Polymorphic form of meisoindigo and modified formulation of meisoindigo

Abstract

To provide a modified meisoindigo formulation that has improved solubility and bioavailability and / or extended shelf-life.SOLUTION: The present invention relates to a novel crystal form, manufacturing procedures, pharmaceutical compositions, formulations and medicaments comprising a N-methylisoindigo crystalline, methods of preparation, and the use of the N-methylisoindigo crystalline to prepare a medicament to prevent cancer, or treat cancer or an inflammatory-related disease associated with pro-inflammatory cytokine expression and / or reduced expression of anti-inflammatory cytokines.SELECTED DRAWING: Figure 5

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a joint application of U.S. Provisional Patent Application No. 62 / 776,9 filed on December 7, 2018. No. 65, which is incorporated herein by reference in its entirety.

[0002] Technical Field

[0002] The present invention relates to pharmaceutical compositions, more particularly to novel crystalline forms of Meisoindigo (crys talline) forms, methods of preparation, and methods of preventing cancer, treating cancer, or treating inflammation-related diseases associated with inhibition of cyclin-dependent kinases, expression of pro-inflammatory cytokines, or reduced expression of anti-inflammatory cytokines. [Background technology]

[0003] 2. Background of the Invention

[0003] Cancer prevention and treatment are driven by advances in epidemiology, therapeutic techniques, and the ability to achieve early diagnosis. The treatment of cancer has improved significantly in the United States in the past decade. However, finding a cure for various cancers such as lung, breast, prostate, colon, etc. remains a major challenge. Current approaches to treat cancer are still limited to extending life span or increasing quality of life. In addition, most meaningful treatments still have significant side effects. Therefore, it is essential to find more effective therapeutic agents with fewer side effects.

[0004] Tumor cells develop inflammatory bowel disease due to a loss of integration and coordination between extracellular signals and the cell cycle machinery. The cell cycle is characterized by unlimited cell proliferation, which is regulated by the cell cycle. A typical cell cycle is divided into G1, S, G2 and M phases. In mammalian cells, proliferation is controlled in the G1 phase of the cell cycle. At the restriction point, the cell can have different fates. Examples of these cell fates include: 1) leaving the cell cycle and entering a reversible quiescent phase; 2) exiting the cell cycle and undergoing apoptosis; 3) differentiating and irreversibly exiting the cell cycle; and 4) passing the restriction point, becoming largely independent of extracellular signals and automatically progressing through the subsequent cell cycles (S, G2, M) to the next G1 phase. Various proteins are then responsible for the regulated progression of the cell through the cell cycle. The key components of the cell cycle machinery are cyclins, cyclin-dependent kinases (CDKs) and their inhibitors. Cyclins are a remarkably diverse family of proteins that are synthesized from mid / late G1 to the M phase of the cell cycle and are then rapidly degraded. CDKs typically contain a catalytic domain of 300 amino acids, which is inactive by itself. Cdks become active by binding to cyclins. The activity of cdks is inhibited by their endogenous inhibitors (CDK inhibitors, or cdkIs, include p15 / p16 / p18 / p19 and p21 / p27). Specific cyclin / CDK complexes form at specific stages of the cell cycle and their activity is required for progression of the cell cycle through S phase and mitosis.

[0005]

[0005] Hyperactivation of CDKs is a hallmark of the majority of human tumor cells. Strategies have been developed to modulate CDK activity for therapeutic intervention by directly targeting CDKs or indirectly affecting CDK regulatory pathways. Small molecule CDK inhibitors such as flavopiridol analogues, polysulfates, toyocamycin derivatives, etc. were designed to specifically interact with the ATP-binding site of CDKs. Clinical trials of these agents have shown anticancer activity. Modulation of CDK activity can be achieved by modulating phosphorylation of CDKSs or by altering the expression of CDKs or their inhibitors (CDKIs). It is difficult to find specific modulators that do not interfere with other cell cycle components and have no effect on normal cells.

[0006] Our previous research has demonstrated that the second generation indirubin, Meisoindigo, We have shown that it arrests leukemic cells in the G phase, inhibits expression of the oncogene c-myb, and induces cell differentiation and maturation at low concentrations (low toxicity) at which cell proliferation is completely inhibited without compromising cell viability.

[0007]

[0007] Furthermore, better solubility and bioavailability and / or longer shelf life are desired. There is a need for a modified Meisoindigo formulation that has a shelf life. Summary of the Invention

[0008]

[0008] The present invention has excellent therapeutic efficacy against various autoimmune diseases / inflammatory diseases and cancers. The present invention further provides a method for preparing said crystalline form (Form 1) as well as pharmaceutical compositions and dosage forms comprising said novel crystalline form.

[0009] In some embodiments, the present invention provides a method for manufacturing a gyro that peaks at approximately 7.71 degrees in 2-theta. The present invention provides a solid form of N-methylisoindigo or a solid crystalline form of N-methylisoindigo (Crystalline Form I) having an X-ray powder diffraction pattern containing

[0010]

[0010] In one embodiment, the solid form or solid crystalline form has an X-ray powder diffraction pattern including peaks at about 7.71°, about 17°, about 18°, and about 29° in 2-theta.

[0011] In another embodiment, the solid form or solid crystalline form has an X-ray powder diffraction pattern substantially as shown in FIG.

[0012] In some embodiments, the solid form or solid crystalline form has an infrared spectrum for N-methylisoindigo substantially as shown in Figure 1. In other embodiments, the solid form or solid crystalline form has an NMR spectrum for N-methylisoindigo substantially as shown in Figure 2.

[0012]

[0013] In yet another embodiment, the solid form or solid crystalline form has a differential scanning calorimetry (DSC) thermogram comprising an endothermic peak at about 235° C. to 237° C. In one embodiment, the solid form or solid crystalline form has a differential scanning calorimetry (DSC) thermogram substantially as shown in Table 1.

[0013]

[0014] In some embodiments, the solid form or solid crystalline form has a particle size distribution with an average particle size d50 of less than 25 μm. In some embodiments, the solid form or solid crystalline form has a particle size distribution with an average particle size d50 of less than 25 μm, less than 24 μm, less than 23 μm, less than 22 μm, less than 21 μm, less than 20 μm, less than 19 μm, less than 18 μm, less than 17 μm, or less than 16 μm. In other embodiments, the solid form or solid crystalline form has a particle size distribution with an average particle size d50 of 1 μm to 25 μm (e.g., 1 μm to 25 μm, 1 μm to 20 μm, 5 μm to 25 μm, 5 μm to 20 μm, 10 μm to 25 μm, or 10 μm to 20 μm).

[0014]

[0015] In some embodiments, the solid form or solid crystalline form has a particle size distribution ratio (d90-d10) / d50 of less than 2.50. In some embodiments, the particle size distribution ratio (d90-d10) / d50 is less than 2.50, less than 2.40, less than 2.30, less than 2.20, less than 2.10, less than 2.00, or less than 1.90.

[0015]

[0016] In other embodiments, the solid form or solid crystalline form has a maximum particle size of less than 100 μm. In certain embodiments, the maximum particle size is less than 100 μm, less than 90 μm, less than 80 μm, less than 70 μm, less than 60 μm, or less than 50 μm.

[0016]

[0017] In some embodiments, the particle size distribution facilitates formulation processing during formulation of the solid or solid crystalline form, while in other embodiments, the particle size distribution enhances the stability and bioavailability of the solid or solid crystalline form.

[0017]

[0018] In some embodiments, the present invention provides a pharmaceutical composition comprising a solid form or solid crystalline form disclosed herein and a pharma- ceutically acceptable carrier.

