N-demethyllubrista as kinase inhibitors

Abstract

Aspects of the present invention relate to the use of N-demethylubrusta and pharmaceutically acceptable formulations thereof to modulate GSK3 [beta] signaling. Some aspects of the invention relate to the use of N-demethylated lubrusta to inhibit protein kinase C. Some aspects of the invention provide methods of treating a subject suffering from a neurological disease and / or psychiatric condition, including Alzheimer's disease, frontotemporal dementia, behavioral complications of dementia, bipolar disorder, depression, schizophrenia, Parkinson's disease, or neuroinflammation, using N-demethylated lubrusta. Some aspects of the invention provide methods of treating conditions associated with diabetes or complications thereof or ischemia, inflammation, pulmonary arterial hypertension, congestive heart failure, cardiovascular disease, skin disease, or cancer using N-demethylubrusta. Some aspects of the present invention relate to the use of N-demethylated lubrusta as a substitute for lubrusta with better pharmacokinetic properties, a lower potential to extend the electrocardiogram QT interval, and / or a lower potential to poor drug interaction. In some embodiments, N-demethylubrusta is administered in combination with lithium or other treatment for bipolar disorders.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application Serial No. 63 / 362,293, filed on March 31, 2022, the disclosure of which is incorporated herein by reference. Field of the Invention

[0003] Aspects of the invention relate to methods of treating neurological diseases or psychiatric disorders including Alzheimer's disease, frontotemporal dementia, behavioral complications of dementia, bipolar disorder, depression, schizophrenia, Parkinson's disease, or neuroinflammation, as well as treating diabetes mellitus and its complications, or ischemia, inflammation, pulmonary hypertension, congestive heart failure, cardiovascular disease, dermatological disease, cancer, or GM2 gangliosidosis, or other conditions where ruboxistaurin is clinically useful. Background of the Invention

[0005] Rubusta has been shown to modulate GSK3 signaling and inhibit protein kinase C.

[0006] As a GSK3 inhibitor, Rubusta has been proposed as a treatment for subjects with neurological diseases 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 pathways that have been widely proposed in regenerative medicine for the treatment of neurological and psychiatric disorders and for reducing neuroinflammation. Inhibition of GSK3 or enhancement of WNT signaling has been linked to potential treatments for type 2 diabetes and renal disorders, including diabetic nephropathy, chronic kidney disease, polycystic kidney disease, and focal segmental glomerulosclerosis, as well as atherosclerosis and alopecia; bone and joint disorders, including osteoarthritis and osteoporosis; inflammatory disorders, including alcoholic hepatitis, inflammatory bowel disease, and septic shock; and ocular disorders, including wet age-related macular degeneration, dry age-related macular degeneration, diabetic macular edema, and cataract. macular edema, Fuch's dystrophy, limbal cell deficiency, dry eye, glaucoma, familial exudative vitreoretinopathy (FEVR), Norrie disease, Coats disease, retinopathy of prematurity, macular telangiectasia, retinal vein occlusion, and Sjögren's syndrome. Inhibition of GSK3 or enhancement of WNT signaling has been linked to potential treatments for ear disorders, including sensorineural hearing loss and conductive hearing loss; lung disorders, including chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis; and short bowel syndrome; and cancers, including melanoma, pancreatic cancer, prostate cancer, colon cancer, and leukemia. As a GSK3 inhibitor, Rubusta has been proposed for use as monotherapy for the treatment of bipolar disorder, in combination with lithium, or in combination with other bipolar disorder treatments.

[0007] As a protein kinase C inhibitor, Rubustadine has been proposed for the treatment of 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 is also thought to be helpful in the treatment of bipolar disorder and Alzheimer's disease.

[0008] However, the pharmacokinetics of rubostat include a high-peak-to-trough ratio, and rubostat has been shown to prolong the QT interval in human subjects. In addition, rubostat levels may be increased by drugs that inhibit CYP3A4 metabolism.

[0009] N-desmethyl ruboxistaurin is a metabolite of ruboxistaurin. SUMMARY OF THE INVENTION

[0011] Aspects of the invention relate to the use of N-desmethylrubostat as a therapeutic agent and as a potentially safer alternative to the use of rubostat in situations where rubostat is clinically useful.

[0012] One aspect of the present invention relates to a method for treating a disorder involving abnormal signaling of GSK3β or protein kinase C by administering a therapeutically effective dose of N-desmethyl rubostat or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof to a subject in need thereof. In other words, the present invention provides N-desmethyl rubostat or a pharmaceutically acceptable salt thereof for treating a disorder characterized by abnormal signaling of GSK3β or protein kinase C by administering a therapeutically effective dose of N-desmethyl rubostat or a pharmaceutically acceptable salt thereof to a subject in need thereof. The method can be applied to the case where N-desmethyl rubostat is administered to a subject who: 1) has never taken rubostat, or 2) has taken rubostat and experienced adverse reactions, or 3) shows a prolonged QT interval, or 4) has been shown to have high plasma levels of rubostat, or 5) is likely to receive drugs that may interfere with rubostat metabolism, or 6) may require a higher dose of rubostat and there are concerns about adverse reactions, QT prolongation, or adverse drug interactions.

[0013] The subject may suffer from 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.

[0014] Subjects may have diabetes, diabetic neuropathy, diabetic retinopathy, diabetic nephropathy, chronic kidney disease, atherosclerosis, alopecia, osteoarthritis, osteoporosis, alcoholic hepatitis, inflammatory bowel disease, wet age-related macular degeneration, dry age-related macular degeneration, diabetic macular edema, Fuch's dystrophy, limbal cell defects, dry eye, glaucoma, familial exudative vitreoretinopathy (FEVR), Norrie's disease, Coats' disease, premature infants Retinopathy, macular telangiectasia, retinal vein occlusion, Sjögren'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.

[0015] N-desmethylrubostat or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof can be administered in combination with lithium for the treatment of bipolar disorder or other conditions in which inhibition of GSK3, protein kinase C, or both is useful. In addition to lithium, combination therapy for bipolar disorder can also include valproic acid, lamotrigine, quetiapine, olanzapine, risperidone, aripiprazole, lurasidone, lumateperone, cariprazine, asenapine, and carbamazepine.

[0016] Lithium can be administered at a sub-effective dose based on monotherapy, and N-desmethylrubostat can be administered at a sub-effective dose based on monotherapy. The sub-effective dose of lithium can be a dose that reduces kidney damage or does not cause kidney damage.

[0017] Subjects may be non-responsive to lithium, or responsive to lithium.

[0018] Another aspect of the present invention relates to a method for determining the diagnosis of bipolar disorder or other conditions in which GSK3 inhibition is clinically useful by administering a therapeutically effective dose of N-desmethylrubostat or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof to a subject to be evaluated and evaluating the subject's clinical response.

[0019] Another aspect of the invention relates to a method of determining an appropriate therapeutic dose of N-desmethylrubostat in a subject by administering increasing doses of N-desmethylrubostat to the subject and assessing the response using GSK3 imaging or GSK3 serology.

