HK1207086B - 2,2-difluoropropionamide derivatives of bardoxolone methyl, polymorphic forms and methods of use thereof - Google Patents

Description

2, 2-difluoropropionamide derivatives of bardoxolone methyl, polymorphs thereof, and methods of use

This application claims benefit of priority from U.S. provisional application No. 61/780,444 filed on day 13 at 2013, U.S. provisional application No. 61/775,288 filed on day 8 at 2013, and U.S. provisional application No. 61/687,669 filed on day 27 at 4/2012; the entire contents of each of these applications are incorporated herein by reference.

In accordance with 37c.f.r.1.821(c), and this is submitted as a sequence listing in the form of an ASCII compatible text file named "REATP 0073WO _ ST 25" created on 24 days 4 months 4 and 2013 and having a size of about 6 kilobytes. The contents of the above documents are hereby incorporated by reference in their entirety.

Technical Field

The present invention generally relates to the following compounds:

n- ((4aS, 6aR, 6bS, 8aR, 12aS, 14aR, 14bS) -11-cyano-2, 2, 6a, 6b, 9, 9, 12 a-heptamethyl-10, 14-dioxo-1, 2, 3, 4, 4a, 5, 6, 6a, 6b, 7, 8, 8a, 9, 10, 12a, 14, 14a, 14 b-octadecahydroabienzene-4 a-yl) -2, 2-difluoropropionamide, which is also referred to herein aS RTA408, 63415 or PP 415. The invention also relates to polymorphs thereof, methods of making and using the same, pharmaceutical compositions thereof, and kits and articles of manufacture thereof.

Background

The anti-inflammatory and anti-proliferative activity of the naturally occurring triterpenoid oleanolic acid has been improved by chemical modification. For example, 2-cyano-3, 12-dioxoolean-1, 9(11) -diene-28-oic acid (CDDO) and related compounds have been developed. See Honda et al, 1997; honda et al, 1998; honda et al, 1999; honda et al, 2000 a; honda et al, 2000 b; honda et al, 2002; suh et al, 1998; suh et al, 1999; place et al, 2003; liby et al, 2005; and U.S. patents 8,129,429, 7,915,402, 8,124,799 and 7,943,778, all of which are incorporated herein by reference. Bardoxolone methyl (CDDO-Me) has been evaluated in phase II and III clinical trials for the treatment and prevention of diabetic nephropathy and chronic kidney disease. See Pergola et al, 2011, which is incorporated herein by reference.

Synthetic triterpenoid analogs of oleanolic acid have also been demonstrated as inhibitors of cellular inflammatory processes such as the induction of Inducible Nitric Oxide Synthase (iNOS) and COX-2 by IFN- γ in mouse macrophages. See Honda et al (2000 a); honda et al (2000 b); honda et al (2002); and U.S. patents 8,129,429, 7,915,402, 8,124,799 and 7,943,778, all of which are incorporated herein by reference. Compounds derived from oleanolic acid have been shown to affect the function of a variety of protein targets and thus modulate the activity of multiple important cellular signaling pathways associated with oxidative stress, cell cycle control, and inflammation (e.g., Dinkova-kostowa et al, 2005; Ahmad et al, 2006; Ahmad et al, 2008; Liby et al, 2007 a; and U.S. patents 8,129,429, 7,915,402, 8,124,799, and 7,943,778).

Given that the biological activity profiles of known triterpenoid derivatives vary, and given that there are a wide variety of diseases that can be treated or prevented with compounds having potent antioxidant and anti-inflammatory effects and that the medical needs that are exhibited within this wide variety of diseases are largely unmet, it is desirable to synthesize new compounds with different biological activity profiles for the treatment or prevention of one or more indications.

Disclosure of Invention

In some aspects of the invention, compounds (also referred to herein as RTA408, 63415, or PP415) having the formula are provided:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is in the form of a pharmaceutically acceptable salt. In some embodiments, the compound is not in the form of a salt.

In some embodiments, the polymorph has an X-ray powder diffraction pattern (CuK α) comprising a halo peak (halo peak) at about 14 ° 2 Θ in some embodiments, the X-ray powder diffraction pattern (CuK α) further comprises a shoulder peak at about 8 ° 2 Θ in some embodiments, the X-ray powder diffraction pattern (CuK α) is substantially as shown in fig. 59 in some embodiments, the polymorph has a T of about 150 ℃ to about 155 ℃_gIncluding, for example, T at about 153 deg.C_gOr T of about 150 ℃_g. In some embodiments, the polymorph has a Differential Scanning Calorimetry (DSC) curve comprising an endotherm centered at about 150 ℃ to about 155 ℃. In some embodiments, the endotherm is centered at about 153 ℃. In some embodiments, the endotherm is centered at about 150 ℃. In some embodiments, a Differential Scanning Calorimetry (DSC) curve is substantially as shown in figure 62.

In some embodiments, the polymorph is a solvate having an X-ray powder diffraction pattern (cuka) comprising peaks at about 5.6 ° 2 Θ, 7.0 ° 2 Θ, 10.6 ° 2 Θ, 12.7 ° 2 Θ, and 14.6 ° 2 Θ. In some embodiments, the X-ray powder diffraction pattern (CuK α) is substantially as shown in the top pattern in figure 75.

In some embodiments, the polymorph is a solvate having an X-ray powder diffraction pattern (cuka) comprising peaks at about 7.0 ° 2 Θ, 7.8 ° 2 Θ, 8.6 ° 2 Θ, 11.9 ° 2 Θ, 13.9 ° 2 Θ (bimodal), 14.2 ° 2 Θ, and 16.0 ° 2 Θ. In some embodiments, the X-ray diffraction pattern (CuK α) is substantially as shown in the second pattern from the top in figure 75.

In some embodiments, the polymorph is an acetonitrile hemisolvate having an X-ray powder diffraction pattern (CuK α) comprising peaks at about 7.5 ° 2 Θ, 11.4 ° 2 Θ, 15.6 ° 2 Θ, and 16.6 ° 2 Θ in some embodiments, the X-ray diffraction pattern (CuK α) is substantiallyAs shown in the second map from the bottom in fig. 75. In some embodiments, the polymorph has a T of about 196 ℃_g. In some embodiments, the polymorph has a Differential Scanning Calorimetry (DSC) curve comprising an endotherm centered at about 196 ℃. In some embodiments, a Differential Scanning Calorimetry (DSC) curve is substantially as shown in figure 116.

In some embodiments, the polymorph is a solvate having an X-ray powder diffraction pattern (cuka) comprising peaks at about 6.8 ° 2 Θ, 9.3 ° 2 Θ, 9.5 ° 2 Θ, 10.5 ° 2 Θ, 13.6 ° 2 Θ, and 15.6 ° 2 Θ. In some embodiments, the X-ray diffraction pattern (CuK α) is substantially as shown in the bottom pattern of figure 75.

In another aspect of the invention, there is provided a pharmaceutical composition comprising an active ingredient consisting of the above compound or a polymorph thereof (e.g. any of the polymorphs herein above and below) and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is formulated for administration in the following manner: oral, intralipidic, intraarterial, intraarticular, intracranial, intradermal, intralesional, intramuscular, intranasal, intraocular, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrarectal, intrathecal, intratracheal, intratumoral, intraumbilicus, intravaginal, intravenous, intravesical (intravesicular), intravitreal, liposomal, topical (locally), transmucosal, parenteral, rectal, subconjunctival, subcutaneous, sublingual, topical (topically), buccal, transdermal, vaginal, cream (creme) form, liquid composition form, via catheter, via lavage, via continuous infusion, via inhalation, via injection, via local delivery, or via local infusion. In some embodiments, the pharmaceutical composition is formulated for oral, intra-arterial, intravenous, or topical administration. In some embodiments, the pharmaceutical composition is formulated for oral administration.

In some embodiments, the pharmaceutical composition is formulated as a hard or soft capsule, a tablet, a syrup, a suspension, an emulsion, a solution, a solid dispersion, a wafer, or an elixir. In some embodiments, the pharmaceutical composition according to the invention further comprises an agent that improves solubility and dispersibility. (e.g., agents that improve solubility and dispersibility, including but not limited to PEG, cyclodextrins (cyclodextrins), and cellulose derivatives.) in some embodiments, the compound or polymorph is suspended in sesame oil.

In other embodiments, the pharmaceutical composition is formulated for topical administration. In other embodiments, the pharmaceutical composition is formulated as a lotion, cream, gel, oil, ointment, salve, emulsion, solution, or suspension. In some embodiments, the pharmaceutical composition is formulated as a lotion, cream, or gel. In some embodiments, the amount of active ingredient is from about 0.01 wt% to about 5 wt%, from about 0.01 wt% to about 3 wt%, or 0.01 wt%, 0.1 wt%, 1 wt%, or 3 wt%.

In another aspect of the invention, there is provided a method of treating or preventing a condition associated with inflammation or oxidative stress in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition as described above or below. The invention likewise relates to the compound N- ((4aS, 6aR, 6bS, 8aR, 12aS, 14aR, 14bS) -11-cyano-2, 2, 6a, 6b, 9, 9, 12 a-heptamethyl-10, 14-dioxo-1, 2, 3, 4, 4a, 5, 6, 6a, 6b, 7, 8, 8a, 9, 10, 12a, 14, 14a, 14 b-octadeca-picene-4 a-yl) -2, 2-difluoropropionamide (or RTA408) or a pharmaceutically acceptable salt thereof or a polymorph of this compound (e.g. any of the polymorphs described herein above or below) for the treatment or prevention of conditions associated with inflammation or oxidative stress, or a pharmaceutical composition comprising any of the above entities and a pharmaceutically acceptable carrier (including, for example, the pharmaceutical compositions described above). The invention also relates to the use of the above-mentioned compounds, polymorphs or pharmaceutical compositions for the manufacture of a medicament for the treatment or prevention of conditions associated with inflammation or oxidative stress. In some embodiments, the condition is associated with inflammation. In other embodiments, the condition is associated with oxidative stress. In some embodiments, the condition is a skin disease or disorder, sepsis, dermatitis, osteoarthritis, cancer, inflammation, an autoimmune disease, inflammatory bowel disease, complications resulting from local or systemic exposure to ionizing radiation, mucositis, acute or chronic organ failure, liver disease, pancreatitis, an ocular disorder, lung disease, or diabetes.

Furthermore, the present invention relates to the compound N- ((4aS, 6aR, 6bS, 8aR, 12aS, 14aR, 14bS) -11-cyano-2, 2, 6a, 6b, 9, 9, 12 a-heptamethyl-10, 14-dioxo-1, 2, 3, 4, 4a, 5, 6, 6a, 6b, 7, 8, 8a, 9, 10, 12a, 14, 14a, 14 b-octadecapicene-4 a-yl) -2, 2-difluoropropionamide (or RTA408) or a pharmaceutically acceptable salt thereof, or a polymorph of this compound (e.g. any of the polymorphs herein above or below), or a pharmaceutical composition comprising any of the above entities and a pharmaceutically acceptable carrier (including, for example, the pharmaceutical compositions described above): a skin disease or disorder, sepsis, dermatitis, osteoarthritis, cancer, inflammation, an autoimmune disease, an inflammatory bowel disease, a complication from local or systemic exposure to ionizing radiation, mucositis, acute or chronic organ failure, a liver disease, pancreatitis, an ocular disorder, a lung disease, or diabetes. Accordingly, the present invention relates to the use of a compound, polymorph or pharmaceutical composition as described above for the manufacture of a medicament for the treatment or prevention of a condition selected from: a skin disease or disorder, sepsis, dermatitis, osteoarthritis, cancer, inflammation, an autoimmune disease, an inflammatory bowel disease, a complication from local or systemic exposure to ionizing radiation, mucositis, acute or chronic organ failure, a liver disease, pancreatitis, an ocular disorder, a lung disease, or diabetes. The present invention also relates to a method of treating or preventing a condition selected from the group consisting of: a skin disease or disorder, sepsis, dermatitis, osteoarthritis, cancer, inflammation, autoimmune diseases, inflammatory bowel disease, complications from local or systemic exposure to ionizing radiation, mucositis, acute or chronic organ failure, liver disease, pancreatitis, ocular disorders, lung disease, or diabetes, comprising administering to said patient a therapeutically effective amount of a compound, polymorph, or pharmaceutical composition described above. In some embodiments, the condition is a skin disease or disorder, such as dermatitis, thermal or chemical burns, chronic wounds, acne, alopecia, other hair follicle disorders, epidermolysis bullosa, sunburn complications, skin pigmentation disorders, aging-related skin conditions; post-surgical trauma, scars from skin injury or burn, psoriasis, dermatological manifestations of autoimmune disease or graft versus host disease, skin cancer, or conditions involving hyperproliferation of skin cells. In some embodiments, the skin disease or disorder is dermatitis. In some embodiments, the dermatitis is allergic dermatitis, atopic dermatitis, dermatitis due to chemical exposure, or radiation-induced dermatitis. In other embodiments, the skin disease or disorder is a chronic wound. In some embodiments, the chronic wound is a diabetic ulcer, a decubitus ulcer, or a venous ulcer. In other embodiments, the skin disease or disorder is alopecia. In some embodiments, the hair loss is selected from baldness or drug-induced hair loss. In other embodiments, the skin disease or disorder is a skin pigmentation disorder. In some embodiments, the skin pigmentation disorder is vitiligo. In other embodiments, the skin disease or disorder is a disorder involving hyperproliferation of skin cells. In some embodiments, the disorder involving hyperproliferation of skin cells is hyperkeratosis.

In other embodiments, the condition is an autoimmune disease, such as rheumatoid arthritis, lupus, Crohn's disease, or psoriasis. In other embodiments, the condition is a liver disease, such as fatty liver disease or hepatitis.

In other embodiments, the condition is an ocular disorder, such as uveitis, macular degeneration, glaucoma, diabetic macular edema, blepharitis, diabetic retinopathy, a corneal endothelial disease or disorder, post-operative inflammation, dry eye, allergic conjunctivitis, or a form of conjunctivitis. In some embodiments, the ocular disorder is macular degeneration. In some embodiments, the macular degeneration is in the dry form. In other embodiments, the macular degeneration is the wet form. In some embodiments, the corneal endothelial disease or disorder is forskohlia corneal endothelial dystrophy (Fuchs endovasculonal dystrophy).

In other embodiments, the condition is a pulmonary disease, such as pulmonary inflammation, pulmonary fibrosis, COPD, asthma, cystic fibrosis, or idiopathic pulmonary fibrosis. In some embodiments, the COPD is induced by cigarette smoke.

In other embodiments, the condition is sepsis. In other embodiments, the condition is mucositis due to radiation therapy or chemotherapy. In some embodiments, the mucositis is present in the oral cavity. In other embodiments, the condition is associated with exposure to radiation. In some embodiments, the radiation exposure results in dermatitis. In some embodiments, the radiation exposure is acute. In other embodiments, the radiation exposure is fractionated (fractionated).

In other embodiments, the condition is cancer. In some embodiments, the cancer is a carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma, or seminoma. In other embodiments, the cancer is bladder cancer, blood cancer, bone cancer, brain cancer, breast cancer, central nervous system cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, gall bladder cancer, genital cancer, genitourinary tract cancer, head cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, muscle tissue cancer, neck cancer, oral or nasal mucosa cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, spleen cancer, small intestine cancer, large intestine cancer, stomach cancer, testicular cancer, or thyroid cancer.

In some embodiments, the pharmaceutical composition is administered prior to or immediately after treatment of a subject with radiation therapy or chemotherapy, wherein the chemotherapy does not comprise RTA408 or its polymorph. In some embodiments, the pharmaceutical composition is administered both before and after treatment of the subject with radiation therapy, chemotherapy, or both. In some embodiments, the treatment reduces side effects of radiation therapy or chemotherapy. In some embodiments, the side effects are mucositis and dermatitis. In some embodiments, the treatment increases the efficacy of radiation therapy or chemotherapy. In some embodiments, the chemotherapy comprises administering to the patient a therapeutically effective amount of 5-fluorouracil or docetaxel (docetaxel).

Additional combination therapy therapies are also contemplated by the present disclosure. For example, in some embodiments, a method of treating cancer in a subject comprising administering to the subject a pharmaceutically effective amount of a compound of the present disclosure may further comprise one or more treatments selected from the group consisting of: administering a pharmaceutically effective amount of a second drug, radiation therapy, immunotherapy, gene therapy, and surgery. In some embodiments, the method may further comprise (1) contacting the tumor cell with the compound prior to contacting the tumor cell with the second drug, (2) contacting the tumor cell with the second drug prior to contacting the tumor cell with the compound, or (3) contacting the tumor cell with both the compound and the second drug. In certain embodiments, the second drug may be an antibiotic, an anti-inflammatory agent, an anti-neoplastic agent, an anti-proliferative agent, an anti-viral agent, an immunomodulatory agent, or an immunosuppressive agent. In other embodiments, the second agent can be an alkylating agent, an androgen receptor modulator, a cytoskeletal disrupting agent, an estrogen receptor modulator, a histone deacetylase inhibitor, an HMG-CoA reductase inhibitor, an prenyl protein transferase inhibitor, a retinoid receptor modulator, a topoisomerase inhibitor, or a tyrosine kinase inhibitor. In certain embodiments, the second drug is 5-azacitidine (5-azacitidine), 5-fluorouracil, 9-cis-retinoic acid, actinomycin D (actinomycin D), alitretinoin (alitretinoin), all-trans retinoic acid, anastamomycin (annamycin), axitinib (axitinib), belinostat (belinostat), bevacizumab (bevacizumab), bexarotene (bexarotene), bosutinib (bosutinib), busulfan (busufan), capecitabine (capecitabine), carboplatin (carboplatin), carmustine (carmustine), CD437, cediranib (cediranib), cetuximab (cetuximab), chlorambucil (loranthus), docetaxel (cytarabine), cyclophosphamide (cytarabine), docetaxel (10-docetaxel), docetaxel (10-daunomycin), docetaxel (10-10), docetaxel (docetaxel), and paclitaxel), docetaxel (docetaxel), a, Deoxyfluorouridine (doxifluridine), doxorubicin (doxorubicin), doxorubicin (epirubicin), erlotinib (erlotinib), etoposide (etoposide), gefitinib (gefitinib), gemcitabine (gemcitabine), gemtuzumab ozogamicin (gemtuzumab ozogamicin), hexamethamine (hexamethlminemide), idarubicin (idarubicin), ifosfamide (ifosfamide), imatinib (imatinib), irinotecan (irinotecan), isotretinoin (isotretinoin), ixabepilone (ixabepilone), lapatinib (laetimib), LBH589, lomustine (lomustine), mechlorethamine (mechlorethamine), melphalan (mellanine), mercaptopurine (thiogalachlorethazine), mitomycin (paclitaxel), mitomycin (oxaliplatin), mitomycin (muramicarbine), mitomycin (methotrexate (oxaliplatin), mitomycin (muramicidin (muramicine), mitomycin (muramicidin (275), mitomycin (mitomycin), mitomycin (muramicidin (mitomycin), mitomycin (muramicidin (e), mitomycin (mitomycin), mitomycin (e), mitomycin (mitomycin), mitomycin (e), mitomycin (mitomycin), mitomycin (mito, Semustine (semustine), sodium butyrate, sodium phenylacetate, streptozotocin (streptozotocin), suberoylanilide hydroxamic acid, sunitinib (sunitinib), tamoxifen (tamoxifen), teniposide (teniposide), thiotepa (thiopeta), thioguanine, topotecan (topotecan), TRAIL, trastuzumab (trastuzumab), tretinoin (tretinoin), trichostatin A (trichostatin A), valproic acid, valrubicin (valrubicin), vandetanib (vandetanib), vinblastine (vinblaststatin), vincristine (vincrisitine), vindesine (vindesine), or vinorelbine (vinorelbine).

Also contemplated are methods of treating or preventing a disease with an inflammatory component in a subject comprising administering to the subject a pharmaceutically effective amount of a compound of the disclosure. In some embodiments, the disease can be, for example, lupus or rheumatoid arthritis. In other embodiments, the disease can be an inflammatory bowel disease, such as crohn's disease or ulcerative colitis. In other embodiments, the disease with an inflammatory component may be a cardiovascular disease. In other embodiments, the disease with an inflammatory component may be diabetes, such as type 1 diabetes or type 2 diabetes. In other embodiments, RTA408, its polymorphs, and pharmaceutical compositions can also be used to treat complications associated with diabetes. Such complications are well known to those skilled in the art and include, but are not limited to, for example, obesity, hypertension, atherosclerosis, coronary heart disease, stroke, peripheral vascular disease, hypertension, nephropathy, neuropathy, muscle necrosis, retinopathy and metabolic syndrome (syndrome X). In other embodiments, the disease with an inflammatory component may be a skin disease, such as psoriasis, acne, or atopic dermatitis. Administration of RTA408, its polymorphs, and pharmaceutical compositions in methods of treatment of such skin diseases may be, but is not limited to, topical administration or oral administration, for example.

In other embodiments, the disease with an inflammatory component may be metabolic syndrome (syndrome X). Patients with this syndrome are characterized by three or more symptoms selected from the group of 5 symptoms: (1) abdominal obesity; (2) hypertriglyceridemia; (3) low high density lipoprotein cholesterol (HDL); (4) hypertension; and (5) elevated fasting glucose, which may be in a range characteristic of type 2 diabetes mellitus if the patient also suffers from diabetes. Each of these symptoms is defined in Third Report of the National Cholesterol reduction Program expert on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults of adults (adult Treatment group III or ATP III), National Institutes of health, 2001, NIH publication No. 01-3670, which is incorporated herein by reference. Patients with metabolic syndrome, whether or not they have or develop overt diabetes, are at increased risk of developing the macrovascular and microvascular complications associated with type 2 diabetes listed above (e.g., atherosclerosis and coronary heart disease).

Another general method of the present disclosure entails a method of treating or preventing a cardiovascular disease in a subject, the method comprising administering to the subject a pharmaceutically effective amount of a compound of the present disclosure. In some embodiments, the cardiovascular disease can be, but is not limited to, atherosclerosis, cardiomyopathy, congenital heart disease, congestive heart failure, myocarditis, rheumatic heart disease, valve disease, coronary artery disease, endocarditis, or myocardial infarction, for example. Also contemplated are methods of using combination therapy for treating or preventing cardiovascular disease in a subject. For example, the method may further comprise administering a pharmaceutically effective amount of one or more cardiovascular drugs. The cardiovascular agent may be, but is not limited to, for example, a cholesterol-lowering agent, an antihyperlipidemic agent, a calcium channel blocker, an antihypertensive agent, or an HMG-CoA reductase inhibitor. In some embodiments, non-limiting examples of cardiovascular drugs include amlodipine (amlodipine), aspirin (aspirin), ezetimibe (ezetimibe), felodipine (felodipine), lacidipine (lacidipine), lercanidipine (lercanidipine), nicardipine (nicardipine), nifedipine (nifedipine), nimodipine (nimodipine), nisoldipine (nisoldipine), or nitrendipine (nitrendipine). In other embodiments, other non-limiting examples of cardiovascular drugs include atenolol (atenolol), bucindolol (bucinndolol), carvedilol (carvedilol), clonidine (clonidine), doxazosin (doxazosin), indoramin (indoramin), labetalol (labetalol), methyldopa (methylopa), metoprolol (metoprolol), nadolol (nadolol), oxprenolol (oxprenolol), phenoxybenzamine (phenoxybenzamine), phentolamine (phenytoamine), pindolol (pindolol), prazosin (prazosin), propranolol (propranolol), terazosin (terazosin), timolol (timolol), or tolazoline (tolazoline). In other embodiments, the cardiovascular drug may be, for example, a statin such as atorvastatin (atorvastatin), cerivastatin (cerivastatin), fluvastatin (fluvastatin), lovastatin (lovastatin), mevastatin (mevastatin), pitavastatin (pitavastatin), pravastatin (pravastatin), rosuvastatin (rosuvastatin), or simvastatin (simvastatin).

Also contemplated are methods of treating or preventing a neurodegenerative disease in a subject, the method comprising administering to the subject a pharmaceutically effective amount of a compound of the present disclosure. In some embodiments, the neurodegenerative disease may be selected, for example, from the group consisting of Parkinson's disease, Alzheimer's disease, Multiple Sclerosis (MS), Huntington's disease, and amyotrophic lateral sclerosis. In a specific embodiment, the neurodegenerative disease is alzheimer's disease. In specific embodiments, the neurodegenerative disease is MS, e.g., primary progressive MS, relapsing-remitting MS, secondary progressive MS, or progressive relapsing MS. In some embodiments, the subject can be, for example, a primate. In some embodiments, the subject may be a human.

In a specific embodiment of a method of treating or preventing a neurodegenerative disease in a subject, the method comprising administering to the subject a pharmaceutically effective amount of a compound of the present disclosure, the treatment inhibits neuronal demyelination in the brain or spinal cord of the subject. In certain embodiments, the treatment inhibits inflammatory demyelination. In certain embodiments, the treatment inhibits neuronal axonal transection in the brain or spinal cord of the subject. In certain embodiments, the treatment inhibits neurite crossing in the brain or spinal cord of the subject. In certain embodiments, the treatment inhibits neuronal apoptosis in the brain or spinal cord of the subject. In certain embodiments, the treatment stimulates remyelination of neuronal axons in the brain or spinal cord of the subject. In certain embodiments, the treatment restores function lost after onset of MS. In certain embodiments, the treatment prevents the onset of new MS. In certain embodiments, the treatment prevents a functional deficiency due to the onset of MS.

One general aspect of the present disclosure contemplates a method of treating or preventing a disorder characterized by overexpression of an iNOS gene in a subject, the method comprising administering to the subject a pharmaceutically effective amount of a RTA408, polymorph, or pharmaceutical composition of the present disclosure.

Another general aspect of the present disclosure contemplates a method of inhibiting IFN- γ -induced nitric oxide production in a cell of a subject, the method comprising administering to the subject a pharmaceutically effective amount of a RTA408, polymorph or pharmaceutical composition of the present disclosure.

Yet another general method of the present disclosure contemplates a method of treating or preventing a disorder characterized by overexpression of a COX-2 gene in a subject, comprising administering to the subject a pharmaceutically effective amount of a RTA408, polymorph, or pharmaceutical composition of the present disclosure.

Also contemplated is a method of treating kidney/renal disease (RKD) in a subject, the method comprising administering to the subject a pharmaceutically effective amount of a compound of the disclosure. See U.S. patent 8,129,429, which is incorporated herein by reference. The RKD may result from, for example, toxic insult. The toxic damage may be caused by, but is not limited to, for example, an imaging agent or drug. The drug may be, for example, a chemotherapeutic agent. In certain embodiments, RKD may result from ischemia/reperfusion injury. In certain embodiments, RKD is caused by diabetes or hypertension. In some embodiments, RKD may be caused by an autoimmune disease. RKD may be further defined as chronic RKD or acute RKD.

In certain methods of treating kidney/renal disease (RKD) in a subject, the method comprises administering to the subject, who has received dialysis or is receiving dialysis, a pharmaceutically effective amount of a compound of the present disclosure. In certain embodiments, the subject has received or is a candidate for receiving a kidney transplant. The subject may be a primate. The primate can be a human. The subject in this or any other method may be, for example, a cow, horse, dog, cat, pig, mouse, rat, or guinea pig.

The present disclosure also contemplates a method for increasing glomerular filtration rate or creatinine clearance in a subject comprising administering to the subject a pharmaceutically effective amount of a RTA408, polymorph or pharmaceutical composition of the present disclosure.

