Polymorphs of N-demethylated lubrusta and salts thereof

Abstract

The present invention relates to novel compositions of N, N-demethylated lubrusta L-lactate. Disclosed are the use of a composition of N-demethylubrusta L-lactate to modulate GSK-3 signaling and the use of a composition of N-demethylubrusta L-lactate to inhibit protein kinase C. The invention further relates to the use of the composition of N-demethylubrusta L-lactate to modulate GSK-3 signaling. Also disclosed are methods of treating a patient suffering from a neurological disease and / or a psychiatric disorder, including Alzheimer's disease, bipolar disorder, depression, schizophrenia, Parkinson's disease, or neuroinflammation, using a composition of N-demethylubrusta L-lactate, and methods of treating a disease associated with diabetes or complications thereof or ischemia, inflammation, pulmonary arterial hypertension, congestive heart failure, cardiovascular disease, skin disease, or cancer using the compositions of N-demethylubrusta L-lactate. In addition, compositions of N-demethylubrusta L-lactate are also disclosed for administration in combination with lithium or other treatment for bipolar disorders.

Description

[0001] Invention Field

[0002] This invention relates to novel compositions of N-desmethyl ruboxistaurin. N-desmethyl ruboxistaurin is a kinase inhibitor used to treat neurological or psychiatric disorders, including Alzheimer's disease, bipolar disorder, depression, schizophrenia, Parkinson's disease, or neuroinflammation, as well as to treat 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 N-desmethyl ruboxistaurin is clinically useful. Background of the Invention

[0004] It has been shown that N-demethylrubesta can regulate GSK-3 signaling and inhibit protein kinase C.

[0005] Rupersta has been investigated in multiple clinical trials for the treatment of diabetes and its complications, including diabetic retinopathy, diabetic neuropathy, and diabetic nephropathy. See A. Girach's U.S. Patent Publication No. 2008 / 0096923, the full text of which is incorporated herein by reference. However, Rupersta has some limitations, including the potential to prolong the QT interval on human electrocardiograms, a short half-life, a high peak-to-trough ratio in plasma when administered once daily, and metabolism via CYP3A4, which may lead to interactions with concomitant drugs that inhibit CYP3A4.

[0006] Recent findings indicate that N-desmethyl rubesta is more effective at inhibiting GSK-3 than rubesta, is more stable, exhibits superior pharmacokinetics, and its metabolism is significantly less affected by CYP3A4 inhibition. Therefore, when inhibition of GSK-3 or protein kinase C (or both) is desired, N-desmethyl rubesta, administered alone or in combination with other drugs, is a preferable alternative to rubesta.

[0007] As a GSK-3 inhibitor, N-desmethylrubesta has been proposed as a treatment for patients 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. GSK-3 inhibitors are known to increase the expression of the WNT protein, thereby enhancing a pathway that has been widely proposed for the treatment of neurological and psychiatric disorders in regenerative medicine.GSK-3 inhibition or enhanced WNT signaling has been associated with potential treatment for the following conditions: type 2 diabetes and kidney diseases (including diabetic nephropathy, chronic kidney disease, polycystic kidney disease, and focal segmental glomerulosclerosis), as well as atherosclerosis, alopecia, bone and joint diseases (including osteoarthritis and osteoporosis), inflammatory diseases (including alcoholic hepatitis, inflammatory bowel disease, and septic shock), eye diseases (including wet age-related macular degeneration, dry age-related macular degeneration, diabetic macular edema), and Fuch's malnutrition. 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.GSK-3 inhibition or enhanced WNT signaling has been associated with potential treatments for the following conditions: ear conditions (including sensorineural and conductive hearing loss), lung conditions (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 GSK-3 inhibitor, N-desmethylrubesta has been proposed as a monotherapy for bipolar disorder, or in combination with lithium, or in combination with other bipolar disorder treatments.

[0008] As a protein kinase C inhibitor, N-desmethylrubesta 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 ganglioside storage disorders. Protein kinase C inhibition has also been proposed for the treatment of bipolar disorder.

[0009] In order to develop N-desmethylrubesta into a therapeutic agent, it must possess favorable physical and pharmacological properties to enable delivery via a preferred route of administration. One such physical property is solubility – a property that can be enhanced by determining suitable salt forms and their crystal forms. Therefore, it is necessary to determine suitable salt forms of N-desmethylrubesta and suitable crystal forms of said salt forms in order to enable its development into a therapeutic agent.

[0010] Invention Summary

[0011] The present invention relates to novel compositions of N-demethylrubesta-L-lactate (identified as N-demethylrubesta-L-lactate crystal form 1, N-demethylrubesta-L-lactate crystal form 2 and N-demethylrubesta-L-lactate crystal form 3).

[0012] For general reference, according to Figure 1 The N-demethylrubesta hydrochloride was prepared according to the method shown. Then, following... Figure 2 The scheme shown achieves the conversion to N-demethylrubesta-L-lactate. Through... Figure 3 shown 1 1H NMR spectroscopy confirmed the separation of the expected salt form.

[0013] On one hand, the present invention is characterized by the crystal form of N-demethylrubesta-L-lactate, which is characterized by... Figure 4 The X-ray powder diffraction (XRPD) pattern shown exhibits peaks at at least 9.4°, 14.6°, 19.3°, 20.8°, and 23.5° when irradiated with CuKa X-rays. The N-demethylrubesta-L-lactate is designated as crystal form 1, and is further characterized by... Figure 5 The DSC and TGA scans shown are shown.

[0014] On one hand, the present invention is characterized by the crystal form of N-demethylrubesta-L-lactate, which is characterized by... Figure 6 The XRPD shown, when irradiated with Cu Ka X-rays, exhibits peaks at at least 2θ angles of approximately 10.6°, 11.0°, 14.7°, 17.3°, and 21.7°. The N-demethylrubesta-L-lactate is designated as crystal form 2 and cannot be further characterized by DSC and TGA because this crystal form is determined to be metastable.

[0015] On one hand, the present invention is characterized by the crystal form of N-demethylrubesta-L-lactate, which is characterized by... Figure 7 The XRPD shown exhibits peaks at 2θ angles of at least about 5.5° and 11.1° when irradiated with Cu Ka X-rays. The N-demethylrubesta-L-lactate is designated as crystal form 3, and is further characterized by... Figure 8 The DSC and TGA scans shown are shown.

[0016] Figure 9 The relationship between N-demethylrubesta-L-lactate crystal forms 1, 2, and 3 is shown, where crystal form 2 is designated as metastable and crystal form 3 is designated as hydrate and lowest energy form.

[0017] The invention also relates to the therapeutic use of the listed N-demethylrubesta-L-lactate compositions.

[0018] Brief description of the attached diagram

[0019] Figure 1 The synthetic scheme for N-demethylrubesta hydrochloride is shown.

[0020] Figure 2 The synthetic scheme for converting N-demethylrubesta hydrochloride to N-demethylrubesta L-lactate is shown.

[0021] Figure 3 Showing N-demethylrubesta-L-lactate 1 H NMR spectrum.

[0022] Figure 4 The XRPD diagram of N-demethylrubesta-L-lactate crystal form 1 is shown.

[0023] Figure 5 The TGA / DSC curves of N-demethylrubesta-L-lactate crystal form 1 are shown.

[0024] Figure 6 The XRPD diagram of N-demethylrubesta-L-lactate crystal form 2 is shown.