[0020] Yet another aspect of the present invention relates to a method of treating a subject having Alzheimer's disease, bipolar disorder or depression and showing signs of elevated GSK3 by administering to the subject a therapeutically effective dose of N-desmethylrubostat or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof and evaluating and monitoring the subject using positron emission tomography (PET) or serology.

[0021] Another aspect of the present invention relates to a method for determining the diagnosis of bipolar disorder or other conditions in which GSK3 inhibition is clinically useful by administering a therapeutically effective dose of N-desmethylrubostat or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof and a therapeutically effective dose of lithium to a subject to be evaluated and evaluating the subject's clinical response. The doses of both N-desmethylrubostat and lithium may be sub-effective on a monotherapy basis.

[0022] Another aspect of the present invention relates to a method of treating a subject with Alzheimer's disease and evidence of elevated GSK3 activity by administering to the subject a therapeutically effective dose of N-desmethylrubostat or a pharmaceutically 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-desmethylrubostat and lithium may be sub-effective on a monotherapy basis.

[0023] Another aspect of the invention relates to a method of determining an appropriate therapeutic dose of N-desmethylrubostat in a subject by administering increasing doses of N-desmethylrubostat and lithium to the subject and assessing the response using positron emission tomography (PET).

[0024] BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The QT interval on an electrocardiogram (ECG) tracing is shown, describing the portion of the heart's contraction cycle from the beginning of left ventricular contraction to the end of left ventricular relaxation.

[0026] Figure 2 A plot of the drug therapeutic window on a graph of % of maximal effect versus drug concentration is shown.

[0027] Figure 3 The chemical structure of Rubustadine is shown.

[0028] Figure 4 The chemical structure of N-desmethylrubestadine is shown.

[0029] Figure 5 Shown is a graph of mean plasma concentration versus time following a single dose of 32 mg of Rubustadine to healthy subjects. The results in the inset were obtained in the fed state.

[0030] Figure 6 A synthetic scheme for N-desmethylrubestadine is shown.

[0031] Figure 7 Shown are the abilities of rubustard and N-desmethylrubustard to inhibit GSK3β and GSK3α at different concentrations.

[0032] Figure 8 The stability of rubestad and N-desmethylrubestad in human liver microsomes was shown.

[0033] Fig. 9 N-desmethylrubostat was shown to provide a pharmacological effect similar to lithium in rats, namely the ability to reduce active ultrasonic vocalizations induced by dextroamphetamine.

[0034] Detailed Description

[0035] Unless otherwise defined, all terms used in the disclosure of the present invention, including technical and scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0036] As disclosed herein, several numerical ranges are provided. It should be understood that, unless the context clearly specifies otherwise, each intermediate value between the upper and lower limits of the range is also specifically disclosed, accurate to one tenth of the unit of the lower limit. The present invention covers each smaller range between any of the values ​​or intermediate values ​​in the range and any other values ​​or intermediate values ​​in the range. The upper and lower limits of these smaller ranges can be independently included in the range or excluded from the range, and each range including any limit, excluding any limit, or including two limits in the smaller range is also included in the present invention, but any specific excluded limit in the range must be observed. When the range includes one or two limits, the range excluding one or two limits in those included limits is also included in the present invention. The term "about" generally includes up to plus or minus 10% of the number shown. For example, "about 10%" can represent a range of 9% to 11%, and "about 20" can mean 18 to 22. Preferably, "about" includes up to plus or minus 6% of the value shown. Alternatively, "about" includes up to plus or minus 5% of the value shown. Other meanings of "about" may be apparent from the context, such as rounding, so that, for example, "about 1" may also mean 0.5 to 1.4.

[0037] The term "pharmaceutically acceptable salt" of a compound means a salt that is pharmaceutically acceptable and possesses the desired pharmacological activity of the parent compound. It is understood that pharmaceutically acceptable salts are 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 organic 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-hydroxyethanesulfonic 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, tert-butylacetic acid, dodecylsulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like. Additional information regarding suitable pharmaceutically acceptable salts may be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, 1985, incorporated herein by reference.

[0038] The term "therapeutically effective amount" as used herein means the amount of the compound of the invention that is capable of alleviating the symptoms of the various pathological conditions described herein. Of course, the specific dosage of the compound administered according to the invention will be determined by the specific circumstances of the case, including, for example, the compound administered, the route of administration, the state of the patient, and the pathological condition being treated. Administration may be once a day, or in multiple sub-doses per day, such as two, three, or more doses per day.

[0039] For monotherapy, an effective dose of N-desmethylrubostat or a pharmaceutically acceptable salt thereof is about 32 to about 320 mg once a day, or about 16 to about 160 mg twice a day. The pharmaceutical composition of N-desmethylrubostat or a pharmaceutically acceptable salt thereof further comprises at least one pharmaceutically acceptable excipient or excipient.

[0040] For combination therapy, the sub-effective dose of N-desmethyl rubostat or its pharmaceutically acceptable salt is about 8 to about 32 mg once a day, or about 4 to about 16 mg twice a day. When N-desmethyl rubostat is combined with lithium, the sub-effective dose of lithium can be about 60 mg to about 600 mg once a day, or about 30 mg to about 300 mg twice a day. Such sub-effective doses of lithium can avoid kidney damage usually caused by lithium treatment. The effective dose of N-desmethyl rubostat or its pharmaceutically acceptable salt once a day can be about 32 or about 64 or about 96 or about 128 or about 160 or about 192 or about 224 or about 256 or about 288 or about 320 mg. The effective dose of N-desmethyl rubostat or its pharmaceutically acceptable salt twice a day can be about 16 or about 32 or about 48 or about 64 or about 80 or about 96 or about 112 or about 128 or about 144 or about 160 mg. The once daily sub-effective dose of N-desmethyl rubostat or a pharmaceutically acceptable salt thereof may be about 8 or about 16 or about 24 or about 32 mg. The twice daily sub-effective dose of N-desmethyl rubostat or a pharmaceutically acceptable salt thereof may be about 4 or about 8 or about 12 or about 16 mg.

[0041] Rubostar has been studied in several clinical trials for the treatment of diabetes and its complications, including diabetic retinopathy, diabetic neuropathy, and diabetic nephropathy. See A. Girach, U.S. Patent Publication No. 2008 / 0096923, which is incorporated herein by reference in its entirety. Its safety profile has been described as excellent, with a lower incidence of serious adverse events than placebo. Due to its clinical safety profile, and due to the growing interest in the use of treatments that inhibit GSK3, further development of Rubostar is encouraged. In addition, it is currently proposed that Rubostar will soon be clinically tested for the treatment of GM2 gangliosidosis.

[0042] However, a careful review of the clinical and preclinical data for Rubostat has shown that it has the potential to prolong the QT interval in human subjects, which could increase the risk of dangerous cardiac arrhythmias, particularly in the setting of an accidental or intentional overdose of Rubostat, when administered with other drugs that prolong the QT interval, or when very high levels of Rubostat are achieved with concurrent administration of drugs that inhibit CYP3A4.

[0043] Evidence of these potential risks comes from a withdrawal assessment prepared by the European Medicines Agency (EMA). The report was prompted by the withdrawal of a marketing authorization application for rubastada that had been submitted to the EMA. The withdrawal assessment showed that rubastada inhibits hERG, a potassium ion channel known for its contribution to the heart's electrical activity. This preclinical assessment is often used to identify compounds with potential QT prolongation risk.