In some embodiments, the pharmaceutical composition is administered in a single dose per day. In other embodiments, the pharmaceutical composition is administered in more than one dose per day. In some embodiments, the pharmaceutical composition is administered in a pharmaceutically effective amount.

In some embodiments, the dose is from about 1mg/kg to about 2000 mg/kg. In other embodiments, the dose is from about 3mg/kg to about 100 mg/kg. In other embodiments, the dose is about 3mg/kg, 10mg/kg, 30mg/kg, or 100 mg/kg.

In other embodiments, the pharmaceutical composition is topically administered. In some embodiments, the topical application is to the skin. In other embodiments, the topical application is to the eye.

In other embodiments, the pharmaceutical composition is administered orally. In other embodiments, the pharmaceutical composition is administered intraocularly.

Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. It should be noted that not just because a compound is assigned to one particular formula means that it cannot be assigned to another formula at the same time.

Drawings

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. One or more German words can be seen in the drawing, includingAnd "temperature," which mean "mass change" and "temperature," respectively. The invention may be better understood by reference to one of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1: the effect of RTA408 on IFN γ -induced nitric oxide production and cell viability in RAW264.7 cells.

Fig. 2a and 2 b: effect of RTA408 on activation of oxidation response element (ARE): (a) NQO1-ARE luciferase activity; (b) GSTA2-ARE luciferase activity.

FIGS. 3 a-f: fold increase in relative Nrf2 GST ARE after cell treatment with: (a) RTA 402; (b)63415(RTA 408); (c)63170, respectively; (d) 63171; (e)63179, respectively; and (f) 63189. The graph also shows cell viability as determined using WST1 cell proliferation reagent and measuring absorbance after 1 hour. All drugs were administered in DMSO and cells were cultured in 10,000 cells/well in 384-well plates in DMEM (low glucose type) supplemented with 10% FBS, 1% penicillin streptomycin, and 0.8mg/mL Geneticin (Geneticin).

FIGS. 4 a-d: effect of RTA408 on Nrf2 target gene expression in HFL1 lung fibroblasts. (a) NQO 1; (b) HMOX 1; (c) GCLM; (d) TXNRD 1.

FIGS. 5 a-d: effect of RTA408 on Nrf2 target gene expression in BEAS-2B bronchial epithelial cells. (a) NQO 1; (b) HMOX 1; (c) GCLM; (d) TXNRD 1.

Fig. 6a and 6 b: effect of RTA408 on Nrf2 target protein levels. (a) SH-SY5Y cells; (b) BV2 cells.

FIG. 7: effect of RTA408 on NQO1 enzyme activity in RAW264.7 cells.

FIG. 8: effect of RTA408 on total glutathione levels in AML-12 hepatocyte cell line.

FIG. 9: effect of RTA408 on WST-1 absorbance as NADPH marker.

FIGS. 10 a-d: effect of RTA408 on the expression of genes involved in NADPH synthesis. (a) H6 PD; (b) PGD; (c) TKT; (d) ME 1.

Fig. 11a and 11 b: (a) effect of RTA408 on TNF- α induced NF- κ B luciferase reporter activation in mouse NIH3T3 cell line, superimposed on WST1 and WST1/2 activity. (b) NF- κ B luciferase reporter gene activation induced by TNF- α in mouse NIH3T3 cell line. The graph shows the relative fold change as a function of the log change in RTA408 concentration.

FIG. 12: effect of RTA408 on TNF- α induced activation of NF-. kappa.B luciferase reporter construct.

Fig. 13a and 13 b: (a) the effect of RTA408 on TNF- α -induced NF- κ B luciferase reporter activation in human A549 cell line superimposed with WST1 and WST1/2 activities. (b) TNF-alpha induced NF-. kappa.B luciferase reporter activation in human A549 cell line. The graph shows the relative fold change as a function of the log change in RTA408 concentration.

FIG. 14: effect of RTA408 on TNF- α induced phosphorylation of I κ B α.

FIGS. 15 a-d: effect of RTA408 on the expression of the following transaminase genes: (a) ALT1(GPT 1); (b) ALT2(GPT 2); (c) AST1(GOT 1); (d) AST1(GOT 2). Asterisks indicate statistically significant differences from the control group (. P < 0.05;. P < 0.01).

FIG. 16: effect of RTA408 on pyruvate levels in cultured myocytes (. P < 0.05).

FIG. 17: 63415 in a model of LPS-mediated pulmonary inflammation (% change in pro-inflammatory cytokines relative to LPS treatment). Compound 63415(QD x 3) was administered at time 0, 24 and 48 hours in female BALB/c mice, followed by LPS administration 1 hour after the last 63415 administration. Animals were sacrificed at 20 hours after LPS administration. BALF was examined for expression of pro-inflammatory cytokines. Compound 63415 reduced proinflammatory cytokines in a dose-dependent manner, with the highest reduction in TNF- α, IL-6 and IL-12 ranging from 50% to 80%.

Fig. 18a and 18 b: effect of RTA408 on LPS-induced lung inflammation in mice. (a) Inflammatory cytokines; (b) nrf2 target. Female BALB/c mice (n ═ 10) were administered RTA408 (QD × 6) at time 0, 24, 48, 72, 96 and 120 hours, followed by LPS at 121 hours and animals sacrificed at 141 hours. Measurement of pro-inflammatory cytokine protein expression in BALF. Nrf2 biomarkers were determined in the lungs. Asterisks indicate statistically significant differences from the saline control group (.;. P < 0.05;. P < 0.01;. P < 0.001).

Fig. 19a and 19 b: 63415 effect on BALF infiltration in bleomycin (bleomycin) -induced pulmonary inflammation: (a) BAL fluid cell count; (b) body weight. Compound 63415(QD × 39) was administered in C57BL/6 mice from day-10 to day 28. Bleomycin was administered on day 0. Body weight was measured daily. BALF cell counts were obtained at sacrifice. A significant reduction in inflammatory infiltration was observed. No significant improvement in chronic inflammation score, interstitial fibrosis or fibrotic foci number was observed.

Fig. 20a and 20 b: effect of RTA408 on bleomycin-induced pulmonary fibrosis in rats: (a) a PMN; (b) hydroxyproline. Asterisks indicate statistically significant differences from the bleomycin control group (. P < 0.05).

FIG. 21: effect of RTA408 on Nrf2 target enzymes in lungs from rats with bleomycin-induced pulmonary fibrosis. Asterisks indicate statistically significant differences from the saline control group (.;. P < 0.05;. P < 0.01;. P < 0.001).

FIGS. 22 a-e: effect of RTA408 on cigarette smoke induced COPD in mice. (a) KC; (b) IL-6; (c) TNF-alpha; (d) IFN-gamma; (e) RANTES. RTA408 (63415) was tested at dose levels of 3mg/kg (low dose), 10mg/kg (medium dose) and 30mg/kg (high dose). AIM analogs (63355) were tested in the same study for comparison. Asterisks indicate statistically significant differences from CS control groups.

FIG. 23: effect of RTA408 on Nrf2 target enzymes in the lungs from mice with cigarette smoke induced COPD. Asterisks indicate statistically significant differences from the saline control group (.;. P < 0.05;. P < 0.01;. P < 0.001). Sword indicates statistically significant differences from mice exposed to cigarette smoke and receiving vehicle (vehicle) administration: (P＜0.05)。

FIGS. 24 a-d: 63415(RTA408) effect on body weight in BALB/c mouse sepsis model. All animals were administered LPS on day 0. (a) Weight: 63415(RTA 408); (b) weight: RTA 405; (c) systemic LPS: survival%: 63415(RTA 408); (d) systemic LPS: survival%: RTA 405. Both RTA 405 and 63415(RTA408) were administered from day-2 to day 2 (QD × 5). Compound 63415(RTA408) improved survival. Body weight as a function of time in 63415-treated BALB/c mice served as a sepsis model.

FIG. 25: 63415 in a radiation-induced model of oral mucositis. RTA 405 or 63415(RTA408) (BID X20) was applied to male Syrian Golden Hamster (Syrian Golden Hamster) on days-5 to-1 and days 1 to 15. Irradiation was performed on day 0. Mucositis scores ranged from 0 to 5 points based on clinical presentation (0 points: complete health; 1-2 points: mild to severe erythema; 3-5 points: ulcers of varying degrees). 63415 at 30mg/kg and 100mg/kg improved mucositis with ulcer reduction of up to 36%.

FIG. 26: 63415 effect on Nrf2 target gene induction in a 14 day mouse toxicity study on C57BL/6 mice. mRNA of Nrf2 target gene was evaluated in the liver of mice receiving oral treatment (QD × 14). Significant increases in mRNA expression of various Nrf2 target genes were observed and consistent with tissue exposure.

Fig. 27a and 27 b: 63415 action on Nrf2 target gene induction in rat liver: (a) a target gene; (b) a negative regulator. mRNA of Nrf2 target gene was evaluated in the liver of rats receiving oral treatment (QD × 14).

Fig. 28a and 28 b: 63415 effect on Nrf2 target genes in monkey tissue: (a) liver; (b) and (4) lung. Using Panomics2.0Plex technique mRNA of Nrf2 target gene was assessed in monkeys receiving oral treatment (QD × 14).

Fig. 29a and 29 b: 63415 effect on Nrf2 target enzyme activity in mouse liver: (a) NQO1 activity; (b) GST activity. Nrf2 target enzyme activity was assessed in the livers of mice receiving oral treatment (QD × 14). NQO1 and GST enzyme activities were induced in a dose-dependent manner.

Fig. 30a and 30 b: 63415 effect on Nrf2 target enzyme activity in rat liver: (a) NQO1 activity; (b) GST activity. Nrf2 target enzyme activity was assessed in the liver of rats receiving oral treatment (QD × 14). NQO1 and GST enzyme activities were induced in a dose-dependent manner.

Fig. 31a and 31 b: 63415 on induction of Nrf2 target enzyme activity in different tissues of cynomolgus monkeys: (a) NQO1 activity; (b) GSR activity.

Fig. 32a and 32 b: concentration of RTA408 in mouse liver, lung and brain and NQO1 activity in mouse liver after daily oral administration for 14 days. (a) Tissue distribution of RTA408 in mice after daily oral administration for 14 days. Data represent mean ± SD of RTA408 concentration in tissues collected at 4 hours after the last dose of the study. The numbers above the error bars represent the mean. (b) Correlation of mouse liver RTA408 content with NQO1 enzyme activity. Individual mouse liver RTA408 liver content was plotted against individual enzyme activity from this report.

Fig. 33a and 33 b: concentration of RTA408 in rat plasma, liver, lung and brain and NQO1 activity in rat liver after daily oral administration for 14 days. (a) Tissue distribution of RTA408 in rats after daily oral administration for 14 days. Data represent mean ± SD of RTA408 concentration in tissues collected at 4 hours after the last dose of the study. The numbers above the error bars represent the mean. These two values are excluded from the mean calculation as being outliers, defined as values that fail the data set to pass the Charpiro-Wilknormatory normality test (Shapiro-Wilknormatory test). (b) Correlation of rat liver RTA408 content with NQO1 enzyme activity. Individual rat liver RTA408 content was plotted against individual enzyme activity from this report. Tissues from the 100mg/kg RTA408 dose group were collected on day 6, and the toxicity observed in this group made it impossible to evaluate liver NQO1 enzyme activity.

Fig. 34a and 34 b: 63415 effect of treatment on Nrf2 activation in monkey PBMCs: (a) PBMC NQO1 versus plasma concentration; (b) lung NQO1 versus PBMC NQO 1.

FIG. 35: 63415 summary of the 14 day monkey toxicity study. All doses were well tolerated without adverse clinical signs. Clinical chemistry data indicate no significant toxicity.

FIG. 36: effect of dosing time on plasma concentration of RTA408 after topical ocular and oral administration. Plasma concentrations of RTA408 were also measured after topical ocular administration of RTA408 daily for 5 days and were determined to remain relatively consistent with measurements obtained after the first day.

Fig. 37a and 37 b: correlation of exposure to RTA408 in monkey plasma with mRNA expression of NQO1 and SRXN1 in PBMC: (a) NQO 1; (b) SRXN 1.

FIG. 38: the concentration of RTA408 in different tissues or fluids in the eye as a function of time after 5 days of topical ocular administration. RTA408 concentrations in plasma were also measured after topical ocular administration.

FIG. 39: effect of RTA408 on the incidence of grade 3 dermatitis caused by acute radiation exposure for different concentrations of topically applied RTA 408.

FIG. 40: effect of RTA408 on the incidence of grade 2 dermatitis caused by acute radiation exposure over a 30 day treatment course for different concentrations of topically applied RTA 408.

FIG. 41: effect of RTA408 on the incidence of grade 2 dermatitis caused by acute radiation exposure over a 28 day treatment course for different concentrations of orally administered RTA 408.

Fig. 42a and 42 b: (a) area under the curve analysis of clinical scores of dermatitis as a function of time for each of the different control groups, including all animals used in the test. (b) Area under the curve analysis of the clinical dermatitis score as a function of the duration of that score for each of the different control groups, including only animals in the trial that completed the entire 30 days.

FIG. 43: mean 1-blind score of acute radiation dermatitis as a function of time without treatment, without treatment and without exposure to radiation, vehicle alone and three oral doses (3mg/kg, 10mg/kg and 30mg/kg) of RTA 408. The dermatitis score was based on the following scale: scores 0 were completely healthy, scores 1-2 indicated mild to moderate erythema with minimal to mild scaling, scores 3-4 indicated moderate to severe erythema and scaling, and scores 5 indicated obvious ulceration.

FIG. 44: mean score of acute radiation dermatitis as a function of time measured every other day from day 4 to day 30 without treatment, without treatment and without exposure to radiation, vehicle alone and three oral doses (3mg/kg, 10mg/kg and 30mg/kg) of RTA 408. The dermatitis score was based on the following scale: scores 0 were completely healthy, scores 1-2 indicated mild to moderate erythema with minimal to mild scaling, scores 3-4 indicated moderate to severe erythema and scaling, and scores 5 indicated obvious ulceration.

FIG. 45: mean score of acute radiation dermatitis as a function of time measured every other day from day 4 to day 30 without treatment, without treatment and exposure to radiation, vehicle alone and three topical local amounts (0.01%, 0.1% and 1%) of RTA 408. The dermatitis score was based on the following scale: scores 0 were completely healthy, scores 1-2 indicated mild to moderate erythema with minimal to mild scaling, scores 3-4 indicated moderate to severe erythema and scaling, and scores 5 indicated obvious ulceration.

FIG. 46: clinical scores of fractionated radiodermatitis plotted against time for each test group, as well as changes in dermatitis scores. The dermatitis score was based on the following scale: scores 0 were completely healthy, scores 1-2 indicated mild to moderate erythema with minimal to mild scaling, scores 3-4 indicated moderate to severe erythema and scaling, and scores 5 indicated obvious ulceration.

FIG. 47: a graph of AUC analysis of dermatitis scores (severity x days) over the entire observation period for each test group is shown. The dermatitis score was assessed every two days from day 4 to day 30 of the study.

Fig. 48a and 48 b: (a) a graph of the absorbance at 595nm of the treated prostate cancer cell line LNCaP shows the relative cytotoxic effect on cells treated with the chemotherapeutic agent and RTA408 relative to RTA408 alone. (b) A graph of the absorbance at 595nm of the treated prostate cancer cell line DU-145 shows the relative cytotoxic effect on cells treated with chemotherapeutic agent and RTA408 relative to RTA408 alone.

FIG. 49: black and white versions of the color photographs of the imaged mice showing the luciferase activity of tumors of the following three mice: control animals that received no treatment, animals that were subjected to ionizing radiation only (single dose, 18Gy) and animals that were given ionizing radiation (single dose, 18Gy, day 0) with RTA408 (17.5mg/kg, intraperitoneal, once daily on days-3 to-1, then given a single dose on days 1, 3 and 5). The color indicated by an arrow indicates the intensity, wherein the intensity is represented in red, yellow, green and blue colors in order of highest intensity to lowest intensity.

FIG. 50: andthe decrease in aqueous humor protein concentration in the case of different formulations of RTA408 (dark bars) following paracentesis induction, compared to literature values for (0.1% dexamethasone (dexamethasone)) and maplacorat (mapracoat) (light bars).

FIG. 51: 63415 dose-dependent inhibition of NO in vivo. CD-1 mice (n ═ 6) were given DMSO or AIM by oral gavage. LPS (5mg/kg) was administered after 24 hours. At 24 hours after administration of LPS, whole blood was collected for NO determination. NO inhibition was determined by the Griess Reaction using reduced deproteinized plasma.

FIG. 52: 63415(RTA408) wide distribution in mouse tissues. Mice were given either 25mg/kg 63415(RTA408) or 25mg/kg RTA 405 orally (QD × 3). Blood (plasma and whole blood) and tissues (brain, liver, lung and kidney) were collected at 6 hours after the last dose. Semi-quantitative analysis of drug content was performed. Significant levels were observed in the CNS.

FIG. 53: 63415 induction of NQO1 activity in mouse liver, lung and kidney. Mice were given orally at 25mg/kg (QD × 3). Tissues were collected at 6 hours after the last dose and analyzed for NQO1 activity. Meaningful NQO1 activation was observed in various tissues.

FIG. 54: 63415 summary of the 14 day mouse toxicity study. C57BL/6 mice were dosed orally (QD × 14). Endpoints included survival, body weight and clinical chemistry. All animals survived to day 14. No significant body weight change occurred compared to the vehicle group, and there were no signs of toxicity at any dose based on clinical chemistry.

FIG. 55: tissue distribution of 63415 in C57BL/6 mice from a 14-day mouse toxicity study. Brain, lung and liver samples were collected at 4 hours after the last dose and 63415 levels were quantified using the sensitive LC/MS method. Exposure in the lungs at 10mg/kg and 100mg/kg, respectively, is NO-induced in vitro IC₅₀55 times and 1138 times. Exposure in brain at 10mg/kg and 100mg/kg respectively is NO-induced in vitro IC₅₀29 times and 541 times.

FIG. 56: 63415 tissue distribution in Sprague Dawley rats. Tissues were collected at 4 hours after the last dose (100mg/kg) on day 14 or day 6, extracted, and 63415 content quantified using the sensitive LC/MS/MS method. Compound 63415 distributed well into the target tissue. Exposure to Lung and brain at 10mg/kg is NO-inhibited in vitro IC₅₀294 times and 240 times.

FIG. 57: target tissue distribution of compound 63415 in cynomolgus monkeys. Tissues were collected at 4 hours after the last dose on day 14. Compound 63415 content was extracted and quantified using a sensitive LC/MS method.

FIG. 58: FT-Raman (Raman) spectra (3400-50 cm) corresponding to sample PP415-P1 in amorphous form (Category 1)^-1)。

FIG. 59: PXRD (1.5 ° -55.5 ° 2 θ) spectra corresponding to sample PP415-P1 in amorphous form (category 1).

FIG. 60: TG-FTIR thermograms (25 ℃ -350 ℃) of samples PP415-P1 corresponding to the amorphous form (category 1).

FIG. 61: sample PP415-P1 corresponding to amorphous form (Category 1) in DMSO-d₆In (1)¹H-NMR spectrum.

FIG. 62: DSC thermogram corresponding to sample PP415-P1 in amorphous form (category 1).

FIG. 63: DVS isotherms corresponding to sample PP415-P1 in amorphous form (Category 1).

FIG. 64: the FT-raman spectrum (top) of sample PP415-P1 corresponding to the amorphous form (category 1) after DVS measurement was unchanged from the FT-raman spectrum (bottom) of the material before DVS measurement. The spectra have been scaled and shifted in the y-axis direction for comparison.

FIG. 65: the PXRD pattern of samples PP415-P1 corresponding to the amorphous form (category 1) after DVS measurement (top) was unchanged from the PXRD pattern of the material before DVS measurement (bottom). The maps were not scaled but shifted in the y-axis direction for comparison.

FIG. 66: the PXRD pattern (top) for samples PP415-P40 corresponds to that of the solvate form (category 2) (bottom, samples PP 415-P19). The maps have been scaled and shifted in the y-axis direction for comparison.

FIG. 67: one week later the PXRD pattern corresponding to the stability samples PP415-P2a (top), PP415-P3a (second from top), PP415-P4a (middle) and PP415-P5a (second from bottom) of the amorphous form (category 1) did not show any correlation with the starting material (bottom, samples PP415-P1) at time point t₀Compared with the PXRD pattern, the difference is. The maps were not scaled but shifted in the y-axis direction for comparison.

FIG. 68: the PXRD patterns corresponding to the stability samples PP415-P2b (top), PP415-P3b (second from top), PP415-P4b (middle) and PP415-P5b (second from bottom) of the amorphous form (category 1) after two weeks did not show any correlation with the starting material (bottom, samples PP415-P1) at time point t₀Compared with the PXRD pattern, the difference is. The maps were not scaled but shifted in the y-axis direction for comparison.

FIG. 69: stability sample PP415-P corresponding to amorphous form (Category 1) after four weeksThe PXRD patterns of 2c (top), PP415-P3c (second from top), PP415-P4c (middle) and PP415-P5c (second from bottom) do not show any correlation with the starting material (bottom, samples PP415-P1) at time t₀Compared with the PXRD pattern, the difference is. The maps were not scaled but shifted in the y-axis direction for comparison.

FIG. 70: FT-Raman spectra (2400-50 cm) of samples (PP 415-P7: top; PP 415-P8: second from top; PP 415-P9: third from top; PP 415-P10: fourth from top; PP 415-P11: middle; PP 415-P15: fourth from bottom; PP 415-P17: third from bottom; PP 415-P21: second from bottom; PP 415-P24: bottom) in solvate form (class 2)^-1). The spectra have been scaled and shifted in the y-axis direction for comparison.

FIG. 71: FT-Raman spectrum (1750-1000 cm) of the solvate form (Category 2) (PP 415-P7: Top)^-1) Clearly different from the amorphous form (category 1) (PP 415-P1: bottom) spectrum. The spectra have been scaled and shifted in the y-axis direction for comparison.

FIG. 72: FT-Raman spectra (1750 and 1000 cm) for class 2 (samples PP 415-P19: top), class 3 (samples PP 415-P6: second from top), class 4 (samples PP 415-P13: second from bottom) and class 5 (samples PP 415-P14: bottom)^-1) Are significantly different from each other. The spectra have been scaled and shifted in the y-axis direction for comparison.

FIG. 73: PXRD pattern (2 ° -32 ° 2 θ) of a sample of solvate form (category 2) (PP 415-P7: top; PP 415-P8: second from top; PP 415-P10: third from top; PP 415-P15: fourth from top; PP 415-P17: middle; PP 415-P18: fourth from bottom; PP 415-P19: third from bottom; PP 415-P21: second from bottom; PP 415-P24: bottom). The maps have been scaled and shifted in the y-axis direction for comparison.

FIG. 74: PXRD patterns (11 ° -21 ° 2 θ) for some samples of the solvate form (Category 2) (PP 415-P7: top; PP 415-P8: second from top; PP 415-P10: middle; PP 415-P21: second from bottom; PP 415-P24: bottom). The maps have been scaled and shifted in the y-axis direction for comparison.

FIG. 75: PXRD patterns (2-32 deg. 2 theta) for class 2 (samples PP 415-P19: top), class 3 (samples PP 415-P6: second from top), class 4 (samples PP 415-P13: second from bottom) and class 5 (samples PP 415-P14: bottom) are distinct. The maps have been scaled and shifted in the y-axis direction for comparison.

FIG. 76: TG-FTIR thermograms corresponding to samples PP415-P7 in the solvate form (category 2).

FIG. 77: TG-FTIR thermograms corresponding to samples PP415-P21 in the solvate form (category 2).

FIG. 78: TG-FTIR thermograms corresponding to samples PP415-P24 in the solvate form (category 2).

FIG. 79: TG-FTIR thermograms corresponding to samples PP415-P29 in the solvate form (category 2).

FIG. 80: TG-FTIR thermograms corresponding to samples PP415-P47 in the solvate form (category 2).

FIG. 81: TG-FTIR thermograms corresponding to samples PP415-P48 in the solvate form (category 2).

FIG. 82: FT-Raman spectra (1800-700 cm) of the solvate form (class 2) (bottom, samples PP415-P7) and of the dried solvate form (class 2) (top, samples PP415-P30)^-1) Are similar and show only small differences that are hardly discernible within the graph. The spectra are scaled for comparison.

FIG. 83: comparison of PXRD patterns (top) for samples PP415-P30 in the form of dried solvates (class 2) with patterns (bottom) for samples PP415-P7 in the form of solvates (class 2). The maps were not scaled but shifted in the y-axis direction for comparison.

FIG. 84: TG-FTIR thermograms corresponding to dried samples PP415-P30 in the solvate form (Category 2).

FIG. 85: the FT-Raman spectrum of the dried samples PP415-P18 (light grey) is similar to the spectrum of the original sample PP415-P15 (dark grey) and shows only small differences that are hardly discernible within the graph. The spectra have been scaled for comparison.

FIG. 86: the PXRD pattern (top) of the dried samples PP415-P18 showed small differences from the pattern (bottom) of the original samples PP415-P15, even though they were all solvate forms (category 2). The maps have been scaled and shifted in the y-axis direction for comparison.

FIG. 87: TG-FTIR thermograms corresponding to samples PP415-P18 in the solvate form (category 2).

FIG. 88: the FT-Raman spectrum of samples PP415-P17 (top) is almost identical to the spectrum of the dried samples PP415-P19 (middle) and PP415-P32 (bottom) and shows only small differences that are hardly discernible within the graph. The spectra have been scaled and shifted in the y-axis direction for comparison.

FIG. 89: the PXRD pattern for the dried samples PP415-P19 (middle) is different from the pattern for the original samples PP415-P17 (top), but still corresponds to the category 2 format. The spectra of the further dried samples PP415-P32 (bottom) show broader peaks with lower S/N ratios. The material, although less crystalline, still corresponds to the class 2 form. The maps have been scaled and shifted in the y-axis direction for comparison.

FIG. 90: TG-FTIR thermograms corresponding to samples PP415-P19 in the solvate form (category 2).

FIG. 91: TG-FTIR thermograms corresponding to samples PP415-P32A in the solvate form (category 2).

FIG. 92: the FT-Raman spectrum of the samples PP415-P21 (top) is identical to the spectrum of the dried samples PP415-P28 (middle) and PP415-P34 (bottom). The spectra have been scaled and shifted in the y-axis direction for comparison.

FIG. 93: the PXRD patterns of the dried samples PP415-P28 (middle) and PP415-P34 (bottom) show broader peaks with lower S/N ratios than the pattern of the original sample PP415-P21 (top), indicating that the samples are less crystalline. The maps are slightly different but still correspond to the class 2 format. They have been scaled and shifted in the y-axis direction for comparison.

FIG. 94: TG-FTIR thermograms corresponding to dried samples PP415-P28 in the solvate form (Category 2).

FIG. 95: TG-FTIR thermograms corresponding to dried samples PP415-P34 in the solvate form (Category 2).

FIG. 96: FT-Raman spectra (2400-50 cm) of samples (PP 415-P6: Top; PP 415-P12: middle; PP 415-P20: bottom) in solvate form (category 3)^-1). The spectra have been scaled and shifted in the y-axis direction for comparison.

FIG. 97: FT-Raman spectroscopy (1750-1000 cm) of samples in solvate form (class 3) (PP 415-P6: Top; PP 415-P12: second from the top; PP 415-P20: second from the bottom)^-1) Very similar to each other with only small differences, e.g. at about 1690cm^-1But clearly different from the FT-Raman spectrum of Category 1(PP 415-P1: bottom). The spectra have been scaled and shifted in the y-axis direction for comparison.