[0019] In some embodiments, the solid form or solid crystalline form is present in the pharmaceutical composition in an amount of at least about 90% by weight, hi other embodiments, the solid form or solid crystalline form is present in the pharmaceutical composition in an amount of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% by weight.

[0018]

[0020] In still other embodiments, the solid form or solid crystalline form is present in the pharmaceutical composition in an amount of 1% to 90% by weight (i.e., 1% to 90% by weight, 1% to 80% by weight, 1% to 70% by weight, 1% to 60% by weight, 1% to 50% by weight, 1% to 40% by weight, 1% to 30% by weight, 1% to 20% by weight, or 1% to 10% by weight).

[0019]

[0021] In some embodiments, the solid form or solid crystalline form is substantially purified, hi other embodiments, the solid form or solid crystalline form is crystalline.

[0022] In some embodiments, the present invention provides a method for preparing a solid or solid crystalline form disclosed herein, the method comprising precipitating the crystalline form from a solution comprising an organic solvent. In one embodiment, the solution comprises glacial acetic acid. In another embodiment, the solution further comprises N-methylisatin, oxindole, and / or HCl.

[0020]

[0023] In another embodiment, the present invention provides a solid form or solid crystalline form of N-methylisoindigo prepared by the methods disclosed herein and recrystallized from any other acceptable organic solvent, preferably glacial acetic acid.

[0021]

[0024] In some embodiments, the present invention provides a method of treating cancer comprising administering to a patient in need thereof a solid or solid crystalline form of N-methylisoindigo disclosed herein or a pharmaceutical composition disclosed herein.

[0022]

[0025] In other embodiments, the present invention provides a method of treating an inflammation-related disease associated with cytokine expression levels, comprising administering to a patient in need thereof a solid or solid crystalline form of N-methylisoindigo disclosed herein or a pharmaceutical composition disclosed herein.

[0023]

[0026] In certain embodiments, the present invention provides a pharmaceutical composition consisting essentially of a solid form or solid crystalline form of N-methylisoindigo disclosed herein. [Brief description of the drawings]

[0024] [Figure 1]

[0027] Figure 1 is the infrared spectrum of N-methylisoindigo. [Diagram 2]

[0028] FIG. 2 is an NMR spectrum of N-methylisoindigo. [Diagram 3]

[0029] FIG. 3 is a mass spectrum of N-methylisoindigo (positive Q1 scan). [Figure 4]

[0030] FIG. 4 is a mass spectrum of N-methylisoindigo (negative Q1 scan). [Diagram 5]

[0031] Figure 5 shows a comparison of X-ray powder diffraction patterns. Unsmoothed XRD data for four N-methylisoindigo samples. The ~40,000 count peak at 7.71 deg has been truncated, allowing a detailed view of the data in the range 5<2theta<55 deg. Note: The extra peak in sample 0501 (blue curve) is shown in more detail in Figure 6 and Table 2. [Figure 6]

[0032] Figure 6 shows plots of the results of the peak selection analysis, detailing the 5<2theta<31deg region of all four XRD patterns for all four N-methylisoindigo samples. The extra peaks for sample 0501 are labeled a-m and are summarized in Table 2. The maximum intensity is observed at approximately 7.71deg (d-spacing = 11.5 Å) with approximately 40,000 counts for all samples, consistent with the idea that the molecular plane of N-methylisoindigo is approximately parallel to this axis within the unit cell of the crystal structure. [Figure 7]

[0033] FIG. 7 shows the calculated powder diffraction pattern of isoindigo (Cu Kα radiation, λ=1.5418 Å) based on the single crystal coordinates, Cambridge structural database reference code “ISOIND”. [Figure 8]

[0034] FIG. 8 shows examples of 1H-NMR spectra of N-methylisoindigo samples 1 to 6. [Figure 9]

[0035] FIG. 9 shows examples of 13C-NMR spectra of N-methylisoindigo samples 1 to 6. [Figure 10A]

[0036] FIG. 10A shows a comparison of the X-ray powder diffraction patterns of Samples 1-6. [Figure 10B] Figures 10B, 10C, 10D, 10E, 10F, and 10G show the individual X-ray powder diffraction patterns for samples 1, 2, 3, 4, 5, and 6, respectively. [Figure 10C] Figures 10B, 10C, 10D, 10E, 10F, and 10G show the individual X-ray powder diffraction patterns for samples 1, 2, 3, 4, 5, and 6, respectively. [Figure 10D]Figures 10B, 10C, 10D, 10E, 10F, and 10G show the individual X-ray powder diffraction patterns for samples 1, 2, 3, 4, 5, and 6, respectively. [Figure 10E] Figures 10B, 10C, 10D, 10E, 10F, and 10G show the individual X-ray powder diffraction patterns for samples 1, 2, 3, 4, 5, and 6, respectively. [Figure 10F] Figures 10B, 10C, 10D, 10E, 10F, and 10G show the individual X-ray powder diffraction patterns for samples 1, 2, 3, 4, 5, and 6, respectively. [Figure 10G] Figures 10B, 10C, 10D, 10E, 10F, and 10G show the individual X-ray powder diffraction patterns for samples 1, 2, 3, 4, 5, and 6, respectively. [Figure 11]

[0037] FIG. 11 shows examples of particle sizes and their distribution for Crystal Form I of Meisoindigo. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025]

[0038] Herein, the inventors disclose a novel and unique crystalline form (Form I) and a method for preparing crystalline N-methylisoindigo that exhibits superior efficacy in treating various autoimmune / inflammatory diseases and cancer.

[0026]

[0039] In an embodiment, the present invention provides a solid form of N-methylisoindigo or a solid crystalline form of N-methylisoindigo having an X-ray powder diffraction pattern substantially as shown in FIG. 10A. In one embodiment, the solid form of N-methylisoindigo or a solid crystalline form of N-methylisoindigo has an X-ray powder diffraction pattern substantially as shown in FIG. 10B. In another embodiment, the solid form of N-methylisoindigo or a solid crystalline form of N-methylisoindigo has an X-ray powder diffraction pattern substantially as shown in FIG. 10C. In another embodiment, the solid form of N-methylisoindigo or a solid crystalline form of N-methylisoindigo has an X-ray powder diffraction pattern substantially as shown in FIG. 10D. In yet another embodiment, the solid form of N-methylisoindigo or a solid crystalline form of N-methylisoindigo has an X-ray powder diffraction pattern substantially as shown in FIG. 10C. The solid form of N-methylisoindigo or the solid crystalline form of N-methylisoindigo has an X-ray powder diffraction pattern substantially as shown in Figure 10E. In another embodiment, the solid form of N-methylisoindigo or the solid crystalline form of N-methylisoindigo has an X-ray powder diffraction pattern substantially as shown in Figure 10F. In another embodiment, the solid form of N-methylisoindigo or the solid crystalline form of N-methylisoindigo has an X-ray powder diffraction pattern substantially as shown in Figure 10G.

[0027]

[0040] In other embodiments, the solid form or solid crystalline form has an NMR spectrum for N-methylisoindigo substantially as shown in FIG. 8 and / or FIG.

[0041] In some embodiments, the solid form or solid crystalline form is in the form of a pharmaceutical composition or formulation ready to be administered to a patient or a patient in need thereof. The solid form or solid crystalline form may be administered by oral, intravenous, topical, localized placement, intraperitoneal or nasal routes. The composition may be utilized to achieve a desired pharmacological effect by administration to a patient in need thereof.

[0028]

[0042] For purposes of the present invention, a patient is a mammal, including a human, in need of treatment for a particular condition or disease. Thus, the present invention includes pharmaceutical compositions comprising a pharma- ceutically acceptable carrier and a pharma- ceutically effective amount of the solid form or solid crystalline form. A pharma- ceutically acceptable carrier is preferably a carrier that is relatively non-toxic and harmless to the patient at a concentration that is compatible with the effective activity of the active ingredient, such that any side effects attributable to the carrier do not impair the beneficial effects of the solid form or solid crystalline form.