[0044] The QT interval is the portion of the electrocardiogram (ECG) tracing that describes the portion of the heart's contraction cycle that begins with left ventricular contraction (the letters "Q" represent the beginning of the "QRS" complex in the waveform) and ends with left ventricular relaxation (the termination of the "T" wave). The QT interval should normally be 425 milliseconds or less. If the QT interval is too long, the heart cannot complete relaxation before the next electrical signal begins, which can result in dangerous arrhythmias. See also Figure 1 .

[0045] In the hERG assay, rubusta blocked the hERG channel, IC 50 The value was 35.6 nM. This resulted in an estimated increase in the QT interval of 5 milliseconds. Further evaluation of cardiac safety in dogs did not show problems, but the results of a "thorough QT" study using Rubusta in human subjects raised concerns. This type of study is conducted using multiple ECG tracings at different time intervals after exposure to the study drug.

[0046] In the full human QT study, rubusta prolonged the QT interval by 6 milliseconds. In addition, several patients in the study had their QT interval prolonged by more than 10 milliseconds. This potential safety issue was the basis for the strong objection from the European Medicines Agency.

[0047] Positive hERG measurements predict QT prolongation, which has been associated with life-threatening arrhythmias, including Torsades de Pointes. These studies alone cannot perfectly predict cardiac risk. However, avoiding QT prolongation is considered clinically desirable. Pharmaceutical companies sometimes stop developing products that prolong the QT interval by more than 5 milliseconds.

[0048] Even when drugs with QT-prolonging properties are approved, they still come with safety warnings in prescribing instructions, such as the safety warning for ziprasidone, which states in its FDA label: “When deciding on treatment options for schizophrenia, prescribers should consider the finding that ziprasidone has a greater ability to prolong the QT / QTc interval than several other antipsychotics (see WARNINGS).” Although torsade de pointes is not in the FDA labeling for ziprasidone, cardiologists prefer other drugs in this class because of concerns about QT prolongation.

[0049] QT prolongation raises greater concern for potential arrhythmias when high doses are administered, in the setting of concomitant use with other drugs that prolong the QT interval, in the event of accidental or intentional overdose, and in situations where plasma exposure of the drug is high, such as when the metabolism of the drug is inhibited by interactions with other drugs. All of these situations apply to Rubusta.

[0050] The use of high doses of Rubostat is currently being investigated for the treatment of congestive heart failure. While 32 mg of Rubostat was widely used during the development of Rubostat to treat diabetic retinopathy, doses up to 256 mg are being explored for the treatment of heart failure. One of the primary outcome measures in the heart failure study is the proportion of patients with QT prolongation.

[0051] The pharmacokinetics of a compound are also important when considering the potential for QT prolongation. The potential for arrhythmia is related to the maximum plasma concentration of the compound. This has been observed in ECG studies where the maximum prolongation of the QT interval occurs at the maximum concentration of the study drug.

[0052] Peak concentrations that cause severe prolongation of the QT interval may not occur in most patients most of the time, but they become a problem when an accidental or intentional overdose occurs. This principle is evident in the instructions for use of tricyclic antidepressants. Physicians are taught to monitor their patients' ECGs during treatment with tricyclic antidepressants because tricyclic antidepressants prolong the QT interval. Importantly, physicians are also taught to reduce the likelihood of dangerous overdose and arrhythmias by prescribing only small amounts of the drug at a time. In principle, an overdose of 30 tablets is safer than an overdose of 100 tablets.

[0053] In normal use, when the half-life is longer, the peak concentration of the drug will be lower (relative to the drug's trough concentration). For example, a drug with a half-life of 24 hours can be administered once daily and its peak drug concentration will be only about 2 times the trough concentration. In contrast, a drug with a half-life of 6 hours may have a peak concentration of about 16 times the trough concentration if administered once daily.

[0054] Lower peak concentrations (relative to trough concentrations) are generally desirable as a principle of drug development because it keeps drug levels within the therapeutic window. There is a dose response for efficacy and there is a dose response for toxicity. A lower peak / trough ratio can help keep drug concentrations at a level that provides efficacy but is not so high as to cause toxicity. See Figure 2 .

[0055] Rubusta has a trough concentration of about 5 nmol / L, compared to a higher peak concentration of about 90 nmol / L (peak-to-trough ratio of about 18, consistent with a half-life of less than 6 hours). This increases the risk not only of QT prolongation but also of other toxicities, among which worsening glycemic control and elevated creatine kinase have raised concerns among the European Medicines Agency.

[0056] A third consideration affecting peak drug concentrations is potential interactions with other drugs. Rubusta is metabolized by CYP3A4, which converts Rubusta to N-desmethyl Rubusta. Figure 3 and Figure 4 Therefore, peak concentrations of Rubustadine may be elevated in the presence of CYP3A4 inhibitors.

[0057] These considerations are particularly relevant in conditions where the patient may be taking one or more medications that prolong the QT interval, including antipsychotics (haloperidol, ziprasidone, quetiapine, thioridazine, olanzapine, risperidone), antiarrhythmics (amiodarone, sotalol, dofetilide, procainamide, quinidine, flecainide), antibiotics (macrolides, fluoroquinolones), antidepressants (amitriptyline, imipramine, citalopram), and other medications (methadone, sumatriptan, ondansetron, cisapride).

[0058] In cases of bipolar disorder, depression, and / or schizophrenia, medications that prolong the QT interval are often prescribed. Patients with these conditions may be prescribed antidepressants. They may also be prescribed atypical antipsychotics, which are indicated for the treatment of bipolar disorder, depression, and schizophrenia.

[0059] QT prolongation is also a concern because patients with bipolar disorder and depression are at increased risk for suicide attempts, which may involve a medication overdose. Additionally, confusion or delusions in patients with mental illness may lead to an accidental overdose.

[0060] In the case of Alzheimer's disease, drugs that prolong the QT interval are frequently prescribed. Depression or agitation (behavioral complications of dementia) may occur, and these conditions can be treated with antidepressants or atypical antipsychotics, or both. Patients with Alzheimer's disease are usually older, which increases their risk of being prescribed antibiotics and other drugs. In addition, confusion or delusions in patients with Alzheimer's disease may lead to accidental overdose.

[0061] Rubusta is metabolized by CYP3A4. CYP3A4 inhibitors 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 drugs may increase peak concentrations of Rubusta and worsen QT prolongation.

[0062] For these reasons, the use of rubostat raises particular concerns in the treatment of neuropsychiatric conditions including bipolar disorder, depression, and Alzheimer's disease, all of which are indications for which new agents are needed and for which research on the use of GSK3 inhibitors and rubostat is encouraged. The use of rubostat also raises concerns when treating elderly subjects, who often take multiple medications and are at higher risk for adverse drug interactions.

[0063] In addition, the half-life of Rubusta is not ideal for its combination with lithium. The elimination half-life of lithium is about 18 hours. A combination product of two drugs should maintain the levels of both products 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 concentrations of the short-acting drug reaching unnecessarily high levels soon after administration, while levels of the same component may be low later in the dosing interval.