[0025] Figure 7 The XRPD diagram of N-demethylrubesta-L-lactate crystal form 3 is shown.

[0026] Figure 8 The TGA / DSC curves of N-demethylrubesta-L-lactate crystal form 3 are shown.

[0027] Figure 9 Transformation diagrams of N-demethylrubesta-L-lactate crystal forms 1, 2, and 3 are shown.

[0028] Figure 10 The kinetic solubility of N-demethylrubesta-L-lactate in water, SGF, FaSSIF, and FeSSIF is shown. Invention Details

[0030] This invention relates to the crystal forms of N-demethylrubesta-L-lactate. In one aspect, the invention is characterized by crystal form 1 of N-demethylrubesta-L-lactate, characterized in that, when irradiated with CuKa X-rays, the X-ray powder diffraction (XRPD) pattern has peaks at least at 2θ angles of about 9.4°, 14.6°, 19.3°, 20.8°, and 23.5°. In another aspect, crystal form 1 of said N-demethylrubesta-L-lactate is characterized in that, when irradiated with CuKa X-rays, the X-ray powder diffraction (XRPD) pattern has peaks at least at 2θ angles of about 9.4°, 14.6°, and 19.3°. In yet another aspect, crystal form 1 of said N-demethylrubesta-L-lactate is characterized in that, when irradiated with CuKa X-rays, the X-ray powder diffraction (XRPD) pattern has peaks at least at 2θ angles of about 14.6°, 19.3°, and 20.8°. In another aspect of the invention, crystal form 1 of the N-demethylrubesta-L-lactate is characterized in that, when irradiated with CuKa X-rays, the X-ray powder diffraction pattern (XRPD) has peaks at least at 2θ angles of about 19.3°, 20.8°, and 23.5°. In another aspect of the invention, crystal form 1 of the N-demethylrubesta-L-lactate is characterized in that, when irradiated with CuKa X-rays, the X-ray powder diffraction pattern (XRPD) has peaks at least at 2θ angles of about 9.4°, 20.8°, and 23.5°. In another aspect of the invention, crystal form 1 of the N-demethylrubesta-L-lactate is characterized in that, when irradiated with CuKa X-rays, the X-ray powder diffraction pattern (XRPD) has peaks at least at 2θ angles of about 9.4°, 14.6°, and 23.5°. In another aspect of the invention, crystal form 1 of the N-demethylrubesta-L-lactate is characterized in that, when irradiated with Cu Ka X-rays, the X-ray powder diffraction pattern (XRPD) has peaks at least at 2θ angles of about 14.6°, 20.8°, and 23.5°. In another aspect of the invention, crystal form 1 of the N-demethylrubesta-L-lactate is characterized in that, when irradiated with Cu Ka X-rays, the X-ray powder diffraction pattern (XRPD) has peaks at least at 2θ angles of about 9.4°, 19.3°, and 23.5°. In another aspect of the invention, crystal form 1 of the N-demethylrubesta-L-lactate is characterized in that, when irradiated with Cu Ka X-rays, the X-ray powder diffraction pattern (XRPD) has peaks at least at 2θ angles of about 9.4°, 14.6°, and 20.8°. In another aspect of the invention, the N-demethylrubesta-L-lactate crystal form 1 is characterized in that, when irradiated with Cu Ka X-rays, the X-ray powder diffraction pattern (XRPD) has peaks at at least 2θ angles of about 14.6°, 19.3°, and 23.5°.In another aspect of the invention, crystal form 1 of the N-demethylrubesta-L-lactate is characterized in that, when irradiated with Cu Ka X-rays, the X-ray powder diffraction pattern (XRPD) has peaks at least at 2θ angles of about 9.4°, 19.3°, and 20.8°. In yet another aspect of the invention, crystal form 1 of the N-demethylrubesta-L-lactate is characterized in that, when irradiated with Cu Ka X-rays, the X-ray powder diffraction pattern (XRPD) has any number and / or combination of peaks at least at 2θ angles of about 9.4°, 14.6°, 19.3°, 20.8°, and 23.5°.

[0031] In one aspect, the present invention is characterized by crystal form 2 of N-demethylrubesta-L-lactate, wherein the X-ray powder diffraction (XRPD) pattern, when irradiated with CuKa X-rays, has peaks at least at 2θ angles of about 10.6°, 11.0°, 14.7°, 17.3°, and 21.7°. In another aspect, crystal form 2 of the N-demethylrubesta-L-lactate is characterized by the X-ray powder diffraction (XRPD) pattern, when irradiated with CuKa X-rays, having peaks at least at 2θ angles of about 10.6°, 11.0°, and 14.7°. In yet another aspect, crystal form 2 of the N-demethylrubesta-L-lactate is characterized by the X-ray powder diffraction (XRPD) pattern, when irradiated with CuKa X-rays, having peaks at least at 2θ angles of about 11.0°, 14.7°, and 17.3°. In another aspect of the invention, crystal form 2 of the N-demethylrubesta-L-lactate is characterized in that, when irradiated with Cu Ka X-rays, the X-ray powder diffraction pattern (XRPD) has peaks at least at 2θ angles of about 14.7°, 17.3°, and 21.7°. In another aspect of the invention, crystal form 2 of the N-demethylrubesta-L-lactate is characterized in that, when irradiated with Cu Ka X-rays, the X-ray powder diffraction pattern (XRPD) has peaks at least at 2θ angles of about 10.6°, 17.3°, and 21.7°. In another aspect of the invention, crystal form 2 of the N-demethylrubesta-L-lactate is characterized in that, when irradiated with Cu Ka X-rays, the X-ray powder diffraction pattern (XRPD) has peaks at least at 2θ angles of about 10.6°, 11.0°, and 21.7°. In another aspect of the invention, crystal form 2 of the N-demethylrubesta-L-lactate is characterized in that, when irradiated with Cu Ka X-rays, the X-ray powder diffraction pattern (XRPD) has peaks at least at 2θ angles of about 11.0°, 17.3°, and 21.7°. In another aspect of the invention, crystal form 2 of the N-demethylrubesta-L-lactate is characterized in that, when irradiated with Cu Ka X-rays, the X-ray powder diffraction pattern (XRPD) has peaks at least at 2θ angles of about 10.6°, 14.7°, and 21.7°. In another aspect of the invention, crystal form 2 of the N-demethylrubesta-L-lactate is characterized in that, when irradiated with Cu Ka X-rays, the X-ray powder diffraction pattern (XRPD) has peaks at least at 2θ angles of about 10.6°, 11.0°, and 17.3°. In another aspect of the invention, the N-demethylrubesta-L-lactate crystal form 2 is characterized in that, when irradiated with Cu Ka X-rays, the X-ray powder diffraction pattern (XRPD) has peaks at at least 2θ angles of about 11.0°, 14.7°, and 21.7°.In another aspect of the invention, the N-demethylrubesta-L-lactate crystal form 2 is characterized in that, when irradiated with Cu Ka X-rays, its X-ray powder diffraction (XRPD) pattern has peaks at least at 2θ angles of about 10.6°, 14.7°, and 17.3°. In yet another aspect of the invention, the N-demethylrubesta-L-lactate crystal form 2 is characterized in that, when irradiated with Cu Ka X-rays, its X-ray powder diffraction (XRPD) pattern has any number and / or combinations of peaks at at least 2θ angles of about 10.6°, 11.0°, 14.7°, 17.3°, and 21.7°.