FIG. 98: PXRD pattern (2 ° -32 ° 2 θ) for samples of solvate form (category 3) (PP 415-P6: top; PP 415-P12: middle; PP 415-P20: bottom). The maps have been scaled and shifted in the y-axis direction for comparison.

FIG. 99: the PXRD pattern (13.5 deg. -18.5 deg. 2 theta) of samples of the solvate form (category 3) (PP 415-P6: top; PP 415-P12: middle; PP 415-P20: bottom) showed small differences. The maps have been scaled and shifted in the y-axis direction for comparison.

FIG. 100: TG-FTIR thermograms corresponding to samples PP415-P6 in the solvate form (category 3).

FIG. 101: TG-FTIR thermograms corresponding to samples PP415-P12 in the solvate form (category 3).

FIG. 102: TG-FTIR thermograms of samples PP415-P25 in the form of dried solvates (category 3).

FIG. 103: TG-FTIR thermograms of samples PP415-P33 in the form of a further dried solvate (class 3).

FIG. 104: FT-Raman spectra (1800-700 cm) of the solvated form (class 3) (top, samples PP415-P6), dried solvated form (class 3) (middle, samples PP415-P25) and further dried solvated form (class 3) (bottom, samples PP415-P33)^-1) Are the same. The spectra have been scaled and shifted in the y-axis direction for comparison.

FIG. 105: PXRD patterns (4 ° -24 ° 2 θ) for solvate form (category 3) (top, samples PP415-P6), dried solvate form (category 3) (middle, samples PP415-P25), and further dried solvate form (category 3) (bottom, samples PP 415-P33). The maps have been scaled and shifted in the y-axis direction for comparison.

FIG. 106: TG-FTIR thermograms of sample PP415-P13 corresponding to acetonitrile solvate form (category 4).

FIG. 107: FT-Raman spectra (1800-700 cm) of dried material (light grey, samples PP415-P26) in acetonitrile solvate form (class 4) (dark grey, samples PP415-P13) and acetonitrile solvate form (class 4)^-1) Are identical and overlap completely. The spectra have been scaled for comparison.

FIG. 108: comparison of the PXRD pattern of the sample PP415-P26 (bottom) of the dried acetonitrile solvate form (category 4) with the reference pattern of the sample PP415-P13 (top) of the acetonitrile solvate form (category 4). The maps were not scaled but shifted in the y-axis direction for comparison.

FIG. 109: TG-FTIR thermograms of samples PP415-P26 in the form of dried acetonitrile solvates (category 4).

FIG. 110: FT-Raman spectra (1800-700 cm) of acetonitrile solvate form (class 4) (top, samples PP415-P35) and dried acetonitrile solvate form (class 4) (middle: samples PP415-P36, and bottom: samples PP415-P37)^-1) Correspond to each other. The spectra have been scaled and shifted in the y-axis direction for comparison.

FIG. 111: the PXRD patterns (4 ° -24 ° 2 θ) of the acetonitrile solvate form (category 4) (top, samples PP415-P35) and the dried acetonitrile solvate form (category 4) (middle: samples PP415-P36, and bottom: samples PP415-P37) were consistent with each other. The maps have been scaled and shifted in the y-axis direction for comparison.

FIG. 112: TG-FTIR thermograms of samples PP415-P36 in the form of dried acetonitrile solvates (category 4).

FIG. 113: TG-FTIR thermograms of samples PP415-P37 in the form of dried acetonitrile solvates (category 4).

FIG. 114: DVS isotherms of desolvated acetonitrile solvate form (category 4) (sample PP 415-P37).

FIG. 115: the PXRD pattern of sample PP415-P37 (acetonitrile solvate form (category 4)) after DVS measurement (bottom) was unchanged from the PXRD pattern of the material before DVS measurement (top). The maps were not scaled but shifted in the y-axis direction for comparison.

FIG. 116: DSC thermogram of desolvated acetonitrile solvate form (category 4) (sample PP 415-P37).

Fig. 117: DSC thermograms of an approximately 1: 1 mixture of sample PP415-P1 in amorphous form (class 1) with sample PP415-P36 in desolvated acetonitrile solvate form (class 4).

FIG. 118: DSC thermograms of an approximately 1: 1 mixture (experimental No.: PP415-P39) of sample PP415-P1 in amorphous form (class 1) with sample PP415-P36 in desolvated acetonitrile solvate form (class 4). The heating sweep (step 1) was stopped at 173 ℃ for 30 minutes (step 2) and then continued (step 3).

FIG. 119: TG-FTIR thermograms of samples PP415-P14 corresponding to THF solvate form (category 5).

FIG. 120: FT-Raman spectra (1800-1100 cm) of the THF solvate form (class 5) (dashed line, samples PP415-P14), dried material of the THF solvate form (class 5) (dotted line, samples PP415-P27) and amorphous form (class 1) (solid line, samples PP415-P1)^-1). The spectra have been scaled for comparison and show small changes in magnitude but little corresponding change in spectral shape.

FIG. 121: comparison of PXRD patterns of samples PP415-P27 (top) of the dried THF solvate form (category 5) with patterns of samples PP415-P14 (bottom) of the THF solvate form (category 5). The maps were not scaled but shifted in the y-axis direction for comparison.

FIG. 122: TG-FTIR thermograms of samples PP415-P27 corresponding to dried THF solvates (category 5).

FIG. 123: the PXRD pattern of samples PP415-P41 (top) corresponds to that of the THF solvate form (category 5) (middle, samples PP415-P14) and does not correspond to that of the heptane solvate form (category 2) (bottom, samples PP 415-P19). The maps have been scaled and shifted in the y-axis direction for comparison.

FIG. 124: the PXRD pattern of samples PP415-P45 (top) corresponds to that of the THF solvate form (category 5) (middle, samples PP415-P14) and does not correspond to that of the heptane solvate form (category 2) (bottom, samples PP 415-P19). The maps have been scaled and shifted in the y-axis direction for comparison.

FIG. 125: the PXRD pattern of samples PP415-P41 (top) corresponds to the THF solvate form (category 5). After drying samples PP415-P41 for 1 day (second from the top, sample: PP415-P44), the material was predominantly amorphous. Some broad peaks with low intensity remained. After further drying overnight (second from the bottom, samples PP415-P44a), the intensity of these broad peaks further decreased. The amorphous form (category 1) is shown as reference (bottom, sample: PP 415-P42). The maps were not scaled but shifted in the y-axis direction for comparison.

FIG. 126: TG-FTIR thermograms corresponding to samples PP415-P44a in amorphous form (category 1).

FIG. 127: the PXRD pattern of samples PP415-P45 (top) corresponds to the THF solvate form (category 5). After drying samples PP415-P45 for 1 day (second from the top, samples PP415-P46), the material was predominantly amorphous. Some broad peaks with low intensity remained. After a total of 4 days of drying (second from the bottom, samples PP415-P46a), the pattern remained unchanged. The amorphous form (category 1) is shown as reference (bottom, samples PP 415-P42). The maps were not scaled but shifted in the y-axis direction for comparison.

FIG. 128: TG-FTIR thermograms corresponding to samples PP415-P46a in amorphous form (category 1).

FIG. 129: the PXRD pattern for samples PP415-P42 (top) corresponds to that of the amorphous form (category 1) (bottom, samples PP 415-P1). The maps have been scaled and shifted in the y-axis direction for comparison.

FIG. 130: the PXRD pattern for samples PP415-P43 (top) corresponds to that of an isomorphic solvate form (category 2) (bottom, samples PP415-P19) and does not correspond to that of a THF solvate form (category 5) (middle, samples PP 415-P14). The maps have been scaled and shifted in the y-axis direction for comparison.

FIG. 131: the PXRD patterns for samples PP415-P47 (top) and PP415-P48 (middle) substantially correspond to the patterns for the isomorphic solvate form (category 2) (bottom, samples PP415-P19), but with some differences. The maps have been scaled and shifted in the y-axis direction for comparison.

FIG. 132: the PXRD pattern for samples PP415-P49 (top) corresponds to that of the amorphous form (category 1) (bottom, samples PP 415-P1). The maps have been scaled and shifted in the y-axis direction for comparison.

Detailed Description

In one aspect, the present invention provides the following compounds:

n- ((4aS, 6aR, 6bS, 8aR, 12aS, 14aR, 14bS) -11-cyano-2, 2, 6a, 6b, 9, 9, 12 a-heptamethyl-10, 14-dioxo-1, 2, 3, 4, 4a, 5, 6, 6a, 6b, 7, 8, 8a, 9, 10, 12a, 14, 14a, 14 b-octadecahydroabienzene-4 a-yl) -2, 2-difluoropropionamide, which is also referred to herein aS RTA408, 63415 or PP 415. In other non-limiting aspects, the present invention also provides polymorphs thereof, including solvates thereof. In other non-limiting aspects, the invention also provides pharmaceutically acceptable salts thereof. Methods of preparation, pharmaceutical compositions, and kits and articles of manufacture of these compounds and polymorphs thereof are also provided, among other non-limiting aspects.

I. Definition of

When used in the case of chemical groups: "Hydrogen" means-H; "hydroxy" means-OH; "oxo" means ═ O; "carbonyl" means-C (═ O) -; "carboxy" means-C (═ O) OH (also written as-COOH or-CO)₂H) (ii) a "halo" independently means-F, -Cl, -Br, or-I; "amino" means-NH₂(ii) a "hydroxyamino" means-NHOH; "nitro" means-NO₂(ii) a Imino means ═ NH; "cyano" means-CN; "isocyanate group" means-N ═ C ═ O; "azido" means-N₃(ii) a In the monovalent case, "phosphate group" means-OP (O) (OH)₂Or a deprotonated form thereof; in the case of divalent, "phosphate group" means-op (O) (oh) O-or its deprotonated form; "mercapto" means-SH; and "thio" means ═ S; "Sulfonyl" means-S (O)₂-; and "sulfinyl" means-S (O) -. Any undefined valency on an atom of a structure shown in this application implicitly denotes a hydrogen atom bonded to said atom.

In the context of the present disclosure, the following formula:

the same structure is shown. When a point is drawn on a carbon, the point indicates that the hydrogen atom attached to the carbon is protruding out of the plane of the page.

The use of the word "a" or "an", when used in conjunction with the term "comprising" in the claims and/or the specification, can mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one" or more than one ".

Throughout this application, the term "about" is used to indicate that a value includes the inherent variation in error of the apparatus, method used to determine the value, or the variation that exists between study subjects. The term "about" when used in the context of X-ray powder diffraction is used to indicate a value of the reported value ± 0.2 ° 2 θ, preferably a value of the reported value ± 0.1 ° 2 θ. The term "about" when used in the context of differential scanning calorimetry or glass transition temperature is used to indicate a value of ± 10 ℃ relative to the maximum of the peak, preferably a value of ± 2 ℃ relative to the maximum of the peak. When used otherwise, the term "about" is used to indicate a value of ± 10% of the reported value, preferably a value of ± 5% of the reported value. It should be understood that whenever the term "about" is used, a specific reference to the exact numerical value shown is also included.

The terms "comprising", "having" and "including" are open-ended linking verbs. Any form or tense of one or more of these verbs, such as "comprising", "having", "including", and "including", is also open-ended. For example, any method that "comprises," "has," or "includes" one or more steps is not limited to having only that one or more steps and also covers other unlisted steps.

The term "effective" when used in this specification and/or claims means sufficient to achieve a desired, expected, or intended result. When used in the context of treating a patient or subject with a compound, "effective amount," "therapeutically effective amount," or "pharmaceutically effective amount" means an amount of the compound that is sufficient to effect such treatment of the disease when administered to a subject or patient to treat the disease.

In the case of X-ray powder diffraction, the term "halo peak" will mean a broad peak, often spanning > 10 ° 2 θ in the X-ray powder diffraction pattern, characteristic of an amorphous solid or system in general.

The term "hydrate," when used as a modifier of a compound, means that the compound has less than one (e.g., hemihydrate), one (e.g., monohydrate), or more than one (e.g., dihydrate) water molecule associated with each molecule of the compound (e.g., the compound in solid form).

The term "IC" as used herein₅₀By "is meant an inhibitory amount that achieves 50% of the maximal response. The quantitative measure indicates a certain drug or other substance required to inhibit a given biological, biochemical or chemical process (or component of a process, i.e., enzyme, cell receptor or microorganism) by halfAmount of proton (inhibitor).

An "isomer" of a first compound is another compound that contains the same constituent atoms as the first compound per molecule, but that differs in the three-dimensional configuration of those atoms.

The term "patient" or "subject" as used herein refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments, the patient or subject is a non-human animal. In certain embodiments, the patient or subject is a primate. In certain embodiments, the patient or subject is a human. Non-limiting examples of human subjects are adults, adolescents, infants and fetuses.

As used generally herein, "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

By "pharmaceutically acceptable salt" is meant a salt of a compound of the invention as defined above which is pharmaceutically acceptable and has the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or acid addition salts with organic acids such as: 1, 2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4 '-methylenebis (3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo [2.2.2] oct-2-ene-1-carboxylic acid, acetic acid, aliphatic monocarboxylic and dicarboxylic acids, aliphatic sulfuric acid, aromatic sulfuric acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, lauryl sulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o- (4-hydroxybenzoyl) benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, methanesulfonic acid, o-chlorobenzenesulfonic acid, o-4' -methylenebis (3-hydroxy-2, Phenyl substituted alkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, t-butylacetic acid, trimethylacetic acid, and the like. Pharmaceutically acceptable salts also include base addition salts which may be formed when an acidic proton present is capable of reacting with an inorganic or organic base. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide, and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. It will be appreciated that the particular anion or cation forming part of any salt of the invention is not critical, so long as the salt as a whole is pharmacologically acceptable. Additional examples of pharmaceutically acceptable Salts and methods of their preparation and use are presented in Handbook of pharmaceutical Salts: properties, and Use (handbook of pharmaceutically acceptable salts: Properties and Use) (edited by P.H.Stahl and C.G.Wermuth, Verlag Helvetica Chimica Acta, 2002).

"prevention (prevention)" includes: (1) inhibiting the onset of a disease in a subject or patient who may be at risk for and/or susceptible to the disease, but who has not experienced or exhibited any or all of the conditions or symptoms of the disease; and/or (2) slowing the onset of symptoms or symptoms of a disease in a subject or patient who may be at risk for and/or susceptible to the disease, but who has not experienced or exhibited any or all of the symptoms or symptoms of the disease.

By "prodrug" is meant a compound that can be metabolically converted in vivo to the inhibitor of the present invention. The prodrug itself may or may not also have activity against a given target protein. For example, a compound comprising a hydroxyl group may be administered in the form of an ester that is converted to the hydroxyl compound by hydrolysis in vivo. Suitable esters that can be converted in vivo to hydroxy compounds include acetate, citrate, lactate, phosphate, tartrate, malonate, oxalate, salicylate, propionate, succinate, fumarate, maleate, methylene-bis- β -hydroxynaphthoate, cholate, isethionate, di-p-toluoyl tartrate, mesylate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate, quinic acid ester (quinate), amino acid ester, and the like. Similarly, compounds comprising amine groups may be administered in the form of amides that are converted to amine compounds by hydrolysis in vivo.

"stereoisomers" or "optical isomers" are isomers of a given compound in which the same atom is bonded to the same other atom, but the three-dimensional configuration of those atoms is different. "enantiomers" are stereoisomers of a given compound that are mirror images of each other as left and right handed. "diastereomer" is a diastereomer of a given compound. Chiral molecules contain a chiral center, also known as a stereocenter or stereogenic (stereogenic) center, which is any point in the molecule that bears groups such that the interchange of any two groups produces a stereoisomer, but is not necessarily an atom. In organic compounds, the chiral center is usually a carbon atom, a phosphorus atom or a sulfur atom, but in organic and inorganic compounds, other atoms may also be stereocenters. The molecule may have multiple stereocenters, giving it many stereoisomers. In compounds where the stereoisomerism is due to tetrahedral stereogenic centers (e.g. tetrahedral carbon), it is assumed that the total number of possible stereoisomers will not exceed 2n, where n is the number of tetrahedral stereocenters. Molecules with symmetry typically have less than the maximum possible number of stereoisomers. A50: 50 mixture of enantiomers is referred to as a racemic mixture. Alternatively, a mixture of enantiomers may be enantiomerically enriched such that one enantiomer is present in an amount greater than 50%. Generally, enantiomers and/or diastereomers may be resolved or separated using techniques known in the art. It is contemplated that for any stereocenter or chiral axis for which stereochemistry has not been determined, the stereocenter or chiral axis may exist in its R form, S form, or a mixture of said R and S forms, including racemic and non-racemic mixtures. The phrase "substantially free of other stereoisomers" as used herein means that the composition contains 15% or less, more preferably 10% or less, even more preferably 5% or less, or most preferably 1% or less of additional stereoisomer(s).

"treating" includes (1) inhibiting the disease (e.g., arresting further development of the condition and/or symptom) in a subject or patient experiencing or exhibiting the condition or symptom of the disease, (2) ameliorating the disease (e.g., reversing the condition and/or symptom) in a subject or patient experiencing or exhibiting the condition or symptom of the disease, and/or (3) causing any measurable reduction in the disease in a subject or patient experiencing or exhibiting the condition or symptom of the disease.

The above definitions supersede any conflicting definition in any reference incorporated herein by reference. However, the fact that certain terms are defined should not be taken to mean that any term that is not defined is indefinite. Rather, all terms used are to be considered as describing the invention so that one of ordinary skill in the art can appreciate the scope of the invention and practice the invention.

RTA408 and Synthesis method

RTA408 can be prepared according to the methods described in the examples section below. These methods can be further modified and optimized using the principles and techniques of organic chemistry applied by those skilled in the art. The principles and techniques are described, for example, in March's Advanced Organic Chemistry: reactions, Mechanisms, and structures (March et al organic chemistry: Reactions, Mechanisms and structures) (2007), which publication is incorporated herein by reference.

It will be appreciated that the particular anion or cation forming part of any salt of the invention is not critical, so long as the salt as a whole is pharmacologically acceptable. Additional examples of pharmaceutically acceptable Salts and methods of their preparation and use are presented in the Handbook of Pharmaceutical Salts: properties, and Use (handbook of salts of pharmaceutical salts: Properties and Use) (2002), which publication is incorporated herein by reference.

RTA408 may also exist in prodrug form. Because prodrugs are known to enhance a number of desirable pharmaceutical qualities, such as solubility, bioavailability, manufacturing, and the like, some of the compounds used in the methods of the invention may be delivered in prodrug form, if desired. Thus, the present invention contemplates prodrugs of the compounds of the present invention as well as methods of delivering the prodrugs. Prodrugs of the compounds used in the present invention may be prepared by modifying functional groups present in the compounds in the following manner: the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound. Thus, prodrugs include, for example, compounds described herein wherein a hydroxy, amino, or carboxy group is bonded to any group that, when the prodrug is administered to a patient, cleaves to form a hydroxy, amino, or carboxylic acid, respectively.

RTA408 may contain one or more asymmetrically substituted carbon or nitrogen atoms, and may be isolated in optically active or racemic forms. Thus, unless a specific stereochemistry or isomeric form is specifically indicated, all chiral, diastereomeric, racemic, epimeric, and all geometric isomeric forms of a structure are intended. RTA408 can exist as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. In some embodiments, a single diastereomer is obtained. The chiral center of RTA408 according to the invention can have either the S configuration or the R configuration.

In addition, the atoms comprising RTA408 of the present invention are intended to include all isotopic forms of the atoms. Isotopes as used herein include those atoms having the same atomic number but different mass numbers. By way of general example, and not limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include¹³C and¹⁴C. similarly, it is contemplated that one or more carbon atoms of the compounds of the present invention may be replaced by one or more silicon atomsAnd (4) replacement. Further, it is contemplated that one or more oxygen atoms of RTA408 may be replaced with one or more sulfur atoms or selenium atoms.

RTA408 and its polymorphs may also have the following advantages: they may be more effective, less toxic, longer acting, more potent, produce fewer side effects, be more easily absorbed, and/or have better pharmacokinetic characteristics (e.g., higher oral bioavailability and/or lower clearance rates), and/or have other useful pharmacological, physical, or chemical advantages than compounds known in the art for use in the indications described herein.

Polymorphic forms of RTA408

In some embodiments, the present invention provides different solid forms of RTA408, including solvates thereof. Preformulation and preliminary polymorphism studies were conducted and RTA408 was found to have a high tendency to solvate. The crystalline forms of classes 2, 3, 4 and 5 are consistent with solvates. For a description of the classes, see table 1 below. Attempts to dry classes 2 and 3 (two groups of isomorphic solvates) were unsuccessful, consistent with tightly bound solvent molecules. In some embodiments, drying the class 4 solid (acetonitrile solvate) produces an isomorphic desolvated form. In some embodiments, drying the class 5 solid (THF solvate) yields the amorphous form class 1. The unsolvated forms of RTA408 include amorphous forms (class 1) and crystalline desolvated solvates of class 4 (isomorphism to the acetonitrile solvates of class 4). In some embodiments, the amorphous form has T_gHigh glass transition at about 153 ℃ (Δ Cp ═ 0.72J/g ℃) and only slightly hygroscopic (Δ m ═ 0.4% 50% → 85% relative humidity). In some embodiments, the amorphous form remains stable under high temperature and high humidity conditions (i.e., open at 40 ℃/about 75% relative humidity or closed at 80 ℃) for at least 4 weeks. In some embodiments, the amorphous form (class 1) is obtained from the class 2 material by a two-step process (conversion to class 5 followed by drying of class 5 to obtain the amorphous form) and a direct one-step process (in cold conditions)Precipitated from acetone solution in a water bath) were successfully prepared. The crystalline desolvated solvate of class 4 (isomorphic with the class 4 solvate) is slightly hygroscopic (from 50% relative humidity to 85% relative humidity, mass gain of about 0.7 wt%) and has a possible melting point of 196.1 ℃ (Δ H ═ 29.31J/g).

By FT-Raman spectroscopy, PXRD, TG-FTIR, Karl Fischer titration (Karl Fischer titration),¹H-NMR, DSC and DVS (see examples section for additional details) to characterize 63415 a sample of amorphous form class 1. The sample was found to contain about 0.9 wt% EtOH with trace amounts of H₂O (according to TG-FTIR). The water content was determined by Karl Fischer titration to be 0.5% by weight. DSC shows T_gA high glass transition temperature (. DELTA.C) of about 153 ℃_p0.72J/g). According to DVS, the material is slightly hygroscopic (Δ m ═ 0.4% 50% → 85% relative humidity). No crystallization was observed in DSC or DVS experiments.

The chemical stability of the amorphous form in organic solvents including acetone, EtOAc, MeOH and MeCN as well as in different aqueous media (e.g. 1% aqueous Tween 80, 1% aqueous SDS, 1% aqueous CTAB) was investigated at time points 6 hours, 24 hours, 2 days and 7 days at a concentration of 1 mg/mL. Decomposition of ≧ 1% was observed only for solutions in MeCN (after 7 days) and for suspensions in 1% aqueous Tween 80 medium (all time points at 254nm, and after 24 hours, 2 days, and 7 days at 242 nm).

In addition, the stability of the amorphous form was studied by storage under high temperature and high humidity conditions (open at 25 ℃/62% relative humidity and 40 ℃/75% relative humidity and closed at 60 ℃ and 80 ℃). After 1 week, 2 weeks and 4 weeks, the stored samples were analyzed by PXRD. None of the samples differed from the amorphous starting material.

Crystallization and drying experiments were performed over 30 times, including suspension equilibration, slow cooling, evaporation and precipitation. In addition to the amorphous form (category 1), 4 new crystalline forms (categories 2, 3, 4 and 5) were obtained.

These 4 new forms (classes 2, 3, 4 and 5) were characterized by FT-raman spectroscopy, PXRD and TG-FTIR. All forms corresponded to solvates (table 1). Under vacuum or N₂Drying experiments were performed under reflux with the aim of obtaining 63415 in crystalline, unsolvated form.

TABLE 1 summary of the obtained categories

Class 2: most crystallization experiments performed yielded solid material of class 2 (see example section below). Its members may correspond to isomorphic non-stoichiometric (< 0.5 equivalents) solvates with tightly bound solvent molecules (solvates of heptane, cyclohexane, isopropyl ether, 1-butanol, triethylamine, and possibly other solvents such as hexane, other ethers, and the like). Raman spectra and PXRD patterns within this class are very similar to each other and therefore the structures may be substantially identical with only small differences due to the different solvents incorporated.

Drying experiments on Category 2 samples did not produce crystalline, unsolvated forms, even at high temperatures (80 ℃) and high vacuum (< 1 × 10)^-3Mbar) still do not completely remove tightly bound solvent molecules; always a solvent content of > 2 wt.% is retained. The crystallinity of these partially dried samples decreased, but neither transformation into a different structure nor significant amorphization was observed.

Class 3: solid substances of class 3 can be obtained by several crystallizations (see the examples section below). A class 3 sample may be an isomorphic solvate of 2PrOH, EtOH and possibly acetone with tightly bound solvent molecules. They may correspond to a stoichiometric hemisolvate or non-stoichiometric solvate with a solvent content of about 0.5 equivalents. As with class 2, the raman spectra and PXRD patterns within this class are very similar to each other, indicating similar structures incorporating different solvents.

Similar to class 2, drying experiments were unsuccessful, very tightly bound solvent molecules can only be partially removed (i.e., at 1 × 10)^-3Mbar and 80 ℃ remove from about 5.4 wt.% to about 4.8 wt.% after up to 3 days). The PXRD pattern remains unchanged.

Category 4 available 7: 3MeCN/H₂O solvent system (see examples section below). It most likely corresponds to a crystalline acetonitrile hemisolvate. By drying (under vacuum or N)₂Flow down at high temperature), most of the solvent molecules can be removed without changing or destroying the crystal structure (PXRD remains unchanged). Thus, a crystalline unsolvated form (or more precisely, a desolvated solvate) is obtained. It is slightly hygroscopic (about 0.7 wt% increase in mass from 50% relative humidity to 85% relative humidity) and has a possible melting point of 196.1 ℃ (Δ H ═ 29.31J/g).

Class 5 uses about 1: 1 THF/H₂And (4) obtaining an O solvent system. Class 5 contains bound THF (and possibly H)₂O). The exact nature of this crystalline solvate has not been determined since the contents of these two components cannot be easily quantified individually.

Drying category 5 causes significant desolvation and a directional transformation to the amorphous form (category 1). In some embodiments, the amorphous form of RTA408 can be prepared by suspending a class 2 heptane solvate in 1: 1 THF/H₂O to form a class 5 solid, followed by drying and amorphization.

Experiments were performed using the class 2 starting material with the aim of preparing the amorphous form (class 1). The predominantly amorphous material (class 1) was prepared in a two-step process via class 5 on a scale of 100mg and 3g (dried at 100 mbar, 80 ℃ for several days) starting from the class 2 material. It was found that it is possible to prepare a completely amorphous material (class 1) in a one-step process avoiding the solvent THF by directly precipitating the amorphous form (class 1) from an acetone solution of the class 2 material in a cold water bath.