[0064] A new drug could be an important alternative to rubostat if it has similar or better potency against protein kinase C or GSK3 while having one or more of the following characteristics: similar or lower inhibition of hERG, longer half-life allowing for lower peak concentrations during the dosing interval (better pharmacokinetics), and lower potential for drug-drug interactions. One aspect of the invention relates to the use of N-desmethylrubostat with these characteristics in situations where rubostat is clinically useful.

[0065] In the hERG assay, N-desmethylrubostat was reported to be slightly less potent than rubostat in blocking hERG channels, requiring higher concentrations, with an IC50 of 62.6 nM for N-desmethylrubostat and 35.6 nM for rubostat. It was also reported that the half-life (t1 / 2) of N-desmethylrubostat was longer than that of rubostat, 23.9 hours versus 5.25 hours, respectively ( Figure 5 ). Inhibitors of CYP3A4 can increase plasma levels of rubusta, but they have not been reported to increase plasma levels of N-desmethylrubusta to the same extent, and the long half-life of N-desmethyl suggests the existence of an alternative metabolism and elimination pathway, namely hydroxylation. Therefore, N-desmethylrubusta may have a slightly lower potential to inhibit hERG, have better pharmacokinetic properties, may have less interaction with CYP3A4 inhibitors, and have a half-life that is more suitable for combination with lithium.

[0066] It has been reported that N-desmethylrubostat has a lower IC for hERG than rubostat 50 The difference is about two-fold, and the peak plasma level of N-desmethylrubast is reported to be only half that of rubast, so this combination of features may reduce the potential of N-desmethylrubast to prolong the QT interval or cause other toxicities to only about one-quarter that of rubast. The difference becomes even greater in the presence of drugs that inhibit CYP3A4.

[0067] The preparation of N-desmethylrubesta (Compound-1) is generally carried out according to Figure 6 The method shown in the figure is carried out. As shown, starting material 1 reacts with a vinyl Grignard reagent in the presence of copper iodide to obtain an alcohol intermediate 2. Those of ordinary skill in the art will recognize that substitutes for vinyl Grignard reagents can be used to achieve the same transformation. These substitutes include, but are not limited to, vinyl zinc reagents, vinyl cuprate reagents, and vinyl lithium reagents. Those of ordinary skill in the art will recognize that substitutes for copper iodide can be used to promote the conversion of intermediate 1 to intermediate 2. Such substitutes include, but are not limited to, alternative Lewis acid reagents and chelating agents, such as crown ethers.

[0068] After isolating intermediate 2, Figure 6Conversion to intermediate 3 by reaction with allyl bromide is shown. One of ordinary skill in the art will recognize that alternative allylation agents can be used to allylate intermediate 2 to intermediate 3. Such allylation agents typically employ substitutes for bromide leaving groups, and include, but are not limited to, allyl chloride, allyl iodide, and allyl methanesulfonate. One of ordinary skill in the art will also recognize that substitutes for the potassium tert-butoxide base shown can be used to achieve the reaction of intermediate 2 with the allylation agent. Such bases include, but are not limited to, hydride reagents, carbonate reagents, bicarbonate reagents, lithium diisopropylamide, sodium hexamethyldisilazide, and the like.

[0069] Figure 6 The two-step conversion of intermediate 3 to intermediate 4 is further shown. As shown, the first step is an ozonolysis reaction, resulting in the cracking of diolefins into dialdehydes, and the second step is the reduction of dialdehydes to diols by sodium borohydride. Those of ordinary skill in the art will recognize that ozonolysis is only one of many reactions or reaction combinations suitable for cracking olefins into aldehydes. Such conversions include, but are not limited to, dihydroxylation of olefins, followed by cracking of the resulting diols into aldehydes. Suitable reagents for dihydroxylation of olefins include, but are not limited to, osmium tetroxide, etc. Suitable reagents for cracking diols into aldehydes include, but are not limited to, sodium periodate, lead tetraacetate, etc. Those of ordinary skill in the art will also recognize that substitutes for sodium borohydride reducing agents are suitable for reducing aldehydes to alcohols. Such reagents include, but are not limited to, lithium aluminum hydride, diisopropyl aluminum hydride, lithium borohydride, borane, etc. Those of ordinary skill in the art will also recognize that substitutes for boron and aluminum-based reducing agents can also be used to reduce aldehydes to alcohols. Such substitutes include, but are not limited to, samarium iodide and triethylsilane.

[0070] like Figure 6 As shown, the diol of intermediate 4 is converted to the bis-mesylate intermediate 5 after reaction with methanesulfonyl chloride and triethylamine. One of ordinary skill in the art will recognize that mesylate is generally useful as a leaving group, as are common alternative leaving groups, including but not limited to chloride, bromide, iodide, tosylate, etc. One of ordinary skill in the art will also recognize that alternatives to triethylamine can be used to convert the alcohol to a mesylate, such alternatives include but are not limited to diisopropylethylamine, pyridine, carbonate reagents, bicarbonate reagents, etc.

[0071] Figure 6 Using cesium carbonate as a base, the reaction of bis-mesylate intermediate 5 with bis-indolemaleimide intermediate 6 to form intermediate 7 is shown. One of ordinary skill in the art will recognize that alternative bases may be used to effectuate the reaction shown to form intermediate 7. Such bases include, but are not limited to, hydrides, alkoxides, carbonates, bicarbonates, and the like.

[0072] The process of converting methylmaleimide to its demethylated form involves first hydrolyzing intermediate 7 to maleic anhydride intermediate 8. Figure 6 As shown, the conversion is achieved using an ethanolic solution of potassium hydroxide. One of ordinary skill in the art will recognize that the conversion of intermediate 7 to intermediate 8 can employ alternatives to potassium hydroxide, including but not limited to sodium hydroxide and lithium hydroxide. In addition, 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.

[0073] according to Figure 6 , maleic anhydride intermediate 8, after reaction with hexamethyldisilazane, generates the corresponding maleimide intermediate 9. One of ordinary skill in the art will recognize that alternative reagents, including but not limited to ammonia, sodium amide, etc., may be used to convert maleic anhydride to maleimide.

[0074] After obtaining maleimide, Figure 6 Cleavage of the trityl protecting group from intermediate 9 to give the alcohol intermediate 10 is shown. Figure 6 Hydrochloric acid is emphasized as the reagent to achieve trityl cleavage, but one of ordinary skill in the art will recognize that alternative acids may be used. Such alternatives include, but are not limited to, hydrobromic acid, trifluoroacetic acid, acetic acid, and the like.

[0075] like Figure 6 As shown, the alcohol of intermediate 10 is converted to mesylate intermediate 11 after reaction with methanesulfonyl chloride and pyridine. One of ordinary skill in the art will recognize that mesylate is generally useful as a leaving group, as are common alternative leaving groups, such alternative leaving groups include but are not limited to chloride, bromide, iodide, tosylate, etc. One of ordinary skill in the art will also recognize that alternatives to triethylamine can be used to convert the alcohol to mesylate, such alternatives include but are not limited to diisopropylethylamine, pyridine, carbonate reagents, bicarbonate reagents, etc.