[0029]

[0043] A pharma- ceutically effective amount of the solid or solid crystalline form is preferably that amount which produces a result or exerts a certain influence on the particular condition being treated. The solid or solid crystalline forms of the present invention can be administered using any effective conventional dosage unit form, e.g., immediate release, delayed release, and sustained release preparations, orally, parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, etc., with pharma- ceutically acceptable carriers well known in the art.

[0030]

[0044] The compounds according to the invention can have systemic and / or local activity, and for this purpose they can be administered in any suitable manner, for example by the oral, parenteral, pulmonary, nasal, sublingual, lingual, buccal, rectal, vaginal, cutaneous, transdermal, conjunctival, auricular route, or by implants or stents.

[0031]

[0045] Among these administration routes, the compounds according to the present invention can be administered in suitable dosage forms.

[0046] For oral administration, the compounds according to the invention can be formulated into dosage forms known in the art that deliver the compounds of the invention in a rapid and / or modified manner, such as tablets (plain or coated, e.g. with a delayed dissolving or insoluble enteric or controlled release coating), orally disintegrating tablets, films / wafers, films / lyophilisates, capsules (e.g. hard or soft gelatin capsules), sugar-coated tablets, granules, pills, powders, emulsions, suspensions, aerosols or liquids. The compounds according to the invention can be incorporated into said dosage forms in crystalline and / or amorphous and / or dissolved form.

[0032]

[0047] Parenteral administration can be carried out by avoiding the absorption step (e.g. intravenous, intraarterial, intracardiac, intrathecal or intralumbal) or by including absorption (e.g. intramuscular, subcutaneous, intradermal, transdermal or intraperitoneal). Suitable dosage forms for parenteral administration are, inter alia, injection and infusion preparations in the form of solutions, suspensions, emulsions, lyophilisates or sterile powders.

[0033]

[0048] Examples of suitable forms for other administration routes are pharmaceutical forms for inhalation (especially powder inhalers, nebulizers), nose drops, nasal solutions, nasal sprays; tablets / films / wafers / capsules for lingual, sublingual or buccal administration; suppositories; eye drops, eye ointments, eye baths, ocular inserts, ear drops, ear sprays, ear powders, ear-rinses, ear tampons, vaginal capsules, aqueous suspensions (lotions, mixturae agitandae), liposoluble suspensions, emulsions, ointments, creams, transdermal therapeutic systems (e.g. patches), milks, pastes, foams, dusting powders, implants or stents.

[0034]

[0049] The compounds according to the invention can be incorporated into defined dosage forms. This can be achieved by mixing them with pharma- ceutical suitable excipients in a known manner. Pharmaceutically suitable excipients include, inter alia: Fillers and carriers (e.g., cellulose, microcrystalline cellulose (e.g., Avicel"), lactose, mannitol, starch, calcium phosphates (e.g., Di-Cafos * )) Ointment bases (e.g. petrolatum, paraffin, triglycerides, waxes, wool fat, wool wax alcohol, lanolin, hydrophilic ointments, polyethylene glycols), Suppository bases (e.g. polyethylene glycol, cocoa butter, hard fats), Solvents (e.g. water, ethanol, isopropanol, glycerol, propylene glycol, medium chain triglyceride fatty oils, liquid polyethylene glycols, paraffin), Surfactants, emulsifiers, dispersing agents or wetting agents (e.g. sodium dodecyl sulfate), lecithins, phospholipids, fatty alcohols (e.g. Lanette * ), sorbitan fatty acid esters (e.g., Span"), polyoxyethylene sorbitan fatty acid esters (e.g., Tween * ), polyoxyethylene fatty acid glycerides (e.g., Cremophor * ), polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, glycerol fatty acid esters, poloxamers (e.g., Pluronic * ), buffers, acids and bases (e.g. phosphates, carbonates, citric acid, acetic acid, hydrochloric acid, sodium hydroxide solution, ammonium carbonate, trometamol, triethanolamine), isotonicity agents (e.g. glucose, sodium chloride), Adsorbents (e.g. highly dispersed silica), Viscosifying, gel-forming, thickening and / or binding agents (e.g. polyvinylpyrrolidone, methylcellulose, hydroxypropylmethylcellulose, hydroxypropyl-cellulose, sodium carboxymethylcellulose, starch, carbomer, polyacrylic acid (e.g. Carbopol * ); alginate, gelatin), Disintegrants (e.g. modified starch, sodium carboxymethylcellulose, sodium starch glycolate (e.g. Explotab * ), cross-linked polyvinylpyrrolidone, croscarmellose-sodium (e.g., AcDiSof), Flow regulators, lubricants, glidants and release agents (e.g. magnesium stearate, stearic acid, talc, highly dispersed silica (e.g. Aerosil * )) Coating materials (e.g. sugar, shellac) and film formers for rapidly or modified dissolving films or diffusion membranes (e.g. polyvinylpyrrolidone (e.g. Kollidon * ), polyvinyl alcohol, hydroxypropyl methylcellulose, hydroxypropyl cellulose, ethyl cellulose, hydroxypropyl-methylcellulose phthalate, cellulose acetate, cellulose acetate phthalate, polyacrylates, polymethacrylates, e.g., Eudragit * )), capsule materials (e.g., gelatin, hydroxypropyl methylcellulose), Synthetic polymers (e.g. polylactides, polyglycolides, polyacrylates, polymethacrylates (e.g. Eudragit * ), polyvinylpyrrolidone (e.g., Kolli don * ), polyvinyl alcohol, polyvinyl acetate, polyethylene oxide, polyethylene glycol and their copolymers and block copolymers), plasticizers (e.g., polyethylene glycol, propylene glycol, glycerol, triacetin, triacetyl citrate, dibutyl phthalate), penetration enhancers, stabilizers (e.g. antioxidants, e.g. ascorbic acid, ascorbyl palmitate, sodium ascorbate, butylated hydroxyanisole, butylated hydroxytoluene, propyl gallate), preservatives (e.g. parabens, sorbic acid, thiomersal, benzalkonium chloride, chlorhexidine acetate, sodium benzoate), colorants (e.g. inorganic pigments, e.g. iron oxide, titanium dioxide), Flavoring, sweetening, flavor- and / or odor-masking agents.

[0035]

[0050] The present invention further relates to pharmaceutical compositions comprising the crystalline forms of N-methylisoindigo disclosed herein, conventionally together with one or more pharma- ceutically suitable excipients, and their uses in accordance with the present invention.

[0036]

[0051] A variety of diseases and disorders can be treated with the crystalline forms of N-methylisoindigo disclosed herein. These diseases and disorders are described in more detail below.

[0037]

[0052] Irregular and / or abnormal inflammation

[0053] Irregular and / or abnormal inflammation is a major component of a wide range of human diseases. People suffering from a variety of degenerative diseases often exhibit excessive blood levels of inflammatory markers. One type of such inflammatory markers is the inflammatory marker cytokines, such as IL-1α, β, IL-2, IL-3, IL-6, IL-7, IL-9, IL-12, IL-17, IL-18, IL-23, TNF-α, LT, LIF, oncostatin, and IFNc1α, β, γ.

