtered, and evaporated under vacuum to obtain the crude compound, which was further purified by silica gel column chromatography (100-200 mesh) eluted with 20-25% ethyl acetate in hexane. The pure fractions were collected and evaporated to obtain the desired compound 4 (58 grams, 57% yield) as a yellow sticky liquid. 1 H NMR(400MHz,DMSO-d6):δ7.42-7.40(m,6H),7.34(t,J=7.6Hz,6H),7.28-7.24(m,3H),4.60(t,J=5.6Hz1H),4.36 (t,J=5.6Hz,1H),3.60-3.56(m,2H),3.52-3.48(m,2H),3.44-3.41(m,3H),2.99-2.97(m,2H),1.61-1.56(m,2H). Step 4: Synthesis of (S)-2-((4-((methylsulfonyl)oxy)-1-(trityloxy)butan-2-yl)oxy)ethyl methanesulfonate (5) [ka]

[0090] To a stirred solution of compound 4 (60 g, 0.15 mol) in DCM (1000 mL) was added triethylamine (66 mL, 0.47 mmol) at 0° C. and stirred for 15 min, followed by methanesulfonyl chloride (32.0 mL, 0.41 mmol). The resulting reaction mixture was stirred at 0° C. for 2 h (reaction monitored by TLC) and quenched with saturated ammonium chloride solution (600 mL). The organic layer was separated, dried over sodium sulfate, filtered, and evaporated under vacuum (≦25° C.) to give the crude compound, which was suspended in a 1:1 mixture of ethyl acetate and heptane (600 mL) and evaporated under vacuum to give a solid. The resulting solid compound was suspended in a 1:1 mixture of ethyl acetate and heptane (600 mL), stirred for 30 min, filtered, the solid was washed with heptane (80 mL), and dried under vacuum to give compound 5 (88 grams, 100% crude yield) as a cream-colored solid. 1 H NMR (400MHz, DMSO-d6): δ7.42-7.39(m,5H),7.37-7.31(m,5H),7.29-7.23(m,3H),7.22-7.18(m,2H),4.34-4.22(m,4H), 3.84-3.83(m,1H),3.69-3.64(m,2H),3.17(s,3H),3.13(s,3H),3.09-3.06(m,1H),3.04-3.02(m,1H),1.88-1.85(m,2H). Step 5: Synthesis of (12E,32E,7S)-21-methyl-7-((trityloxy)methyl)-22,25-dihydro-11H,21H,31H-6-oxa-1,3(3,1)-diindra-2(3,4)-pyrrolacyclononaphane-22,25-dione (7) [ka]

[0091] To a stirred solution of compound 6 (41.5 g, 0.12 mol) in DMF (850 mL) was added cesium carbonate (86.0 g, 0.26 mol), the reaction mixture was heated to 100 °C, and compound 5 (85.0 g (crude), 0.15 mol) was added dropwise at the same temperature. The resulting reaction mixture was stirred at 100 °C for 24 h. After the reaction was completed (monitored by TLC), it was cooled to 50 °C, celite (25 g) was added and stirred for 15 min. The reaction product was filtered through celite and the filtrate was partitioned between ethyl acetate (800 mL) and water (400 mL). The organic layer was separated, dried over sodium sulfate, filtered and evaporated under vacuum to obtain the crude compound, which was further purified by silica gel column chromatography (100-200 mesh) eluted with 25-30% ethyl acetate in hexane. The pure fractions were collected and evaporated to obtain the desired compound 7 (55 grams, 51% yield) as a brick red solid. 1 H NMR (400MHz, DMSO-d6): δ7.83(d,J=7.6Hz1H),7.75(d,J=8.0Hz1H),7.49(d,J=8.4Hz1H) ,7.45(s,1H),7.41(s,1H),7.34-7.26(m,10H),7.25-7.22(m,6H),7.18-7.15(m,2H),7. 12-7.06(m,2H),4.25-4.24(m,1H),4.17-4.04(m,3H),3.71-3.67(m,1H),3.55-3.50(m, 1H),3.31-3.30(m,1H),3.07(s,3H),3.05-3.00(m,2H),2.10-2.07(m,1H),2.02(m,1H). Step 6: Synthesis of (12E,32E,7S)-7-((trityloxy)methyl)-22,25-dihydro-11H,31H-6-oxa-1,3(3,1)-diindra-2(3,4)-furanicylcyclononaphane-22,25-dione (8) [ka]