[0076] In the final stage of synthesis, Figure 6 It is shown that intermediate 11 is converted to compound-1 after reaction with methylamine. Figure 6 Not shown, the methylamine is further converted to its corresponding hydrochloride salt. One of ordinary skill 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 recognized by those skilled in the art and generally yield structures such as ComprehensiveOrganicTra nsformations (Larock, Wiley) resources.

[0077] Those skilled in the art will recognize that, in addition to the above description and Figure 6In addition to the reaction shown in, there are many other reactions that can be used to prepare N-desmethylrubesta (Compound-1). Suitable reactions are readily determined by those skilled in the art and can be used in, for example, Comprehensive Organic Transformations (Larock, Wiley). Strategies for introducing and cleaving protecting groups are readily determined by those skilled in the art and can be found in, for example, Protective Groups in Organic Synthesis (Greene and Wutz, Wiley). A person of ordinary skill in the art will also recognize that generally applicable Figure 6 as well as Figure 6 Any and all alternatives and variations of the pathways described in, alternative combinations of reagents, solvents, temperature conditions and reaction times will provide similar chemical results, thereby enabling the preparation of Compound-1.

[0078] The ability of N-desmethylrubostat to inhibit GSK3β, a specific form of GSK associated with bipolar disorder and other neuropsychiatric conditions, was previously unknown. Surprisingly, N-desmethylrubostat was found to be approximately twice as potent as rubostat in inhibiting GSK3β. Figure 7 ). This greater potency provides the ability to use N-desmethylrubostat at lower doses than rubostat.

[0079] N-desmethyl rubostat was found to have a higher stability in human liver microsomes compared to rubostat. It was found that after 15 minutes in the liver, 76.38% of N-desmethyl rubostat remained, which was in contrast to 3.51% of rubostat remaining ( Figure 8 ). In this study, the CYP3A4 inhibitor troleandomycin increased rubustard levels 23-fold at 15 minutes, while N-desmethyl rubustard levels were only increased 1.2-fold. This suggests that CYP3A4 has less effect on N-desmethyl rubustard metabolism than on rubustard metabolism (Table 1).

[0080] Table 1. Metabolic stability in pooled human liver microsomes with and without inhibitors

[0081]

[0082] The ability of N-desmethylrubostat to cross the blood-brain barrier was previously unknown. Pharmacokinetic studies in rats showed brain penetration of N-desmethylrubostat, with a brain / plasma ratio of 1.18 after 4 hours, supporting the potential for therapeutic use of N-desmethylrubostat in the treatment of conditions of the central nervous system (Table 2).

[0083] Table 2. Concentrations of N-desmethylrubustard in rat plasma and brain

[0084]

[0085] The ability of N-desmethylrubotar to have pharmacological effects similar to those of lithium was previously unknown. N-desmethylrubotar was able to reduce ultrasonic appetite vocalizations in rats treated with dextroamphetamine, and the magnitude of this effect was similar to that of lithium ( Fig. 9 ). This supports the potential of N-desmethylrubotarin to provide similar clinical benefits as lithium.

[0086] As an alternative to Rubostat, N-desmethyl Rubostat may be administered to subjects who have never used Rubostat. As an alternative to Rubostat, N-desmethyl Rubostat may be administered to subjects who have experienced adverse effects of Rubostat, have a prolonged QT interval, have demonstrated high Rubostat drug levels, or have the potential to receive medications that may interfere with Rubostat metabolism, or in whom higher doses of Rubostat may be required and may raise concerns about adverse effects, QT prolongation, or adverse drug interactions.

[0087] The risk of QT prolongation and other potential toxicities can be further reduced by administering N-desmethyl rubostat with lithium. Both N-desmethyl rubostat and lithium inhibit GSK3, and lithium has been shown to have a synergistic effect with another GSK3 inhibitor in the treatment of bipolar disorder in animal models. If N-desmethyl rubostat is administered in combination with lithium, the desired amount of GSK3 inhibition can be achieved with a lower concentration of N-desmethyl rubostat. Therefore, N-desmethyl rubostat or a pharmaceutically acceptable salt thereof can be administered in combination with lithium for the treatment of bipolar disorder or other conditions in which inhibition of GSK3, protein kinase C, or both is useful.

[0088] This combination can not only reduce the dose of N-desmethyl rubostat, but also reduce the dose of lithium required for treating bipolar disorder, Alzheimer's disease and other conditions where it is clinically desirable to inhibit GSK3 by lithium, thereby improving safety. The dose of N-desmethyl rubostat in the combination can be a dose lower than the dose required as a monotherapy (sub-effective dose), and the dose of lithium in the combination can be a dose lower than the dose required as a monotherapy (sub-effective dose). The combination can be used to provide therapeutic effects for subjects who are unresponsive to lithium or intolerant to standard doses of lithium. N-desmethyl rubostat can be used to provide additional therapeutic effects for subjects who only partially respond to lithium, as an alternative to higher lithium doses.

[0089] In addition, rubostat 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-desmethylrubostat has superior pharmacokinetics and a lower potential for QT prolongation and can therefore be used as an alternative to rubostat in combination with valproic acid, lamotrigine, quetiapine, olanzapine, risperidone, aripiprazole, lurasidone, lumepirone, cariprazine, asenapine, and carbamazepine.

[0090] Antipsychotics that are known not to prolong the QT interval include olanzapine, risperidone, aripiprazole, lumepirone, xanomeline-trospium, iloperidone, and lurasidone, which have also been used to treat neurological and / or psychiatric disorders including bipolar disorder, depression, Parkinson's disease, and schizophrenia. N-desmethyllubotar can be combined with these antipsychotics to treat these conditions.

[0091] In addition, the response to N-desmethyl rubostat or to the combination of N-desmethyl rubostat and lithium can be used to determine 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 is being developed as a diagnostic method. N-desmethyl rubostat, alone or in combination with lithium, can be administered to subjects with excessive GSK3β activity on PET to treat Alzheimer's disease, and a decrease in GSK3β activity on PET after administration of N-desmethyl rubostat can support its use (alone or in combination with lithium) as an appropriate therapy administered at an appropriate dose.

[0092] The compositions and treatment methods disclosed herein are relevant to any condition for which Rubostar 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, neuropathy) and chemotherapy-induced cognitive impairment.

[0093] Rubostat and N-desmethylrubostat are reported to be equally potent in inhibiting protein kinase C, and thus these compositions and methods of treatment are relevant to indications in which rubostat may be used 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.

[0094] These compositions and treatment methods are also relevant to conditions in which 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, Fuch's dystrophy, limbal cell defects, dry eye, glaucoma, familial exudative vitreoretinopathy (FEVR), Norrie's disease, Coats' disease, retinopathy of prematurity, macular telangiectasia, retinal vein occlusion, Sjögren'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 relevant to GM2 gangliosidosis, for which the use of rubustadine has been proposed.