[0038]

[0054] A non-exhaustive list of common medical problems directly caused by inflammatory cytokines includes: arthritis, where inflammatory cytokines cause damage within the synovial membrane leading to destruction of cartilage and bone in the joints; kidney failure, where inflammatory cytokines restrict blood circulation and damage nephrons; lupus, where inflammatory cytokines trigger an autoimmune attack; asthma, where inflammatory cytokines close the airways; psoriasis, where inflammatory cytokines trigger dermatitis; pancreatitis, where inflammatory cytokines trigger pancreatic cell damage; allergies, where inflammatory cytokines trigger an autoimmune response; fibrosis, where inflammatory cytokines attack traumatic tissue; surgical complications, where inflammatory cytokines prevent healing; anemia, where inflammatory cytokines attack erythropoietin production; and fibromyalgia, where inflammatory cytokines are elevated in fibromyalgia patients. Other diseases associated with chronic inflammation include cancer caused by chronic inflammation; heart attack, where chronic inflammation contributes to coronary atherosclerosis; Alzheimer's disease, where chronic inflammation destroys brain cells; congestive heart failure, where chronic inflammation causes myocardial weakness; stroke, where chronic inflammation promotes thromboembolic events; and aortic stenosis, where chronic inflammation damages heart valves. Arteriosclerosis, osteoporosis, Parkinson's disease, infections, inflammatory bowel disease, such as Crohn's disease and ulcerative colitis, and multiple sclerosis (a classic autoimmune inflammation-related disease), are also associated with inflammation. Some diseases in advanced stages can be life-threatening. Although several methods are available for the treatment of such inflammatory diseases, the results are generally unsatisfactory, as evidenced by the lack of efficacy and the associated drug-related side effects.

[0039]

[0055] inflammatory bowel disease

[0056] Inflammatory bowel disease (IBD) includes Crohn's disease (CD) and ulcerative colitis (UC), both of which are idiopathic chronic diseases occurring with increasing frequency in many parts of the world. In the United States, it affects more than 600,000 people annually. IBD can involve either or both the small and large intestine. CD can involve any part of the gastrointestinal tract. It usually involves the distal small intestine and colon. It either does not use the rectum or causes inflammation or infection with drainage around the rectum. UC usually causes ulcers in the lower part of the large intestine, often starting from the rectum. Symptoms vary but can include diarrhea, fever, and pain. Patients with prolonged UC are at increased risk of developing colon cancer. Currently, there is no satisfactory treatment as the cause of IBD is unknown, although infectious and immune mechanisms have been proposed. IBD treatment traditionally aims to control the inflammatory symptoms with corticosteroids, aminosalicylates, and standard immunosuppressants such as azathioprine (6-mercaptopurine), methotrexate, and cyclosporine. Of course, the best disease-modifying therapies are the immunosuppressants: azathioprine and methotrexate, both of which have a slow onset of action and only moderate efficacy. Prolonged treatment can cause liver damage (fibrosis or cirrhosis) and bone marrow suppression. Also, patients often become refractory to such treatment. Other treatments simply address the symptoms.

[0040]

[0057] psoriasis

[0058] Psoriasis is one of the most common immune-mediated chronic skin diseases that appears in various forms and levels of severity, affecting approximately 2% or over 4.5 million people in the United States, of which 1.5 million are thought to have a moderate to severe form of the disease. Ten to 30 percent of psoriasis patients also develop a form of arthritis, i.e., psoriatic arthritis, which damages the bones and connective tissues near the joints. Psoriasis appears as raised red patches of skin covered with flaky white buildup. It may also have an acne-like (pustular psoriasis) or burned (erythrodermic) appearance. Psoriasis can also cause intense itching and burning. Patients are affected both physically and mentally. Several modalities are currently available for the treatment of psoriasis, such as topical treatments, phototherapy, and systemic applications. However, they are generally considered to only suppress and modify the disease, and none are curative. Also, many treatments are cosmetically undesirable, inconvenient for long-term use, or associated with significant toxicity.

[0041]

[0059] Over the past 20 years, the understanding of the biology of psoriasis has improved and biological therapies have become available that target the activity of T lymphocytes and cytokines responsible for the inflammatory properties of the disease.Currently, drugs prescribed for psoriasis include the TNF-α inhibitors ENBREL (etanercept), REMICADE (infliximab) and HUMIRA (adalimumab), which were first used to treat rheumatoid arthritis (RA), and the T-cell inhibitors AMEVIVE (alefacept) from Biogen, approved in 2002, and RAPTIVA (efalizumab) from Genentech / Xoma, approved in 2003.More recently, biologics have been developed, such as antibodies against IL-17 (e.g., ixekizumab, Eli Lilly) or IL-23 (e.g., guselkumab, Johnson & Johnson). AMEVIVE® (Alefacept) is an immunoglobulin fusion protein consisting of the first extracellular domain of human LFA-3 fused to the hinge, C(H)2 and C(H)3 domains of human IgG(1). It inhibits T-cell proliferation via NK cells. RAPTIVA® (efalizumab) is a humanized monoclonal antibody known as anti-CD11a that targets the T-cell adhesion molecule, leukocyte function-associated antigen-1 (LFA-1). Preventing the binding of LFA-1 to its ligand (ICAM-1, intercellular adhesion molecule-1) inhibits lymphocyte activation and migration, thereby generating reduced lymphocyte infiltration, thereby limiting the cascade of events that ultimately lead to the signs and symptoms of psoriasis. However, potential side effects to TNF-α inhibitors are severe, such as the development of lymphomas and worsening congestive heart failure, resulting in severe infections and sepsis, as well as exacerbations of multiple sclerosis and central nervous system problems. Although the side effects of the T-cell inhibitors AMEVIVE® (alefacept) or RAPTIVA® (efalizumab) are better tolerated in the treatment of psoriasis, these treatments act as immunosuppressants, which can increase the risk of infection and reactivate underlying chronic infections. may activate immune systems or increase the risk of developing cancer.

[0042]

[0060] Although the past two decades have seen great advances in understanding the biology of psoriasis, and unconventional treatments for psoriasis have become available, as described above, much of the suffering it generates remains inadequately addressed. A survey of over 40,000 American patients with psoriasis conducted by the National Psoriasis Foundation in 1998 showed that 79% of young patients were frustrated by the ineffectiveness of their treatment. Of those with severe disease, 32% felt that their treatment was not effective enough.

[0043]

[0061] Rheumatoid arthritis

[0062] Rheumatoid arthritis (RA) is another example of a troublesome inflammatory disorder. It is a common chronic inflammation-related disease characterized by chronic inflammation of the lining (synovium) of joints and / or other internal organs. Inflammatory cells can also invade and damage bone and cartilage. The involved joints can lose their shape and alignment, resulting in loss of motion. Patients with RA have joint pain, stiffness, warmth, redness, and swelling, as well as other systemic symptoms such as fever, fatigue, and anemia. Currently, approximately 1% of the population or 2.1 million people in the United States are affected, with more women (1.5 million) than men (0.6 million). The pathology of RA is not well understood, but a cascade of inappropriate immunological responses has been hypothesized as a mechanism. Unfortunately, conventional treatments are ineffective for RA (29). The disease does not respond completely to symptomatic drugs, including corticosteroids and nonsteroidal anti-inflammatory drugs (NSAIDs), which have been used since the 1950s. These drugs also carry the risk of significant side effects. The therapeutic effects of disease-modifying antirheumatic drugs (DMARDs) such as methotrexate (MTX) are often inconsistent and short-lived.