Diseases associated with inflammation and/or oxidative stress

Inflammation is a biological process that provides resistance to infectious or parasitic organisms and repairs damaged tissues. Inflammation is often characterized by local vasodilation, redness, swelling, and pain, recruitment of leukocytes to the site of infection or injury, production of inflammatory cytokines such as TNF-a and IL-1, and production of reactive oxygen or nitrogen clusters such as hydrogen peroxide, superoxide, and peroxynitrite. In the later stages of inflammation, tissue remodeling, angiogenesis, and scarring (fibrosis) can occur as part of the wound healing process. Under normal circumstances, the inflammatory response is regulated, transient, and resolves in a coordinated manner after the infection or injury has been properly treated. However, if the regulatory mechanisms fail, acute inflammation can become excessive and life threatening. Alternatively, inflammation can become chronic and cause cumulative tissue damage or systemic complications. Based at least on the evidence presented herein, RTA408 can be used to treat or prevent inflammation or inflammation-related diseases.

Many serious and refractory human diseases involve dysregulation of inflammatory processes, including diseases such as cancer, atherosclerosis, and diabetes, which are not considered inflammatory conditions in the traditional sense. In the case of cancer, inflammatory processes are associated with processes including tumor formation, progression, metastasis and resistance to therapy. In some embodiments, RTA408 can be used to treat or prevent cancer, including carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma or seminoma, or bladder, blood, bone, brain, breast, central nervous system, cervical, colon, endometrial, esophageal, gallbladder, genital, genitourinary, head, kidney, throat, liver, lung, muscle, neck, oral or nasal mucosa, ovarian, pancreatic, prostate, skin, spleen, small intestine, large intestine, stomach, testis, or thyroid. Atherosclerosis, which has long been viewed as a disorder of lipid metabolism, is now understood to be primarily an inflammatory condition in which activated macrophages play an important role in the formation and eventual rupture of atherosclerotic plaques. Activation of inflammatory signaling pathways has also been shown to play a role in the development of insulin resistance and in peripheral tissue damage associated with diabetic hyperglycemia. Overproduction of reactive oxygen species and reactive nitrogen species, such as superoxide, hydrogen peroxide, nitric oxide and peroxynitrite, are hallmarks of inflammatory conditions. Evidence of deregulation of peroxynitrite production has been reported in a wide variety of diseases (Szabo et al, 2007; Schulz et al, 2008; Forstermann, 2006; Pall, 2007).

Autoimmune diseases such as rheumatoid arthritis, lupus, psoriasis and multiple sclerosis involve inappropriate and long-term activation of inflammatory processes in affected tissues, caused by dysfunction of self versus non-self recognition and response mechanisms in the immune system. In neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, nerve damage is associated with activation of microglia and elevated levels of proinflammatory proteins such as Inducible Nitric Oxide Synthase (iNOS). Chronic organ failure, such as renal failure, heart failure, liver failure and chronic obstructive pulmonary disease, is closely associated with the presence of chronic oxidative stress and inflammation leading to the generation of fibrosis and eventual loss of organ function. Oxidative stress in vascular endothelial cells lining large and small blood vessels can lead to endothelial dysfunction and is considered to be an important contributing factor in the development of systemic cardiovascular disease, diabetic complications, chronic kidney disease and other forms of organ failure, as well as many other age-related diseases including degenerative diseases of the central nervous system and retina.

Many other conditions involve oxidative stress and inflammation in affected tissues, including inflammatory bowel disease; inflammatory skin diseases; mucositis and dermatitis associated with radiotherapy and chemotherapy; eye diseases, such as uveitis, glaucoma, macular degeneration and various forms of retinopathy; graft failure and rejection; ischemia-reperfusion injury; chronic pain; degenerative conditions of bone and joints, including osteoarthritis and osteoporosis; asthma and cystic fibrosis; seizure disorders; and neuropsychiatric conditions including schizophrenia, depression, bipolar disorder, post-traumatic stress disorder, attention deficit disorder, autism spectrum disorder (autism-spectrum disorder), and eating disorders such as anorexia nervosa. Dysregulation of the inflammatory signaling pathway is considered to be a major factor in the pathology of muscle wasting diseases including muscular dystrophy and various forms of cachexia.

Inflammatory signaling disorders are also implicated in a variety of life-threatening acute conditions, including acute organ failure involving the pancreas, kidneys, liver or lungs, myocardial infarction or acute coronary syndrome, stroke, septic shock, trauma, severe burns and allergic reactions.

Many complications of infectious diseases also involve dysregulation of the inflammatory response. While inflammatory responses can kill invading pathogens, excessive inflammatory responses can also be quite destructive and in some cases can be a major cause of damage in infected tissues. In addition, excessive inflammatory responses can also lead to systemic complications due to the overproduction of inflammatory cytokines such as TNF- α and IL-1. This is considered to be a cause of death from severe influenza, severe acute respiratory syndrome and sepsis.

Aberrant or overexpression of iNOS or cyclooxygenase-2 (COX-2) has been implicated in the pathogenesis of many disease processes. For example, it is clear that NO is a potent mutagen (Tamir and Tannebaum, 1996) and that nitric oxide can also activate COX-2(Salvemini et al, 1994). Furthermore, iNOS is significantly increased in the colon tumor of rats induced by the carcinogen azoxymethane (Takahashi et al, 1997). A series of synthetic triterpenoid analogs of oleanolic acid have been demonstrated to be potent inhibitors of cellular inflammatory processes such as the induction of Inducible Nitric Oxide Synthase (iNOS) and COX-2 by IFN-. gamma.in mouse macrophages. See Honda et al (2000 a); honda et al (2000 b); and Honda et al (2002), all of which are incorporated herein by reference.

In one aspect, RTA408 disclosed herein is characterized, in part, in that it is capable of inhibiting nitric oxide production induced by exposure to interferon-gamma in macrophage-derived RAW264.7 cells. RTA408 is further characterized by the ability to induce the expression of antioxidant proteins such as NQO1 and to reduce the expression of pro-inflammatory proteins such as COX-2 and Inducible Nitric Oxide Synthase (iNOS). These properties are relevant for the treatment of a wide range of diseases and conditions involving oxidative stress and dysregulation of inflammatory processes, such diseases and conditions include cancer, complications from local or systemic exposure to ionizing radiation, mucositis and dermatitis from radiation therapy or chemotherapy, autoimmune diseases, cardiovascular diseases (including atherosclerosis), ischemia-reperfusion injury, acute and chronic organ failure (including renal failure and heart failure), respiratory diseases, diabetes and diabetic complications, severe allergies, transplant rejection, graft-versus-host disease, neurodegenerative diseases, diseases of the eye and retina, acute and chronic pain, degenerative bone diseases (including osteoarthritis and osteoporosis), inflammatory bowel disease, dermatitis and other skin diseases, sepsis, burns, seizure disorders and neuropsychiatric disorders.

In another aspect, RTA408 can be used to treat a subject having a condition such as an eye disease. For example, uveitis, macular degeneration (both dry and wet forms), glaucoma, diabetic macular edema, blepharitis, diabetic retinopathy, diseases and disorders of the corneal endothelium (e.g., forskohlii corneal endothelial dystrophy), post-operative inflammation, dry eye, allergic conjunctivitis, and other forms of conjunctivitis are non-limiting examples of eye diseases that may be treated with RTA 408.

In another aspect, RTA408 can be used to treat a subject having a condition, such as a skin disease or disorder. For example, dermatitis, including allergic dermatitis, atopic dermatitis, dermatitis due to chemical exposure, and radiation-induced dermatitis; thermal or chemical burns; chronic wounds including diabetic ulcers, bedsores and varicose ulcers; acne; alopecia, including baldness and drug-induced alopecia; other disorders of the hair follicle; epidermolysis bullosa; sunburn and its complications; skin pigmentation disorders, including vitiligo; an aging-related skin condition; wound healing after surgery; prevention or reduction of scarring due to skin injury, surgery or burn; psoriasis; cutaneous manifestations of autoimmune disease or graft versus host disease; prevention or treatment of skin cancer; disorders involving hyperproliferation of skin cells, such as hyperkeratosis, are non-limiting examples of skin diseases that can be treated with RTA 408.

Without being bound by theory, it is believed that activation of the antioxidant/anti-inflammatory Keap1/Nrf2/ARE pathway is implicated in both anti-inflammatory and anti-cancer properties of the compounds disclosed herein.

In another aspect, RTA408 can be used to treat a subject having a condition caused by an elevated level of oxidative stress in one or more tissues. Oxidative stress is caused by abnormally high or sustained levels of reactive oxygen species, such as superoxide, hydrogen peroxide, nitric oxide, and peroxynitrite (formed by the reaction of nitric oxide with superoxide). Oxidative stress may be accompanied by acute or chronic inflammation. Oxidative stress can be caused by the following factors: mitochondrial dysfunction; activation of immune cells such as macrophages and neutrophils; acute exposure to external agents such as ionizing radiation or cytotoxic chemotherapeutic agents (e.g., doxorubicin); trauma or other acute tissue injury; ischemia/reperfusion; poor circulation or anemia; hypoxia or hyperoxia, either local or systemic; elevated levels of inflammatory cytokines and other inflammation-related proteins; and/or other abnormal physiological states such as hyperglycemia or hypoglycemia.

In animal models of many such conditions, stimulation-induced heme oxygenase (HO-1), a target gene of the Nrf2 pathway, expression has been demonstrated to have significant therapeutic effects, including in models of myocardial infarction, renal failure, graft failure and rejection, stroke, cardiovascular disease, and autoimmune disease (e.g., Sacerdoti et al, 2005; Abraham and Kappas, 2005; Bach, 2006; Araujo et al, 2003; Liu et al, 2006; Ishikawa et al, 2001; Kruger et al, 2006; Satoh et al, 2006; Zhou et al, 2005; Morse and Choi, 2002). This enzyme breaks down free heme into iron, carbon monoxide (CO) and biliverdin, which is subsequently converted into the potent antioxidant molecule bilirubin.

In another aspect, RTA408 can be used to prevent or treat acute and chronic tissue damage or organ failure due to oxidative stress exacerbated by inflammation. Examples of diseases falling into this category include heart failure, liver failure, graft failure and rejection, renal failure, pancreatitis, fibrotic pulmonary diseases (especially cystic fibrosis, COPD, and idiopathic pulmonary fibrosis), diabetes (including complications), atherosclerosis, ischemia-reperfusion injury, glaucoma, stroke, autoimmune diseases, autism, macular degeneration, and muscular dystrophy. For example, in the case of autism, studies have shown that increased oxidative stress in the central nervous system can lead to the disease (Chauhan and Chauhan, 2006).

There is also evidence linking oxidative stress and inflammation to the development and pathology of many other conditions of the central nervous system, including psychiatric conditions such as psychosis, major depression, and bipolar disorder; seizure disorders, such as epilepsy; pain and sensory syndromes, such as migraine, neuropathic pain or tinnitus; and behavioral syndromes, such as attention deficit disorder. See, e.g., Dickerson et al, 2007; hanson et al, 2005; kendall-tatkett, 2007; lencz et al, 2007; dudhgaonkar et al, 2006; lee et al, 2007; morris et al, 2002; ruster et al, 2005; McIver et al, 2005; sarcoleilli et al, 2006; kawakami et al, 2006; ross et al, 2003, both of which are incorporated herein by reference. For example, elevated levels of inflammatory cytokines including TNF- α, interferon- γ, and IL-6 are associated with severe psychiatric disorders (Dickerson et al, 2007). Microglial activation has also been associated with severe psychiatric disorders. Therefore, down-regulation of inflammatory cytokines and inhibition of excessive activation of microglia may be beneficial to patients with schizophrenia, major depression, bipolar disorder, autism spectrum disorder, and other neuropsychiatric conditions.

Thus, in conditions involving oxidative stress alone or exacerbated by inflammation, treatment may comprise administering to the subject a therapeutically effective amount of a compound of the invention, such as those described above or throughout this specification. Treatment may be administered prophylactically prior to a predictable oxidative stress state (e.g., organ transplantation or administration of radiation therapy to a cancer patient), or may be administered therapeutically in a setting involving established oxidative stress and inflammation. In some embodiments, when the compounds of the present invention are used to treat a patient receiving radiation therapy and/or chemotherapy, the compounds of the present invention may be administered prior to, concurrently with, and/or after the radiation therapy or chemotherapy, or the compounds may be administered in combination with other therapies. In some embodiments, the compounds of the present invention can prevent and/or reduce the severity of side effects associated with radiation therapy or chemotherapy (using different agents) without reducing the anti-cancer effects of the radiation therapy or chemotherapy. Because such side effects may be dose limiting for radiation therapy and/or chemotherapy, in some embodiments, the compounds of the present invention may be used to allow for higher and/or more frequent administration of such radiation therapy and/or chemotherapy, for example, resulting in greater therapeutic efficacy. In some embodiments, the compounds of the present invention, when administered in combination with radiation therapy and/or chemotherapy, may increase the efficacy of a given dose of radiation therapy and/or chemotherapy. In some embodiments, when administered in combination with radiation therapy and/or chemotherapy, the compounds of the present invention can increase the efficacy of a given dose of radiation therapy and/or chemotherapy and reduce (or at least not increase) the side effects of the radiation therapy and/or chemotherapy. In some embodiments, and without being bound by theory, this combined efficacy may result from inhibition of the activity of the proinflammatory transcription factor NF- κ B by the compounds of the invention. NF- κ B is typically activated long-term in cancer cells, and the activation is associated with resistance to therapy and promotion of tumor progression (e.g., Karin, 2006; Aghajan et al, 2012). In some embodiments, the compounds of the invention may also inhibit other transcription factors that contribute to inflammation and cancer, such as STAT3 (e.g., He and Karin, 2011; Grivennikov and Karin, 2010).

RTA408 can be used to treat or prevent inflammatory conditions such as sepsis, dermatitis, autoimmune diseases, and osteoarthritis. RTA408 may also be used to treat or prevent inflammatory pain and/or neuropathic pain, for example, by inducing Nrf2 and/or inhibiting NF- κ B.

RTA408 may also be used to treat or prevent diseases such as: cancer, inflammation, alzheimer's disease, parkinson's disease, multiple sclerosis, autism, amyotrophic lateral sclerosis, huntington's disease, autoimmune diseases (e.g. rheumatoid arthritis, lupus, crohn's disease and psoriasis), inflammatory bowel disease, all other diseases whose pathogenesis is thought to involve overproduction of nitric oxide or prostaglandins, and pathologies involving oxidative stress alone or exacerbated by inflammation. RTA408 can be used to treat or prevent cancer, including carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma or seminoma, or bladder, blood, bone, brain, breast, central nervous system, cervical, colon, endometrial, esophageal, gall bladder, genital, genitourinary tract, head, kidney, throat, liver, lung, muscle, neck, oral or nasal mucosa, ovarian, pancreatic, prostate, skin, spleen, small intestine, large intestine, stomach, testicular, or thyroid cancer.

Another aspect of inflammation is the production of inflammatory prostaglandins, such as prostaglandin E. RTA408 may be used to promote vasodilation, plasma extravasation, local pain, elevated temperature, and other inflammatory symptoms. Inducible forms of the enzyme COX-2 are associated with their production and high levels of COX-2 are present in inflamed tissues. Thus, inhibition of COX-2 may alleviate many symptoms of inflammation, and many important anti-inflammatory drugs, such as ibuprofen (ibuprofen) and celecoxib (celecoxib), act by inhibiting COX-2 activity. A class of cyclopentenone prostaglandins (cypgs), such as 15-deoxy prostaglandin J2, also known as PGJ2, has been shown to play a role in stimulating coordinated regression of inflammation (e.g., Rajakariar et al, 2007). COX-2 is also associated with the production of cyclopentenone prostaglandins. Thus, inhibition of COX-2 may interfere with complete resolution of inflammation, potentially promoting the persistence of activated immune cells in the tissue and leading to chronic "smoldering" inflammation. This effect can lead to an increased incidence of cardiovascular disease in patients who have long-term use of selective COX-2 inhibitors.

In one aspect, RTA408 can be used to control the production of proinflammatory cytokines in a cell by selectively activating Regulatory Cysteine Residues (RCRs) on proteins that modulate the activity of redox-sensitive transcription factors. Activation of RCR by cyPG has been shown to lead to a pro-resolution program in which the activity of the antioxidant and cytoprotective transcription factor Nrf2 is strongly induced and the activity of the pro-oxidative and pro-inflammatory transcription factors NF- κ B and STAT are inhibited. In some embodiments, RTA408 can be used to increase the production of molecules that are antioxidant and reducing (NQO1, HO-1, SOD1, γ -GCS) and reduce oxidative stress and the production of pro-oxidative and pro-inflammatory molecules (iNOS, COX-2, TNF- α). In some embodiments, RTA408 can be used to restore cells that host an inflammatory event to a non-inflammatory state by promoting resolution of inflammation and limiting excessive tissue damage to the host.

A. Cancer treatment

In some embodiments, RTA408, polymorphs, and methods of the present disclosure can be used to induce apoptosis of tumor cells, induce cell differentiation, inhibit cancer cell proliferation, inhibit inflammatory responses, and/or function in chemopreventive capacity. For example, the present invention provides novel polymorphs having one or more of the following properties: (1) capable of inducing apoptosis and differentiating both malignant and non-malignant cells, (2) activity at submicromolar or nanomolar levels as inhibitors of proliferation of many malignant or premalignant cells, (3) capable of inhibiting de novo synthesis of the inflammatory enzyme Inducible Nitric Oxide Synthase (iNOS), (4) capable of inhibiting NF- κ B activation, and (5) capable of inducing expression of heme oxygenase-1 (HO-1).

Levels of iNOS and COX-2 are elevated in certain cancers and carcinogenesis has been implicated, and COX-2 inhibitors have been shown to reduce the incidence of primary colon adenomas in humans (Rostom et al, 2007; Brown and DuBois, 2005; Crowlel et al, 2003). Expression of iNOS in Myeloid Derived Suppressor Cells (MDSC) (Angulo et al, 2000) and COX-2 activity in cancer cells has been shown to result in prostaglandin E₂(PGE₂) Production of the prostaglandin E₂Arginase expression has been shown to be induced in MDSCs (Sinha et al, 2007). Arginase and iNOS are enzymes that utilize L-arginine as a substrate and produce L-ornithine and urea and L-citrulline and NO, respectively. Depletion of arginine from the tumor microenvironment by MDSCs, coupled with the production of NO and peroxynitrite, has been shown to inhibit T cell proliferation and induce apoptosis (Bronte et al, 2003). Inhibition of COX-2 and iNOS has been shown to reduce MDSC accumulation, restore cytotoxic activity of tumor-associated T cells, and delay tumor growth (Sinha et al, 2007; Mazzoni et al, 2002; Zhou et al, 2007).

Inhibition of the NF-. kappa.B and JAK/STAT signaling pathways has been implicated as a strategy to inhibit proliferation of epithelial cancer cells and induce their apoptosis. Activation of STAT3 and NF-. kappa.B has been shown to cause inhibition of apoptosis and promotion of proliferation, invasion and metastasis of cancer cells. Many target genes involved in these processes have been shown to be transcriptionally regulated by both NF-. kappa.B and STAT3 (Yu et al, 2007).

In addition to direct effects in epithelial cancer cells, NF-. kappa.B and STAT3 also have important effects in other cells present within the tumor microenvironment. Experiments in animal models have demonstrated that NF- κ B is required in both cancer and hematopoietic cells to amplify the effects of inflammation on cancer development and progression (Greten et al, 2004). Inhibition of NF-. kappa.B in cancer cells and bone marrow cells decreased the number and size of the resulting tumors, respectively. Activation of STAT3 in cancer cells results in the production of various cytokines (IL-6, IL-10) that inhibit tumor-associated Dendritic Cell (DC) maturation. In addition, these cytokines in dendritic cells themselves activate STAT 3. Inhibition of STAT3 in a mouse cancer model restored DC maturation, promoted anti-tumor immunity, and inhibited tumor growth (Kortylewski et al, 2005). In some embodiments, RTA408 and its polymorphs can be used to treat cancer, including, for example, prostate cancer. In some embodiments, RTA408 and its polymorphs can be used in combination therapy to treat cancer, including, for example, prostate cancer. See, e.g., example H below.

B. Multiple sclerosis and other neurodegenerative conditions

In some embodiments, RTA408, polymorphs, and methods of the invention are useful for treating Multiple Sclerosis (MS) or other neurodegenerative conditions in a patient, such as alzheimer's disease, parkinson's disease, or amyotrophic lateral sclerosis. MS is known as an inflammatory condition of the central nervous system (Williams et al, 1994; Merrill and Benvenist, 1996; Genain and Nauser, 1997). Based on several studies, there is evidence that inflammatory, oxidative and/or immunological mechanisms are involved in the pathogenesis of Alzheimer's Disease (AD), Parkinson's Disease (PD), Amyotrophic Lateral Sclerosis (ALS) and MS (Bagasra et al, 1995; McGeer and McGeer, 1995; Simonian and Coyle, 1996; Kaltschmidt et al, 1997). Epidemiological data suggest that chronic use of NSAIDs that block prostaglandin synthesis from arachidonic acid significantly reduces the risk of AD production (McGeer et al, 1996; Stewart et al, 1997). Accordingly, agents that block the formation of NO and prostaglandins are useful in methods of preventing and treating neurodegenerative diseases. Successful therapeutic candidates for treating the disease often need to be able to cross the blood-brain barrier. See, e.g., U.S. patent publication 2009/0060873, which is incorporated herein by reference.

C. Inflammation of nerve

In some embodiments, RTA408, polymorphs, and methods of the invention can be used to treat patients with neuroinflammation. Neuroinflammation encompasses the following concepts: the responses and roles of microglia and astrocytes in the central nervous system have fundamentally inflammatory-like characteristics, and these responses are critical for the pathogenesis and progression of a wide variety of neurological disorders. These concepts have been extended from Alzheimer's disease to other neurodegenerative diseases (Eikelenboom et al, 2002; Ishizawa and Dickson, 2001), ischemic/toxic diseases (Gehrmann et al, 1995; Touzani et al, 1999), oncology (Graeber et al, 2002), and even to normal brain development. Neuroinflammation combines numerous complex cellular responses including activation of microglia and astrocytes and induction of cytokines, chemokines, complement proteins, acute phase proteins, oxidative damage and related molecular processes, and such events can have deleterious effects on neuronal function, leading to neuronal damage, further glial activation and ultimately neurodegeneration.

D. Renal diseases

In some embodiments, RTA408, as well as polymorphs thereof, can be used to treat patients with kidney diseases and disorders, including renal failure and Chronic Kidney Disease (CKD), based on methods taught, for example, by US8,129,429, which is incorporated herein by reference. Renal failure, which results in inadequate removal of metabolic waste products from the blood and abnormal concentrations of electrolytes in the blood, is a major medical problem facing the world, especially in developed countries. Diabetes and hypertension are among the most important causes of chronic renal failure, also known as Chronic Kidney Disease (CKD), but it is also associated with other conditions such as lupus. Acute renal failure can result from exposure to certain drugs (e.g., acetaminophen) or toxic chemicals, or from ischemia-reperfusion injury associated with shock or surgical procedures (e.g., transplantation), and can lead to chronic renal failure. In many patients, renal failure progresses to a stage where the patient needs regular dialysis or kidney transplantation to continue life. Both of these procedures are highly invasive and associated with significant side effects and quality of life issues. While effective treatments are available for some of the complications of renal failure (e.g., hyperparathyroidism and hyperphosphatemia), no available treatment has proven to stop or reverse the potential progression of renal failure. Thus, agents that can improve impaired renal function would represent a significant advance in the treatment of renal failure.

Inflammation significantly contributes to the pathology of CKD. There is also a strong mechanistic link between oxidative stress and renal dysfunction. The NF-. kappa.B signaling pathway plays an important role in the progression of CKD, because NF-. kappa.B regulates the transcription of MCP-1, a chemokine responsible for monocyte/macrophage recruitment leading to an inflammatory response that ultimately damages the kidney (Wardle, 2001).

The Keap1/Nrf2/ARE pathway controls transcription of various genes encoding antioxidant enzymes, including heme oxygenase-1 (HO-1). Excision of the Nrf2 gene in female mice results in the development of lupus-like glomerulonephritis (Yoh et al, 2001). Furthermore, several studies have demonstrated that HO-1 expression is induced in response to renal damage and inflammation and that this enzyme and its products (bilirubin and carbon monoxide) play a protective role in the kidney (Nath et al, 2006).

Acute Kidney Injury (AKI) can occur following ischemia-reperfusion, treatment with certain pharmacological agents such as cisplatin and rapamycin (rapamycin), and intravenous injection of radiocontrast media for use in medical imaging. As in CKD, inflammation and oxidative stress lead to the pathology of AKI. The underlying molecular mechanisms for radiocontrast-induced nephropathy (RCN) are not fully understood; however, a combination of events including prolonged vasoconstriction, impaired renal autoregulation, and direct toxicity of contrast media are all likely to lead to renal failure (Tumlin et al, 2006). Vasoconstriction results in reduced renal blood flow and causes ischemia-reperfusion and the production of reactive oxygen species. HO-1 is strongly induced under these conditions and has been shown to prevent ischemia-reperfusion injury in several different organs, including the kidney (Nath et al, 2006). Specifically, the induction of HO-1 has been demonstrated to be protective in a rat model of RCN (Goodman et al, 2007). Reperfusion also induces inflammatory responses in part by activating NF- κ B signaling (Nichols, 2004). Targeting NF- κ B has been proposed as a therapeutic strategy to prevent organ damage (Zingarelli et al, 2003).

E. Cardiovascular diseases

In some embodiments, RTA408, polymorphs, and methods of the invention are useful for treating patients with cardiovascular disease. The etiology of CV disease is complex, but most of the causes are associated with inadequate or complete disruption of blood supply to critical organs or tissues. Typically, the condition results from rupture of one or more atherosclerotic plaques, resulting in thrombosis that blocks blood flow in critical blood vessels.

In some cases, atherosclerosis may be so extensive in critical blood vessels that stenosis (narrowing of the artery) occurs and blood flow to critical organs, including the heart, is chronically inadequate. The chronic ischemia can lead to a variety of end organ damage, including cardiac hypertrophy associated with congestive heart failure.

Atherosclerosis occurs when a physical defect or injury to the arterial lining (endothelium) triggers an inflammatory reaction involving vascular smooth muscle cell proliferation and leukocyte infiltration into the affected area, a potential defect leading to many forms of cardiovascular disease. Eventually, a complex lesion called atherosclerotic plaque may form, consisting of a combination of the above cells with deposits of cholesterol-bearing lipoproteins and other substances (e.g., Hansson et al, 2006). While current therapeutic treatments provide significant benefits, mortality due to cardiovascular disease remains high and the need to treat cardiovascular disease remains significantly unmet.

The induction of HO-1 has been shown to be beneficial in various models of cardiovascular disease, and low levels of HO-1 expression have been clinically associated with increased risk of CV disease. Thus, RTA408, polymorphs, and methods of the invention are useful for treating or preventing a variety of cardiovascular disorders, including but not limited to atherosclerosis, hypertension, myocardial infarction, chronic heart failure, stroke, subarachnoid hemorrhage, and restenosis. In some embodiments, RTA408, polymorphs, and methods of the invention can be used as a combination therapy with other known cardiovascular therapies such as, but not limited to, anticoagulants, thrombolytics, streptokinase, tissue plasminogen activator, surgery, coronary artery bypass grafting, balloon angioplasty, the use of stents, drugs that inhibit cell proliferation, or drugs that reduce cholesterol levels.

F. Diabetes mellitus

In some embodiments, RTA408, and polymorphs thereof, can be used to treat patients with diabetes based on methods such as those taught by US8,129,429, which is incorporated herein by reference. Diabetes mellitus is a complex disease characterized by the body's inability to regulate circulating levels of glucose. This disorder may result from a deficiency in insulin, a peptide hormone that regulates the production and absorption of glucose in various tissues. Insulin deficiency impairs the ability of muscle tissue, adipose tissue, and other tissues to properly absorb glucose, resulting in hyperglycemia (abnormally high levels of glucose in the blood). Most commonly, the insulin deficiency results from an under-production in the islet cells of the pancreas. In most cases, this results from the destruction of these cells by autoimmunity, a condition known as type 1 or juvenile onset diabetes, but also may be due to physical trauma or some other cause.