[0095] The compositions and treatment methods disclosed herein may also be used in veterinary applications to improve the health and well-being of livestock and companion animals by treating the animals for any of the above-mentioned indications. Example

[0096] Materials and methods

[0097] Example 1. Compound synthesis: N-desmethylrubestra was synthesized according to the following procedure:

[0098] Step-1: Synthesis of (S)-1-(trityloxy)pent-4-en-2-ol (2):

[0099]

[0100] To a solution of vinylmagnesium bromide (1M in THF, 840mL, 0.84mol) was added copper iodide (4.5g, 23.62mmol) at -40°C under a nitrogen atmosphere. After stirring for 20 minutes at -40°C, compound-1 (150g, 0.46mmol) dissolved in anhydrous THF (750mL) was added dropwise to the reaction mixture, and the resulting reaction mixture was stirred for 2 hours at -40°C. After the reaction was completed (monitored by TLC), saturated ammonium chloride (1000mL) was added. The reaction was warmed to room temperature while stirring and extracted with ethyl acetate (1000mL). The organic layer was separated and washed with ammonia water (250mL). The organic layer was separated, dried over sodium sulfate, filtered and evaporated in vacuo to obtain compound-2 as a dark brown viscous substance (166 grams, crude yield 100%). 1H 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).

[0101] Step-2: Synthesis of (S)-(((2-(allyloxy)pent-4-en-1-yl)oxy)methanetrityl)triphenyl (3):

[0102]

[0103] Under nitrogen atmosphere, potassium tert-butoxide (70.0 g, 0.62 mmol) was added in batches to a stirred solution of compound-2 (165 g, 0.48 mol) in anhydrous THF (1500 mL). The resulting reaction content was heated to 45 ° C and stirred for 2 hours, then cooled to room temperature, followed by addition of allyl bromide (145.5 g, 1.22 mol) at room temperature and continued stirring for 1 hour 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 in vacuo to obtain crude compound-3, which was further purified by silica gel column chromatography (100-200 mesh) and eluted with a hexane solution of 0.5-1% ethyl acetate. Pure fractions were collected and evaporated under reduced pressure to obtain the desired compound-3, which was a light yellow semi-solid (106 grams, yield 58%). 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).

[0104] Step-3: Synthesis of (S)-3-(2-hydroxyethoxy)-4-(trityloxy)butan-1-ol (4):

[0105]

[0106] At -45 ° C, ozone gas was bubbled for 18 hours in a stirred solution of compound-3 (100g, 0.26mol) in MeOH:DCM (1: 1) (800mL). After the reaction was completed (monitored by TLC), it was poured into a solution of sodium borohydride (21.5g, 0.57mol) in 0.5N NaOH solution (370mL) at 0 ° C. The resulting reaction mixture was stirred at room temperature for 16 hours. After the reaction was completed (monitored by TLC), it was quenched with 1N HCl solution until the pH value was 6-7. The resulting solution was then extracted with ethyl acetate (750mL). The organic layer was separated, dried over sodium sulfate, filtered and evaporated under vacuum to obtain a crude compound, which was further purified by silica gel column chromatography (100-200 mesh) and eluted with a hexane solution of 20-25% ethyl acetate. Pure fractions were collected and evaporated to obtain the desired compound-4, which was a yellow colloidal liquid (58 grams, yield 57%). 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).

[0107] Step-4: Synthesis of (S)-2-((4-((methylsulfonyl)oxy)-1-(trityloxy)butan-2-yl)oxy)ethyl methanesulfonate (5):

[0108]

[0109] Triethylamine (66 mL, 0.47 mmol) was added to a stirred solution of compound-4 (60 g, 0.15 mol) in DCM (1000 mL) at 0 ° C and stirred for 15 minutes, followed by the addition of methanesulfonyl chloride (32.0 mL, 0.41 mmol). The resulting reaction mixture was stirred at 0 ° C for 2 hours, (the reaction was monitored by TLC), and quenched with a saturated ammonium chloride solution (600 mL). The organic layer was separated, dried over sodium sulfate, filtered and evaporated under vacuum (≤ 25 ° C) to obtain a crude compound, which was suspended in a 1: 1 mixture (600 mL) of ethyl acetate and heptane, and evaporated under vacuum to obtain a solid. The resulting solid compound was suspended in a 1: 1 mixture (600 mL) of ethyl acetate and heptane, stirred for 30 minutes, filtered, washed with heptane (80 mL) and dried under vacuum to obtain compound-5, which was a cream solid (88 grams, crude yield 100%). 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).

[0110] Step-5: Synthesis of (12E,32E,7S)-21-methyl-7-((trityloxy)methyl)-22,25-dihydro-11H,21H,31H-6-oxa-1,3(3,1)-diindol-2(3,4)-pyrrole heterocyclic 9-fan-22,25-dione (7):

[0111]

[0112] To a stirred solution of compound-6 (41.5 g, 0.12 mol) in DMF (850 mL), cesium carbonate (86.0 g, 0.26 mol) was added, and the reaction mixture was heated to 100 ° C, and then 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 hours. After the reaction was completed (monitored by TLC), it was cooled to 50 ° C, diatomaceous earth (25 g) was added and stirred for 15 minutes. The reaction mass was filtered on diatomaceous earth and the filtrate was distributed 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 a crude compound, which was further purified by silica gel column chromatography (100-200 mesh) and eluted with a hexane solution of 25-30% ethyl acetate. Pure fractions were collected and evaporated to obtain the desired compound-7, which was a brick red solid (55 grams, yield 51%). 1 H NMR (400MHz, DMSO-d6): δ7.83(d,J=7.6Hz 1H),7.75(d,J=8.0Hz 1H),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).

[0113] Step-6: Synthesis of (12E,32E,7S)-7-((trityloxy)methyl)-22,25-dihydro-11H,31H-6-oxa-1,3(3,1)-diindol-2(3,4)-furanheterocyclopenta-22,25-dione (8).

[0114]

[0115] Potassium hydroxide (68.0 g, 1.22 mol) was added to a stirred solution of compound-7 (85.0 g, 0.12 mol) in ethanol (850 mL) and heated to 80 ° C. The resulting reaction mixture was stirred for 24 hours. After the reaction was completed (monitored by TLC), the reaction mixture was evaporated in vacuo to obtain a residue, which was distributed in DCM (850 mL) and 20% citric acid solution (450 mL). The organic layer was separated, dried over sodium sulfate, filtered and evaporated in vacuo to obtain crude compound-8 as a dark brown solid (62 grams, 74% yield). 1 HNMR (400MHz, DMSO-d6): δ7.88(d,J=7.6Hz 1H), 7.82(d,J=7.6Hz 1H), 7.65(d,J=2.0Hz 2H), 7.55(d,J=8.0Hz 1H), 7.41(d,J=7.6Hz 1H), 7.34-7.26 (m, 12H), 7.25-7.20 (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). (Additional protons in the aromatic region are not included)

[0116] Step-7: Synthesis of (12E,32E,7S)-7-((trityloxy)methyl)-22,25-dihydro-11H,21H,31H-6-oxa-1,3(3,1)-diindol-2(3,4)-pyrrole heterocyclic 9-fan-22,25-dione (9):

[0117]

[0118] To compound-8 (95.0g, 0.25mol) in DMF (950mL) stirring solution, add HMDS (294.0mL, 2.47mol), methanol (6.0mL), and be heated to 80 ℃.Reaction mixture is stirred at 80 ℃ for 5 hours.After reaction is completed (monitored by TLC), cool to room temperature and quench with 1N HCl solution (950mL), and extract with DCM (1500mL).Separate organic layer, dry over sodium sulfate, filter and vacuum evaporate, obtain crude compound (84g), it is further purified by silica gel column chromatography (100-200 mesh), eluted with the hexane solution of 20-25% ethyl acetate.Collect pure fraction and evaporate under vacuum, obtain desired compound-9, be purple solid (70 grams, yield 74%). 1H NMR (400MHz, DMSO-d6): δ10.91(s,1H),7.81(d,J=8.0Hz 1H),7.73(d,J=8.0Hz 1H),7.48(d,J=8.4Hz 2H),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).