[0044]

[0063] Recently, a new class of biological DMARDs (disease-modifying antirheumatic drugs) has been developed for the treatment of RA based on understanding the role of cytokines, TNF-α, and IL-1 in the inflammatory process. The FDA has approved several such DMARDs, for example, ENBREL® (etanercept) from Immunex / Amgen Inc. in 1998, REMICADE® (infliximab) from Centocor / Johnson & Johnson, HUMIRA® (adalimumab) from Abbott Laboratories Inc. in 2002, and KINERET® (anakinra) from Amgen in 2001. ENBREL® (etanercept) is a soluble TNF receptor (TNFR) recombinant protein. REMICADE® (infliximab) is a humanized murine (chimeric) anti-TNF-α monoclonal antibody. HUMIRA® (adalimumab) is a fully human anti-TNF monoclonal antibody created using phage display technology resulting in an antibody with human-derived heavy and light chain variable regions and a human IgG1:k constant region. All three protein-based drugs target and bind to TNF-α, blocking its effects. KINERET® (anakinra) is a recombinant IL-1 receptor antagonist that is similar to native human IL-1Ra except for the addition of a single methionine residue at its amino terminus. KINERET® (anakinra) blocks the biological activity of IL-1 by competitively inhibiting IL-1 binding to the IL-1 type I receptor (IL-1RI), thereby reducing the proinflammatory effects of IL-1.

[0045]

[0064] Treatment with these biologic DMARDs relieves symptoms, inhibits the progression of structural damage, and improves physical function in moderately to severely active RA. Three marketed TNF-α blockers have similar efficacy when combined with MTX, a widely used DMARD in the treatment of RA patients. Although these biologic therapies offer significant short- and mid-term efficacy and a good overall safety profile in many RA patients, they can cause significant problems and long-term side effects, e.g., in the liver, that remain to be evaluated. There has been a disturbing association between the use of both ENBREL® (etanercept) or REMICADE® (infliximab) and the development of lymphoma. As noted above, several reports indicate that patients treated with ENBREL® (etanercept) or REMICADE® (infliximab) have worsening congestive heart failure, developed serious infections and sepsis, and experienced increased exacerbations of multiple sclerosis and other central nervous system problems.

[0046]

[0065] Urological chronic pelvic pain syndrome

[0066] Interstitial cystitis or painful bladder syndrome and chronic prostatitis or chronic pelvic pain syndrome have recently been renamed urological chronic pelvic pain syndrome (UCPPS). UCPPS is a chronic condition characterized by a combination of uncomfortable bladder pressure, bladder pain, and / or pelvic pain that can range from mild burning or discomfort to severe pain. It affects approximately one million Americans. Children and men can be affected, but most are women. UCPPS can have a long-lasting and significant impact on the patient's quality of life. Despite continuing efforts, there is no effective treatment or mechanistic understanding of the pathogenesis of UCPPS.

[0047]

[0067] A variety of medications and therapies are available that are based solely on symptom relief. These include: oral medications; bladder instillation with dimethylsulfoxide (directly into the bladder through a thin, flexible tube inserted through the urethra) or injection of a solution containing a combination of drugs, such as a combination of heparin, lidocaine, and sodium bicarbonate; nerve stimulation using mild electricity (transcutaneous electrical nerve stimulation or TENS) to relieve pelvic pain and, in some cases, reduce urination frequency; bladder distension; and surgery (bladder augmentation, radiofrequency therapy, or resection).

[0048]

[0068] Oral medications are the most convenient treatments, aiming to relieve pain and / or other symptoms by relaxing and protecting the bladder from irritation with painkillers (ibuprofen, and nonsteroidal anti-inflammatory drugs); tricyclic antidepressants, such as amitriptyline or imipramine; or antihistamines, such as diphenhydramine and loratadine; or pentosan, a semisynthetically produced heparin-like macromolecular carbohydrate derivative that is chemically and structurally similar to glycosaminoglycans. Of these, pentosan (Elmiron) is the only oral drug approved by the Food and Drug Administration (FDA) specifically for interstitial cystitis. It is not known how pentosan works, but it may reinforce or otherwise restore the lining of the bladder and protect the bladder wall from substances in the urine that can irritate it. However, it may take several months for the patient to begin to feel a reduction in pain and as long as six months to feel a reduction in urinary frequency.

[0049]

[0069] The etiology of UCPPS is unknown. However, several hypotheses exist, including inflammation / autoimmunity, abnormalities in cell proliferation, mysterious infection, genetic, chemical, neurological, psychological, hormonal, and multifactorial. Of these, proliferation and inflammation are probably the most important. Histologically, it is characterized by thinning or ulceration of the bladder epithelial layer. Cystoscopic abnormalities of the bladder of patients with UCPPS include pinpoint hemorrhages, termed "glomerulations," and ulcers extending into the lamina propria (Hunner's ulcer). The most consistent histologic abnormalities include denudation or thinning of the bladder epithelium to 1-2 cell layers. These findings suggest that UCPPS may be caused by inhibition of normal bladder epithelial cell proliferation and the resulting loss of epithelial barrier integrity and subsequent exposure of intramural sensory neurons to urinary constituents. This hypothesis was supported by the recent identification of a glycosylated frizzled-related peptide inhibitor of cell proliferation derived from bladder epithelial cells in patients with UCPPS. This antiproliferative factor (APF) significantly inhibits bladder cell proliferation by modulating cell adhesion molecules and growth factor production.

[0050]

[0070] Multiple sclerosis

[0071] Multiple sclerosis (MS) is an autoimmune disease diagnosed in 350,000-500,000 people in the United States. Multiple areas of myelin inflammation and scarring in the brain and spinal cord indicate the disease. MS patients show varying degrees of neurological dysfunction depending on the location and extent of myelin scarring. Common symptoms of MS include fatigue, weakness, spasticity, balance problems, bladder and bowel problems, numbness, blindness, tremors, and depression. Current treatments for MS merely relieve symptoms or slow the progression of disability, and some newer treatments for MS, including stem cell transplantation and gene therapy, are conservative. Although anti-TNF antibodies have shown protective effects in experimental autoimmune encephalomyelitis (EAE), they exacerbate the disease in MS patients, suggesting that blocking TNF-α alone is not sufficient.

[0051]

[0072] Neurodegenerative diseases

[0073] Alzheimer's disease (AD) and Parkinson's disease (PD) are the two most common neurodegenerative diseases. AD is a brain disorder. It severely affects a person's ability to carry out daily activities. It involves the parts of the brain that control thinking, memory, and language. It is estimated that about 4 million Americans, usually over the age of 60, suffer from AD.

[0052]

[0074] PD is a progressive disease of the central nervous system that affects over 1.5 million people in the United States. Clinically, the disease is characterized by decreased locomotor activity, gait difficulty, postural instability, rigidity, and tremor. PD is caused by degeneration of pigmented neurons in the substantia nigra of the brain, resulting in reduced dopamine availability. The causes of these neurodegenerative diseases are unknown, and there is currently no cure for the disease.

[0053]

[0075] Thus, there is a need for new approaches to treat the above and other inflammation-related diseases. Although the mechanisms by which inflammation-related diseases are caused remain unclear and often vary from one another, it has been shown that immune system dysfunction caused by cytokine deregulation plays an important role in the initiation and progression of inflammation.

[0054]

[0076] Cytokines can generally be classified into three types: inflammatory (IL-1α, β, IL-2, IL-3, IL-6, IL-7, IL-9, IL-12, IL-17, IL-18, IL-23, TNF-α, LT, LIF, oncostatin, and IFNc1α, β, γ); anti-inflammatory (IL-4, IL-10, IL-11, W-13, and TGFB); and chemokines (IL-8, Groα, MIP-1, MCP-1, ENA-78, and RANTES).

[0055]

[0077] In many inflammatory conditions, inflammatory cytokines, especially TNF-α, IL-1β, and IL-6, as well as the anti-inflammatory cytokine IL-10, appear to play important roles in the pathogenesis of various inflammation-related diseases and therefore may be potential therapeutic targets. For example, elevated levels of several inflammatory cytokines (TNF-α, IFNγ, IL-1, IL-2, IL-6, and IL-12) and chemokines (IL-8, MCP-1, and RANTES) have been observed in several inflammation-related diseases, such as CD, psoriasis, RA, Graves' disease, and Hashimoto's thyroiditis, which are paralleled by increases in soluble TNF receptors, IL-1 receptor antagonists, and the anti-inflammatory cytokine IL-10. IL-10 has been shown to suppress elevated inflammatory cytokine production both in vitro in LPMC cultures and in vivo in patients. The positive response of CD patients treated with IL-10 indicates that there may also be an imbalance in the production of inflammatory and anti-inflammatory cytokines in CD.