[0092] To a stirred solution of compound 7 (85.0 g, 0.12 mol) in ethanol (850 mL) was added potassium hydroxide (68.0 g, 1.22 mol) and heated to 80° C. The resulting reaction mixture was stirred for 24 h. After the reaction was completed (monitored by TLC), the reaction mixture was evaporated under vacuum to give a residue, which was partitioned between DCM (850 mL) and 20% citric acid solution (450 mL). The organic layer was separated, dried over sodium sulfate, filtered, and evaporated under vacuum to give crude compound 8 (62 grams, 74% yield) as a dark brown solid. 1 H NMR (400MHz, DMSO-d6): δ7.88(d,J=7.6Hz1H),7.82(d,J=7.6Hz1H),7.65(d,J=2. 0Hz2H),7.55(d,J=8.0Hz1H),7.41(d,J=7.6Hz1H),7.34-7.26(m,12H),7.25-7.2 0(m,5H), 7.19-7.13(m,2H), 4.33-4.28(m,1H), 4.20-4.06(m,3H), 3.73-3.69(m,1H), 3.58-3.54(m,1H), 3.09-3.07(m,2H), 2.17-2.12(m,1H), 2.01-1.97(m,1H). (Extra protons in the aromatic region are not included.) Step 7: Synthesis of (12E,32E,7S)-7-((trityloxy)methyl)-22,25-dihydro-11H,21H,31H-6-oxa-1,3(3,1)-diindra-2(3,4)-pyrrolacyclononaphane-22,25-dione (9) [ka]

[0093] To a stirred solution of compound 8 (95.0 g, 0.25 mol) in DMF (950 mL) was added HMDS (294.0 mL, 2.47 mol) and methanol (6.0 mL) and heated to 80° C. The reaction mixture was stirred at 80° C. for 5 h. After completion of the reaction (monitored by TLC), it was cooled to room temperature, quenched with 1N HCl solution (950 mL) and extracted with DCM (1500 mL). The organic layer was separated, dried over sodium sulfate, filtered and evaporated under vacuum to give crude compound (84 g), which was further purified by silica gel column chromatography (100-200 mesh) eluted with 20-25% ethyl acetate in hexane. The pure fractions were collected and evaporated under vacuum to give the desired compound 9 (70 grams, 74% yield) as a purple solid. 1 H NMR (400MHz, DMSO-d6): δ10.91(s,1H),7.81(d,J=8.0Hz1H),7.73(d,J=8.0Hz1H),7.48(d ,J=8.4Hz2H),7.43(s,1H),7.39(s,1H),7.33-7.23(m,12H),7.23-7.21(m,3H),7.18-7.14 (m,2H),7.11-7.06(m,2H),4.27-4.23(m,1H),4.13-4.00(m,3H),3.70-3.67(m,1H),3.55 -3.47(m,1H),3.33-3.26(m,1H),3.02-2.99(m,2H),2.13-2.08(m,1H),2.01-1.98(m,1H). Step 8: Synthesis of (12E,32E,7S)-7-(hydroxymethyl)-22,25-dihydro-11H,21H,31H-6-oxa-1,3(3,1)-diindra-2(3,4)-pyrrolacyclononaphane-22,25-dione (10) [ka]