[0119] Step-8: Synthesis of (12E,32E,7S)-7-(hydroxymethyl)-22,25-dihydro-11H,21H,31H-6-oxa-1,3(3,1)-diindol-2(3,4)-pyrrole heterocyclic 9-fan-22,25-dione (10):

[0120]

[0121] At room temperature, 6N HCl (700 mL) was added to a stirred solution of compound-9 (70.0 g, 0.18 mol) in ethanol (700 mL). The resulting reaction contents were heated to 80 ° C and maintained for 3 hours. After the reaction was completed (monitored by TLC), it was cooled to room temperature and stirred for 1 hour. The resulting solid was filtered and washed with water (350 mL) and dried in vacuo at 45 ° C to obtain compound-10 as a purple solid (40 grams, crude yield 88%). 1 H NMR (400MHz, DMSO-d6): δ10.92(s,1H),7.82(d,J=7.6Hz 1H),7.78(d,J=7.6Hz 1H),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),7.13-7.10(m,2H),4.69(t,J=5.2Hz 1H),4.35-4.33(m,1H),4.24-4.15(m,3H),3.91-3.87(m,1H),3.65-3.60(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).

[0122] Step-9: Synthesis of ((12E,32E,7S)-22,25-dioxo-22,25-dihydro-11H,21H,31H-6-oxa-1,3(3,1)-diindol-2(3,4)-pyrroleheterocyclopentadien-7-yl)methyl methanesulfonate (11):

[0123]

[0124] Pyridine (33.2mL, 0.39mol) is added to the stirring solution of compound-10 (39.0g, 0.09mol) in THF (400mL) at room temperature, stirred for 20 minutes, then methanesulfonic anhydride (46.0g, 0.26mol) is added to the reaction at room temperature. The obtained reaction mixture is stirred for 4 hours. After the reaction is completed (monitored by TLC), the reactant is distributed between ethyl acetate (100mL) and water (50mL), and the organic layer is separated, dried over sodium sulfate, filtered and evaporated in vacuo to obtain a crude compound (37.0g), which is further purified by silica gel column chromatography (100-200 mesh), eluted with DCM. Collect pure fractions and evaporate under reduced pressure to obtain desired compound-11, which is a purple solid (30 grams, yield 65%). 1 H NMR (400MHz, DMSO-d6): δ10.92(s,1H),7.83(d,J=7.6Hz 1H),7.78(d,J=7.6Hz 1H),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),3.93-3.90(m,1H),3.66-3.61(m,1H),3.17(s,3H),2.19-2.14(m,1H),2.03-1.98(m,1H).

[0125] Step-10: Synthesis of (12E,32E,7S)-7-((methylamino)methyl)-22,25-dihydro-11H,21H,31H-6-oxa-1,3(3,1)-diindol-2(3,4)-pyrrole heterocyclic 9-fan-22,25-dione hydrochloride (Compound-1):

[0126]

[0127] In an autoclave, a solution of 2M methylamine in THF (400mL) was added to a stirred solution of compound-11 (10.0g, 0.019mol) in THF (400mL) at -40°C. The reaction was gradually heated to 70°C and stirred for 24 hours. After the reaction was completed (monitored by TLC), vacuum evaporation gave a crude compound (12.0g). This batch was merged with 4 other batches of the same scale to give 60.0g of crude product. These 60 grams were purified by silica gel column chromatography (230-400 mesh, 2% MeOH / DCM). Pure fractions were collected and concentrated to give the desired free alkali of compound-1 (22.0g), which was a red solid. The free alkali was suspended in ether (220mL) and cooled to 0°C. Ethanol HCl (33mL) was added at 0°C. The resulting suspension was stirred at 0 °C for 30 min, filtered, washed with ether (50 mL) and dried under vacuum at 40 °C for 1 h to afford compound-1 as a brick red solid (16.9 g, 36% yield). 1 H NMR (400 MHz, DMSO-d6): δ 10.93 (s, 1H, exchanged in D2O), 8.72-8.71 (m, 2H, exchanged in D2O), 7.81 (t, J = 8.0 Hz, 2H), 7.55 (d, J = 8.0 Hz 1H),7.49(s,2H),7.47(d,J=8.4Hz,1H),7.23(t,J=7.2Hz,2H),7.14(t,J=7 .2Hz,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=9.2Hz,1H),3.27-3.24(m,1H),3.0 1-2.98(m,1H),2.53(t,J=5.6Hz,3H),2.22-2.20(m,1H),2.06-2.03(m,1H).

[0128] Example 2. Kinase assay:

[0129] Rubusta was purchased from a commercial laboratory, and N-desmethylrubusta was synthesized according to Example 1. Kinase reactions were performed in a 384-well format. Reaction conditions included 0.25 ng GSK3α or GSK3β (final enzyme concentrations of 0.62 and 0.68 nM, respectively), 0.25 μg 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% Brij-35, and 3 mM DTT). Compounds or 1% DMSO were added. 5X stock solutions of the above buffers were prepared (without DTT) and stored at room temperature.

[0130] Two solutions were prepared. The first contained 1X buffer and 2X enzyme and substrate. The second contained 1X buffer and 2X ATP. Each plate was loaded with 2.5 μL of the first solution, followed by the addition of compounds, followed by incubation at room temperature for 15 minutes, and then 2.5 μL of the second solution was added (followed by pulse spinning). The plates were then incubated at room temperature for 60 minutes (using another plate as a lid in the dark). To stop the reaction and consume the remaining ATP, 5 μL of ADP-Glo ​​reagent was added to each well, and the plates were then left at room temperature for another 40 minutes. To detect the production of ADP, 10 μL of ADP-Glo ​​Kinase Assay Substrate was added to each well. After 5 to 30 minutes, the luminescence values ​​of the plates were read.

[0131] Example 3. Microsome stability:

[0132] Prepare a master solution containing 100mM phosphate buffer, 5mM MgCl2 solution and 0.5mg / mL human microsomes. Add 40μL of 10mM NADPH solution to each well to produce a final concentration of 1mM NADPH. Preheat the mixture at 37°C for 5 minutes. Negative control samples are prepared by replacing the NADPH solution with 40μL ultrapure H2O. Prepare samples containing NADPH in duplicate. Prepare one negative control. Start the reaction by adding 2μL of 200μM control compound or test compound solution. Use Verapamil as a positive control. The final concentration of the test compound or control compound is 1μM.