[0056]

[0078] In summary, approaches to treat inflammation-related diseases have emerged in recent years, partly as a result of increasing concern regarding the severity of these diseases and partly as a result of the identification of sites in their immunopathogenesis. Due to considerable progress in understanding the key role of IL-1, it has undergone evolutionary changes. Most of the efforts have focused on targeting TNF-α and IL-1, and several products (TNF-α inhibitors: infliximab, a monoclonal anti-TNF-α antibody; and etanercept, a p75 TNF-α receptor) are currently on the market or in clinical trials for the treatment of RA, psoriasis, and IBD, as mentioned above. Several other drug candidates or strategies targeting IL-1, IL-6, IL17, IL23, or IL-10 have been developed or are in development. These biological treatments offer significant short- and mid-term efficacy in many patients with RA or other autoimmune / inflammatory diseases. These drugs are well tolerated and have a good overall safety profile, but active pharmacovigilance is required. Based on their mechanism of action and previous notice of a wide range of side effects, the long-term risk of side effects, including hematological, infectious, neurological, oncological, and immunological effects, should be considered.

[0057]

[0079] The strategy of targeting a single inflammatory cytokine as an anti-inflammatory therapy ignores the very important fact that inflammation-related diseases involve a very sophisticated cytokine network "system". For example, chemokines, a family of immune molecules related to IL-8, contain approximately 50 ligands and 20 receptors, and often act redundantly, thus making it difficult to select a suitable specific antagonist, as well as lacking long-term efficacy. In addition, the products currently on the market or in development are mainly protein-based drugs, which are expensive to produce and inconvenient to administer (i.e., injection). Therefore, since the functioning of the immune system is delicately balanced by the activity of inflammatory and anti-inflammatory mediators or cytokines, modulation of multiple inflammatory / anti-inflammatory cytokines instead of blocking only one specific inflammatory cytokine with a small molecule should not only achieve better therapeutic efficacy with fewer side effects, but also have many advantages of small molecule drugs.

[0058]

[0080] Based on this concept, applicants have previously investigated several types of small molecules to test their abilities in regulating various cytokines and explore their potential clinical applications for the treatment of various inflammation-related diseases.

[0059]

[0081] Meisoindigo is an indirubin derivative with few side effects that has been used in the treatment of chronic myeloid leukemia (CML) in China. Applicants have shown that Meisoindigo and its derivatives are active against solid tumors through their ability to inhibit cyclin-dependent kinases, induce cell differentiation, and promote apoptosis. In this invention, applicants demonstrate the novel therapeutic activity of this type of molecule in the treatment of various inflammation-related diseases in rodents as well as humans, such as inflammatory bowel disease and psoriasis. Applicants show that this type of substance inhibits the secretion and expression of multiple inflammatory cytokines, including IL-1β, IL-6, and TNF-α, in cell lines, and promotes the production of the anti-inflammatory cytokine IL-10. In recent clinical trials, Meisoindigo has been demonstrated to be highly effective against ulcerative colitis, while no significant side effects were observed.

[0060]

[0082] Meisoindigo has been shown to be effective in preventing cancer, treating cancer, and treating inflammation-related diseases associated with the expression of pro-inflammatory cytokines, reduced expression of anti-inflammatory cytokines, or both.

[0061]

[0083] In some embodiments, the crystalline forms of Meisoindigo disclosed herein are stable under conditions of light, high temperature and high humidity. The crystalline forms of Meisoindigo disclosed herein are also stable under conditions of grinding, pressure and heat that meet the requirements of production, transportation and storage of the formulation.

[0062]

[0084] In other embodiments, the crystalline forms of Meisoindigo disclosed herein are, e.g. For example, the crystalline forms of the present invention are superior in terms of purity, ease of handling (lower hygroscopicity), flowability, ease of polishing, and / or quality control compared to other crystalline forms, and are useful as crystals suitable for pharmaceutical preparations. These crystalline forms exhibit excellent stability when in contact with heat, light, oxygen, humidity, or other molecules. Furthermore, the crystalline forms of the present invention have excellent filtration performance, drying characteristics, and flowability, and can be advantageously produced industrially.

[0063]

[0085] In addition, the crystalline form of the present invention, which has a residual solvent amount less than the standard value set forth in the ICH (International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use) guideline on residual solvents, is safe as a medicine. Furthermore, the disclosed crystalline form has a relatively small charge and is easy to handle in pharmaceutical production and packaging.

[0064]

[0086] In yet other embodiments, the crystalline forms of the present invention have one or more beneficial properties, particularly the following advantages: improved stability, improved solubility and dissolution rate in water or aqueous systems, improved thermodynamic stability, and / or improved storage stability.

[0065]

[0087] In one embodiment, the preparation methods disclosed herein are stable, repeatable and controllable, and therefore suitable for industrial production. Preparation of Crystal Form I

[0088] Equipment and starting materials

[0089] Reaction vessel: The preparation is carried out in a 12 liter round bottom flask equipped with a thermowell, reflux condenser, and necks for charging reagents and solvents.

[0066]

[0090] Filtration flask: 20 liter glass filtration flask equipped with a vacuum connection.

[0091] Benchtop Filtration Funnel: A high-density polyethylene funnel with an inner diameter of 10.25” (26.035 cm), total height of 8” (20.32 cm), containing a fixed porous filter plate and fitted with a 12.7 mm vacuum fitting.

[0067]

[0092] Starting materials: N-methylisatin and oxindole.

[0093] Main Process Steps

[0094] The manufacturing process for the production of bulk N-methylisoindigo (Natura-alpha, NAT) involves the following main steps:

[0068]

[0095] 1. A 12 L 3-neck round bottom flask on a heating mantle equipped with an overhead agitator is charged with 3.75 L of glacial acetic acid, followed by 0.75 kg (4.65 mol) of N-methylisatin and 0.63 kg (4.73 mol) of oxindole and 30 mL of concentrated HCl.

[0069]

[0096] 2. Heat the reaction mixture to 85-95°C for 2-3 hours.

[0097] 3. The reaction mixture is cooled to 25-30°C and the precipitated product is suction filtered.

[0098] 4. Wash the filter cake with 0.75 L and 0.38 L of glacial acetic acid and suck dry.

[0070]

[0099] 5. The filter cake is redissolved in 4.5 L of refluxing glacial acetic acid and the recrystallized product is suction filtered, washed with 0.75 L and 0.38 L portions of glacial acetic acid and dried under vacuum at 50°C.

[0071]

[0100] Natura-alpha (NAT) is a catalyst in acetic acid with concentrated HCl as follows: It is prepared by condensing N-methylisatin with oxindole using

[0072] [ka]

[0073]

[0101] Herein, we also disclose N-methylisoindigo crystalline compositions. do.

[0102] N-Methylisoindigo is a dark red crystalline powder. N-Methylisoindigo is sparingly soluble in acetone, chloroform, and ethanol. N-Methylisoindigo is sparingly soluble in water.

[0074]

[0103] The structure elucidation and confirmation of N-methylisoindigo was carried out using the first standard batch SR- I 205a. The solid state of N-methylisoindigo is shown below (in the "Other Properties" section).