[0094] To a stirred solution of compound 9 (70.0 g, 0.18 mol) in ethanol (700 mL) was added 6N HCl (700 mL) at room temperature. The resulting reaction contents were heated to 80° C. for 3 h. After the reaction was complete (monitored by TLC), cooled to room temperature and stirred for 1 h, the resulting solid was filtered, washed with water (350 mL) and dried under vacuum at 45° C. to give compound 10 (40 grams, 88% crude yield) as a purple solid. 1 H NMR(400MHz,DMSO-d6):δ10.92(s,1H),7.82(d,J=7.6Hz1H),7.78(d,J=7.6Hz1H),7.53(d, J=8.0Hz,1H),7.51(s,1H),7.46(d,J=8.4Hz,1H),7.45(s,1H),7.25-7.22(m,2H),7.13-7.1 0(m,2H),4.69(t,J=5.2Hz1H),4.35-4.33(m,1H),4.24-4.15(m,3H),3.91-3.87(m,1H),3.6 5-3.60(m,1H),3.53-3.49(m,1H),3.43-3.39(m,1H),2.09-2.07(m,1H),1.98-1.97(m,1H). Step 9: Synthesis of ((12E,32E,7S)-22,25-dioxo-22,25-dihydro-11H,21H,31H-6-oxa-1,3(3,1)-diindra-2(3,4)-pyrrolacyclononaphan-7-yl)methyl methanesulfonate (11) [ka]

[0095] To a stirred solution of compound 10 (39.0 g, 0.09 mol) in THF (400 mL) was added pyridine (33.2 mL, 0.39 mol) at room temperature and stirred for 20 min. Methanesulfonic anhydride (46.0 g, 0.26 mol) was added to the reaction at room temperature. The resulting reaction mixture was stirred for 4 h. After the reaction was completed (monitored by TLC), the reaction was partitioned between ethyl acetate (100 mL) and water (50 mL), the organic layer was separated, dried over sodium sulfate, filtered and evaporated under vacuum to give crude compound (37.0 g), which was further purified by silica gel column chromatography (100-200 mesh) eluted with DCM. The pure fractions were collected and evaporated under reduced pressure to give the desired compound 11 (30 grams, 65% yield) as a purple solid. 1 H NMR(400MHz,DMSO-d6):δ10.92(s,1H),7.83(d,J=7.6Hz1H),7.78(d,J=7.6Hz1H ),7.54(d,J=8.4Hz,1H),7.52(s,1H),7.48(d,J=8.4Hz,1H),7.46(s,1H),7.22- 7.17(m,2H),7.14-7.10(m,2H),4.44-4.38(m,2H),4.22-4.14(m,4H),3.93-3.9 0(m,1H),3.66-3.61(m,1H),3.17(s,3H),2.19-2.14(m,1H),2.03-1.98(m,1H). Step 10: Synthesis of (12E,32E,7S)-7-((methylamino)methyl)-22,25-dihydro-11H,21H,31H-6-oxa-1,3(3,1)-diindra-2(3,4)-pyrrolacyclononaphane-22,25-dione hydrochloride (compound 1) [ka]

[0096] To a stirred solution of compound 11 (10.0 g, 0.019 mol) in THF (400 mL) in an autoclave at -40 °C was added 2 M methylamine in THF (400 mL). The reaction was gradually heated to 70 °C and stirred for 24 h. After the reaction was complete (monitored by TLC), it was evaporated under vacuum to give crude compound (12.0 g). This batch was combined with four additional batches of the same scale to give 60.0 g of crude product. 60 grams of crude product was purified by silica gel column chromatography (230-400 mesh, 2% MeOH / DCM). Pure fractions were collected and concentrated to give the desired free base of compound 1 (22.0 g) as a red solid. The free base was suspended in diethyl ether (220 mL) and cooled to 0 °C. Ethanol-HCl (33 mL) was added at 0 °C. The resulting suspension was stirred at 0° C. for 30 min, filtered, washed with diethyl ether (50 mL), and dried under vacuum at 40° C. for 1 h to give compound 1 (16.9 g, 36% yield) as a brick-red solid. 1 H NMR (400 MHz, DMSO-d6): δ 10.93 (s, 1H, exchanged in DO), 8.72-8.71 (m, 2H, exchanged in DO), 7.81 (t, J = 8.0 Hz, 2H), 7.55 (d, J = 8.0 Hz 1H), 7.49 (s, 2H), 7.47 (d, J = 8.4 Hz, 1H), 7.23 (t, J = 7.2 Hz, 2H), 7.14 (t, J = 7.2 Hz, 2H), 4.46 -4.41(m,1H),4.33-4.25(m,2H),4.15-4.10(m,1H),3.86-3.84(m,1H),3.73-3.71(m,1H),3.62(t,J=9.2H) z,1H),3.27-3.24(m,1H),3.01-2.98(m,1H),2.53(t,J=5.6Hz,3H),2.22-2.20(m,1H),2.06-2.03(m,1H). Example 2 Kinase Assay