Diabetes may also occur when muscle cells and fat cells become less responsive to insulin and inappropriately absorb glucose, resulting in hyperglycemia. This phenomenon is known as insulin resistance, and the resulting condition is known as type 2 diabetes. Type 2 diabetes is the most common type, highly associated with obesity and hypertension. Obesity is associated with an inflammatory state of adipose tissue that is thought to play a major role in the development of insulin resistance (e.g., Hotamisigil, 2006; Guilherm et al, 2008).

Diabetes is associated with damage to many tissues, primarily because hyperglycemia (and hypoglycemia, which can result from excessive or inappropriate insulin administration) are significant sources of oxidative stress. Because the RTA408, polymorphs, and methods of the present invention are able to prevent oxidative stress, particularly by inducing HO-1 expression, they are useful in treating many complications of diabetes. As noted above (Cai et al, 2005), chronic inflammation and oxidative stress in the liver are suspected to be major contributing factors in the development of type 2 diabetes. Further, PPAR such as thiazolidinedione_γAgonists are capable of reducing insulin resistance and are known to be effective in the treatment of type 2 diabetes. In some embodiments, RTA408, polymorphs, and methods of the invention can be used with PPARs such as thiazolidinediones_γAgonists are used as a combination therapy.

G. Arthritis (arthritis)

In some embodiments, the RTA408, polymorphs, and methods of the invention can be used to treat a patient with some form of arthritis. In some embodiments, the arthritis forms that can be treated with RTA408 and polymorphs of the invention are Rheumatoid Arthritis (RA), psoriatic arthritis (PsA), spondyloarthropathies (SpA) (including Ankylosing Spondylitis (AS), reactive arthritis (ReA) and Enteropathic Arthritis (EA)), Juvenile Rheumatoid Arthritis (JRA) and early inflammatory arthritis.

For rheumatoid arthritis, the initial signs usually appear in the synovial lining layer, synovial fibroblasts proliferate and they attach to the articular surface at the joint margins (Lipsky, 1998). Macrophages, T cells and other inflammatory cells are subsequently recruited into the joint where they produce a number of mediators, including the following cytokines: interleukin-1 (IL-1) which causes chronic sequelae, thereby causing bone and cartilage destruction, and tumor necrosis factor (TNF-. alpha.) which plays a role in inflammation (Dinarello, 1998; Ared and Dayer, 1995; van den Berg, 2001). The concentration of IL-1 in plasma is significantly higher in RA patients than in healthy individuals, and it is worth noting that plasma IL-1 levels have a correlation with RA disease activity (Eastgate et al, 1988). In addition, synovial fluid levels of IL-1 have been correlated with various radiographic and histological features of RA (Kahle et al, 1992; Rooney et al, 1990).

Other forms of arthritis include psoriatic arthritis (PsA), which is a chronic inflammatory joint disease characterized by arthritis combined with psoriasis. Studies have shown that PsA shares many genetic, pathogenic and clinical features with other spondyloarthropathies (SpA), a group of diseases including ankylosing spondylitis, reactive arthritis and enteropathic arthritis (Wright, 1979). The notion that PsA belongs to the group of SpA has recently been further supported by imaging studies that demonstrate the presence of extensive onset and death inflammation in PsA, but not in RA (McGonagle et al, 1999; McGonagle et al, 1998). More specifically, onset and arrest inflammation has been considered as one of the earliest events occurring in SpA, leading to bone remodeling and joint stiffness in the spine, and synovitis of the joint when the inflamed onset and arrest point is near the peripheral joint. Increased amounts of TNF-alpha have been reported in both psoriatic skin (Ettehadi et al, 1994) and synovial fluid (Partsch et al, 1997). Recent trials have demonstrated the positive benefits of anti-TNF treatment in both PsA (Mease et al, 2000) and ankylosing spondylitis (Brandt et al, 2000).

Juvenile Rheumatoid Arthritis (JRA) is the term used for the most common form of arthritis in children, and applies to a family of diseases characterized by chronic inflammation and hypertrophy of the synovium. The term overlaps with, but is not entirely synonymous with, a family of diseases referred to in europe as juvenile chronic arthritis and/or juvenile idiopathic arthritis.

Polyarticular JRA is a unique clinical subtype characterized by inflammation and synovial hyperplasia in multiple (4 or more) joints, including the facet joints of the hand (Jarvis, 2002). This JRA subtype can be severe because it involves multiple joints and can progress rapidly over time. Although clinically unique, polyarticular JRA is not identical and patients differ in disease presentation, age of onset, prognosis and response to treatment. These differences are likely to reflect a range of changes in the immune nature and inflammatory attack that may occur in this disease (Jarvis, 1998).

Ankylosing Spondylitis (AS) is a subset of diseases within the broader disease classification of spondyloarthropathies. Patients with different subsets of spondyloarthropathies often have very different causes of the disease, ranging from bacterial infection to genetic factors. However, in all subpopulations, the end result of the disease process is axial arthritis.

AS is a chronic systemic inflammatory rheumatic disorder of the axial skeleton with or without extraosseous manifestations. The sacroiliac joint and spine are affected primarily, but the hip and shoulder joints may also be involved, as well as the peripheral joints or certain extra-articular structures, such as the eye, vasculature, nervous system, and gastrointestinal system, which are less commonly involved. The etiology of the disease is not fully understood (Wordsworth, 1995; Calin and Taurog, 1998). The etiology is strongly associated with the major histocompatibility class I (MHC I) HLA-B27 allele (Calin and Taurog, 1998). AS inflicts aggression in individuals during their adulthood and is alarming because AS may cause chronic pain and irreversible damage to tendons, ligaments, joints and bones (Brewerton et al, 1973 a; Brewerton et al, 1973 b; Schlosstein et al, 1973).

H. Ulcerative colitis

In some embodiments, the RTA408, polymorphs, and methods of the invention can be used to treat patients with ulcerative colitis. Ulcerative colitis is a disease that causes inflammation and sores called ulcers in the lining of the large intestine. The inflammation usually occurs in the rectum and lower colon, but it can affect the entire colon. Ulcerative colitis may also be referred to as colitis or proctitis. The inflammation frequently empties the colon, causing diarrhea. Ulcers form at sites where inflammation has killed the cells lining the colon, and the ulcers bleed and produce pus.

Ulcerative colitis is an Inflammatory Bowel Disease (IBD) which is the common name for diseases that cause inflammation in the small intestine and colon. Ulcerative colitis can be difficult to diagnose because its symptoms are similar to other intestinal disorders and another type of IBD, crohn's disease. Crohn's disease is different from ulcerative colitis in that it causes deeper inflammation within the intestinal wall. In addition, crohn's disease usually occurs in the small intestine, but the disease may also occur in the mouth, esophagus, stomach, duodenum, large intestine, appendix and anus.

I. Crohn's disease

In some embodiments, RTA408, polymorphs, and methods of the invention can be used to treat patients with crohn's disease. Symptoms of crohn's disease include enteritis and the development of intestinal stenosis and fistulas; neuropathy is often associated with these symptoms. Anti-inflammatory drugs such as 5-aminosalicylates (e.g. mesalamine) or corticosteroids are usually prescribed, but these drugs are not always effective (reviewed in Botoman et al, 1998). Immunosuppression with cyclosporine is sometimes beneficial for patients who are resistant or intolerant to corticosteroids (Brynskov et al, 1989).

In the active case of Crohn's disease, elevated concentrations of TNF- α and IL-6 are secreted into the blood circulation and mucosal cells locally overproduce TNF- α, IL-1, IL-6 and IL-8 (supra; Funakoshi et al, 1998). These cytokines can have a wide range of effects on physiological systems including bone development, hematopoiesis, and liver, thyroid, and neuropsychiatric functions. In addition, an imbalance in the IL-1 β/IL-1ra ratio where the pro-inflammatory IL-1 β predominates has been observed in patients with Crohn's disease (Rogler and Andus, 1998; Saiki et al, 1998; Dionne et al, 1998; but see Kuboyama, 1998).

Treatments that have been proposed for Crohn's disease include the use of various cytokine antagonists (e.g., IL-1ra), inhibitors (e.g., IL-1. beta. invertase inhibitors and antioxidants), and anti-cytokine antibodies (Rogler and Andus, 1998; van Hogezand and Verspagent, 1998; Reimund et al, 1998; Lugering et al, 1998; McAllndon et al, 1998). In particular, anti-TNF- α monoclonal antibodies have been tried and met with some success in the treatment of Crohn's disease (Targan et al, 1997; Stack et al, 1997; van Dullemen et al, 1995). These compounds can be used in combination therapy with RTA408, polymorphs, and methods of the present disclosure.

J. Systemic lupus erythematosus

In some embodiments, RTA408, polymorphs, and methods of the invention can be used to treat patients with SLE. Systemic Lupus Erythematosus (SLE) is an autoimmune rheumatic disease characterized by the deposition of autoantibodies and immune complexes in tissues, resulting in tissue damage (Kotzin, 1996). SLE potentially directly affects multiple organ systems, and its clinical manifestations are diverse and variable (reviewed by Kotzin and O' Dell, 1995), compared to autoimmune diseases such as MS and type 1 diabetes. For example, some patients may exhibit primarily skin rash and joint pain, exhibit spontaneous relief and require little drug treatment. On the other end of the range are patients who require treatment with high doses of steroids and cytotoxic drugs such as cyclophosphamide, which exhibit severe and progressive renal involvement (Kotzin, 1996).

One of the antibodies produced by SLE, IgG anti-dsDNA, plays a major role in the production of lupus Glomerulonephritis (GN) (Hahn and Tsao, 1993; Ohnishi et al, 1994). Glomerulonephritis is a serious condition in which the capillary wall of the glomerulus that purifies the blood of the kidney becomes thickened due to hyperplasia on the epithelial side of the glomerular basement membrane. The disease is usually chronic and progressive and can lead to eventual renal failure.

K. Irritable bowel syndrome

In some embodiments, RTA408, polymorphs, and methods of the invention are useful for treating patients with Irritable Bowel Syndrome (IBS). IBS is a functional disorder characterized by changes in abdominal pain and bowel habits. This syndrome may begin in early adulthood and may be associated with significant functional deficits. This syndrome is not the same condition. Rather, subtypes of IBS have been described based on the primary symptoms, i.e., diarrhea, constipation, or pain. Limited treatment is required in the absence of "warning" symptoms such as fever, weight loss, and gastrointestinal bleeding.

There is increasing evidence for the origin of IBS indicating a relationship between infectious enteritis and subsequent development of IBS. Inflammatory cytokines may play a role. In a survey of patients with a history of diagnosed bacterial gastroenteritis (Neal et al, 1997), 25% reported persistent changes in bowel habits. The persistence of symptoms can be attributed to psychological stress upon acute infection (Gwee et al, 1999).

Recent data indicate that bacterial overgrowth in the small intestine may also have a role in IBS symptoms. In one study (Pimentel et al, 2000), 157 of 202 IBS patients scheduled for a hydrogen breath test (78%) had positive test results for bacterial overgrowth. Of the 47 subjects with follow-up testing, 25 (53%) reported improvement in symptoms (i.e., abdominal pain and diarrhea) with antibiotic treatment.

Sjogren's syndrome (Syndrome)

In some embodiments, RTA408, polymorphs, and methods of the invention can be used to treat patients with sjogren's syndrome. Primary Sjogren's Syndrome (SS) is a slowly progressive chronic systemic autoimmune disease that affects mainly middle-aged women (female to male ratio 9: 1), although the disease can be seen in all age stages including childhood (Jonsson et al, 2002). The disease is characterized by infiltration and destruction of exocrine glands by lymphocytes, which are infiltrated by monocytes including CD4+ lymphocytes, CD8+ lymphocytes, and B cells (Jonsson et al, 2002). In addition, extraglandular (systemic) manifestations are seen in 1/3 patients (Jonsson et al, 2001).

In other systemic autoimmune diseases, such as RA, factors critical for ectopic Germinal Centers (GCs) have been identified. Rheumatoid synovial tissue with GC was shown to produce the chemokines CXCL13, CCL21 and Lymphotoxin (LT) - β (detected on follicular center and mantle layer B cells). Multivariate regression analysis of these analytes identified CXCL13 and LT- β as isolated cytokines predictive of GC in rheumatoid synovitis (Weyand and Goronzy, 2003). More recently, CXCL13 and CXCR5 in salivary glands have been shown to play an important role in inflammatory processes by recruiting B and T cells, thereby promoting lymphopoiesis and ectopic GC formation in SS (salomonson et al, 2002).

Psoriasis disease

In some embodiments, the RTAs 408, polymorphs, and methods of the invention can be used to treat patients with psoriasis. Psoriasis is a chronic skin disease of desquamation and inflammation that affects 2% to 2.6% or 580 to 750 million people of the united states population. Psoriasis occurs when skin cells rapidly rise from their starting point below the skin surface and accumulate on the surface before they have a chance to mature. Typically this shift, also known as a metabolic switch, takes about a month, but in psoriasis, a metabolic switch may only occur within a few days. In the typical form of psoriasis, it produces a red (inflamed) thick skin plaque covered by silvery scales. These plaques, sometimes referred to as plaques, are often itchy or painful. The plaques most commonly appear on the elbows, knees, other parts of the legs, scalp, lower back, face, palm and sole, but they may also appear on the skin at any location on the body. The disease can also affect the nails, toenails, and the soft tissues inside the genitals and mouth.

Psoriasis is an immune system driven skin disorder, involving in particular the type of white blood cells known as T cells. Generally, T cells help protect the body against infection and disease. In the case of psoriasis, T cells mistakenly begin to function and become so active that they trigger other immune responses, leading to inflammation and rapid metabolic switching of skin cells.

Infectious diseases

In some embodiments, RTA408, polymorphs, and methods of the present disclosure can be used to treat infectious diseases, including viral infections and bacterial infections. As noted above, the infection may be associated with a severe local or systemic inflammatory response. For example, influenza can cause severe lung inflammation and bacterial infection can cause a systemic excessive inflammatory response, including the overproduction of various inflammatory cytokines, which are hallmarks of sepsis. In addition, the compounds of the present invention are useful for directly inhibiting the replication of viral pathogens. Previous studies have demonstrated that related compounds such as CDDO inhibit HIV replication in macrophages (vazzez et al, 2005). Other studies have shown that inhibition of NF-. kappa.B signaling can inhibit influenza virus replication, and that cyclopentenone prostaglandins can inhibit virus replication (e.g., Mazur et al, 2007; Pica et al, 2000).

The present invention relates to the treatment or prevention of each of the diseases/disorders/conditions mentioned above in section 1V using compound RTA408 or a pharmaceutically acceptable salt thereof, or a polymorph of the compound (e.g., any of the polymorphs described herein above or below), or a pharmaceutical composition comprising any of the above entities and a pharmaceutically acceptable carrier (including, e.g., the pharmaceutical composition described above).

Pharmaceutical formulations and routes of administration

RTA408 can be administered by a variety of methods, such as orally or by injection (e.g., subcutaneously, intravenously, intraperitoneally, etc.). Depending on the route of administration, the active compound may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound. They may also be administered by continuous infusion to the site of disease or trauma.

To administer RTA408 by means other than parenteral administration, it may be necessary to coat the compound with a substance that prevents its inactivation, or to co-administer the compound with the substance. For example, the therapeutic compound may be administered to the patient in a suitable carrier such as a liposome or diluent. Pharmaceutically acceptable diluents include saline and buffered aqueous solutions. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes (Strejan et al, 1984).

RTA408 may also be administered parenterally, intraperitoneally, intraspinally, or intracerebrally. Dispersions can be prepared in glycerol, liquid polyethylene glycols and mixtures thereof, as well as in oils. Under normal conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

Sterile injectable solutions can be prepared as desired by incorporating the required amount of RTA408 in an appropriate solvent with one or a combination of ingredients enumerated above, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the therapeutic compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient (i.e., the therapeutic compound) plus any additional desired ingredient from a previously sterile-filtered solution thereof.

RTA408 can be made completely amorphous using a direct spray drying procedure. RTA408 can be administered orally using, for example, an inert diluent or an assimilable edible carrier. The therapeutic compound and other ingredients may also be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into the patient's diet. For oral therapeutic administration, the therapeutic compound may be incorporated with, for example, excipients and used in the following forms: ingestible tablets, buccal tablets, troches, capsules (including hard or soft capsules), elixirs, emulsions, solid dispersions, suspensions, syrups, wafers and the like. Of course, the percentage of therapeutic compound in the compositions and formulations can vary. The amount of therapeutic compound in the therapeutically useful composition is such that a suitable dosage will be obtained.

It is particularly advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suitable as unitary dosages for the patients to be treated, each unit containing a predetermined quantity of a therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications of the unit dosage form of the present invention are determined and directly dependent on the following factors: (a) the unique characteristics of a therapeutic compound and the particular therapeutic effect to be achieved, and (b) limitations inherent in the technology of formulating the therapeutic compound to treat a selected condition in a patient.

RTA408 can also be topically applied to the skin, eye, or mucosa. In some embodiments, the compounds can be prepared as lotions, creams, gels, oils, ointments, salves, solutions, suspensions, or emulsions. Alternatively, if topical delivery to the lung is desired, the therapeutic compound may be administered by inhalation in the form of a dry powder or aerosol formulation.

RTA408 will typically be administered at a therapeutically effective dose sufficient to treat a condition associated with a given patient. For example, the efficacy of a compound can be evaluated in an animal model system (e.g., the model systems shown in the examples and figures) that can predict efficacy in treating human diseases.

The actual dosage of RTA408 or a composition comprising RTA408 administered to a patient can be determined based on physical and physiological factors such as age, sex, body weight, severity of the condition, type of disease being treated, previous or concurrent therapeutic intervention, specific disease of the patient, and the route of administration. These factors can be determined by one skilled in the art. The practitioner responsible for administration will typically determine the concentration of the active ingredient in the composition and the appropriate dosage for an individual patient. The dosage can be adjusted by a single physician if any complications occur.

An effective amount will typically range from about 0.001mg/kg to about 1000mg/kg, from about 0.01mg/kg to about 750mg/kg, from about 100mg/kg to about 500mg/kg, from about 1.0mg/kg to about 250mg/kg, from about 10.0mg/kg to about 150mg/kg, administered in one or more doses per day for one or more days (depending, of course, on the mode of administration and the factors discussed above). Other suitable dosage ranges include 1mg to 10000mg per day, 100mg to 10000mg per day, 500mg to 10000mg per day, and 500mg to 1000mg per day. In some specific embodiments, the amount is less than 10,000mg per day, in the range of 750mg to 9000mg per day.

The effective amount may be less than 1 mg/kg/day, less than 500 mg/kg/day, less than 250 mg/kg/day, less than 100 mg/kg/day, less than 50 mg/kg/day, less than 25 mg/kg/day, or less than 10 mg/kg/day. Alternatively, it may be in the range of 1 mg/kg/day to 200 mg/kg/day. In some embodiments, the amount may be 10mg/kg, 30mg/kg, 100mg/kg, or 150mg/kg, formulated as a suspension in sesame oil, as described below in example C1. In some embodiments, the amount may be 3mg/kg, 10mg/kg, 30mg/kg, or 100mg/kg, administered daily via oral gavage, as described below in examples C2 and C3. In some embodiments, the amount may be 10mg/kg, 30mg/kg, or 100mg/kg, administered orally, as described below in example C6. For example, for treatment of a diabetic patient, the unit dose may be an amount that reduces blood glucose by at least 40% compared to an untreated patient. In another embodiment, the unit dose is an amount that lowers blood glucose to a level of ± 10% of the blood glucose level of the non-diabetic patient.

In other non-limiting examples, the dose may further comprise about 1 μ g per kilogram body weight, about 5 μ g per kilogram body weight, about 10 μ g per kilogram body weight, about 50 μ g per kilogram body weight, about 100 μ g per kilogram body weight, about 200 μ g per kilogram body weight, about 350 μ g per kilogram body weight, about 500 μ g per kilogram body weight, about 1mg per kilogram body weight, about 5mg per kilogram body weight, about 10mg per kilogram body weight, about 50mg per kilogram body weight, about 100mg per kilogram body weight, about 200mg per kilogram body weight, about 350mg per kilogram body weight, about 500mg per kilogram body weight to about 1000mg per kilogram body weight or more per kilogram body weight per administration, and any range derivable therein. In non-limiting examples of ranges derivable from the numbers listed herein, ranges of about 5mg per kg body weight to about 100mg per kg body weight, about 5 μ g per kg body weight to about 500mg per kg body weight, and the like may be administered based on the numbers described above.

In certain embodiments, a pharmaceutical composition of the present disclosure can comprise, for example, at least about 0.01% RTA 408. In other embodiments, RTA408 may comprise, for example, from about 0.01% to about 75% or from about 0.01% to about 5% of the unit weight, and any range derivable therein. In some embodiments, RTA408 may be used in the form of a formulation of, for example, 0.01%, 0.1%, or 1% suspension in sesame oil, as described below in examples F and G. In some embodiments, RTA408 can be formulated for topical application to the skin or ocular surface using a pharmaceutically suitable carrier or in the form of a suspension, emulsion, or solution at a concentration in the range of about 0.01% to 10%. In some embodiments, the concentration is in the range of about 0.1% to about 5%. The optimal concentration may vary depending on the target organ, the particular formulation, and the condition to be treated.

Single or multiple doses of an agent comprising RTA408 are contemplated. The desired time interval for delivery of multiple doses can be determined by one of ordinary skill in the art using only routine experimentation. As an example, the patient may be administered two doses per day at about 12 hour intervals. In some embodiments, the agent is administered once daily. The agents may be administered according to conventional schedules. A conventional schedule, as used herein, refers to a predetermined specified period of time. A regular schedule may cover periods of time that are the same or different lengths of time, as long as the schedule is predetermined. For example, a conventional schedule may include twice daily, once every two days, once every three days, once every four days, once every five days, once every six days, once weekly, once monthly, or any set number of days or weeks therebetween. Alternatively, the predetermined regular schedule may include twice daily administration for the first week, once daily administration for months thereafter, and the like. In other embodiments, the invention provides that the agents can be administered orally and that the time of administration is dependent or independent of food intake. Thus, for example, the medicament may be taken every morning and/or every night, whether when the patient has eaten or is about to eat.

Combination therapy

In addition to use as monotherapy, RTA408 and polymorphs described in this invention can also be used in combination therapy. Effective combination therapy can be achieved with a single composition or pharmacological agent that includes both agents, or with two different compositions or agents administered simultaneously, where one composition includes RTA408 or its polymorph, and the other composition includes a second agent. This alternative treatment modality may be administered prior to, concurrently with, or subsequent to the administration of RTA408 or its polymorph. Treatment with RTA408 or its polymorph can be separated from administration of the other agent, either following or preceding, by a time interval in the range of minutes to weeks. In embodiments where another agent is administered separately from RTA408 or its polymorph, it will generally be ensured that the effective time period has not expired between the time of each delivery, such that each agent will still be able to exert an advantageous combined effect. In such cases, it is contemplated that RTA408 or polymorph and another therapeutic agent will typically be administered within about 12-24 hours of each other, more preferably within about 6-12 hours of each other, with a delay time of only about 12 hours being most preferred. However, in some cases, it may be desirable to significantly extend the period of treatment with intervals of days (2 days, 3 days, 4 days, 5 days, 6 days, or 7 days) to weeks (1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks) between corresponding administrations.

It is also contemplated that it will be desirable to administer RTA408 or its polymorph or another agent more than once. In this regard, various combinations may be employed. By way of illustration, provided RTA408 or its polymorph is "a" and the other agent is "B," the following permutations based on a total of 3 and 4 administrations are exemplary:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B

A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A

A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B。

other combinations are also contemplated. Non-limiting examples of pharmacological agents that may be used in the present invention include any pharmacological agent known to be beneficial in the treatment of cancer. In some embodiments, combinations of RTA408 or its polymorphs with cancer targeted immunotherapy, gene therapy, radiotherapy, chemotherapeutic agents, or surgery are contemplated. Combinations of RTA408 or its polymorph with more than one of the above methods, including more than one type of specific therapy, are also contemplated. In some embodiments, the immunotherapy may be other cancer targeting antibodies, such as, but not limited to trastuzumabAlemtuzumab (alemtuzumab)BevacizumabCetuximabAnd panitumumab (panitumumab)Or conjugated antibodies, e.g. ibritumomab tiuxetanTositumomab (tositumomab)Butuximab (brentuximab vedotin)Adriazumab (ado-trastuzumab emtansine) (Kadcyla)^TM) Or dini interleukin 2(denileukin dititox)Furthermore, in some embodiments, it is contemplated that RTA408 or its polymorphs can be combined with dendritic cell-based immunotherapy (e.g., Sipuleucel-T)) Or adoptive T cell immunotherapy for use in combination therapy.

Furthermore, it is contemplated that RTA408 or its polymorph is used in combination with chemotherapeutic agents such as, but not limited to, anthracyclines, taxanes, methotrexate, mitoxantrone, estramustine, doxorubicin, etoposide, vinblastine, carboplatin, vinorelbine, 5-fluorouracil, cisplatin, topotecan, ifosfamide, cyclophosphamide, epirubicin, gemcitabine, vinorelbine, irinotecan, etoposide, vinblastine, pemetrexed, melphalan, capecitabine, and oxaliplatin. In some embodiments, RTA408, or its polymorph, is used in combination with radiation therapy, including but not limited to the use of ionizing radiation. In some embodiments, the effect of the cancer therapeutic is synergistically enhanced by administration with RTA408 and its polymorphs. In some embodiments, a combination therapy comprising RTA408 is used to treat cancer, including, for example, prostate cancer. See, e.g., example H below.

In some embodiments, the method may further comprise (1) contacting the tumor cell with the compound prior to contacting the tumor cell with the second chemotherapeutic, (2) contacting the tumor cell with the second chemotherapeutic prior to contacting the tumor cell with the compound, or (3) simultaneously contacting the tumor cell with the compound and the second chemotherapeutic. In certain embodiments, the second chemotherapeutic agent may be an antibiotic, an anti-inflammatory agent, an antineoplastic agent, an antiproliferative agent, an antiviral agent, an immunomodulatory agent, or an immunosuppressive agent. In other embodiments, the second chemotherapeutic agent may be an alkylating agent, an androgen receptor modulator, a cytoskeletal disrupting agent, an estrogen receptor modulator, a histone deacetylase inhibitor, an HMG-CoA reductase inhibitor, an prenyl protein transferase inhibitor, a retinoid receptor modulator, a topoisomerase inhibitor, or a tyrosine kinase inhibitor. In certain embodiments, the second chemotherapeutic agent is 5-azacitidine, 5-fluorouracil, 9-cis-retinoic acid, actinomycin D, alitretinoin, all-trans retinoic acid, anamycin, axitinib, belinostat, bevacizumab, bexarotene, bosutinib, busulfan, capecitabine, carboplatin, carmustine, CD437, cediranib, cetuximab, chlorambucil, cisplatin, cyclophosphamide, cytarabine, dacarbazine, dasatinib, daunomycin, decitabine, docetaxel, dolastatin-10, doxifluridine, doxorubicin, epirubicin, erlotinib, etoposide, gefitinib, gemcitabine, gemtuzumab ozolomide, altretamine, idarubicin, ifosfamide, imatinib, irinotecan, and another, Isotretinoin, ixabepilone, lapatinib, LBH589, lomustine, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitomycin, mitoxantrone, MS-275, neratinib, nilotinib, nitrosourea, oxaliplatin, paclitaxel, plicamycin, procarbazine, semaxanib, semustine, sodium butyrate, sodium phenylacetate, streptozotocin, suberoylanilide hydroxamic acid, sunitinib, tamoxifen, teniposide, thiotepa, thioguanine, topotecan, TRAIL, trastuzumab, tretinoin, trichostatin A, valproic acid, valrubicin, vandetanib, vinblastine, vincristine, vindesine, or vinorelbine.