[0075]

[0104] Elucidation of structure and other properties

[0105] Structure elucidation: The structure elucidation and structure of the compound N-methylisoindigo are provided. The analytical techniques used for structure elucidation were infrared spectroscopy, nuclear magnetic resonance spectroscopy, and mass spectrometry.

[0076]

[0106] Infrared spectrum

[0107] Infrared spectra of compounds were obtained on pellets of 1% mixtures of compounds in anhydrous KBr. 1685 and 1695 cm -1 The IR absorption of was characteristic of the carbonyl absorption of the expected product. The results are provided below in Figure 1.

[0077]

[0108] Nuclear magnetic resonance spectroscopy

[0109] Nuclear magnetic resonance spectra were obtained from NuMega Resonance Labs, I The spectrum was recorded by N.C. (San Diego, CA). The spectrum was obtained in deuterated chloroform solution using the residual proton resonance at δ 7.27 ppm as the reference peak; the proton resonances at δ 3.29 (s, 3H), 6.79 (d, J=8Hz, 1H), 6.81 (d, J=8Hz, 1H), 7.06 (dd, J=8, 8Hz, 1H), 7.08 (dd, J=8, 8Hz, 1H), 7.31 (dd, J=8, 8Hz, 1H), 7.39 (dd, J=8, 8Hz, 1H), 7.85 (bs, 1H), 9.12 (d, J=8Hz, 1H), and 9.19 (d, J=8Hz, 1H) were consistent with the expected product structure. The results are provided below in Figure 2.

[0078]

[0110] mass spectrometry

[0111] Mass spectra were also obtained from NuMega Resonance Labs, Inc. Ionization was achieved by using electrospray ionization. The positive ion at 277 mass units in the positive Q1 scan (Figure 3) and the negative ion at 275 mass units in the negative Q1 scan (Figure 4) were characteristic of the M+H and MH ions of the expected product, respectively.

[0079]

[0112] Other characteristics

[0113] Solid State

[0114] Melting point

[0115] Melting points were obtained using a capillary melting point apparatus. The capillary tubes had an OD of 0.8–1. The melting temperatures were measured with an electronic thermometer calibrated with a K-type thermocouple.

[0080]

[0116] The data in Table 1 demonstrates batch-to-batch comparability.

[0117] polymorphism research

[0118] Polymorphism screening

[0119] X-ray diffraction patterns were obtained for four batches of drug substance, NAT-0501, NAT-0502 , NAT-0503 and NAT-0601 were used.

[0081]

[0120] The raw X-ray diffraction patterns for the four samples are shown in Figure 5, with the exception that for clarity, The y-axis has been intentionally shifted for clarity and the 100% peak has been cut (2θ=7.71°). From these four X-ray diffraction profiles, the following becomes evident:

[0121] 1. All four samples from the same manufacturing procedure have one peak at 2θ=7.71°. The compounds contain the same crystalline form in which the peak is predominant (approximately 40,000 counts versus the next highest peak at 2,000).

[0082]

[0122] 2. Samples NAT-0502 and NAT-0503 have almost the same crystalline components. be.

[0123] 3. Sample NAT-0601 is slightly more crystalline than the others, 17, 18 and and weaker peaks such as the shoulder of the 29° peak are more prevalent.

[0083]

[0124] 4. Sample NAT-0501 contains additional peaks and additional components in small amounts in the powder. This means that there are peaks at 11, 16 and 23° (e.g., several multiple but small peaks at 11, 16 and 23°). A possible explanation for this could be that lot #0501 was reprocessed. When lot #NAT-0501 was prepared, there was no recrystallization step and the purity was found to be less than 98%. A recrystallization step was added and the resulting product met specifications.

[0084]

[0125] Figure 6 shows a detailed view of the peak selection analysis in the program Jade. A list of the 2θ values ​​found for the samples is summarized in Table 2.

[0126] Searching / matching characteristic peaks in the powder X-ray diffraction pattern of a sample is done by powder X-ray diffraction. The results of this study did not result in any known phases in the databases (PDF 2006 and ICSD 2007). Therefore, no phase determination could be made. However, as can be seen from the appearance of the powder pattern of isoindigo (obtained from the Cambridge structural database), the pattern of the sample is consistent with an unfolded molecule in a plane of the approximate size and orientation (within the crystal unit cell) of isoindigo, with one major peak at exactly 7.7°, as can also be seen in Figure 7. It has a "finger-like" region at 13° < 2θ < 30°, and a much weaker "finger-like" region at 13° < 2θ < 30°).

[0085]

[0127] The data are from graphite monochromated Cu Kα x radiation (λ = 1.5418 Å) with a scan step size and speed of 0.02 deg and 0.01 deg sec, respectively. -1 The data were collected on a Philips XPert system as a logarithmic plot. XY plots were analyzed using the programs WinplotR and Jade.

[0086] [Table 2-1]

[0087] [Table 2-2]

[0088]

[0128] The terms "about" and "substantially represented" generally refer to X-ray powder diffraction (XRPD) peaks. ±0.2 for temperature measurements and ±3°C for differential scanning calorimetry (DSC). Particle Size and Distribution

[0129] Form I was analyzed for its particle size and distribution. Five batches were All samples were tested using a Mastersizer 2000 (MIIA14730, Malvern, UK).

[0089]

[0130] As shown in Table 3, the particle size of the five batches of crystalline form I was very uniform. The particles were found to be uniformly distributed. Figure 11 shows an example of the particle distribution of the crystalline Form I of the compound. These physical properties facilitate formulation processing such as formulation into various solid and topical formulations as required without additional processing of the raw active pharmaceutical ingredient (API) material. The physical properties also contribute to equivalent and stable bioavailability from batch to batch.

[0090] [Table 3]

[0091]

[0131] Experimental data show that Form I has uniform particle size with ideal distribution. (See FIG. 11.) This uniform particle size facilitates the formulation of pharmaceutical compositions from crystalline Form I. The uniform particle size also indicates the reproducibility of the process for forming crystalline Form I. Preparation of additional crystalline forms

[0132] Various forms of crystalline N-methylisoindigo (Natura-alpha After studying various synthetic routes and various crystallization / recrystallization conditions to produce a) To this end, we have identified the following additional synthesis and crystallization / recrystallization conditions: The resulting products were examined using X-ray powder diffraction (XRD).

[0092] [ka]

[0093]

[0133] Indole reacts with methyl iodide using NaH as a catalyst to give methyl indol The compound was then purified by column chromatography and recrystallized under various conditions as described below, and the structure was confirmed. 1 H-NMR, 13 The compounds were characterized using C-NMR and LC-MS, and representative spectra are shown in Figures 8-9.

[0094]

[0134] Sample #1: Recrystallization in methanol / water: 300 mg of freshly synthesized N-methyl Chili isoindigo was heated to 80°C, 80ml of methanol was added and heated under reflux until clear. 200ml of water was then slowly added to the solution while heating to reflux. The solution became cloudy again, but it was continuously heated under reflux until it became clear again, and then filtered while still hot. The solution was allowed to stand and cool to room temperature. The compound gradually precipitated, which was then filtered and dried.

[0095]

[0135] Sample #2: Recrystallization in methanol: 300 mg of freshly synthesized N-methyl isopropyl alcohol. The indigo was heated to 80°C, 80ml of methanol was added and heated to reflux until clear. The solution was then filtered while still hot and allowed to cool to room temperature. The compound gradually precipitated, which was then filtered and dried.

[0096]

[0136] Sample #3: Recrystallization with ethyl acetate and ligroin: 100 mg of freshly mixed The resulting N-methylisoindigo was heated to 80°C and 40 ml of ethyl acetate was added while heating at reflux until it became clear. 120 ml of ligroin was then slowly added to the solution. The solution became cloudy and was heated at reflux again until it became clear. The solution was filtered while still hot, allowed to stand, and then cooled to room temperature. The compound gradually precipitated, which was then filtered and dried.