[0032] On one hand, the present invention is characterized by crystal form 3 of N-demethylrubesta-L-lactate, wherein the X-ray powder diffraction (XRPD) pattern, when irradiated with CuKa X-rays, has peaks at least at 2θ angles of about 5.5° and 11.1°. On another hand, the crystal form 2 of the N-demethylrubesta-L-lactate is characterized by the X-ray powder diffraction (XRPD) pattern, when irradiated with CuKa X-rays, having peaks at least at 2θ angles of about 5.5°. On yet another hand, the crystal form 2 of the N-demethylrubesta-L-lactate is characterized by the X-ray powder diffraction (XRPD) pattern, when irradiated with CuKa X-rays, having peaks at least at 2θ angles of about 11.1°. In another aspect of the invention, the N-demethylrubesta-L-lactate crystal form 2 is characterized in that, when irradiated with Cu Ka X-rays, the X-ray powder diffraction pattern (XRPD) has any number and / or combination of peaks at at least 2θ angles of about 5.5° and 11.1°.

[0033] Unless otherwise defined, all terms used in this disclosure, including technical and scientific terms, shall have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0034] As disclosed herein, multiple numerical ranges are provided. It should be understood that, unless the context explicitly specifies otherwise, each intermediate value (accurate to one-tenth of the lower limit unit) between the upper and lower limits of the range is also explicitly disclosed. This invention covers every smaller range between any of the stated values ​​or intermediate values ​​within the range and any other stated values ​​or intermediate values ​​within the range. The upper and lower limits of these smaller ranges may be independently included in or excluded from the range, and each range that includes, excludes, or includes two limits is also covered in this invention, subject to any explicitly excluded limits within the range. Where the range includes one or two limits, ranges that exclude one or both of those included limits are also included in this invention. The term “about” generally includes at most +10% or -10% of the indicated number. For example, “about 10%” may represent a range of 9% to 11%, and “about 20” may mean 18 to 22. Preferably, “about” includes at most +6% or -6% of the indicated value. Alternatively, “about” includes at most +5% or -5% of the indicated value. Other meanings of “about” may be obvious from the context, such as rounding, so “about 1” could also mean 0.5 to 1.4.

[0035] The term "pharmaceutically acceptable salt" for a compound refers to a salt that is pharmaceutically acceptable and possesses the desired pharmacological activity of the parent compound. It should be understood that acids that pair with bases to form pharmaceutically acceptable salts are generally considered safe for use in pharmaceutical medicines. These acids include inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid; or 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, glucoheponic acid, 4,4'-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, and mucoconic acid. More information on suitable pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, 1985, which is incorporated herein by reference.

[0036] As used herein, the term "therapeuticly effective amount" refers to the amount of the compound of the invention 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 depend on the specific circumstances relating to the case, including, for example, the compound administered, the route of administration, the patient's condition, and the pathological condition being treated. Administration may be once daily or in multiple sub-dose doses daily, such as two, three, or more doses daily.

[0037] For monotherapy, the effective dose of N-desmethylrubesta or a pharmaceutically acceptable salt, solvate, or polymorph thereof is about 32 to about 320 mg once daily, or about 16 to about 160 mg twice daily. Pharmaceutical compositions of N-desmethylrubesta or a pharmaceutically acceptable salt, solvate, or polymorph thereof also contain at least one pharmaceutically acceptable adjuvant or excipient. For combination therapy, the sub-effective dose of N-desmethylrubesta or a pharmaceutically acceptable salt, solvate, or polymorph thereof is about 8 to about 32 mg once daily, or about 4 to about 16 mg twice daily. When N-desmethylrubesta is combined with lithium, the sub-effective dose of lithium may be about 60 mg to about 600 mg once daily, or about 30 mg to about 300 mg twice daily. This sub-effective dose of lithium can avoid kidney damage commonly caused by lithium treatment. A once-daily effective dose of N-desmethylrubesta or a pharmaceutically acceptable salt, solvate, or polymorph thereof may be about 32, about 64, about 96, about 128, about 160, about 192, about 224, about 256, about 288, or about 320 mg. A twice-daily effective dose of N-desmethylrubesta or a pharmaceutically acceptable salt, solvate, or polymorph 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. A once-daily sub-effective dose of N-desmethylrubesta or a pharmaceutically acceptable salt, solvate, or polymorph thereof may be about 8, about 16, about 24, or about 32 mg. A twice-daily sub-effective dose of N-desmethylrubesta or a pharmaceutically acceptable salt, solvate, or polymorph thereof may be about 4, about 8, about 12, or about 16 mg.

[0038] For the application of N-desmethylrubesta for therapeutic purposes, it is necessary to identify and characterize the salt form and crystal form. Aspects of the present invention include the discovery, characterization, and methods for producing novel crystal forms of N-desmethylrubesta L-lactate. These crystal forms exhibit superior solubility compared to N-desmethylrubesta hydrochloride.

[0039] The preparation of N-demethylrubesta hydrochloride (compound-1) generally follows the... Figure 1The method is illustrated. As shown, starting material 1 reacts with a vinyl Grignard reagent in the presence of copper iodide to give alcohol intermediate 2. Those skilled in the art will recognize that substitutes for the vinyl Grignard reagent can also be used to achieve the same conversion. Such substitutes include, but are not limited to, vinyl zinc reagents, vinyl cuprate reagents, and vinyl lithium reagents. Those skilled in the art will also recognize that substitutes for copper iodide can be used to facilitate the conversion of intermediate 1 to intermediate 2. Such substitutes include, but are not limited to, substituted Lewis acid reagents and chelating agents, such as crown ethers.

[0040] After separating intermediate 2 Figure 1 The conversion to intermediate 3 via reaction with allyl bromide is illustrated. Those skilled in the art will recognize that alternative allylating agents can be used for the allylation reaction of intermediate 2 to intermediate 3. Such allylating agents are typically substitutes for bromide leaving groups, including but not limited to allyl chloride, allyl iodide, and allyl methanesulfonate. Those skilled 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 allylating agent. Such bases include, but are not limited to, hydride reagents, carbonate reagents, bicarbonate reagents, lithium diisopropylamino, sodium hexamethyldisilazane, etc.

[0041] Figure 1 The two-step conversion of intermediate 3 to intermediate 4 is also illustrated. As shown, the first step is an ozone decomposition reaction, resulting in the cleavage of the diene to a dialdehyde; the second step is the reduction of the dialdehyde to a diol using sodium borohydride. Those skilled in the art will recognize that ozone decomposition is only one of many reactions or combinations of reactions suitable for cleaving olefins to aldehydes. Such conversions include, but are not limited to, the dihydroxylation of olefins followed by the cleavage of the resulting diol to an aldehyde. Suitable reagents for the dihydroxylation of olefins include, but are not limited to, osmium tetroxide. Suitable reagents for the cleavage of diols to aldehydes include, but are not limited to, sodium periodate, lead tetraacetate, etc. Those skilled in the art will also recognize that alternatives to sodium borohydride reducing agents are also suitable for reducing aldehydes to alcohols. Such reagents include, but are not limited to, lithium aluminum hydride, diisopropyl aluminum hydride, lithium borohydride, boranes, etc. Those skilled in the art will also recognize that alternatives to boron- and aluminum-based reducing agents can also be used to reduce aldehydes to alcohols. Such alternatives include, but are not limited to, samarium iodide and triethylsilane.