In addition, combination therapies utilizing the RTAs 408, polymorphs, and pharmaceutical compositions of the present disclosure to treat cardiovascular diseases are contemplated. For example, in addition to the RTA408, polymorphs, and pharmaceutical compositions of the present disclosure, the methods can further comprise administering a pharmaceutically effective amount of one or more cardiovascular drugs. The cardiovascular agent may be, but is not limited to, for example, a cholesterol-lowering agent, an antihyperlipidemic agent, a calcium channel blocker, an antihypertensive agent, or an HMG-CoA reductase inhibitor. In some embodiments, non-limiting examples of cardiovascular drugs include amlodipine, aspirin, ezetimibe, felodipine, lacidipine, lercanidipine, nicardipine, nifedipine, nimodipine, nisoldipine, or nitrendipine. In other embodiments, other non-limiting examples of cardiovascular agents include atenolol, bucindolol, carvedilol, clonidine, doxazosin, indoramin, labetalol, methyldopa, metoprolol, nadolol, oxprenolol, phenoxybenzamine, phentolamine, pindolol, prazosin, propranolol, terazosin, timolol, or tolazoline. In other embodiments, the cardiovascular drug may be, for example, a statin, such as atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, or simvastatin.

VII. examples

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Synthesis of RTA408 (63415)

Reagents and conditions: (a) (PhO)₂PON₃(DPPA)、Et₃N, toluene, at 0 ℃ for 5 minutes, then at room temperature overnight, about 94%; (b) benzene, at 80 ℃ for 2 hours; (c) HCl, CH₃CN, at room temperature for 1 hour; (d) CH (CH)₃CF₂CO₂H、DCC、DMAP、CH₂Cl₂At room temperature overnight, starting from RTA401 (4 steps), 73%.

Compound 1: to the toluene solution (400mL) in the reactor, RTA401 (which can be prepared according to the methods taught, for example, by Honda Et al, 1998; Honda Et al, 2000 b; Honda Et al, 2002; Yates Et al, 2007; and U.S. Pat. Nos. 6,326,507 and 6,974,801, which are incorporated herein by reference) (20.0g, 40.6mmol) and Et are added with stirring₃N (17.0mL, 122.0mmol) and cooled to 0 ℃. Diphenylphosphoryl azide (DPPA) (13.2mL, 61.0mmol) was added over 5 minutes with stirring at 0 ℃ and the mixture was stirred continuously overnight at room temperature (HPLC-MS check showed no RTA401 remaining). The reaction mixture was added directly to a silica gel column and purified by column chromatography (silica gel, CH with 0% to 5% EtOAc in CH)₂Cl₂) Purification gave compound 1 as a white foam (19.7g, ca 94%, partial conversion to compound 2).

Compound 2: compound 1(19.7g, about 38.1mmol) and benzene (250mL) were added to the reactor and heated to 80 ℃ with stirring for 2 hours (HPLC-MS check showed no compound 1 remaining). The reaction mixture was concentrated under reduced pressure to give crude compound 2as a solid residue, which crude compound 2 was used in the next step without purification.

Compound 3: under stirring, crude compound 2 (less than or equal to 38.1mmol) and CH are added₃CN (200mL) was added to the reactor and cooled to 0 ℃. HCl (12N, 90mL) was added over 1 min at 0 ℃ and the mixture was stirred continuously at room temperature for 1 h (HPLC-MS check showed no compound 2 remaining). The reaction mixture was cooled to 0 ℃ and 10% NaOH (about 500mL) was added with stirring. Then, saturated NaHCO was added with stirring₃(1L.) extraction of the aqueous phase by EtOAc (2 × 500mL) by H₂The combined organic phases were washed with O (200mL), saturated NaCl (200mL), and Na₂SO₄Dried and concentrated to give crude compound 3(16.62g) as a pale yellow foam, which crude compound 3 was used in the next step without purification.

RTA 408: crude amine 3(16.62g, 35.9mmol), CH, was added under stirring at room temperature₃CF₂CO₂H (4.7388g, 43.1mmol) and CH₂Cl₂(360mL) was added to the reactor. Dicyclohexylcarbodiimide (DCC) (11.129g, 53.9mmol) and 4- (dimethylamino) -pyridine (DMAP) (1.65g, 13.64mmol) were then added and the mixture was stirred continuously at room temperature overnight (HPLC-MS check showed no compound 3 remaining). The reaction mixture was filtered to remove solid by-products and the filtrate was added directly to a silica gel column and purified twice by column chromatography (silica gel, 0% to 20% EtOAc in hexanes) to give compound RTA408 as a white foam (16.347g, 73%, 4 steps from RTA 401):¹H NMR(400MHz，CD₃Cl)8.04(s，1H)，6.00(s，1H)，5.94(s，br，1H)，3.01(d，1H，J＝4.8Hz)，2.75-2.82(m，1H)，1.92-2.18(m，4H)，1.69-1.85(m，7H)，1.53-1.64(m，1H)，1.60(s，3H)，1.50(s，3H)，1.42(s，3H)，1.11-1.38(m，3H)，1.27(s，3H)，1.18(s，3H)，1.06(s，3H)，1.04(s，3H)，0.92(s，3H)；m/z 555(M+1)。

B. pharmacodynamics

The following provides a summary of in vitro and in vivo studies evaluating the major pharmacodynamic effects of RTA 408.

In vitro Effect of RTA408 on Keap1-Nrf2 and NF- κ B

AIM inhibition of IFN γ -induced NO production was Nrf 2-dependent (Dinkova-Kostova, 2005). RAW264.7 mouse macrophages were seeded into 96-well plates (in triplicate) at 30,000 cells/well in RPMI 1640 supplemented with 0.5% FBS and at 37 ℃ at 5% CO₂And (4) incubating. The next day, with DMSO (vehicle) or RTA408 cells were pre-treated for 2 hours, followed by treatment with 20ng/mL mouse IFN γ for 24 hours. Nitrite (NO) as a substitute for nitric oxide in the culture medium was measured using the GriessReagent System (catalog No.: G2930, Promega) according to the manufacturer's instructions₂ ^-) Because nitrite is the main stable decomposition product of NO. Cell viability was assessed using the wST-1 Cell Proliferation Reagent (catalog No. 11644807001, Roche applied Science) according to the manufacturer's instructions. Determination of IC based on inhibition of IFN γ -induced nitric oxide production normalized for cell viability₅₀The value is obtained. Treatment with RTA408 caused dose-dependent inhibition of IFN γ -induced NO production, average IC₅₀The value was 3.8. + -. 1.2 nM. Results from representative experiments are shown in figure 1. IC for discovering RTA408₅₀Values compared to IC for compounds 63170 (8. + -.3 nM), 63171 (6.9. + -. 0.6nM), 63179 (11. + -.2 nM) and 63189 (7. + -.2 nM)₅₀The value is 45-65% lower. 63170. 63171, 63179 and 63189 are compounds having the formula:

effect of RTA408 on Nrf2 target genes

RTA408 was tested in two different luciferase reporter assays to assess the activation of ARE. The first luciferase reporter gene tested was under the control of a single ARE derived from the promoter of the human NQO1 gene, which allowed quantitative assessment of the endogenous activity of Nrf2 transcription factors in cultured mammalian cells. NQO 1-expression of firefly luciferase by ARE luciferase reporter plasmid NQO 2 with a dna corresponding to the sequence of luciferase in human NADPH: control of binding of specific enhancer sequences for Antioxidant Response Elements (AREs) identified in the promoter region of the quinone oxidoreductase 1(NQO1) gene (Xie et al, 1995). The NQO 1-ARE-luciferase reporter plasmid was constructed by inserting human NQO1-ARE (5'-CAGTCACAGTGACTCAGCAGAATCTG-3') into the pLuc-MCS vector using the HindIII/XhoI cloning site (GenScript Corp., Piscataway, N.J.). HuH-7 human hepatoma cell lines maintained in dmem (Invitrogen) supplemented with 10% FBS and 100U/mL (each) of penicillin and streptomycin were transiently transfected with NQO1-ARE luciferase reporter plasmid and pRL-TK plasmid, which constitutively expresses Renilla (Renilla) luciferase and was used as an internal control for normalization of transfection levels, using Lipofectamine (Lipofectamine)2000 (Invitrogen). 30 hours after transfection, cells were treated with RTA408 for 18 hours. The activities of firefly Luciferase and Renilla Luciferase were determined by the Dual-Glo Luciferase Assay (Luciferase Assay) (Cat. No.: E2920, Promega) and luminescence signals were measured on an L-Max II luminometer (Molecular Devices). Firefly luciferase activity was normalized for renilla luciferase activity, and fold induction of normalized firefly activity relative to vehicle control group (DMSO) was calculated. Figure 2a shows the dose-dependent induction of luciferase activity by RTA408 in this cell line. Values represent the average of three independent experiments. 20% less RTA408 (12nM) than 63189(14.9nM) was required to increase transcription of NQO1ARE in HuH-7 cells by a factor of 2. Similarly, RTA408 requiring 5/12-10/21 of 63170(25.2nM) and 63179(29.1nM), respectively, increased transcription of NQO1ARE in HuH-7 cells by a factor of 2.

The effect of RTA408 on luciferase reporter activation was also assessed in the AREc32 reporter cell line. This cell line was derived from human breast cancer MCF-7 cells and was stably transfected with a firefly luciferase reporter gene under transcriptional control of 8 copies of the rat GSTA2ARE sequence (Wang et al, 2006, incorporated herein by reference). After 18 hours of treatment with RTA408, firefly luciferase activity was measured using the ONE-Glo luciferase assay system (Promega, catalog No.: E6110) according to the manufacturer's instructions. A dose-dependent response was observed in the AREc32 reporter cell line (fig. 2 b). About 2-fold induction of luciferase activity was evident after treatment with 15.6nM RTA408 in both the NQO1-ARE reporter assay system and the GSTA2-ARE reporter assay system. When looking at the results from the GSTA2-ARE (AREc32) luciferase activity study and the WST1 viability study, the effect of 63415(RTA408) on GSTA2-ARE induction can be directly compared to the effects of RTA402, 63170, 63171, 63179 and 63189 (fig. 3 a-f). In the luciferase reporter assay, 63415 showed the fastest induction of GSTA2-ARE mediated transcription among the 5 comparative compounds compared to the value of RTA402, which required a concentration of 93nM to achieve 4-fold induction. All other compounds showed similar induction only at much higher concentrations, 63170 requiring a concentration of 171nM, 63171 requiring a concentration of 133nM, 63179 requiring a concentration of 303nM and 63189 requiring a concentration of 174nM to achieve 4-fold induction of luciferase activity. These values correspond to the amount of active compound that needs to be increased by a factor of 1.86 (63415), 3.40 (63170), 2.65 (63171), 6.05 (63179) and 3.47 (63189) compared to RTA402 in order to produce the same amount of activity.

RTA408 was also demonstrated to increase transcript levels of a known Nrf2 target gene in HFL1 human fetal lung fibroblasts and BEAS-2B human bronchial epithelial cell lines HFL1 cells were cultured in F-12K medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin BEAS-2B cells were cultured in DMEM/F-12 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin 2.5 × 10.10⁵The density of individual cells/well was seeded into 6-well culture dishes. The next day, cells were treated with DMSO (vehicle) or RTA408 (7.8nM, 15.6nM, 31.3nM, 62.5nM or 125nM) for 18 hours. Each well received the same amount of vehicle. After processing, the medium was removed and the cells were harvested using RLT buffer (Qiagen). The lysate was homogenized using a QIAShreder column (Qiagen, Cat. No.: 79654) and RNeasyMini kit (Qiagen, mesh)Recording the number: 74104) RNA was isolated. For reverse transcription, RNA (1. mu.g) was reacted with oligo (dT)_12-18Primers and H₂O combination, final volume 23.25. mu.L the mixture was heated to 70 ℃ for 10 minutes and then placed on ice containing 8. mu.L of 5 × Strand 1 buffer, 2. mu.L of 1mg/mL BSA, 2. mu.L of 20mM DTT, 4. mu.L of 5mM dNTP chelate, 0.25. mu.L of LRNaseOUT^TMAnd 0.5. mu.LII the master mix of reverse transcriptase was added to the RNA mixture and incubated for 1 hour at 42 ℃. The reaction was deactivated by heating to 70 ℃ for 10 minutes. The reaction mixture was washed with H before use in qPCR₂O1: 3 dilution 2.5. mu.L of diluted reverse transcription reaction was mixed with a set of PCR primers (0.36. mu.M final concentration), 2 × iQ^TMGreen Supermix (Bio-Rad, Cat. No.: 170-₂The O-pool reached a final volume of 20. mu.L. The sequences of the PCR primers were as follows: glutamic acid-cysteine ligase modification subunit (GCLM) forward primer 5'-GCTGTGGCTACTGCGGTATT-3' (SEQ ID NO: 1), reverse primer 5'-ATCTGCCTCAATGACACCAT-3' (SEQ ID NO: 2); heme oxygenase-1 (HMOX1) forward primer 5'-TCCGATGGGTCCTTACACTC-3' (SEQ ID NO: 3), reverse primer 5'-TAGGCTCCTTCCTCCTTTCC-3' (SEQ ID NO: 4); NAD (P) H dehydrogenase quinone 1(NQO1) forward primer 5'-AAAACACTGCCCTCTTGTGG-3' (SEQ ID NO: 5), reverse primer 5'-GTGCCAGTCAGCATCTGGTA-3' (SEQ ID NO: 6); ribosomal protein S9(RPS9) forward primer 5'-GATGAGAAGGACCCCACGGCGTCTG-3' (SEQ ID NO: 7), reverse primer 5'-GAGACAATCCAGCAGCCCAGGAGGG-3' (SEQ ID NO: 8); thioredoxin reductase 1(TXNRD1) forward primer 5'-ATTGCCACTGGTGAAAGACC-3' (SEQ ID NO: 9) and reverse primer 5'-ACCAATTTTGTTGGCCATGT-3' (SEQ ID NO: 10). The specificity and amplification efficiency of all primers have been previously confirmed. cDNA was amplified using the following cycling conditions: (95 ℃ for 3 minutes, 44 cycles of 95 ℃ for 30 seconds, 60 ℃ for 15 seconds, 72 ℃ for 15 seconds, followed by a melting curve from 55 ℃ to 95 ℃,the increment was 0.5 ℃). Using the comparative CT method (Δ Δ C)_T) The relative abundance of each Nrf2 target gene was determined. PCR reactions were run on each sample in three replicate wells. Two independent experiments were performed using the above conditions. Treatment of HFL1 lung fibroblasts with RTA408 for 18 hours resulted in increased expression of several Nrf2 target genes including NQO1, HMOX1, GCLM and TXNRD1 as measured by quantitative PCR (fig. 4 a-d). The induction of RTA408 was dose-dependent for all genes tested and was also evident at concentrations as low as 15.6 nM. Treatment of BEAS-2B bronchial epithelial cells with RTA408 for 18 hours resulted in similar dose-dependent increases in all of the Nrf2 target genes evaluated (FIGS. 5 a-d). RTA408 also increased expression of Nrf2 target gene in normal human mesangial cells (nHMC), mouse BV2 microglia cell line, and human SH-SY5Y neuroblastoma cell line at similar concentrations.

Protein levels of Nrf2 targets NQO1 and HMOX1 in SH-5Y5Y cells and BV-2 cells were measured by Western blotting after treatment with RTA408 SH-SY5Y cells were treated with 4 × 10⁵The density of individual cells/well was seeded in 6-well plates BV-2 cells at 2.5 × 10⁴The density of individual cells/well was plated into 6-well plates 24 hours (BV-2) or 48 hours (SH-SY5Y) after plating, the cells were treated with RTA408 for 24 hours after treatment, the cells were washed twice with cold PBS and harvested in lysis buffer, the cells were sonicated and debris was cleared by centrifugation (10 minutes at 18,000rcf, microcentrifuge 18 centrifuge of Beckmann Coulter.) the total protein in the supernatant was quantified using Bio-Rad protein reagent with BSA as standard. the total cellular proteins were separated on SDS-PAGE and the proteins were transferred to nitrocellulose membrane, the membrane was blocked in TBST containing 5% milk (1 TBS 1 × containing 0.1% Tween-20) for 1 hour, washed 3 times with TBST and incubated with primary antibody at 4 ℃ Nn 1 antibody from Abcam (accession number: 2346); HMOX1(HO-1) from Sanbata antibody 10789, from milk-bios protein reagent + Happy antibody was added overnight at room temperatureThen (c) is performed. Affinity pure (Affinipure) goat anti-rabbit or anti-mouse IgG secondary antibody was from Jackson ImmunoResearch (catalog numbers 111. sup. 035. sup. 144 and 115. sup. 035. sup. 146. sup. 035). The film was washed in TBST, developed using ECL, and exposed to X-ray film. Treatment with RTA408 also increased the level of NQO1 protein in SH-SY5Y cells in a dose-dependent manner (fig. 6 a). HMOX1 protein was not detected in untreated SH-SY5Y cells or SH-SY5Y cells treated with RTA 408. Treatment with RTA408 increased protein levels of NQO1 and HMOX1 at concentrations up to 125nM in BV2 cells (fig. 6 b). EC of RTA408 for Induction of Nrf2 protein expression in SK-N-SH cells₅₀Values (56.4nM) vs EC of 63171(122nM), 63189(102nM) and 63179(126nM)₅₀The value is 45-65% lower. The same amount of 63170(54.6nM) was required.

Measurement of EC using an intracellular Western blot NQO1 assay₅₀Wherein the cells are incubated with the compound being evaluated for three days. After incubation with the compound of interest, the cells were reacted with mouse NQO1 antibody, and then reacted with IRDye-800 CW-anti-mouse IgG antibody the next day. The target signal is visualized and then analyzed.

Consistent with the induction of Nrf2 target genes and corresponding protein products, treatment of RAW264.7 mouse macrophages for 24 hours also enhanced NQO1 enzyme activity in a dose-dependent manner, with an increase at 7.8nM being evident (fig. 7). NQO1 enzyme activity was measured by a modified Prochasca assay (Prochaska assay) (Prochaska and Santamaria, Anal biochem.169: 328-336, 1988, which is incorporated herein by reference).

Taken together, these data from various cell lines demonstrate that treatment with RTA408 enhances transcriptional activity controlled by antioxidant response elements, increases expression of Nrf2 target gene and enhances activity of Nrf2 target gene product NQO 1.

Effect of RTA408 on markers of the Redox Capacity of cells

Glutathione and NADPH are the means for maintaining the redox ability of cellsThe key factor required. Involved in the synthesis of glutathione (e.g., GCLC and GLCM) and NADPH [ e.g., hexose-6-phosphate dehydrogenase (H6PD) and malic enzyme 1(ME1) ]]Have been shown to be regulated by Nrf2 (Wu, 2011). Use of GSH-Glo in mouse AML-12 hepatocyte cell line^TMGlutathione assay kit (Promega, catalog number: V6912), the effect of RTA408 treatment on total glutathione levels was evaluated according to the manufacturer's instructions. Treatment of AML-12 cells with RTA408 for 24 hours increased total cellular glutathione levels in a dose-dependent manner (figure 8). The data shown are representative of two independent experiments. An increase of > 2 fold in total glutathione was observed at RTA408 concentrations as low as 15.6 nM. Induced EC of glutathione levels by RTA408 using the RAW264.7 mouse model₅₀EC for values (9.9nM) vs 63170(12.1nM), 63171(23.2nM) and 63189(16nM)₅₀The value is 22 to 57 percent lower.

The effect of RTA408 treatment on NADPH levels was evaluated in HCT-116 cells as measured by the absorbance of the redox sensitive dye WST-1(Roche Applied Science, Cat. No.: 11644807001). WST-1 absorbance is commonly used to assess cell viability by measuring glycolytic production of NAD (P) H by living cells. Thus, WST-1 absorbance will also increase in the absence of any effect on cell viability but rather in the presence of increased NADPH production (Berridge et al, 1996, which is incorporated herein by reference). A variety of key genes involved in NADPH production have also been shown to be regulated by Nrf2 (Thimmulappa et al, 2002; Wu et al, 2011, both of which are incorporated herein by reference). RTA408 treatment for 24 hours increased WST-1 absorbance in a dose-dependent manner (FIG. 9), indicating an increase in NADPH levels.

HCT-116 cells were treated with RTA408 for 24 hours and the mRNA levels of H6PD, phosphogluconate dehydrogenase (PGD), transketolase (TKT) and ME1 were measured using quantitative PCR, HCT-116 cells were plated at3 × 10⁵The density of individual cells/well was seeded into 6-well culture dishes. The next day, cells were treated with DMSO (vehicle), 10nM RTA408 or 50nM RTA408 for 24 hours. Each one of which isThe wells received the same amount of vehicle. After processing, the medium was removed and the cells were harvested using RLT buffer (Qiagen). The lysates were homogenized using QIAShreder column (Qiagen, Cat. No.: 79654) and RNA was isolated using RNeasy Mini kit (Qiagen, Cat. No.: 74104). For reverse transcription, RNA (1. mu.g) was reacted with oligo (dT)_12-18Primers and H₂O combination, final volume 23.25. mu.L the mixture was heated to 70 ℃ for 10 minutes and then placed on ice containing 8. mu.L of 5 × Strand 1 buffer, 2. mu.L of 1mg/mL BSA, 2. mu.L of 20mM DTT, 4. mu.L of 5mM dNTP mix, 0.25. mu.L of LRNaseOUT^TMAnd 0.5. mu.LII the master mix of reverse transcriptase was added to the RNA mixture and incubated for 1 hour at 42 ℃. The reaction was deactivated by heating to 70 ℃ for 10 minutes. The reaction mixture was washed with H before use in qPCR₂O1: 3 dilution 2.5. mu.L of diluted reverse transcription reaction was mixed with a set of PCR primers (0.36. mu.M final concentration), 2 × iQ^TMGreen Supermix (Bio-Rad, Cat. No.: 170-₂The O-pool reached a final volume of 20. mu.L. The sequences of the PCR primers were as follows: ribosomal protein S9(RPS9) forward primer 5'-GATGAGAAGGACCCCACGGCGTCTG-3' (SEQ ID NO: 7), reverse primer 5'-GAGACAATCCAGCAGCCCAGGAGGG-3' (SEQ ID NO: 8); hexose-6-phosphate dehydrogenase (H6PD) forward primer 5'-GAGGCCGTGTACACCAAGAT-3' (SEQ ID NO: 11), reverse primer 5'-AGCAGTGGGGTGAAAATACG-3' (SEQ ID NO: 12); phosphogluconate dehydrogenase (PGD) forward primer 5'-AAGGCACTCTACGCTTCCAA-3' (SEQ ID NO: 13), reverse primer 5'-AGGAGTCCTGGCAGTTTTCA-3' (SEQ ID NO: 14); transketolase (TKT) forward primer 5'-CATCTCCGAGAGCAACATCA-3' (SEQ ID NO: 15), reverse primer 5'-TTGTATTGGCGGCTAGTTCC-3' (SEQ ID NO: 16); malic enzyme 1(ME1) forward primer 5'-TATATCCTGGCCAAGGCAAC-3' (SEQ ID NO: 17) reverse primer 5'-GGATAAAGCCGACCCTCTTC-3' (SEQ ID NO: 18). Has already been done to all guides in advanceThe specificity and amplification efficiency of the product were confirmed. cDNA was amplified using the following cycling conditions: (95 ℃ for 3 minutes, 44 cycles of 95 ℃ for 30 seconds, 60 ℃ for 15 seconds, 72 ℃ for 15 seconds, followed by a melting curve from 55 ℃ to 95 ℃ in 0.5 ℃ increments). The relative abundance of each Nrf2 target gene was determined using the comparative CT method (Δ Δ CT). PCR reactions were run on each sample in three replicate wells. Two independent experiments were performed using the above conditions. Treatment with RTA408 resulted in a dose-dependent increase in the expression of genes involved in NADPH synthesis (fig. 10 a-d).

In conclusion, treatment with RTA408 increased the total glutathione level in AML-12 hepatocytes and increased the WST-1 absorbance, a marker of NADPH production, in HCT-116 cells. This observation has relevance to the increased expression of a number of key genes encoding enzymes involved in NADPH synthesis.

Effect of RTA408 on TNF α -induced NF- κ B Signaling

NF-. kappa.B is a transcription factor that plays an important role in the regulation of many immune and inflammatory responses. RTA402 and other AIMs have been shown to inhibit pro-inflammatory NF- κ B signaling in a variety of cell lines (Shishodia, 2006; Ahmad, 2006; Yore, 2006). The effect of RTA408 and compounds 63171, 63179, 63170 and 63189 on NF-. kappa.B-Luc reporter genes was explored using the mouse NIH3T 3/NF-. kappa.B-Luc cell line (Panomics). The NIH3T 3/NF-. kappa.B-luc cell line possessed chromosomal integration of a firefly luciferase reporter construct regulated by 8 copies of the NF-. kappa.B response element. Can be measured by NF-kB IC₅₀The values of (c) quantify the effect of these compounds. RTA408 showed an IC of 1.2. mu.M₅₀Shows an IC of 1.4. mu.M when normalized for viability₅₀. The other 4 compounds showed NIH3T 3/NF-. kappa.B IC at 1.7. mu.M, 0.2. mu.M, 1.1. mu.M and 1.1. mu.M₅₀Values, when normalized for viability, show IC of 1.8. mu.M, 0.6. mu.M, 1.1. mu.M and 1.0. mu.M, respectively₅₀The value is obtained. In FIGS. 11a and B, RTA408 and its effect on NF- κ B are plotted as a function of the administered dose and the relative fold change, and WST1 and WST1/2 are shown. Evaluation of RTA in HeLa/NF-. kappa.B-Luc cells408 on TNF α -induced NF-. kappa.B signalling in human cervical adenocarcinoma cell lines stably transfected with luciferase reporter constructs under the control of multiple NF-. kappa.B transcriptional response elements HeLa/NF-. kappa.B-Luc cells were pretreated with RTA408 for 1 hour, followed by additional treatment with TNF- α (10ng/mL) for 5 hours after treatment, luminescence was measured and the effect of RTA408 pretreatment on TNF- α -induced luciferase activity was determined the mean results and standard deviation from three independent experiments are shown in FIG. 12, RTA408 was shown with an IC of 517. + -.83 nM₅₀Dose-dependent inhibition of TNF- α -induced NF-. kappa.B activation similar results were observed in another NF-. kappa.B reporter cell line (A549/NF-. kappa.B-Luc) in which RTA408 had an IC of 627nM (in the range of 614-649 nM)₅₀The TNF- α -induced NF- κ B activation was inhibited by RTA408 reduced expression of the NF- κ B promoter reporter in HeLa/NF- κ B-Luc cells 1.6-1.8 times as efficiently as 63189(854nM) and 63170(953nM), respectively, further experiments with the human A549 cell line showed IC of RTA408₅₀The value that was 1.7 μ M and that had been normalized for viability was 1.7 μ M. IC of RTA408₅₀Shows activity similar to 63189, 63179, 63171 and 63170, and 63189, 63179, 63171 and 63170 show IC of 1.1. mu.M, 1.4. mu.M, 2.0. mu.M and 1.0. mu.M, respectively₅₀The value is obtained. When those values were normalized for viability, the assay showed IC's of 1.2. mu.M, 1.5. mu.M, 2.1. mu.M and 1.1. mu.M, respectively₅₀. Fold changes in NF- κ B as a function of RTA408 concentration and WST1 and WST1/2 curves were plotted and are shown in FIGS. 13a and 13B.