[0097]

[0137] Sample #4: Recrystallization in ethyl acetate: 100 mg of freshly synthesized N-methylisocyanate The indigo was heated to 80°C and 30 ml of ethyl acetate was added under reflux until it became clear. The solution was then filtered while still hot, allowed to stand and cooled to room temperature. The compound gradually precipitated, which was then filtered and dried.

[0098]

[0138] Sample #5: Recrystallization in ethyl alcohol and water: 100 mg of newly synthesized Heat the N-methylisoindigo to 80℃ and reflux for 30 minutes until it becomes transparent. 1 of ethyl alcohol was added. 60 ml of distilled water was then added to the solution. The solution became moderately cloudy and then clear again. The solution was filtered while still hot, allowed to stand, and cooled to room temperature. The compound gradually precipitated, which was then filtered and dried.

[0099]

[0139] Sample #6: Recrystallization in ethyl alcohol: 100 mg of freshly synthesized N-methyl Chilled isoindigo was heated to 80°C and 30 ml of ethyl alcohol was added under reflux until it became clear. The solution was filtered while still hot, allowed to stand, and cooled to room temperature. The compound gradually precipitated, which was then filtered and dried.

[0100]

[0140] The purity of samples 1-6 was analyzed and each sample was found to be >98.00% pure. It was.

[0141] Some physical properties of samples #1 to #6 are shown in Table 4. The appearance of their crystal morphology The powders are different: Samples #1 and #5 are powder-like; Samples #2 and #4 are dendritic, while Samples #3 and #6 are needle-like. Both dendritic and needle-like are known to have slow flow rates and are therefore not ideal for formulation.

[0101] [Table 4]

[0102]

[0142] Structural characterization of the newly synthesized N-methylisoindigo 1 H NMR, 1 3 The analysis was performed by C-NMR and LC-MS, and representative spectra are shown in Figures 8 and 9.

[0103]

[0143] 10A-10G show the X-ray spectra (PXRD) of six crystalline samples. None of them repeated each other. PXRD suggests that samples 1-6 are mixtures of various crystalline forms.

[0104]

[0144] Compared with the other samples, the XRD spectra of sample 1 and sample 5 show that the It showed many narrow and sharp peaks, which indicates a relatively good crystalline state of Meisoindigo. However, a relatively broad peak occurred at a larger θ, implying that a non-crystalline form was also present. In comparison, sample 1 produced more sharp and intense peaks with a shorter melting range than sample 5, thus indicating a better crystalline state of Meisoindigo than sample 5.

[0105]

[0145] Samples 2 and 4 show fewer narrow peaks and fewer tall peaks. and that the crystalline morphology was not only of larger grain size but also a mixture of crystal types. show.

[0106]

[0146] Samples 3 and 6 have peaks that are not only narrower but also relatively impure compared to the other samples. Many peaks of these samples overlapped with each other, indicating a typical mixture of crystal types of Meisoindigo. The number of crystal types of samples 3 and 6 was much higher than that of the other four samples. This indicates that many crystalline forms can occur rapidly under given re-crystalline conditions, and that the crystalline forms may contain solvent. These results suggest that the re-crystalline conditions of samples 3 and 6 were not suitable for producing a uniform crystalline powder.

[0107]

[0147] N-Methylisoindigo is chemically classified as a "conjugated aromatic lactam" Since the crystals are oriented in a crystalline form, hydrogen bonds (H-bonds) and π-stack structures are easily formed between the molecules. Therefore, it is difficult to dissolve these compounds in a solvent, and it is easy to form crystals and atypical crystal forms that may contain solvent during crystallization. The crystal forms of Samples 1-6 contain particles that are not uniform and have poor flow rates. This makes it more difficult to formulate the crystal forms of Samples 1-6 without additional processing steps. In contrast, crystalline form I is unique in its nature, has uniform particle size, flowability, and is reproducible in its production and isolation.

[0108]

[0148] Unless otherwise defined, all technical and scientific terms used herein are intended to be understood as meaning those to which the present invention pertains. The terms "subject matter" and "materials" have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications, patents, and patent applications cited herein are incorporated by reference in their entirety for all purposes.

[0109]

[0149] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

[0110]

[0150] While this invention has been described in connection with specific embodiments thereof, it is capable of further modifications. It is understood that this application is intended to cover any variations, uses, or adaptations of the invention in accordance with the principles of the invention in general, including such departures from the present disclosure as come within the knowledge or practice in the art to which the invention pertains and as applicable to the essential features as set forth above and as defined in the appended claims.

Claims

1. A solid crystalline form of N-methylisoindigo (Form I) having an X-ray powder diffraction pattern that includes a peak at 7.71° in 2-theta.

2. 2. The solid crystalline form of N-methylisoindigo of claim 1 having an X-ray powder diffraction pattern including peaks at about 7.71°, about 17°, about 18°, and about 29° in units of 2-theta.

3. (a) an X-ray powder diffraction pattern including peaks at 7.71°±0.2°, 17°±0.2°, 18°±0.2°, and 29°±0.2° in 2-theta; (b) In agreement with the infrared spectrum in Figure 1, the following spectra are obtained in wave numbers: 457.33, 542.45, 610.05, 734.01, 773.14, 1052.31, 1075.01, 1095.87, 1154.70, 1331.53, 1376.54, 1463.82, 1474.27, 1617.41, 1618.39, 1684.00, 2923.19, 3074.83, 3137.94, 3183.81, and 3400.17 cm －１ The infrared spectrum of N-methylisoindigo, including peaks at (c) an NMR spectrum for N-methylisoindigo, including chemical shifts in ppm at 1.58554, 3.29446, 6.80109, 7.05415, 7.07039, and 7.26075, in accordance with the NMR spectrum of FIG. 2; and (d) Particle size distribution of average particle size d50 less than 25 μm 3. The solid crystalline form of N-methylisoindigo according to claim 1 or 2, having one or more of the following:

4. 3. A solid crystalline form of N-methylisoindigo according to claim 1 or 2, having a particle size distribution ratio (d90-d10) / d50 of less than 2.50 and a maximum particle size of less than 100 μm.

5. 3. The solid crystalline form of N-methylisoindigo according to claim 1 or 2, wherein the solid crystalline form of N-methylisoindigo has a particle size distribution with an average particle size d50 of less than 25 μm, said particle size distribution facilitating formulation processing during formulation and enhancing the stability and bioavailability of the solid crystalline form of N-methylisoindigo.

6. A pharmaceutical composition comprising the solid crystalline form of N-methylisoindigo according to any one of claims 1 to 5 and a pharma- ceutically acceptable carrier.

7. 7. The pharmaceutical composition of claim 6, wherein the solid crystalline form of N-methylisoindigo is present in the composition in an amount of at least 90% by weight.

8. 6. The solid crystalline form of N-methylisoindigo according to any one of claims 1 to 5, which is purified.

9. The solid crystalline form of N-methylisoindigo according to any one of claims 1 to 8, which is crystalline.

10. A method for preparing the solid crystalline form of N-methylisoindigo according to any one of claims 1 to 5, comprising precipitating the crystalline form of N-methylisoindigo from a solution comprising glacial acetic acid.

11. The method of claim 10, wherein the solution further comprises N-methylisatin, oxindole, and / or HCl.

12. A pharmaceutical composition comprising the solid crystalline form of N-methylisoindigo according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 6 or 7 for use in a method of treating cancer.

13. A pharmaceutical composition for use in a method for treating an inflammation-related disease associated with cytokine expression levels comprising the solid crystalline form of N-methylisoindigo according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 6 or 7.

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