[0097] Ruboxistaurin was purchased from a commercial laboratory and N-desmethyl ruboxistaurin was synthesized as in Example 1. Kinase reactions were performed in a 384-well format. Reaction conditions included 0.25 ng of GSK3α or GSK3β (final enzyme concentrations of 0.62 and 0.68 nM, respectively), 0.25 μg of GSK substrate, ATP (19 or 12 μM for GSK3α or β, respectively), and 50 mM Tris buffer (pH 7.5, 5 mM MgCl2, 0.01% Bridge 35, and 3 mM DTT). Compound or 1% DMSO was added. A5X stocks (without DTT) of the above buffers were prepared and stored at room temperature.

[0098] Two solutions were prepared. The first contained 1X buffer and 2X enzyme and substrate. The second contained 1X buffer and 2X ATP. After placing 2.5 μL of the first solution on each plate, compounds were added and incubated at room temperature for 15 minutes, then 2.5 μL of the second solution was added (followed by a pulse spin). The plates were then incubated at room temperature for 60 minutes (in the dark, using another plate as a lid). To stop the reaction and deplete residual ATP, 5 μL of ADP-Glo ​​reagent was added to each well, after which the plates were left at room temperature for another 40 minutes. To detect ADP production, 10 μL of ADP-Glo ​​kinase detection substrate was added to each well. After 5-30 minutes, the plates were read for luminescence. Example 3 Microsomal Stability

[0099] A master solution was prepared with 100 mM phosphate buffer, 5 mM MgCl2 solution, and 0.5 mg / mL human microsomes. 40 μL of 10 mM NADPH solution was added to each well to give a final concentration of 1 mM NADPH. The mixture was pre-warmed at 37°C for 5 minutes. A negative control sample was made by replacing the NADPH solution with 40 μL of ultrapure H2O. Two samples containing NADPH were made. A single negative control was made. The reaction was started by adding 2 μL of 200 μM control or test compound solution. Verapamil was used as a positive control. The final concentration of test or control compound was 1 μM.

[0100] Aliquots of 50 μL were taken from the reaction solution at 0, 15, 30, 45 and 60 min. The reaction was stopped by adding 4 volumes of cold acetonitrile with IS (100 nM alprazolam, 200 nM imipramine, 200 nM labetalol and 2 μM ketoprofen). The samples were centrifuged at 3220 g for 40 min. An aliquot of 90 μL of the supernatant was mixed with 90 μL of ultrapure H2O and used for LC-MS / MS analysis. Peak areas were determined from extracted ion chromatograms. Example 4 Blood-brain barrier penetration

[0101] Male Sprague Dawley (SD) rats were administered 1 mg / kg N-desmethylruboxistaurin intravenously (via the tail vein). Blood samples were collected from three animals at 0.25, 1, and 4 h after administration. Brain samples were collected from three animals each at 0.25, 1, and 4 h after administration from the other nine rats at 0.25, 1, and 4 h after administration. Brain samples were weighed and homogenized in phosphate-buffered saline. For precipitation, samples (20 μL) were added to a 200 μL mixture containing acetonitrile and vortexed for 30 s. After centrifugation at 4000 rpm for 15 min at 4 °C, the supernatant was diluted in a 1:2 ratio with ultrapure H2O, and 15 μL of the supernatant was injected into an LC / MS / MS system (liquid chromatography and tandem mass spectrometry) to analyze the levels of N-desmethylruboxistaurin. Example 5 Ultrasonic vocalizations in rats