The effect of RTA408 on TNF- α induced phosphorylation of I κ B α, a key step in NF- κ B pathway activation, was also evaluated in HeLa cells. HeLa cells were pre-treated with RTA408 for 6 hours, followed by treatment with TNF-. alpha. (20ng/mL) for 5 minutes. The total level and phosphorylation level of I κ B α were assessed by western blot. I κ B α primary antibody was from Santa-Cruz (sc-371), pI κ B α antibody was from Cell Signaling (9246), and actin antibody was from Millipore (MAB 1501). Affinity-pure (affini-pure) goat anti-rabbit (IgG) conjugated to peroxidase and affinity-pure goat anti-mouse IgG secondary antibody conjugated to peroxidase were purchased from Jackson ImmunoResearch. Western blots were developed using ECL and X-ray films were exposed. Consistent with the results from the luciferase reporter assay, RTA408 inhibited TNF- α induced phosphorylation of I κ B α in a dose-dependent manner (fig. 14).

RTA408 has also been shown to inhibit other proinflammatory signaling pathways, such as IL-6-induced phosphorylation of signal transducers and activator of transcription 3(STAT3) and NF-. kappa.B receptor activator ligand (RANKL) -induced osteoclastogenesis. IL-6-induced phosphorylation of STAT3 was inhibited in HeLa cells by pretreatment with 1 μ M RTA408 for 6 hours. Monoclonal antibodies to STAT3(124H6) and phospho-STAT 3(Tyr705) were from Cell Signaling Technology. Affinity pure goat anti-rabbit IgG conjugated to peroxidase and affinity pure goat anti-mouse IgG conjugated to peroxidase were from Jackson ImmunoResearch. Osteoclastogenesis is a multistep differentiation process caused by binding of RANKL to its receptor RANK located on cells of hematopoietic origin. This leads to activation of NF- κ B and MAPK, which in turn increases transcription of osteoclast-specific target genes, including tartrate-resistant acid phosphatase (TRAP). The effect of RTA408 on RANKL-induced osteoclastogenesis was evaluated in the mouse macrophage cell line RAW 264.7. RAW264.7 cells were seeded into 24-well plates at a density of 5,000 cells/well. The following day, cells were treated with RTA408 for 2 hours, followed by 50ng/mL recombinant mouse RANKL (R)&D systems). The treated cells were incubated for 4 days to allow differentiation into osteoclasts. Differentiation into osteoclasts was assessed by measuring TRAP activity. Briefly, 90 μ L of conditioned cell culture medium was removed from each test well and aliquoted into three replicate wells (30 μ L/well) of a 96-well plate. Then 170 μ L of TRAP assay buffer (Kamiya Biomedical) was added to each well and the plates were incubated at 37 ℃ for 3 hours. After incubation, absorbance at 540nm was measured using a Spectramax M2 plate reading spectrophotometer. RTA408 inhibited RANKL-induced TRAP activity and osteoclast formation, IC, in a dose-dependent manner₅₀Is about 5-10 nM.

Effect of RTA408 on the expression of the Gene encoding transaminase

To assess whether treatment with RTA408 affects transaminase mRNA levels, mouse AML-12 hepatocytes were treated with RTA408 for 18 hours and mRNA levels of the gene encoding transaminase were measured using quantitative PCR, AML-12 cells were treated with 3 × 10 using 2mL per well of medium, AML-12 cells were treated with 3 × 10⁵Individual cells/well were seeded into 6-well culture dishes. The next day, cells were treated with DMSO (vehicle) or 250nM and 500nM rta408 for 18 hours at 37 ℃. Each well received 0.1% DMSO. Three independent replicates were performed. After processing, the medium was removed and the cells were harvested using RLT buffer (Qiagen). The lysate was homogenized using QIAShreder column (Qiagen, Cat. No.: 79654) and RNA was isolated using RNeasy Mini kit (Qiagen, Cat. No.: 74104). For reverse transcription, RNA (1. mu.g) was reacted with oligo (dT)_12-18Primers and H₂O combination, final volume 23.25. mu.L the mixture was heated to 70 ℃ for 10 minutes and then placed on ice containing 8. mu.L of 5 × Strand 1 buffer, 2. mu.L of 1mg/mLBSA, 2. mu.L of 20mM DTT, 4. mu.L of 5mM dNTP chelate, 0.25. mu.L of LRNaseOUT^TMAnd 0.5. mu.LII the master mix of reverse transcriptase was added to the RNA mixture and incubated for 1 hour at 42 ℃. The reaction was deactivated by heating to 70 ℃ for 10 minutes. The reaction mixture was washed with H before use in qPCR₂O1: 3 dilution 2.5. mu.L of diluted reverse transcription reaction was mixed with a set of PCR primers (0.36. mu.M final concentration), 2 × iQ^TMGreen Supermix (Bio-Rad, Cat. No.: 170-₂Group OThe final volume of 20. mu.L was reached. The sequences of the PCR primers were as follows: ribosomal protein L19(Rpl19) forward primer 5'-TCAGGCTACAGAAGAGGCTTGC-3' (SEQ ID NO: 19), reverse primer 5'-ACAGTCACAGGCTTGCGGATG-3' (SEQ ID NO: 20); NAD (P) H dehydrogenase quinone 1(Nqo1) forward primer 5'-TCGGGCTAGTCCCAGTTAGA-3' (SEQ ID NO: 21), reverse primer 5'-AAAGAGCTGGAGAGCCAACC-3' (SEQ ID NO: 22); glutamate pyruvate transaminase 1(Gpt1 or Alt1) forward primer 5'-CACGGAGCAGGTCTTCAACG-3' (SEQ ID NO: 23), reverse primer 5'-AGAATGGTCATCCGGAAATG-3' (SEQ ID NO: 24); glutamate pyruvate transaminase 2(Gpt2 or Alt2) forward primer 5'-CGCGGTGCAGGTCAACTACT-3' (SEQ ID NO: 25), reverse primer 5'-CCTCATCAGCCAGGAGAAAA-3' (SEQ ID NO: 26); glutamate oxaloacetate transaminase 1(Got1 or Ast1) forward primer 5'-GGCTATTCGCTATTTTGTGT-3' (SEQ ID NO: 27), reverse primer 5'-GACCAGGTGATTCGTACAAT-3' (SEQ ID NO: 28); glutamate oxaloacetate transaminase 2(Got2 or Ast2) forward primer 5'-AGAGTCCTCTTCAGTCATTG-3' (SEQ ID NO: 29), reverse primer 5'-ATGATTAGAGCAGATGGTGG-3' (SEQ ID NO: 30). The specificity and amplification efficiency of all primers have been previously confirmed. cDNA was amplified using the following cycling conditions: (95 ℃ for 3 minutes, 44 cycles of 95 ℃ for 30 seconds, 60 ℃ for 15 seconds, 72 ℃ for 15 seconds, followed by a melting curve from 55 ℃ to 95 ℃ in 0.5 ℃ increments). The relative abundance of each Nrf2 target gene was determined using the comparative CT method (Δ Δ CT). PCR reactions were run on each sample in three replicate wells. Treatment with RTA408 increased the mRNA levels of alanine aminotransferase 1(Alt1 or Gpt1) and aspartate aminotransferase 1(Ast1 or Got1) (fig. 15a, 15 c). RTA408 had no effect on the mRNA level of alanine aminotransferase 2(Alt2 or Gpt2) and reduced the mRNA level of aspartate aminotransferase 2(Ast2 or Got2) (FIGS. 15b, 15 d). These results demonstrate that RTA408 affects transaminase gene expression in vitro at the concentrations tested (250nM or 500 nM).

Effect of RTA408 on the level of glycolytic intermediates

Studies in diabetic mice have demonstrated that bardoxolone methyl increases muscle-specific insulin-stimulated glucose uptake (Saha, 2010). In humans, a higher percentage of patients receiving bardoxolone methyl reported experiencing muscle spasms than patients receiving placebo (Pergola, 2011). Following administration of insulin, muscle spasms have also been reported in diabetic patients, suggesting a possible link to muscle glucose metabolism. The effect of RTA408 on glycolytic metabolism was evaluated by assessing lactate and pyruvate levels in cultured rodent C2C12 myocytes. To measure lactate levels, differentiated C2C12 myotubes were treated with 1 μ M or 2 μ M RTA408 or insulin for 3 hours at 37 ℃. The buffer was removed and retained for measurement of extracellular lactate levels. Cell debris was pelleted by centrifugation (10 min at 14,000 rpm) before measuring lactate. To measure intracellular lactate, cells were suspended in PBS containing 0.1% Triton X-100 and lysed by shearing with a 25 gauge needle. The cell lysate was centrifuged (10 min at 14,000rpm, 4 ℃) and the lactate in the supernatant was measured. Intracellular and extracellular lactate were measured using a lactate assay kit (BioVision, Cat. No.: K607-100). Similar to treatment with insulin, treatment of differentiated C2C12 myotubes with 1 μ M or 2 μ M RTA408 for 3 hours significantly increased intracellular and extracellular lactate levels in a dose-dependent manner.

To measure pyruvate levels, differentiated C2C12 myotubes were treated with 250nM or 500nM RTA408 nM or 100nM insulin for 18 hours. After drug treatment, the medium was removed and the cells were washed with PBS. The cells were lysed in pyruvate assay buffer (pyruvate assay kit, BioVision, Cat. No.: K609-100). The cell lysate was centrifuged (10 min at 14,000rpm, 4 ℃) and the pyruvate level in the supernatant was measured. Treatment of differentiated C2C12 myotubes with 250nM or 500nM RTA408 for 18 hours also significantly (P < 0.0001, marked with an asterisk) increased intracellular pyruvate levels in a dose-dependent manner (figure 16). Taken together, these results demonstrate that RTA408 can affect muscle glycolytic intermediates in vitro at the tested concentrations; however, it is not clear how the results from this ex vivo system at the RTA408 test concentration correlate with the potential effect on human glucose metabolism at clinically relevant dose levels.

7. In vitro evaluation of RTA408 efflux by MRP-1

One of the characteristics of a drug candidate is the exo-ratio of the compound. The efflux ratio measures the ease with which a compound is transported across a membrane. The MRP-1 protein, multi-drug resistance helper protein 1, is one of a family of proteins that help facilitate the transport of organic anions and other small molecules across cell membranes. A larger exclusion ratio generally means that the drug candidate is more easily transported out of the cell membrane and less available for modulating intracellular processes. Similar proteins also regulate transport of compounds across the blood brain barrier. The exclusion ratio of MRP-1 to RTA408 (1.3) was experimentally determined to be about 1/10 and less than 1/40 for 63179(56.5) and 63189(57.1) for 63170(10) and 63171 (11.2). Without being bound by theory, RTA408 may not be a good substrate for MRP-1 and/or a candidate for p-glycoprotein mediated efflux at the blood brain barrier. In some embodiments, RTA408 can be used to treat a Central Nervous System (CNS) disorder.

Protective effects of rta408 in animal models of lung disease

RTA408 was tested in various animal models of lung disease to evaluate its potential efficacy in the lung. For all studies, RTA408 was orally administered in sesame oil at a dosage level ranging from 3mg/kg to 150mg/kg daily. In most cases, RTA408 is administered starting days before the induction of a response to lung injury.

LPS-induced pulmonary inflammation in mice

RTA408 was tested in two studies of LPS-induced pulmonary inflammation in mice. In the first study, to find the initial dose range, RTA408 (30mg/kg, 100mg/kg, or 150mg/kg) was administered orally once a day for three days, followed by LPS administration 1 hour after the last dose. Bronchoalveolar lavage fluid (BALF) was collected 20 hours after LPS administration (21 hours after final administration of RTA 408) and Luminex was used^TMTechnical evaluation of proinflammatory marker (i.e., IL-6, IL-12p40, TNF- α, and RANTES) levels RTA408 treatment resulted in IL-12p40 (at all doses) andTNF- α (at 100mg/kg and 150mg/kg doses) was significantly reduced (FIG. 17). in the second study, RTA408 (10mg/kg, 30mg/kg or 100mg/kg) was administered daily for 6 days, followed by LPS administration 1 hour after the last dose.in this study, significant weight loss was observed starting from day 3 at the 100mg/kg dose level.A significant reduction in TNF- α was observed at the 10mg/kg dose, and significant reductions in IL-12p40, TNF- α and RANTES were observed at the 30mg/kg dose (FIG. 18 a). further evaluation of the lungs from mice in this study showed significant involvement of related Nrf2 target genes at 10mg/kg and 30mg/kg, including significant induction of NQO1 enzyme activity (by measuring the rate of 2, 6-dichlorophenol-indophenol reduction) and total GSH (GSH-Glo)^TMIncrease in Promega corporation (Promega, Madison, WI)) in Madison, wisconsin (fig. 18 b).

2. Bleomycin-induced pulmonary fibrosis

The effect of RTA408 was also evaluated in a bleomycin-induced pulmonary fibrosis model in mice and rats. In the first pilot study, mice were administered RTA408 (10mg/kg, 30mg/kg or 100mg/kg) daily via oral gavage for 39 days with a bleomycin challenge (challenge) (intranasal) on day 10. On the last day of administration, lung tissue was collected and histologically analyzed to assess the degree of inflammation and interstitial fibrosis. In this model, no statistically significant effect was observed at the RTA408 dose tested (fig. 19a and 19 b). Additional evaluations were performed using a rat model of pulmonary fibrosis that has been extensively characterized at the lovley respiratory Institute (LovelaceRespiratory Research Institute). In this study, rats were challenged with bleomycin or saline by intratracheal administration on day 0. Following challenge, animals received RTA408 (3mg/kg, 10mg/kg, or 30mg/kg) via oral gavage daily for 28 days. The 30mg/kg dose was stopped on day 14 due to excessive dehydration and diarrhea in the animals. For the remaining animals, bronchoalveolar lavage fluid was collected on day 28 for assessment of proinflammatory infiltration by flow cytometry, and lung tissue analyzed for hydroxyproline levels by LC-MS and histopathology. Challenge with bleomycin sulphate induced massive release of neutrophils in BALF and an increase in soluble collagen, as well as an increase in hydroxyproline in the lung. Treatment with 3mg/kg and 10mg/kg RTA408 significantly inhibited the infiltration of Polymorphonuclear (PMN) cells into the lungs and also resulted in a meaningful reduction (about 10% -20%) of hydroxyproline deposition (fig. 20a and 20 b).

Importantly, histopathological evaluation showed a significant reduction in collagen deposition in rats treated with RTA408 as assessed by trichrome staining. Whereas bleomycin control animals predominantly showed moderate staining, animals treated with 10mg/kg RTA408 predominantly had minimal to mild staining (table 2).

Table 2: effect of RTA408 on collagen deposition in rat Lung as assessed by trichrome staining intensity

^aValues represent the intensity of staining of interstitial trichrome staining in areas of pulmonary alteration induced by bleomycin in animals.

Further evaluation of the lungs of rats in this study also showed meaningful involvement of the relevant Nrf2 target gene as determined by Quantigene Plex 2.0 multiplex assay (Affymetrix, Santa Clara, CA) of Santa Clara, california (figure 21). RTA408 significantly and in a dose-dependent manner increased NQO1, Txnrd, Gsr, and Gst enzyme activities in the lungs of rats exposed to bleomycin, demonstrating that RTA408 activates Nrf2 in this disease context. NQO1 enzyme activity was assessed by measuring the rate of reduction of dcppip. Txnrd, Gst, and Gst enzyme activities were measured using a kit commercially available from cayman chemical company (Ann Arbor, MI).

3. Cigarette smoke induced COPD in mice

RTA408 was also tested in a cigarette smoke induced mouse COPD model. Mice received RTA daily via oral gavage408(3mg/kg, 10mg/kg or 30mg/kg) for two weeks and exposed to cigarette smoke 5 days per week during administration of RTA408 at the end of the study, lung tissue and BALF were collected for analysis of inflammatory infiltration and cytokines in this experiment, multiple dose administration of RTA408 at doses as low as 3mg/kgRTA 408 caused significant inhibition of pro-inflammatory cytokines including KC (mouse functional homolog of human IL-8) and TNF- α, such as with Luminex^TMMeasured by the technique. A summary of the results from this study is presented in fig. 22 a-e. AIM analogs (63355) were tested in the same study for comparison. 63355 is a compound having the formula:

further evaluation of lungs from mice in this study also showed significant involvement of the relevant Nrf2 target gene (fig. 23). Cigarette smoke exposure significantly reduced the enzymatic activity of NQO1 in the lungs (measured as the rate of reduction of DCPIP); applying RTA408 rescues this loss. RTA408 at a dose of 30mg/kg also induced Txnrd enzymatic activity. In general, treatment did not alter Gsr enzyme activity, but decreased Gst enzyme activity, both of which may be the result of transient reactions by these enzymes. Txnrd, Gst, and Gst enzyme activities were measured using a kit commercially available from Cayman Chemical company (AnAb, Mich.).

4. Ovalbumin-induced asthma in mice

The potential activity of RTA408 was also evaluated in a preliminary study in an ovalbumin-induced mouse asthma model. Mice were sensitized with intraperitoneal injections of ovalbumin and aluminum hydroxide on days 0 and 14 and were challenged intranasally with ovalbumin in saline on days 14, 25, 26 and 27. Mice received RTA408 (3mg/kg, 10mg/kg, or 30mg/kg) via oral gavage daily on days 1-13 and 15-27. Following sensitization and challenge with ovalbumin, total white blood cell counts were significantly increased in vehicle-treated mice compared to positive control (dexamethasone) -treated mice. An increase in the number of T cells and B cells was also observed in vehicle-treated mice. Treatment with 30mg/kg RTA408 significantly reduced the number and percentage of B cells in the airways. RTA408 (3mg/kg and 30mg/kg) also significantly reduced the number of macrophages detected in the airways, but did not significantly reduce the average percentage of macrophages. These observations suggest potential efficacy in this model.

Effect of RTA408 on LPS-induced sepsis in mice

Sepsis was induced with LPS (21mg/kg) injected intraperitoneally on day 0, and survival was observed until day 4. RTA408 (10mg/kg, 30mg/kg, or 100mg/kg) was administered daily via oral gavage from day-2 to day 2. In the vehicle control group, 60% of the animals survived until day 4 (higher than the expected survival rate of about 40% in this model). In the RTA408 treatment group, 80% of the animals in the 10mg/kg dose group and 90% of the animals in the 30mg/kg dose group survived until day 4 (FIGS. 24c and 24 d). For the 100mg/kg dose group, 90% of the animals survived until day 4, with only one death occurring on day 4. While these effects induced by RTA408 indicate great efficacy in this model, the relatively high survival in the vehicle control group makes no statistically significant difference between the control and RTA408 treated groups possible. The results obtained using compound RTA 405 are also presented (fig. 24a and 24 b). RTA 405 is a compound having the formula:

effect of RTA408 on radiation-induced oral mucositis

Exposure to acute radiation directed at the cheek pouch of the hamster produces an effect similar to that observed in human oral ulcerative mucositis. These effects include moderate to severe mucositis characterized by severe erythema and vasodilation, superficial mucosal erosion, and the formation of ulcers. A single study was conducted to evaluate the role of RTA408 in this model. On day 0, each hamster was given a40 Gy acute radiation dose to the left cheek pouch. RTA408 (10mg/kg, 30mg/kg or 100mg/kg) was orally administered twice daily from day-5 to day-1 and from day 1 to day 15. Oral mucositis was assessed every other day starting on day 6 and continuing until day 28 using a standard 6-point scale. Both the 30mg/kg dose of RTA408 and the 100mg/kg dose of RTA408 resulted in a significant reduction in the duration of ulcerative mucositis (figure 25). In addition, a dose-dependent decrease in the percentage of animals with a mucositis score of > 3 was observed. However, administration of 30mg/kg or 100mg/kg of RTA408 resulted in a significant dose-dependent decrease in body weight gain in the irradiated hamsters. On day 2, 2 out of 8 hamsters in the 100mg/kg dose group were euthanized due to weight loss of more than 20%.

Effect of RTA408 on in vivo Induction of the Nrf2 biomarker

As described above, the key molecular target of RTA408 is Nrf2, and Nrf2 is a core transcriptional regulator of antioxidant cytoprotection. Activation of Nrf2 induces the upregulation of a group of cytoprotective genes including NQO1, enzymes involved in GSH synthesis [ i.e., glutamate-cysteine ligase catalytic and modified subunits (Gclc and Gclm) ], enzymes involved in detoxification [ i.e., glutathione S-transferase [ Gst ]), and efflux transporters [ i.e., multidrug resistance-associated proteins (Mrp) ]. Induction of these genes produces coordinated cellular forces to prevent oxidative damage, and is characterized by increased antioxidant capacity, induced glutathione synthesis, and conjugation and elimination of potentially harmful molecules from the cell. In addition to efficacy endpoints and Nrf2 target gene expression evaluated in the various animal models described above, the ability of RTA408 to induce Nrf2 target gene expression was also evaluated using tissues collected from RTA 408-treated healthy mice, rats, and monkeys.

As part of the non-GLP 14-day toxicity study on RTA408 performed in mice, rats and monkeys, tissues were collected for the purpose of measuring mRNA and enzyme activity levels of selected Nrf2 target genes. For mice andfor rats, liver samples were collected 4 hours after the last dose on day 14. For monkeys, blood (for PBMC isolation), liver, lung, and brain tissue were collected 24 hours after the last dose on day 14. The enzymatic activities of NQO1, Gst, and glutathione reductase (Gsr) as described above were measured in tissue homogenates. The level of mRNA was determined according to the manufacturer's protocol using Quantigene Plex 2.0 technology, which involves the use ofMagnetic beads were subjected to hybridization-based assays for direct quantification of mRNA targets. In addition, RTA408 concentrations in plasma and tissues were measured by LC/MS method on a TQD mass spectrometer (Waters, Milford, MA).

RTA408 increased expression of various Nrf2 target genes substantially in a dose-dependent manner at doses of 10mg/kg, 30mg/kg, and 100mg/kg (fig. 26, 27a, 28a, and 28 b). RTA408 upregulated Nrf2 target gene transcription also resulted in a functional increase in antioxidant responses, as indicated by dose-dependent increases in NQO1, Gst and Gsr enzyme activity in the rodent liver and monkey liver and lung (fig. 29a and 29b, fig. 30a and 30b, fig. 31a and 31 b). Furthermore, liver exposure of RTA408 correlated with the level of enzymatic activity of NQO1 (prototype target gene of Nrf 2) in rodents (fig. 32b, fig. 33 b). In monkeys, mRNA expression levels of both NQO1 and thioredoxin 1(SRXN1) in PBMC correlated with plasma exposure to RTA408 (fig. 37a and 37 b). Overall, RTA408 increased mRNA levels and activity of Nrf2 target, and the increase was generally correlated with tissue and plasma exposure, suggesting that Nrf2 target may serve as a viable biomarker for Nrf2 activation (fig. 34a and b) and may be used to assess the pharmacological activity of RTA408 in healthy human subjects.

D. Safety pharmacology

A GLP compliant safety pharmacology program was completed using RTA 408. This includes in vitro and in vivo (monkey) studies of the cardiovascular system, as well as studies of the respiratory and central nervous systems in rats.

1. Evaluation of the Effect of RTA408 on the cloned hERG channel expressed in HEK293 cells

This study was conducted to evaluate the rapid-activation inward rectifying potassium current (I) of RTA408_Kr) The rapid activating inward rectifier potassium current is conducted by an hERG (human ether-a-go-go related gene) channel stably expressed in a human embryonic kidney (HEK293) cell line. Whole cell patch clamp electrophysiological methods were used to assess the effect of RTA408 on hERG-related potassium currents. Determination of RTA408 has an IC of 12.4. mu.M in the hERG QPatch _ Kv11.1 assay₅₀The value is obtained. This value is 2.5-3 times the value of 63170 (4.9. mu.M) and 63189 (3.8. mu.M), respectively. IC of RTA408₅₀The value was similar to that of 63171 (15.7. mu.M).

2. Cardiovascular evaluation of RTA408 in Cynomolgus monkeys (Cynomolgus Monkey)

A single study was conducted in conscious, freely-moving cynomolgus monkeys to evaluate the potential cardiovascular effects of RTA 408. The 4 male and 4 female cynomolgus monkeys were administered vehicle (sesame oil) and RTA408 at dose levels of 10mg/kg, 30mg/kg and 100mg/kg according to a Latin square design (Latin square design), with one animal/sex/treatment dosed weekly, followed by a 14 day washout period (washout) between administrations until each animal received all treatment. All animals were administered vehicle and RTA408 via oral gavage in a dose volume of 5 mL/kg.

Animals were equipped with telemetry transmitters to measure body temperature, blood pressure, heart rate and perform Electrocardiogram (ECG) evaluations. Body temperature, systolic, diastolic and mean arterial blood pressure, heart rate and ECG parameters (QRS duration and RR, PR and QT intervals) were monitored continuously from at least 2 hours before dosing until at least 24 hours after dosing. ECG traces were printed from cardiovascular monitoring data at designated time points and qualitatively evaluated by a committee certified veterinary cardiologist. The cardiovascular endpoint of untreated animals was continuously monitored for at least 24 hours prior to the first administration at study time, and these data were used to calculate the corrected QT interval throughout the study.

All animals were observed for morbidity, mortality, injury, and food and water availability at least twice daily. Clinical observations were made before dosing, at about 4 hours post-dosing, and after completion of the cardiovascular monitoring period. Body weights were measured and recorded the day before each treatment was administered.

RTA408 caused no death, adverse clinical signs, or significant changes in body weight, body temperature, blood pressure, or qualitative or quantitative (PR interval, RR interval, QRS interval, QT interval) ECG parameters at dosage levels of 10mg/kg, 30mg/kg, and 100mg/kg (FIG. 35; Table 45). In the 100mg/kg dose group, a small (average 1.6%) but statistically significant increase in the corrected QT interval was observed; however, the individual animal data did not show a consistent increase in QTc that would indicate a relevant effect for the test article. Thus, these slight increases in QTc are not considered to be relevant to RTA408 treatment due to the small magnitude of the change and the lack of consistent response in each animal. Thus, oral administration of RTA408 at doses up to and including 100mg/kg had no effect on cardiovascular function in cynomolgus monkeys.

3. Neurobehavioral assessment of RTA408 in rats

RTA408 was evaluated for potential acute neurobehavioral toxicity in rats. 3 each of 10 males and 10 females[(SD)]The treatment groups of rats received RTA408 at dose levels of 3mg/kg, 10mg/kg or 30 mg/kg. Another group of 10 animals/sex served as control and received vehicle (sesame oil). Vehicle or RTA408 was administered once on day 1 to all groups via oral gavage at a dose volume of 10 mL/kg.

All animals were observed for morbidity, mortality, injury, and food and water availability twice daily. Clinical signs were observed prior to dosing on day 1 and after each functional observation combination (FOB) evaluation. The FOB evaluation was performed before dosing (day-1) and at about 4 hours and 24 hours after dosing. Body weight was measured and recorded before dosing on day 1.