[0042] like Figure 1As shown, the diol of intermediate 4 is converted to dimethanesulfonate intermediate 5 after reacting with methanesulfonyl chloride and triethylamine. Those skilled in the art will recognize that methanesulfonates, as leaving groups, are generally as useful as common alternative leaving groups, including but not limited to chlorides, bromides, iodides, toluenesulfonates, etc. Those skilled in the art will also recognize that substitutes for triethylamine are useful in the conversion of alcohols to methanesulfonates; such substitutes include, but are not limited to, diisopropylethylamine, pyridine, carbonate reagents, bicarbonate reagents, etc.

[0043] Figure 6 The reaction of dimethanesulfonate intermediate 5 with bisindolylmaleimide intermediate 6 to form intermediate 7 is shown, using cesium carbonate as a base. Those skilled in the art will recognize that alternative bases can be used to achieve the shown reaction to form intermediate 7. Such bases include, but are not limited to, hydrides, alkoxides, carbonates, bicarbonates, etc.

[0044] The process of converting methylmaleimide to its demethylated form involves first hydrolyzing intermediate 7 to maleic anhydride intermediate 8. For example... Figure 1 As shown, this conversion is achieved using an ethanol solution of potassium hydroxide. Those skilled in the art will recognize that the conversion of intermediate 7 to intermediate 8 can be achieved using alternatives to potassium hydroxide, including but not limited to sodium hydroxide and lithium hydroxide. Furthermore, those skilled in the art will recognize that ethanol can be replaced with any protic solvent, including but not limited to methanol and water.

[0045] according to Figure 1 Maleic anhydride intermediate 8 reacts with hexamethyldisilazane to form the corresponding maleimide intermediate 9. Those skilled in the art will recognize that alternative reagents, including but not limited to ammonia and sodium amide, can be used to convert maleic anhydride to maleimide.

[0046] After maleimide is formed Figure 1 The cleavage of the triphenylmethyl protecting group from intermediate 9 to form alcohol intermediate 10 is shown. Although Figure 6 Hydrochloric acid is highlighted as the reagent for achieving the cleavage of triphenylmethyl groups, but those skilled in the art will recognize that alternative acids can be used. These alternatives include, but are not limited to, hydrobromic acid, trifluoroacetic acid, and acetic acid.

[0047] like Figure 1As shown, the alcohol in intermediate 10 is converted to methanesulfonyl chloride and pyridine upon reaction. Those skilled in the art will recognize that methanesulfonates, as leaving groups, are generally as useful as common alternative leaving groups, including but not limited to chlorides, bromides, iodides, toluenesulfonates, etc. Those skilled in the art will also recognize that triethylamine substitutes can be used in the conversion of alcohols to methanesulfonates, including but not limited to diisopropylethylamine, pyridine, carbonate reagents, bicarbonate reagents, etc.

[0048] In the final stage of synthesis, Figure 1 This shows that intermediate 11 is converted into compound-1 after reacting with methylamine. Although Figure 6 Not shown, but methylamine is further converted to its corresponding hydrochloride salt after treatment with hydrochloric acid. Those skilled in the art will understand that there are alternative strategies for converting compounds such as intermediate 11 into structures such as compound-1. Such strategies are generally recognized by those skilled in the art, and said strategies typically yield structures such as... Comprehensive Organic Transformations Supported by resources from (Larock, Wiley). Those skilled in the art will also recognize that, in addition to forming the hydrochloride salt at this stage, any alternative salt form can be prepared by replacing hydrochloric acid with an alternative acid. Such alternative acids include, but are not limited to, L-lactic acid.

[0049] Those skilled in the art will recognize that, in addition to the above description and Figure 1 Besides the reaction shown, many other reactions can be used to prepare N-demethylrubesta (compound-1). Suitable reactions are readily determined by those skilled in the art and can be performed in, for example... Comprehensive Organic Transformations The strategies for introducing and cleaving protecting groups can be readily determined by those skilled in the art and can be found in resources such as (Larock, Wiley). Protective Groups in Organic Synthesis The resources (Greene and Wutz, Wiley) were found. Those skilled in the art will also recognize that this is generally applicable to... Figure 1 as well as Figure 1 Any and all alternatives and variations of the route described herein, and alternative combinations of reagents, solvents, temperature conditions, and reaction times, will provide similar chemical results, thereby enabling the preparation of compound-1.

[0050] The preparation of N-demethylrubesta-L-lactate from N-demethylrubesta hydrochloride generally follows the following steps: Figure 2 The method shown.

[0051] like Figure 2 As shown, from Figure 1Compound-1 was treated with an aqueous solution of sodium bicarbonate to release N-demethylrubesta in its free base form. Those skilled in the art will recognize that alternatives to sodium bicarbonate will provide suitable results for the conversion of N-demethylrubesta hydrochloride to the free N-demethylrubesta base. Suitable alternatives to sodium bicarbonate include, but are not limited to, inorganic bases such as sodium carbonate, potassium carbonate, potassium bicarbonate, and cesium carbonate. Furthermore, suitable alternatives to sodium bicarbonate include, but are not limited to, organic bases such as pyridine, triethylamine, and diisopropylethylamine.

[0052] Further as Figure 2 As shown, N-demethylrubesta free base was treated with L-lactic acid to generate N-demethylrubesta L-lactic acid salt. Once generated, N-demethylrubesta L-lactic acid salt was subjected to polymorph screening experiments to identify and characterize its various crystal forms.

[0053] Once prepared, the solubility of N-demethylrubesta-L-lactate was studied, and compared with existing supplies of hydrochloride and free base (both based on...). Figure 1 The solubility of L-lactate was compared with that of hydrochloride. Kinetic solubility studies were conducted over 24 hours in water, simulated gastric juice (SGF), fasting simulated intestinal juice (FaSSIF), and fed simulated intestinal juice (FeSSIF). As shown in Table 1, L-lactate showed approximately a 10-20 fold improvement in solubility in water after 4 hours compared to hydrochloride. The kinetic solubility of L-lactate is shown in Table 1. Figure 10 As shown.

[0054] Table 1: Comparison of kinetic solubility of N-demethylrubesta-L-lactate with free base and hydrochloride

[0055]

[0056] Once N-demethylrubesta-L-lactate is observed to have superior solubility compared to existing hydrochloride and free base, its polymorphism is evaluated using standard polymorph screening methods. Those skilled in the art will recognize that these polymorph screening methods include, but are not limited to, antisolvent addition temperature cycling, room temperature slurry, heated (e.g., 50°C) slurry, slow evaporation, solid-vapor diffusion, liquid-vapor diffusion, slow cooling, grinding, and polymer-induced crystallization.

[0057] Following standard polymorph screening experiments on N-demethylrubesta-L-lactate, three distinct, previously unknown polymorphs were isolated and characterized by XRPD, DSC, and TGA. Figure 4 The XRPD diagram shown and Figure 5 The crystal form 1 characterized by the DSC and TGA data shown is displayed as follows Figure 9 The anhydrous substance shown. (Through...) Figure 6The crystal form 2, characterized by the XRPD plot shown, is displayed as follows: Figure 9 The metastable anhydrous substance is shown. (Through...) Figure 7 The XRPD diagram shown and Figure 8 The crystal form 3, characterized by the DSC and TGA data shown, is displayed as follows: Figure 9 The anhydrous form shown was displayed as a hydrate and was the lowest energy form of the three identified crystal forms. These assessments were conducted through... Figure 9 The competitive transformation studies summarized in the illustrations are as follows. Table 2 summarizes the characteristics of the identified crystal forms.