[0102] Ninety-eight male Wistar rats were acclimated to the testing facility and handled daily (10 min / day) for 7 days prior to the start of the experiment. Male Wistar rats (~200 g) were used in this study. There were 8 rats in each group in 5 groups (lithium and 0, 10, 30, 100 mg / kg N-desmethylruboxistaurin before administration of dextroamphetamine or D-AMP). There were also 9 rats that did not receive D-AMP. Rats were placed in white Plexiglas boxes (50 × 50 × 50 cm) and video-recorded for 10 min to acclimate to the testing apparatus. Ultrasonic vocalizations (USVs) were recorded with a microphone mounted 45 cm above the open-field box to establish a baseline, non-treatment-related USV response (50 kHz vocalizations) for each rat. The next day, rats were injected with saline (1 mL / kg body weight, IP) and immediately placed in the open-field test box. USVs were recorded for 10 min. This data served as the basis for selecting equivalent groups for drug testing. On day 3, rats were administered positive treatment controls (lithium carbonate 100 mg / kg in saline administered subcutaneously, or N-desmethylruboxistaurin 0, 10, 30, or 100 mg / kg IP administered intraperitoneally). 60 min later, D-amphetamine (in saline) was administered at 2.5 mg / kg IP, and rats were immediately placed in the open field test box. USVs were recorded for 10 min.

Claims

1. N-desmethyllboxystaurine or a pharmaceutically acceptable salt thereof for use in the treatment of disorders characterized by abnormal signaling of GSK3β or protein kinase C, wherein the use is characterized by administering a therapeutically effective dose of N-desmethyllboxystaurine or a pharmaceutically acceptable salt thereof to a subject in need thereof.

2. The subject is 1) has never taken ruboxystaurine, 2) has taken ruboxystaurine and experienced side effects, 3) has shown an extended QT interval, 4) has shown a high plasma concentration of ruboxystaurine, 5) may be administered a drug that may interfere with the metabolism of ruboxystaurine, or 6) may require a high dose of ruboxystaurine and is of concern for side effects, QT prolongation, or adverse drug interactions, N-desmethylruboxystaurine or a pharmaceutically acceptable salt thereof for use according to claim 1.

3. The subject has a neurological disorder and / or mental disorder, N-desmethyllboxystaurine or a pharmaceutically acceptable salt thereof for use according to claim 1 or 2.

4. The neurological and / or mental disorders are selected from Alzheimer's disease, frontotemporal dementia, behavioral complications of dementia, bipolar disorder, depression, schizophrenia, Parkinson's disease, neuroinflammation, autism spectrum disorder, fragile X syndrome, Pitt-Hopkins syndrome, Rett syndrome, traumatic brain injury, stroke, acute spinal cord injury, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), neurofibromatosis type 1, neuronal ceroid lipofuscinosis, chronic pain, neuropathic pain, chemotherapy-induced neuropathy, and chemotherapy-induced cognitive impairment, and N-desmethyllboxystaurine or a pharmaceutically acceptable salt thereof for use according to claim 3.

5. The disease / disorder is selected from type 2 diabetes mellitus, diabetic retinopathy, diabetic neuropathy, diabetic macular edema, diabetic nephropathy, chronic kidney disease, polycystic kidney disease, and focal segmental glomerulosclerosis, and is N-desmethyllboxystaurine or a pharmaceutically acceptable salt thereof for use according to claim 1 or 2.

6. The disease / disorder is selected from bone and joint disorders including atherosclerosis, alopecia, osteoarthritis and osteoporosis, inflammatory disorders including alcoholic hepatitis and inflammatory bowel disease, and septic shock, and is N-desmethyllboxystaurine or a pharmaceutically acceptable salt thereof for use according to claim 1 or 2.

7. The disease / disorder is selected from eye disorders including exudative age-related macular degeneration, atrophic age-related macular degeneration, Fuchs corneal dystrophy, corneal epithelial cell defects, dry eye, glaucoma, familial exudative vitreoretinopathy (FEVR), Norie's disease, Coats' disease, retinopathy of prematurity, macular telangiectasia, retinal vein occlusion, and Sjögren's syndrome, and / or ear disorders including sensorineural hearing loss and conductive hearing loss, N-desmethyllboxystaurine or a pharmaceutically acceptable salt thereof for use according to claim 1 or 2.