[0133] Aliquots of 50 μL were taken from the reaction solution at 0, 15, 30, 45 and 60 minutes. The reaction was stopped by adding 4 volumes of cold acetonitrile containing IS (100 nM alprazolam, 200 nM imipramine, 200 nM labetalol and 2 μM ketoprofen). The samples were centrifuged at 3,220 g for 40 minutes. An aliquot of 90 μL of the supernatant was mixed with 90 μL ultrapure H2O and then used for LC-MS / MS analysis. Peak areas were determined from the extracted ion chromatograms.

[0134] Example 4. Blood / brain barrier penetration:

[0135] 1 mg / kg of N-desmethyl Rubostat was administered intravenously to male Sprague Dawley (SD) rats (via the tail vein). Blood samples were collected from 3 animals at 0.25, 1 and 4 hours after administration. Brain samples were collected from another 9 rats, 3 rats at a time at 0.25, 1 and 4 hours after administration. Brain samples were weighed and homogenized with phosphate buffered saline. Samples (20 μL) were added to 200 μL of a mixture containing acetonitrile for precipitation and then vortexed for 30 seconds. After centrifugation at 4 degrees Celsius and 4000 rpm for 15 minutes, the supernatant was diluted with ultrapure H2O in a ratio of 1:2, and 15 μL of the supernatant was then injected into an LC / MS / MS system (liquid chromatography with tandem mass spectrometry) to analyze the level of N-desmethyl Rubostat.

[0136] Example 5. Ultrasonic vocalization of rats:

[0137] Before the start of the experiment, 98 male Wistar rats were adapted to the test facility and treated every day (10 minutes / day) for 7 days. Male Wistar rats (~200g) were used in this study: 5 groups of 8 rats in each group (lithium and 0, 10, 30, 100mg / kg N-desmethylrubestad were administered before dextroamphetamine or D-AMP). There were also 9 rats that did not receive D-AMP. The rats were placed in a white Plexiglas box (50x 50x 50cm) and recorded for 10 minutes to adapt them to the test device. Ultrasonic vocalizations (USVs) were recorded using a microphone installed 45cm above the open field box to establish the baseline of each rat, i.e., non-treatment-related USV reactions (50-kHz calls). The next day, rats were injected with saline (1mL / kg body weight, IP) and immediately placed in an open field test box. USVs were recorded for 10 minutes. These data were used as the basis for selecting equivalent groups for drug testing. On day 3, rats were given a positive treatment control (100 mg / kg lithium carbonate in saline subcutaneously), or 0, 10, 30, or 100 mg / kg N-desmethylrubustard intraperitoneally (IP). 60 minutes later, D-amphetamine was given IP at 2.5 mg / kg (in saline), and the rats were immediately placed in an open field test box. USVs were recorded for 10 minutes.

Claims

1. N-desmethyl rubostat or a pharmaceutically acceptable salt thereof for use in treating a disorder characterized by abnormal signal transduction of GSK3β or protein kinase C, wherein the use is characterized in that a therapeutically effective dose of N-desmethyl rubostat or a pharmaceutically acceptable salt thereof is administered to a subject in need thereof.

2. N-desmethylrubostat or a pharmaceutically acceptable salt thereof for use according to claim 1, wherein the subject 1) has never taken rubostat, or 2) has taken rubostat and experienced adverse reactions, or 3) exhibits a prolonged QT interval, or 4) has been shown to have elevated plasma levels of rubostat, or 5) is likely to receive medications that may interfere with the metabolism of rubostat, or 6) may require a higher dose of rubostat and there are concerns about adverse reactions, QT prolongation or adverse drug interactions.

3. N-desmethylrubostat or a pharmaceutically acceptable salt thereof for use according to claim 1 or 2, wherein the subject suffers from a neurological disease and / or a psychiatric disorder.

4. N-desmethylrubostatin or a pharmaceutically acceptable salt thereof for use according to claim 3, wherein the disease / disorder is 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.

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

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

7. N-desmethylrubostatin or a pharmaceutically acceptable salt thereof for use according to claim 1 or 2, wherein the disease / disorder is selected from an ocular disorder including wet age-related macular degeneration, dry age-related macular degeneration, Fuch's dystrophy, limbal cell defects, dry eye, glaucoma, familial exudative vitreoretinopathy (FEVR), Norrie's disease, Coats' disease, retinopathy of prematurity, macular telangiectasia, retinal vein occlusion and Sjögren's syndrome, and / or an ocular disorder including sensorineural hearing loss and conductive hearing loss.

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

9. N-desmethylrubostat or a pharmaceutically acceptable salt thereof for use according to any one of the preceding claims, wherein N-desmethylrubostat or a pharmaceutically acceptable salt thereof is administered in an amount of about 32 to about 320 mg once daily or about 16 to about 160 mg twice daily.

10. N-desmethylrubostat or a pharmaceutically acceptable salt thereof for use according to any one of claims 1 to 8, wherein N-desmethylrubostat or a pharmaceutically acceptable salt thereof is administered in combination with lithium.

11. N-desmethylrubostat or a pharmaceutically acceptable salt thereof for use according to any one of claims 1 to 8, wherein the subject is unresponsive to lithium.

12. N-desmethylrubostat or a pharmaceutically acceptable salt thereof for use according to claim 10, wherein the subject is responsive to lithium.

13. N-desmethylrubostat or a pharmaceutically acceptable salt thereof for use according to claim 10, wherein lithium is administered at a sub-effective dose based on a monotherapy, and wherein N-desmethylrubostat or a pharmaceutically acceptable salt thereof is administered at a sub-effective dose based on a monotherapy.

14. N-desmethylrubostat or a pharmaceutically acceptable salt thereof for use according to claim 13, wherein the sub-effective dose of lithium is from about 60 mg to about 600 mg once daily, or from about 30 mg to about 300 mg twice daily.

15. N-desmethylrubostat or a pharmaceutically acceptable salt thereof for use according to claim 11, wherein the sub-effective dose of N-desmethylrubostat or a pharmaceutically acceptable salt thereof is administered at about 8 to about 32 mg once daily or about 4 to about 16 mg twice daily.

16. N-desmethyl rubostat or a pharmaceutically acceptable salt thereof for use according to claim 3, wherein N-desmethyl rubostat or a pharmaceutically acceptable salt thereof is administered in combination with valproic acid, lamotrigine, quetiapine, olanzapine, risperidone, aripiprazole, lurasidone, lumepirone, cariprazine, asenapine, carbamazepine, xanomeline-trospium chloride, iloperidone or a combination thereof.

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

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

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

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

21. N-desmethylrubostat or a pharmaceutically acceptable salt thereof for use according to claim 20, wherein the dosages of both N-desmethylrubostat and lithium are sub-effective on a monotherapy basis.

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

23. N-desmethylrubostat or a pharmaceutically acceptable salt thereof for use according to claim 19, wherein the dosages of both N-desmethylrubostat and lithium are sub-effective on a monotherapy basis.

24. N-desmethyl rubostat or a pharmaceutically acceptable salt thereof for use in determining an appropriate therapeutic dose of N-desmethyl rubostat in a subject, wherein the use is characterized by administering increasing doses of N-desmethyl rubostat or a pharmaceutically acceptable salt thereof and lithium to the subject, and assessing the response using positron emission tomography (PET).

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