[0058] Table 2: Summary of characterization of N-demethylrubesta-L-lactate crystal forms

[0059]

[0060] --: Exothermic; --: Insufficient quantity to test.

[0061] In one implementation, the listed N-desmethylrubesta crystal forms can be used as potential therapeutic agents for subjects suffering from neurological disorders and / or psychiatric conditions.

[0062] In any case where rubosta is clinically useful as a GSK-3 inhibitor, the compositions and treatments disclosed herein are relevant, including mental and neurological disorders such as bipolar disorder, depression, Alzheimer's disease, autism spectrum disorder, Fragile X syndrome, Pitt Hopkins syndrome, traumatic brain injury, stroke, acute spinal cord injury, schizophrenia, Parkinson's disease, amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). These compositions and treatments are relevant to indications in which rubosta has been 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 ganglioside storage disorders. These are associated with conditions for which GSK-3 inhibition and / or 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' dystrophy, limbal cell agenesis, 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 associated with GM2 ganglioside storage diseases for which the use of rubesta has been proposed.

[0063] In one implementation, the clinical response to N-desmethylrubesta or a combination of N-desmethylrubesta and lithium can be used to diagnose bipolar disorder and other conditions in which GSK-3 inhibition is clinically useful. In Alzheimer's disease, positron emission tomography (PET) of GSK-3β activity is being developed as a diagnostic method. In some implementations, N-desmethylrubesta can be administered, alone or in combination with lithium, to subjects with excessively high GSK-3β activity on PET to treat Alzheimer's disease, and the reduction in GSK-3β activity on PET following administration of N-desmethylrubesta can support its use as an appropriate therapy when administered at an appropriate dose (alone or in combination with lithium).

[0064] In one embodiment, the compositions and treatments disclosed herein can also be used in veterinary applications to improve the health and well-being of livestock and companion animals by treating any of the aforementioned indications in animals. Rubesta and N-desmethylrubesta have the same potency in inhibiting protein kinase C.

[0065] Those skilled in the art will understand that many different modifications can be made without departing from the spirit of the invention. Therefore, it should be clearly understood that the various embodiments of the invention described herein are merely illustrative and not intended to limit the scope of the invention.

[0066] Example

[0067] instrument

[0068] Powder X-ray diffraction (XRPD)

[0069] For XRPD analysis, a PANalytical Empyrean and an X'Pert3 X-ray powder diffractometer were used. The XRPD parameters used are listed in Table 3.

[0070] Table 3: Parameters for XRPD Testing

[0071]

[0072] Thermogravimetric analysis and differential scanning calorimetry (TGA and DSC)

[0073] TGA data was collected using a Discovery TGA 5500 from TA Instruments. DSC was performed using a Discovery DSC 2500 from TA Instruments. Detailed parameters used are listed in Table 4.

[0074] Table 4: Parameters for TGA and DSC tests

[0075]

[0076] Solution nuclear magnetic resonance (NMR) spectroscopy

[0077] Use DMSO- d 6 Solution NMR was collected using a Bruker 400M NMR spectrometer as a solvent.

[0078] Ion chromatography (IC)

[0079] The Thermo Scientific™ Dionex™ Aquion™ Ion Chromatography (IC) System 1100 with a conductivity detector was used, and detailed chromatographic conditions are listed in Table 5.

[0080] Table 5: Chromatographic conditions and parameters for ion content testing

[0081]

[0082] High-performance liquid chromatography (HPLC)

[0083] Waters H-Class was used, and detailed chromatographic conditions are listed in Table 6.

[0084] Table 6: Chromatographic conditions and parameters for purity and solubility testing

[0085]

[0086] Example 1. Compound Synthesis: N-Demethylrubesta hydrochloride was synthesized according to the following procedure:

[0087] Step-1: Synthesis of (S)-1-(triphenylmethyloxy)pent-4-en-2-ol (2):

[0088]

[0089] Under a nitrogen atmosphere, at -40°C, copper iodide (4.5 g, 23.62 mmol) was added to a solution of vinyl magnesium bromide (1 M THF solution, 840 mL, 0.84 mol). After stirring at -40°C for 20 min, compound-1 (150 g, 0.46 mmol) dissolved in anhydrous 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 heated to room temperature with stirring and extracted with ethyl acetate (1000 mL). The organic layer was separated and washed with ammonia (250 mL). The organic layer was separated, dried with sodium sulfate, filtered, and evaporated under vacuum to give compound 2, a dark brown viscous substance (166 g, 100% crude yield). 1 H NMR (400 MHz, CDCl3): δ 7.45-7.42 (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 =9.6 Hz, J =4.0 Hz, 1H), 3.09 (dd, J =9.2 Hz, J =6.8 Hz, 1H), 2.27-2.22 (m, 3H).

[0090] Step-2: Synthesis of (S)-(((2-(allyloxy)pent-4-en-1-yl)oxy)methanetriphenylmethyl)triphenyl(3):

[0091]

[0092] Under a nitrogen atmosphere, potassium tert-butoxide (70.0 g, 0.62 mmol) was added fractionally to a stirred solution of compound 2 (165 g, 0.48 mol) in anhydrous THF (1500 mL). The resulting reaction mixture was heated to 45 °C and stirred for 2 h, then cooled to room temperature. Allyl bromide (145.5 g, 1.22 mol) was then added at room temperature, and stirring was continued for 1 h at room temperature. After the reaction was complete (monitored by TLC), saturated ammonium chloride (1500 mL) was added to the reaction mixture, and the mixture was 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) eluting with a hexane solution of 0.5-1% ethyl acetate. The pure fraction was collected and evaporated under reduced pressure to give the desired compound 3 as a pale yellow semi-solid (106 g, 58% yield). 1 H NMR (400MHz, CDCl3): δ 7.48-7.44 (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 =17.2 Hz, J =2.0 Hz, 1H), 5.15(dd, J =10.4 Hz, J =2.0 Hz, 1H), 5.03 (dd, J =17.2 Hz, J =2.0 Hz, 1H), 4.96 (dt, J =10.4 Hz, J =1.2 Hz, 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).

[0093] Step 3: Synthesis of (S)-3-(2-hydroxyethoxy)-4-(triphenylmethyloxy)but-1-ol (4):

[0094]

[0095] 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 hours. 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 stirred at room temperature for 16 hours. After the reaction was complete (monitored by TLC), it was quenched with 1 N HCl solution until pH 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 give a crude compound, which was further purified by silica gel column chromatography (100-200 mesh) eluting with a 20-25% ethyl acetate solution in hexane. The pure fraction was collected and evaporated to give the desired compound 4 as a yellow gelatinous liquid (58 g, 57% yield). 1 H NMR (400 MHz, DMSO- d 6 ): δ 7.42-7.40 (m, 6H), 7.34 (t, J =7.6 Hz, 6H), 7.28-7.24 (m, 3H), 4.60 (t, J =5.6 Hz 1H), 4.36 (t, J =5.6 Hz, 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).