8. The disease / disorder is selected from lung disorders including chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, and pulmonary hypertension, and / or cancers including melanoma, pancreatic cancer, prostate cancer, colon cancer, and leukemia, and / or short bowel syndrome, ischemia, inflammation, cardiovascular disease, congestive heart failure, skin disease, inflammation, or GM2 gangliosidosis, N-desmethyllboxystaurine or a pharmaceutically acceptable salt thereof for use according to claim 1 or 2.

9. N-desmethyllboxystaurine or a pharmaceutically acceptable salt thereof for use according to claim 1 or 2, administered once daily in an amount of about 32 to about 320 mg, or twice daily in an amount of about 16 to about 160 mg.

10. N-desmethyllboxystaurine or a pharmaceutically acceptable salt thereof for use according to claim 1 or 2, administered in combination with lithium.

11. The subject is N-desmethyllboxystaurine or a pharmaceutically acceptable salt thereof for use according to claim 1 or 2, which does not react with lithium.

12. The subject is N-desmethylruboxystaurine or a pharmaceutically acceptable salt thereof for use according to claim 10, which reacts with lithium.

13. The use of N-desmethyllboxystaurine or a pharmaceutically acceptable salt thereof for the use according to claim 10, wherein lithium is administered in a non-effective dose for monotherapy, and N-desmethyllboxystaurine or a pharmaceutically acceptable salt thereof is administered in a non-effective dose for monotherapy.

14. N-desmethyllboxystaurine or a pharmaceutically acceptable salt thereof for use according to claim 13, wherein the non-effective dose of lithium is administered once daily at approximately 60 mg to approximately 600 mg, or twice daily at approximately 30 mg to approximately 300 mg.

15. The non-effective dose of N-desmethyllboxystaurine or a pharmaceutically acceptable salt thereof for use according to claim 11 is approximately 8 to approximately 32 mg administered once daily, or approximately 4 to approximately 16 mg administered twice daily.

16. N-desmethyllboxystaurine or a pharmaceutically acceptable salt thereof for use according to claim 3, administered in combination with valproic acid, lamotrigine, quetiapine, olanzapine, risperidone, aripiprazole, lurasidone, lumateperone, caliprazine, asenapine, carbamazepine, xanomellin-trospium, iloperidone, or a combination thereof.

17. N-desmethyllboxystaurine or a pharmaceutically acceptable salt thereof for use in establishing the diagnosis of bipolar disorder or other conditions for which GSK3β inhibition is clinically useful, wherein the use is characterized by administering a therapeutically effective dose of N-desmethyllboxystaurine or a pharmaceutically acceptable salt thereof to a subject to be evaluated and evaluating the subject's clinical response.

18. N-desmethyllboxystaurin or a pharmaceutically acceptable salt thereof for use in establishing an appropriate therapeutic dose of N-desmethyllboxystaurin in a subject, wherein the use is characterized by administering an increasing dose of N-desmethyllboxystaurin or a pharmaceutically acceptable salt thereof to the subject and evaluating the response using GSK3β imaging or GSK3β serology.

19. N-desmethyllboxystaurine or a pharmaceutically acceptable salt thereof for use in the treatment of a subject having a condition characterized by Alzheimer's disease, frontotemporal dementia, behavioral complications of dementia, bipolar disorder or depression, and evidence of elevated GSK3β, wherein such use is characterized by administering a therapeutically effective dose of N-desmethyllboxystaurine or a pharmaceutically acceptable salt thereof to the subject and evaluating and monitoring the subject using positron emission tomography (PET) or serology.

20. N-desmethyllboxystaurine or a pharmaceutically acceptable salt thereof for use in the treatment of a subject having Alzheimer's disease, frontotemporal dementia, behavioral complications of dementia, bipolar disorder or depression, and a condition characterized by elevated GSK3β activity, wherein the use is characterized by administering to the subject a therapeutically effective dose of N-desmethyllboxystaurine or a pharmaceutically acceptable salt thereof and a therapeutically effective dose of lithium, and monitoring the subject using positron emission tomography (PET).

21. N-desmethyllboxystaurine or a pharmaceutically acceptable salt thereof for use according to claim 20, wherein the doses of both N-desmethyllboxystaurine and lithium are non-effective doses in the case of monotherapy.

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