[0096] Step 4: Synthesis of (S)-2-((4-((methanesulfonyl)oxy)-1-(triphenylmethyloxy)but-2-yl)oxy)ethyl methanesulfonate (5):

[0097]

[0098] At 0 °C, triethylamine (66 mL, 0.47 mmol) was added to a stirred solution of compound 4 (60 g, 0.15 mol) in DCM (1000 mL), and the mixture was stirred for 15 min, followed by the addition of methanesulfonyl chloride (32.0 mL, 0.41 mmol). The resulting reaction mixture was stirred at 0 °C for 2 h (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 give a 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, washed with heptane (80 mL), and dried under vacuum to give compound 5 as a cream-colored solid (88 g, 100% crude yield). 1 H NMR (400 MHz, DMSO- d 6 ): δ 7.42-7.39 (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).

[0099] Step 5: Synthesis of (12E,32E,7S)-21-methyl-7-((triphenylmethyloxy)methyl)-22,25-dihydro-11H,21H,31H-6-oxa-1,3(3,1)-diindolaza-2(3,4)-pyrrolocyclononadenosine-22,25-dione (7):

[0100]

[0101] Cesium carbonate (86.0 g, 0.26 mol) was added to a stirred solution of compound 6 (41.5 g, 0.12 mol) in DMF (850 mL), and the reaction mixture was heated to 100 °C. Then, compound 5 (85.0 g (crude product), 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 complete (monitored by TLC), it was cooled to 50 °C, diatomaceous earth (25 g) was added, and the mixture was stirred for 15 minutes. The reaction mixture was filtered through diatomaceous earth, 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 give the crude compound, which was further purified by silica gel column chromatography (100-200 mesh) eluting with a 25-30% ethyl acetate solution in hexane. The pure fraction was collected and evaporated to give the desired compound-7 as a brick-red solid (55 g, 51% yield). 1 H NMR (400 MHz, DMSO- d 6 ): δ 7.83 (d, J =7.6 Hz 1H), 7.75 (d, J =8.0 Hz 1H), 7.49 (d, J =8.4 Hz 1H), 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).

[0102] Step-6: Synthesis of (12E,32E,7S)-7-((triphenylmethyloxy)methyl)-22,25-dihydro-11H,31H-6-oxa-1,3(3,1)-diindola-2(3,4)-furanhexacyclononaeno-22,25-dione (8):

[0103]

[0104] 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 the mixture was heated to 80 °C. The resulting reaction mixture was stirred for 24 hours. After the reaction was complete (monitored by TLC), the reaction mixture was evaporated under vacuum to give a residue, which was partitioned between dichloromethane (850 mL) and a 20% citric acid solution (450 mL). The organic layer was separated, dried with sodium sulfate, filtered, and evaporated under vacuum to give crude compound 8 as a dark brown solid (62 g, 74% yield). 1 H NMR (400 MHz, DMSO- d 6 ): δ 7.88 (d, J =7.6 Hz 1H), 7.82 (d, J =7.6 Hz 1H), 7.65 (d, J =2.0 Hz 2H), 7.55 (d, J =8.0 Hz 1H), 7.41 (d, J =7.6 Hz (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.)

[0105] Step 7: Synthesis of (12E,32E,7S)-7-((triphenylmethyloxy)methyl)-22,25-dihydro-11H,21H,31H-6-oxa-1,3(3,1)-diindola-2(3,4)-pyrrolocyclononadenosine-22,25-dione (9):

[0106]

[0107] HMDS (294.0 mL, 2.47 mol) and methanol (6.0 mL) were added to a stirred solution of compound 8 (95.0 g, 0.25 mol) in DMF (950 mL), and the mixture was heated to 80 °C. The reaction mixture was stirred at 80 °C for 5 hours. After the reaction was complete (monitored by TLC), the mixture 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) eluting with 20-25% ethyl acetate in hexane. The pure fraction was collected and evaporated under vacuum to give the desired compound 9 as a purple solid (70 g, 74% yield). 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.91 (s, 1H), 7.81 (d, J =8.0 Hz 1H), 7.73 (d, J =8.0 Hz 1H), 7.48(d, J =8.4 Hz 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).

[0108] Step 8: Synthesis of (12E,32E,7S)-7-(hydroxymethyl)-22,25-dihydro-11H,21H,31H-6-oxa-1,3(3,1)-diindola-2(3,4)-pyrrolocyclononadenosine-22,25-dione (10):

[0109]

[0110] 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 mixture was heated to 80 °C and maintained for 3 hours. After the reaction was complete (monitored by TLC), the mixture was cooled to room temperature, stirred for 1 hour, filtered to obtain a solid, washed with water (350 mL), and dried under vacuum at 45 °C to give compound 10 as a purple solid (40 g, 88% crude yield). 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.92 (s, 1H), 7.82 (d, J =7.6 Hz 1H), 7.78 (d, J =7.6 Hz 1H), 7.53 (d, J =8.0 Hz, 1H), 7.51(s, 1H), 7.46 (d, J =8.4 Hz, 1H), 7.45 (s, 1H), 7.25-7.22 (m, 2H), 7.13-7.10(m, 2H), 4.69 (t, J =5.2 Hz 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).

[0111] Step 9: Synthesis of ((12E,32E,7S)-22,25-dioxo-22,25-dihydro-11H,21H,31H-6-oxa-1,3(3,1)-diindola-2(3,4-pyrrolocyclononaeno-7-yl)methylmethanesulfonate (11):

[0112]

[0113] At room temperature, pyridine (33.2 mL, 0.39 mol) was added to a stirred solution of compound 10 (39.0 g, 0.09 mol) in THF (400 mL), and the mixture was stirred for 20 min. Then, methanesulfonic anhydride (46.0 g, 0.26 mol) was added to the reaction mixture at room temperature. The resulting reaction mixture was stirred for 4 h. After the reaction was complete (monitored by TLC), the reaction mixture was partitioned between ethyl acetate (100 mL) and water (50 mL), the organic layer was separated, dried over sodium sulfate, filtered, and evaporated under reduced pressure to give a crude product (37.0 g), which was further purified by silica gel column chromatography (100-200 mesh) eluting with DCM. The pure fraction was collected and evaporated under reduced pressure to give the desired compound 11 as a purple solid (30 g, 65% yield). 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.92 (s, 1H), 7.83 (d, J =7.6 Hz 1H), 7.78 (d, J =7.6 Hz 1H), 7.54 (d, J =8.4 Hz, 1H), 7.52 (s, 1H), 7.48 (d, J =8.4 Hz, 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).

[0114] Step-10: Synthesis of (12E,32E,7S)-7-((methylamino)methyl)-22,25-dihydro-11H,21H,31H-6-oxa-1,3(3,1)-diindola-2(3,4)-pyrrolocyclononaenopane-22,25-dione hydrochloride (compound-1):

[0115]

[0116] In an autoclave at -40°C, a 2M methylamine THF solution (400 mL) was added to a stirred solution of compound 11 (10.0 g, 0.019 mol) in THF (400 mL). The reaction was gradually heated to 70°C and stirred for 24 hours. After the reaction was complete (monitored by TLC), the mixture was evaporated under vacuum to give a crude compound (12.0 g). This batch was combined with four other batches of the same size to give 60.0 g of crude product. 60 g of the crude product was purified by silica gel column chromatography (230-400 mesh, 2% MeOH / DCM). The purified fraction was collected and concentrated to give the desired compound-1 free base (22.0 g) as a red solid. The free base was suspended in diethyl ether (220 mL) and cooled to 0°C. Ethanol hydrochloric acid (33 mL) was added at 0°C. The resulting suspension was stirred at 0°C for 30 minutes, filtered, washed with ether (50 mL), and dried under vacuum at 40°C for 1 hour to give compound-1 as a brick-red solid (16.9 g, 36% yield). 1 H NMR (400 MHz, DMSO- d 6 ): 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 =8.0Hz 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 =9.2 Hz, 1H), 3.27-3.24 (m,1H), 3.01-2.98 (m, 1H), 2.53 (t, J =5.6 Hz, 3H), 2.22-2.20 (m, 1H), 2.06-2.03(m, 1H).

[0117] Example 2. Compound Synthesis: N-Demethylrubesta-L-Lactate was prepared from N-Demethylrubesta hydrochloride according to the following procedure:

[0118] Step 1: Synthesis of demethylated rubesta (free base):

[0119]

[0120] At 0–5 °C, an aqueous solution of sodium bicarbonate was added to a suspension of (desmethylrubesta)-HCl salt (10 g, 20.36 mmol) in 10% MeOH DCM solution (3 L) until the pH of the reaction mixture was between 7.5 and 8.0. The reaction mixture was stirred for 1 hour (with continuous pH monitoring), and the organic layer was separated. The separated organic layer was washed with water (3 x 500 mL), separated, dried over sodium sulfate, and evaporated under vacuum to give desmethylrubesta (free base) as a red solid (8.7 g, 94% yield).

[0121] Step 2: Synthesis of (demethyllubosta)-L-lactate:

[0122]

[0123] A solution of L-lactic acid (1.84 g, 20.42 mmol) in acetone (10 mL) was added dropwise to a stirred solution of demethylrubesta (free base) (8.7 g, 19.14 mmol) in acetone (240 mL) over 15–20 minutes. Then, 50 mg of (demethylrubesta)-L-lactate was added as a seed crystal. The resulting reaction mixture was stirred in the dark (with the reaction flask wrapped in aluminum foil) for 7 days. The precipitated solid was then filtered and washed with acetone (40 mL) to give a wet solid. This solid was then freeze-dried (by freezing in CAN / water 3:1) for 16 hours to give the lactate as a brick-red solid compound (7.8 g, 75% yield). 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.99 (br s, 1H), 7.83 (t, J =8.4 Hz, 2H), 7.56 (d, J =8.0 Hz 1H), 7.52 (s, 1H), 7.49-7.47 (m, 2H), 7.24-7.19 (m,2H), 7.15-7.11 (m, 2H), 4.43-4.38 (m, 1H), 4.27-4.20 (m, 2H), 4.16-4.14 (m,1H), 3.92 (q, J =6.8 Hz, 1H), 3.87-3.84 (m, 1H), 3.60 (t, J=8.8 Hz, 1H), 3.50(br s, 1H), 2.79-2.76 (m, 1H), 2.69-2.64 (m, 1H), 2.33 (s, 3H), 2.17-2.16 (m,1H), 2.04-2.02 (m, 1H), 1.21 (d, J =6.8 Hz, 1H).

[0124] Example 3. Preparation and characterization of N-demethylrubesta-L-lactate crystal form 1

[0125] Crystal form 1 was obtained from a 5-day room temperature slurry (N2, dark) containing 50.0 mg N-demethylrubesta free base and 11.2 mg L-lactic acid (1.0 eq.) in 1.0 mL EtOAc. The solid was separated by centrifugation and characterized after vacuum drying at room temperature. XRPD ( Figure 4 The peaks shown in the table below are displayed. TGA / DSC curves ( Figure 5 The results show that the weight loss up to 120.0℃ is 4.19%, there is an endothermic peak at 87.7℃ and an exothermic peak (peak temperature) at 112.7℃.

[0126]

[0127] Example 4. Preparation and characterization of N-demethylrubesta-L-lactate crystal form 2

[0128] Crystal form 2 was obtained from a 6-day room temperature slurry (N2, dark) in acetone / H2O (857:143, v:v) in the L-lactate form. The sample was converted to L-lactate crystal form 3 after centrifugation and air drying at room temperature overnight.

[0129] Three batches of L-lactate crystal form 2 were subsequently re-prepared using L-lactate crystal form 1 in acetone / H2O (857:143, v:v) for 3 days at room temperature (N2, dark). XRPD (XRPD) was recorded prior to conversion to crystal form 3. Figure 6 The peaks shown in the table below are displayed. Due to the tendency to rapidly transform into crystal form 3, crystal form 2 is considered metastable.

[0130]

[0131] Example 5. Preparation and characterization of N-demethylrubesta-L-lactate crystal form 3

[0132] Crystal form 3 was obtained from a 6-day slurry of crystal form 1 in H2O (N2, dark). XRPD data were collected after centrifugation and air-drying overnight at room temperature. Figure 7 The following table shows the peaks.

[0133] Crystal form 3 was reconstituted using a 3-day room temperature slurry of crystal form 1 in H2O (N2, dark) and air-dried at room temperature for 4 hours. TGA / DSC curves ( Figure 8 The results show that the weight loss is 3.95% up to 110.0℃, with four endothermic peaks at 98.2, 126.6, 150.8, and 221.5℃, and one exothermic peak (peak temperature) at 165.0℃.

[0134]

[0135] Example 6. Solubility assessment of N-methylrubesta-L-lactate

[0136] The kinetic solubility of N-methylrubesta-L-lactate, hydrochloride, and free base was evaluated in H2O and three biological media: SGF, FaSSIF, and FeSSIF. In the experiments, samples were mixed and dissolved in H2O, SGF, FaSSIF, and FeSSIF using a rolling method at 37 °C, with a solid loading of ~10 mg / mL (based on free base). Solubility tests were performed at different time points (1 h, 4 h, and 24 h). Samples at each time point were centrifuged and filtered (using a 0.45 μm PTFE membrane) to separate the liquid phase for free base concentration and pH determination. N-methylrubesta-L-lactate showed the highest solubility in water, ranging from 7 to 10 mg / mL, compared to the corresponding hydrochloride and free base.

[0137]

Claims

1. A crystalline form of N-desmethyl Rubsta L-lactate having at least one peak at a diffraction angle 2 theta (°) of at least about 9.4°, 14.6°, or 19.3° as measured by X-ray diffraction using Cu Ka X-ray radiation or calculated from X-ray diffraction.

2. The crystalline form of N-desmethyl Rubsta L-lactate of claim 1 having at least one peak at a diffraction angle 2 theta (°) of at least about 9.4°, 14.6°, or 19.3° as measured by X-ray diffraction using Cu Ka X-ray radiation or calculated from X-ray diffraction.

3. The crystalline form of N-desmethyl Rubsta L-lactate of claim 1 having at least one peak at a diffraction angle 2 theta (°) of at least about 9.4°, 14.6°, 19.3°, 20.8°, or 23.5° as measured by X-ray diffraction using Cu Ka X-ray radiation or calculated from X-ray diffraction.

4. The crystalline form of N-desmethyl Rubsta L-lactate of any one of claims 1 to 3 having an X-ray powder diffraction spectrum as shown in FIG.

4.

5. A crystalline form of N-desmethyl Rubsta L-lactate having at least one peak at a diffraction angle 2 theta (°) of at least about 10.6°, 11.0°, or 14.7° as measured by X-ray diffraction using Cu Ka X-ray radiation or calculated from X-ray diffraction.

6. The crystalline form of N-desmethyl Rubsta L-lactate having at least one peak at a diffraction angle 2 theta (°) of at least about 10.6°, 11.0°, or 14.7° as measured by X-ray diffraction using Cu Ka X-ray radiation or calculated from X-ray diffraction.

7. The crystalline form of N-desmethyl Rubsta L-lactate having at least one peak at a diffraction angle 2 theta (°) of at least about 10.6°, 11.0°, 14.7°, 17.3°, or 21.7° as measured by X-ray diffraction using Cu Ka X-ray radiation or calculated from X-ray diffraction.

8. The crystalline form of N-desmethyl Rubsta L-lactate of any one of claims 5 to 7 having an X-ray powder diffraction spectrum as shown in FIG.

6.

9. A crystalline form of N-desmethyl Rubsta L-lactate having at least one peak at a diffraction angle 2 theta (°) of at least about 5.5° or 11.1° as measured by X-ray diffraction using Cu Ka X-ray radiation or calculated from X-ray diffraction.

10. The crystalline form of N-desmethyl Rubsta L-lactate having at least one peak at a diffraction angle 2 theta (°) of at least about 5.5° or 11.1° as measured by X-ray diffraction using Cu Ka X-ray radiation or calculated from X-ray diffraction.

11. The crystalline form of N-desmethyl Rubsta L-lactate of any one of claims 9 to 10 having an X-ray powder diffraction spectrum as shown in FIG.

7.

12. A method of treating a disorder involving abnormal signaling of GSK-3 or protein kinase C, the method comprising administering to a subject in need thereof a therapeutically effective dose of a crystalline form of N-desmethyl rubusta as set forth in any one of claims 1 to 11.

13. The method of claim 12, wherein the N-desmethyl rubusta crystalline form is administered to a subject who: 1) has never taken rubusta; or 2) has taken rubusta and experienced an adverse reaction; or 3) shows a prolonged QT interval; or 4) has shown to have high plasma levels of rubusta; or 5) is likely to receive a drug that can interfere with rubusta metabolism; or 6) can require a higher dose of rubusta and is at risk of experiencing an adverse reaction, prolonged QT interval, or adverse drug interaction.

14. The method of claim 12 or 13, wherein the subject has a neurological disease and / or a psychiatric disorder.

15. The method of claim 14, 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 / or chemotherapy-induced cognitive impairment.

16. The method of claim 12 or 13, 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 / or focal segmental glomerulosclerosis.

17. The method of claim 12 or 13, wherein the disease / disorder is selected from atherosclerosis, hair loss, bone and joint disorders including osteoarthritis and osteoporosis, inflammatory diseases including alcoholic hepatitis, inflammatory bowel disease, and septic shock.

18. The method of claim 12 or 13, 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 stem cell deficiency, dry eye, glaucoma, familial exudative vitreoretinopathy (FEVR), Norrie disease, Coats’ disease, retinopathy of prematurity, macular telangiectasia, retinal vein occlusion, and Sjogren’s syndrome; and / or an ear disorder including sensorineural hearing loss and conductive hearing loss.

19. The method of claim 12 or 13, wherein the disease / condition is selected from the group consisting of a pulmonary condition, including chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, pulmonary arterial 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, skin disease, inflammation, or GM2 gangliosidosis.

20. The method of any one of claims 12 to 19, wherein the N-desmethyl rubusoside crystalline form is administered in an amount of about 32 to about 320 mg once daily or about 16 to about 160 mg twice daily.

21. The method of any one of claims 12 to 20, wherein the N-desmethyl rubusoside crystalline form is administered in combination with lithium.

22. The method of any one of claims 12 to 20, wherein the subject is non-responsive to lithium.

23. The method of claim 21, wherein the subject is responsive to lithium.

24. The method of claim 21, wherein lithium is administered in a sub-effective dose based on monotherapy, and wherein the N-desmethyl rubusoside crystalline form is administered in a sub-effective dose based on monotherapy.

25. The method of claim 24, wherein the sub-effective dose of lithium is about 60 mg to about 600 mg once daily or about 30 mg to about 300 mg twice daily.

26. The method of claim 24, wherein the sub-effective dose of the N-desmethyl rubusoside crystalline form is administered in about 8 to about 32 mg once daily or about 4 to about 16 mg twice daily.

27. A method for determining the diagnosis of bipolar disorder or other condition in which GSK-3 inhibition is clinically useful comprising administering to a subject to be evaluated a therapeutically effective dose of N-desmethyl rubusoside crystalline form and evaluating the subject for clinical response.

28. A method of determining the appropriate therapeutic dose of N-desmethyl rubusoside in a subject comprising administering increasing doses of N-desmethyl rubusoside crystalline form and assessing response using GSK-3 imaging or GSK-3 serology.

29. A method of treating a subject having Alzheimer's disease, bipolar disorder, or depression and exhibiting signs of elevated GSK-3 comprising administering to the subject a therapeutically effective dose of N-desmethyl rubusoside crystalline form and evaluating and monitoring the subject using positron emission tomography (PET) or serology.

30. A method for determining the diagnosis of bipolar disorder or other condition in which GSK-3 inhibition is clinically useful comprising administering to a subject to be evaluated a therapeutically effective dose of N-desmethyl rubusoside crystalline form and a therapeutically effective dose of lithium and evaluating the subject for clinical response.

31. The method of claim 30, wherein the doses of N-desmethyl rubusoside and lithium are both sub-effective based on monotherapy.

32. A method of treating a subject having Alzheimer's disease and exhibiting elevated GSK-3p activity, comprising administering to the subject a therapeutically effective dose of N- desmethyl ruboxyl or a pharmaceutically acceptable salt, solvate or polymorph thereof, and a therapeutically effective dose of lithium, and monitoring the subject using positron emission tomography (PET).

33. The method of claim 32, wherein the dose of the crystalline form of N- desmethyl ruboxyl and lithium are both sub-effective as monotherapies.

34. A method of determining an appropriate therapeutic dose of a crystalline form of N-desmethyl ruboxyl in a subject, comprising administering increasing doses of the crystalline form of N-desmethyl ruboxyl and lithium to the subject, and assessing response using positron emission tomography (PET).

35. A crystalline form of N-desmethyl ruboxyl L-lactate.

36. The crystalline form of N-desmethyl ruboxyl L-lactate of claim 35, having the X-ray powder diffraction pattern shown in Figure 4, and prepared by slurrying N- desmethyl ruboxyl free base with L-lactic acid in ethyl acetate and recovering the lactate salt.

37. The crystalline form of N-desmethyl ruboxyl L-lactate of claim 35, having the X-ray powder diffraction pattern shown in Figure 6, and prepared by slurrying N- desmethyl ruboxyl free base with L-lactic acid in a mixture of acetone and water and recovering the lactate salt.

38. The crystalline form of N-desmethyl ruboxyl L-lactate of claim 35, having the X-ray powder diffraction pattern shown in Figure 7, and prepared by slurrying N- desmethyl ruboxyl free base with L-lactic acid in water and recovering the lactate salt.

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