RTA408 did not cause death, adverse clinical observations, or effect on any of the neurobehavioral measures tested at doses of 3mg/kg, 10mg/kg, and 30 mg/kg. A slight decrease in body weight gain was observed at about 24 hours post-dose in the 30mg/kg group, which could potentially be correlated with the test article. With respect to the primary neurobehavioral endpoints evaluated in this study, RTA408 did not produce any adverse effects in rats at doses up to and including 30 mg/kg.

4. Pulmonary evaluation of RTA408 in rats

The potential effect of RTA408 on lung function was evaluated in rats. 3 each of 8 males and 8 females[(SD)]The treatment groups of rats received RTA408 at dose levels of 3mg/kg, 10mg/kg or 30 mg/kg. Another group of 8 animals/sex served as control and received vehicle (sesame oil). Vehicle or RTA408 was administered once on day 1 to all groups via oral gavage at a dose volume of 10 mL/kg.

All animals were observed for mortality, morbidity, injury, and food and water availability twice daily. Clinical observations were made before dosing, at about 4 hours post-dosing, and after completion of the 8-hour lung monitoring period. Body weight was measured and recorded on the day of RTA408 administration. Lung function (respiratory rate, tidal volume, and minute volume) was monitored for at least 1 hour prior to dosing to establish baseline and at least 8 hours post-dosing.

RTA408 did not cause death, adverse clinical observations, or effects on any of the lung parameters evaluated at doses of 3mg/kg, 10mg/kg, and 30 mg/kg. Thus, with respect to the primary lung endpoint evaluated in this study, RTA408 did not produce any adverse effects in rats at doses up to and including 30 mg/kg.

E. Non-clinical overview

1. Pharmacokinetics

RTA408 has been studied in vitro and in vivo to assess its PK and metabolic properties. In vitro studies have been performed to determine plasma protein binding and blood/plasma partition of RTA408, inhibition and induction of cytochrome P450(CYP450), and to identify metabolites formed by liver microsomes in mice, rats, monkeys, and humans. Data relating to in vivo absorption and distribution following repeated administration of RTA408 has been obtained primarily by monitoring drug levels in plasma and selected tissues from toxicology studies. Bioanalytical methods (LC/MS) based on sensitive and selective liquid chromatography-mass spectrometry have been used to measure the concentration of RTA408 in plasma, blood and tissues with appropriate accuracy and precision. Measurements were performed on TQD and QToF mass spectrometers (Waters).

a. Absorption of

Absorption and systemic pharmacokinetic behavior of RTA408 was studied in mice, rats and monkeys after single and repeated (daily) oral administration. After oral administration of the suspension formulation at a dose of 10mg/kg to 100mg/kg, maximum concentrations were observed in mice over 1 hour to 2 hours, and in rats and monkeys over 1 hour to 24 hours. Systemic exposure of RTA408 tended to be highest in rats, with lower levels observed in mice and monkeys. Estimates of the apparent terminal half-life of RTA408 observed after oral administration typically range from 6 hours to 26 hours, but in some cases, the significantly extended absorption period makes it impossible to calculate a definitive half-life estimate.

Systemic exposure of RTA408 is substantially similar in males and females. The exposure to RTA408 after repeated daily oral administrations tends to be slightly higher (< 1 fold) than the exposure observed after a single dose. Administration of RTA408 in the form of a suspension formulation in a dosage range of 3mg/kg to 100mg/kg generally results in a dose-proportional increase in systemic exposure. However, administration of higher doses (100 mg/kg to 800mg/kg in monkeys; 500mg/kg to 2000mg/kg in rats) did not result in a similar increase in exposure, indicating that absorption was saturated at doses above 100 mg/kg. Following oral administration of an unoptimized (loosely-filled) capsule formulation of RTA408 (3mg/kg) to monkeys, the systemic exposure normalized for dose tended to be slightly lower than that observed with the suspension formulation.

Single and repeated topical local applications were used in rats to study RTA408 absorption and systemic pharmacokinetic behavior. Administration of RTA408 in the range of 0.01% to 3% showed lower plasma concentrations relative to similar oral dosing. Systemic exposure to RTA408 generally increases in a dose-dependent manner. Topical application was formulated as a suspension in sesame oil.

Rabbits were used to evaluate ocular absorption and systemic pharmacokinetic behavior of RTA 408. RTA408 was topically applied to the eyes once a day for 5 days. Ocular administration showed a lower plasma concentration of RTA408 relative to that of RTA408 when RTA408 was administered orally (fig. 36). The amount of RTA408 in plasma showed only a small change even after 5 consecutive days compared to the concentration after the first dose, relative to when RTA408 was orally administered, the plasma concentration was almost 100-fold higher when RTA408 was orally administered (fig. 36).

b. Distribution of

Plasma protein binding of RTA408 was evaluated in plasma of mice, rats, rabbits, dogs, piglets, monkeys, and humans using ultracentrifugation methods at RTA408 concentrations of 10-2000 ng/mL. RTA408 binds extensively to plasma proteins. Plasma protein binding ranged from 93% (mouse) to > 99% (mini-pig) in non-clinical species, 95% in toxicological species (rat and monkey), and 97% in humans. There is no evidence of concentration-dependent protein binding in any of the species tested. The results from the experiments on the blood to plasma partition indicate that RTA408 tends to distribute primarily in the plasma fraction of the blood in a linear fashion for all species and all concentrations tested, with blood to plasma ratios all < 1.0.

Distribution of RTA408 in tissues has been studied after oral administration to mice, rats and monkeys. In a 14-day non-GLP toxicity study, selected tissues (liver, lung and brain) were collected at a single time point (4 hours for rats and mice; 24 hours for monkeys) after administration of the last dose of the study and analyzed for RTA408 content using LC/MS. RTA408 is readily distributed in the lungs, liver and brain. The RTA408 concentration was similar to or slightly higher (< 1-fold) than the concentration in plasma in mouse and rat lungs at 4 hours, while the RTA408 concentration was 6 to 16-fold higher than the plasma concentration in monkey lungs at 24 hours. A similar pattern was observed for the brain. In contrast, RTA408 concentrations in the livers of mice and rats were 5 to 17 times the plasma concentration at 4 hours, and RTA408 concentrations in the livers of monkeys were 2 to 5 times the plasma concentration at 24 hours.

The pharmacodynamic effect of RTA408 in tissues was assessed in mice, rats and monkeys by monitoring induction of Nrf2 target gene in the same tissues collected from 14-day toxicity studies for drug exposure. Induction of Nrf2 target genes by RTA408 resulted in an increase in antioxidant responses as indicated by dose-dependent increases in NQO1, glutathione S-transferase (Gst) and glutathione reductase (Gsr) enzyme activities in the tissues examined. Enzyme activity was measured as described above. Furthermore, in rodents, the liver content of RTA408 correlates with the level of enzymatic activity of NQO1 (the prototype target gene of Nrf 2). In monkeys, mRNA expression levels of both NQO1 and thioredoxin 1(SRXN1) in Peripheral Blood Mononuclear Cells (PBMCs) correlated with plasma exposure of RTA408 (fig. 37a and 37 b). In summary, RTA408 induced a biomarker for Nrf2 in rodents and monkeys, and the induction generally correlated well with tissue and plasma exposure to RTA 408.

When RTA408 was administered to rabbits via topical ocular administration, the highest concentration of the compound was found in the cornea, retina or iris, while the vitreous humor, aqueous humor and plasma showed significantly lower concentrations of RTA408 (fig. 38).

C. Metabolism

The metabolism of RTA408 has been studied after incubating RTA408 in vitro with liver microsomes from mice, rats, monkeys, and humans for 60 minutes in the presence of a Nicotinamide Adenine Dinucleotide Phosphate (NADPH) regeneration system and a uridine diphosphate glucuronosyltransferase (UGT) reaction mixture. Extensive metabolic switching of RTA408 was observed with primate microsomes, with < 10% of the parent molecule remaining at the end of the 60 min incubation in both monkey and human microsomes. In contrast, the degree of metabolism in rodent microsomes is lower, with > 65% of the parent molecule remaining at the end of the incubation. The lack of available, trusted standards for the various potential metabolites of RTA408 makes it impossible to quantitatively evaluate the observed metabolites. Qualitatively, a similar pattern of RTA408 metabolites is observed across species, and includes peaks of mass consistent with the reduction and hydroxylation of RTA408 and glucuronidation of RTA408 or its reduced/hydroxylated metabolites. No unique human metabolites were observed, and all peaks in human microsomal incubation were also observed in one or more preclinical species. In particular, based on in vitro microsomal data, all human metabolites are present in rats or monkeys, i.e. selected rodent and non-rodent toxic species.

d. Pharmacokinetic drug interactions

The potential of RTA408 to inhibit cytochrome P450(CYP450) -mediated metabolism was evaluated using pooled human liver microsomes and standard substrates for specific CYP450 enzymes. RTA408 direct inhibition of CYP2C8 and CYP3A4/5, K for each enzyme_iThe values were all about 0.5. mu.M. No significant inhibition was observed for the other enzymes tested (CYP1a2, CYP286, CYP2C9, CYP2C19 or CYP2D6), with inhibition at the highest tested concentration (3 μ M) < 50%. In addition, there is little or no evidence of metabolic-dependent inhibition of any of the enzymes tested. Based on these data, as well as the potentially high concentrations that can be achieved locally in the Gastrointestinal (GI) tract after oral administration, future studies investigating the potential for CYP3a4/5 mediated drug-drug interactions may need to be conducted.

Cultured human hepatocytes were used to evaluate the potential of RTA408 to induce CYP450 enzyme expression. RTA408 (up to 3 μ M) is not an inducer of CYP1a2, CYP286 or CYP3a4 enzyme activity in cultured human hepatocytes under conditions in which the prototype inducer causes the expected increase in CYP activity. Enzyme activity was measured by monitoring substrate conversion by CYP1a2, CYP286, and CYP3a4 to phenacetin (phenacetin), bupropion (bupropion), and testosterone, respectively, in isolated microsomes.

Effect of RTA408 on acute radiation dermatitis

The role of RTA408 as a topical local or oral prophylactic agent for acute radiation dermatitis has been studied. Male BALB/c mice were used and irradiated at day 0 with a 30Gy dose (Table 3). Rats are administered either sesame oil vehicle or RTA408 on days-5 to-1 and days 1 to 30. RTA408 was administered orally at 3mg/kg, 10mg/kg, and 30mg/kg in sesame oil and topically at 0.01%, 0.1%, and 1% composition percentage in sesame oil. The dermatitis was evaluated blindly every other day from day 4 to day 30. On day 12, a typical peak in dermatitis was observed and 4 mice were sacrificed at 4 hours after administration of the dose. The remaining mice were sacrificed at 4 hours after the administration on day 30. Plasma and irradiated skin samples were collected on day 12 and day 30 for mRNA and histological examination.

Table 3: research design for acute radiation dermatitis model

In the test group of mice treated with RTA408, the severity of dermatitis that occurred appeared to be slightly reduced when RTA408 was administered orally or topically (fig. 39-42). Furthermore, the curves plotting the mean clinical scores of dermatitis over time for the test groups showed some variation when RTA408 was administered orally or topically in comparison to the untreated test groups (fig. 43-45), especially where RTA408 was given by oral administration. Furthermore, as can be seen in tables 4 and 5 below, the percentage of mice suffering from dermatitis with a clinical score higher than 3 among mice treated with RTA408 by oral administration was significantly lower, while the percentage of mice suffering from dermatitis with a clinical score higher than 2 among the test groups given to RTA408 with topical administration was slightly lower.

Effect of RTA408 on fractionated radiation dermatitis

In the case where RTA408 was utilized by topical application, the effect of RTA408 on improving the effect of fractionated radiation dermatitis was measured. Balb/c mice were used to which RTA408 in the form of a topical formulation was administered daily from day-5 to day 30 at three doses ranging from 0.01% to 1%. Mice were irradiated on days 0-2 and 5-7 with a dose of 10Gy 6 times per day. Mice were blindly evaluated for clinical dermatitis scores every two days from day 4 onward until the end of the study. In fig. 46, the graph shows the variation of the mean clinical score for each group plotted as a function of time. The graph shows statistically significant improvement in the score of mice treated with 0.1% to 1% RTA408 surface topical formulation. The study and treatment parameters can be found in table 6.

Table 6: study conditions for fractionated radiation-induced dermatitis

Area under the curve (AUC) analysis was performed by analyzing the mean clinical scores shown in figure 46 to obtain the severity of the dermatitis relative to the duration of dermatitis. This AUC analysis allowed a direct comparison of the effect of different percent compositions between mice of different groups and RTA408 (fig. 47 and table 7). Application of the topical RTA408 formulation reduced grade 2 and grade 3 lesions from 60% and 33% when mice were exposed to vehicle alone to 21% and 6% respectively with 1% concentration of RTA 408. Other RTA compositions showed some activity, but not as significant as that shown by the 1% formulation.

Table 7: percentage of dermatitis score per treatment group

Group of	% of days is more than or equal to 2	% of days is more than or equal to 3
			No radiation, no treatment	0％	0％
With radiation and without treatment	66％	31％
			With radiation, sesame oil	60％	33％

Group of	% of days is more than or equal to 2	% of days is more than or equal to 3
			With radiation, RTA408 (0.01%)	54％	29％
With radiation, RTA408 (0.1%)	40％	13％
			With radiation, RTA408 (1%)	21％	6％

Synergistic effects of RTA408 and cancer therapeutics on tumor growth

Studies of the effects of RTA408 used in combination with traditional chemotherapeutic agents were conducted to determine the efficacy of potential treatments. An in vitro study was performed to determine the effect of RTA408 on two different prostate cancer cell lines LNCaP and DU-145. As can be seen in figure 48a, 5-fluorouracil-treated prostate cancer cell line (LNCaP) in vitro showed a statistically significant increase in cytotoxicity when combined with RTA408 at doses ranging from 0.125 μ M to 0.5 μ M. Using the prostate cell lines DU-145 and docetaxel, RTA408 amplified the cytotoxicity of chemotherapeutic agents in a statistically significant manner for RTA408 doses from 0.125 μ M to 0.75 μ M, as shown in fig. 48 b. This evidence supports the notion that RTA408 may act synergistically with cancer therapeutics and may be used in some embodiments to provide greater efficacy in treating cancer patients.

After successful results of the in vitro assay were obtained, an in vivo preliminary assay was performed using LNCaP/C4-2B and DU145 human prostate cancer (hereinafter referred to as C4-2B-Luc and DU145-Luc, respectively) engineered to express firefly luciferase. Notably, both cell lines grew in an androgen-independent manner. Cells were cultured in RPMI1640 supplemented with 10% FBS. Cells were harvested using TrypLE Express (Invitrogen) and washed in PBS and counted. Cells were reconstituted in PBS to reach a final concentration of 3 × 106 cells/30 μ Ι _ (unless otherwise specified) and aliquoted in separate tubes. Growth factor-reduced Matrigel (Matrigel) (BD Bioscience) was thawed overnight at +4 ℃ and transferred to tubes in 30 μ L aliquots. The cell/matrigel solution was transferred to an animal feeding room (vivarium) and injected immediately prior to 1: 1 ratio mixing. Each mouse (n ═ 1/group, three animals in total) received a single subcutaneous injection of tumor cells. Tumors were allowed to pre-form for 4 weeks. One animal was then treated with RTA408 (17.5mg/kg, i.p.) once daily for 3 days (days-3 to-1). The next day (day 0), the RTA408 treated animals and another animal were treated with a single dose of 18Gy IR, which was localized to the pelvic region where the tumor was implanted. Over the following week, mice pretreated with RTA408 received three additional doses of RTA408 (17.5mg/kg, i.p.) once every other day. The third animal received no treatment and served as a positive control. Tumor progression was monitored weekly via real-time imaging. To detect luciferase-expressing tumor cells, mice were injected intraperitoneally with D-luciferin 5 minutes prior to imaging according to the manufacturer's protocol (Caliper life science). Prior to imaging, mice were anesthetized by isoflurane inhalation and imaged on the IVIS lumine XR system (calipers life science). For normalization, the minimum exposure time necessary to image control tumors was determined and all animals were imaged under these conditions. On day 7, no significant reduction in tumor size was seen in the IR-treated animals compared to the controls, while animals receiving both RTA408 and IR showed smaller tumor images. Control animals showed sustained tumorigenesis and growth on days 14 and 21, while animals treated with ionizing radiation showed some improvement, most notably on day 21. On the other hand, animals treated with RTA408 and ionizing radiation showed no progression from day 7 to day 14 and no visible tumor on day 21. Weekly progression of tumors can be seen in fig. 49. Both in vitro and in vivo data demonstrate that RTA408 appears to complement the activity of different cancer therapeutic agents, thereby improving the efficacy of the agents.

Effect of RTA408 on models of ocular inflammation

A study was conducted on the effect of RTA408 on ocular inflammation using a new zealand albino strain of rabbits. Rabbits were divided into 5 groups (12 rabbits each group) and given three different concentrations (0.01%, 0.1% and 1%) of RTA408, 0.1% ofEye drops and vehicle (sesame oil). Three instillations were given to each rabbit within 60 minutes prior to puncture induction and two instillations were given within 30 minutes after puncture induction. Each instillation was 50 μ L and was given in both eyes. Aqueous humor was collected from 6 animals at each time point 30 minutes after puncture induction and again at 2 hours. The amount of inflammation is determined by the protein concentration in the aqueous humor. As shown in FIG. 50, only at 0.01% RTA408 in the formulation, RTA408 showed similar aqueous humor protein reduction to any of the highest concentrations of other reference compounds (MaxiDex or Matlacco-latt)Has less effect. The effect of increasing RTA408 concentration appears to be negligible, since all concentrations of RTA408 appear to show relatively similar effects within the error range in reducing the concentration of aqueous humor protein.

J. Polymorph screening

Compound 63415 was pre-formulated and polymorphism studied. As part of this study, a preliminary polymorphism protocol was carried out with the aim of identifying, with a rather high probability, the anhydrous form and the possible hydrates that are most stable at room temperature. A total of 30 crystallization experiments were performed including phase equilibration, drying experiments and other techniques. All obtained solids were characterized by FT-raman spectroscopy. All new forms were characterized by PXRD and TG-FTIR and optionally by DSC and DVS.

In addition, amorphous forms were prepared and characterized. Several experiments using different techniques and methods were performed to prepare the amorphous form. The amorphous form was characterized by FT-raman spectroscopy, PXRD, TG-FTIR, DSC, DVS and karl-fischer titration. The stability of the amorphous form was tested over a4 week process under high humidity and high temperature conditions.

1. Starting materials and nomenclature

Two batches of 63415 were used as starting material (table 8). 63415 is also referred to in this disclosure as PP 415. All samples obtained or generated during this planning process obtain a unique identification code in the form of PP415-Px, where Px refers to the sample/experiment number (x ═ 1, 2.... n).

Table 8: starting materials

2. Compound 63415, batch No. 0414-66-1(PP 415-P1):

amorphous form

By FT-Raman spectroscopy, PXRD, TG-FTIR, Karl-Fischer titration,¹H-NMR, DSC, DVS and approximate solubility measurements 63415 runs 0414-66-1 starting material were characterized. The results are summarized in table 9.

Table 9: 63415 characterization of the starting Material (PP415-P1)

The FT-raman spectrum (fig. 58) will be used as a reference spectrum for the starting material. PXRD (fig. 59) did not show a spike pattern. The broad halo peak at about 10 ° -20 ° 2 θ is characteristic of amorphous material.

TG-FTIR thermogram (FIG. 60) shows about 0.9 wt% EtOH (i.e. about 0.1 equiv.) with trace H from 25 ℃ to 200 ℃₂Gradual loss of O. Decomposition started at T > 290 ℃.

The water content of 0.5% by weight was determined by Karl-Fischer titration.

¹The H-NMR spectrum (fig. 61) is consistent with the structure and shows about 0.08 equivalents of EtOH, which is consistent with the TG-FTIR thermogram.

DSC thermogram (FIG. 62) showing amorphous material at T in the first heating scan_g＝152.7℃(ΔC_pGlass transition occurred at 0.72J/g ℃). In the second scan after quenching, at T_g＝149.7℃(ΔC_pGlass transition occurred at 0.45J/g ℃).

DVS isotherms (fig. 63) shows that a gradual mass loss of 1.0 wt.% occurs after decreasing the relative humidity from 50% relative humidity to 0% relative humidity; equilibrium was reached at 0% relative humidity. After increasing the relative humidity to 95% relative humidity, a gradual mass increase of 2.1 wt% (relative to the mass at 0% relative humidity) occurred; equilibrium is reached at 95% relative humidity. After lowering the relative humidity from 95% relative humidity to 50% relative humidity, the final mass was 0.2 wt% lower than the starting mass. A mass gain of 0.4 wt% at 85% relative humidity (relative to the starting mass) classifies the sample as slightly hygroscopic.

The FT-raman spectrum (fig. 64) and PXRD pattern (fig. 65) of the sample after DVS measurement were unchanged from the spectrum and pattern of the sample before the measurement.

The approximate solubility of the PP415-P1 starting material was measured in a mixture of 12 solvents and 4 solvents by manual dilution plus visual observation at room temperature (table 10). Due to experimental error inherent in this approach, these solubility values are intended to be considered rough estimates and are only used in the design of crystallization experiments. All solvent mixtures are listed in volume ratio (v/v).

Table 10: approximate solubility of PP415-P1 (amorphous) starting Material

Solvent(s)

Solubility S [ mg/mL]

Solvent(s)	Solubility S [ mg/mL]
		Toluene	S＞200
DCM	S＞200
		EtOAc	S＞210
Acetone (II)	S＞230
		MeCN	S＞230
DMF	S＞210
		MeOH	S＜210
EtOHa	105＜S＜210
		2PrOH	16＜S＜19
DEE	S≥1d
		Heptane (Heptane)	S＜1
H2O	S＜1
		2PrOH/H2O(9∶1)b	7.9＜S＜8.5
MeCN/H2O(2∶3)c	S＜1
		EtOAc/heptane (1: 1)a	100＜S＜200
toluene/DEE (1: 1)a	S＞220

^aPrecipitation was observed after about 1 day;

^bwater Activity a (H) at 25 ℃₂O) is about 0.7;

^cwater Activity a (H) at 50 ℃₂O)＞0.9；

^dInitially, it did not dissolve completely (S < 1), but the solid residue dissolved completely overnight (S > 1).

3. Compound 63415, run No. 2083-69-DC (PP 415-P40): class 2

63415 run Nos. 2083-69-DC are heptane solvates. This material (PP415-P40) was characterized by PXRD and found to correspond to class 2 (fig. 66).

Class 2 may correspond to isomorphic non-stoichiometric (< 0.5 equivalents) solvates with tightly bound solvents (solvates of heptane, cyclohexane, isopropyl ether, 1-butanol, triethylamine, and possibly other solvents such as hexane and other ethers).

The small peaks visible at 7.9 ° 2 θ and 13.8 ° 2 θ in the maps of PP415-P40 do not correspond to the peaks of categories 3, 4 or 5. Their origin is unclear at this point.

4. Chemical stability of the amorphous form

The chemical stability of the amorphous form in different solvents was investigated over the course of 7 days.

Solutions/suspensions were prepared at a concentration of 1mg/mL in 4 organic solvents (acetone, MeOH, MeCN, EtOAc) and three aqueous surfactant media (1% aqueous SDS, 1% aqueous Tween 80, 1% aqueous CTAB).

4 separate solutions/suspensions were prepared for each solvent, allowed to equilibrate for 6 hours, 24 hours, 2 days and 7 days and then analyzed by HPLC.

The relative area% obtained from the HPLC chromatogram is given in table 11. The compound appears to be somewhat unstable in the diluent (MeCN with 0.1% formic acid); during the course of the sequence (i.e. within about 24 hours), the area% of the reference sample (PP415-P1, run at the beginning and end of the sequence) decreased from 99.9% to 99.3% at 254nm and from 99.9% to 99.5% at 242 nm. Due to this effect, the samples measured towards the end of the sequence (set in the following order: 7 days, 2 days, 24 hours, 6 hours) may be affected and the area% obtained may be underestimated.

Table 11: 63415 chemical stability of the amorphous form (PP415-P1)

^aAt the third wavelength (210nm), the signal strength is weak and the signal-to-noise ratio is large, so no integration is performed

^bSuspensions, for all time points, not all substances were dissolved

^cSuspension, not all solids dissolved for time points 24 hours and 6 hours

Decomposition of ≧ 1% was observed for the solution in MeCN (after 7 days) and for the suspension in 1% aqueous Tween 80 medium (254 nm at all time points, and 242nm after 24 hours, 2 days, and 7 days).

5. Storage stability of the amorphous form

To gain more insight into the basic properties and physical stability of the amorphous form of 63415, it was stressed by storage at high temperature and high relative humidity.

Samples of the amorphous form (PP415-P1 starting material) were tested at 25 deg.C/about 62% relative humidity (at saturated NH)₄NO₃On aqueous solution) and 40 ℃/about 75% relative humidity (on saturated aqueous NaCl solution) and closed storage at 60 ℃ and 80 ℃ (table 12). At time points 0, 1, 2 and 4 weeks, the samples were examined by PXRD and compared to the starting material PP 415-P1.

Storage stability testing of the amorphous form of Table 12.63415 (PP415-P1)

Sample (I)	Condition	Point in time	PXRD results
				PP415-P2a	Open, 25 deg.C/about 62% relative humidity	1 week	Amorphous form
PP415-P2b	Open, 25 deg.C/about 62% relative humidity	2 weeks	Amorphous form
				PP415-P2c	Open, 25 deg.C/about 62% relative humidity	4 weeks	Amorphous form
PP415-P3a	Open, 40 deg.C/about 75% relative humidity	1 week	Amorphous form
				PP415-P3b	Open, 40 deg.C/about 75% relative humidity	2 weeks	Amorphous form
PP415-P3c	Open, 40 deg.C/about 75% relative humidity	4 weeks	Amorphous form
				PP415-P4a	Closed type, 60 deg.C	1 week	Amorphous form
PP415-P4b	Closed type, 60 deg.C	2 weeks	Amorphous form
				PP415-P4c	Closed type, 60 deg.C	4 weeks	Amorphous form
PP415-P5a	Closed type, 80 deg.C	1 week	Amorphous form
				PP415-P5b	Closed type, 80 deg.C	2 weeks	Amorphous form
PP415-P5c	Closed type, 80 deg.C	4 weeks	Amorphous form

After 1 week (time point 1 week, fig. 67), 2 weeks (time point 2 weeks, fig. 68) and 4 weeks (time point 4 weeks, fig. 69), all 4 samples were still amorphous, since the powder X-ray diffraction patterns showed no difference compared to the starting material at time point 0 week.

6. Crystallization and drying experiments

a. Crystallization experiment

Phase equilibrium, crystallization from hot solution and evaporation experiments were performed with the amorphous form as starting material in order to identify with a rather high probability the most suitable anhydrous form and possible hydrates at room temperature. All obtained species were characterized by FT-raman spectroscopy; selected samples were also characterized by PXRD.

FT-raman spectra are classified into classes according to the similarity of their peak positions. The original sample (PP415-P1, see table 8) was classified together with the crystalline product. However, the spectra within a class are not identical, but are similar. There may be small differences and peak shifts. Considering only FT-raman spectra, it is difficult to determine whether a class of spectra belongs to the same polymorph.

Peaks in PXRD patterns were determined and then classified into individual clusters using PANalytical X' pert (highscore plus) software. These clusters identify spectra with high similarity. However, there are small but significant differences within a cluster. Thus, the patterns within a cluster do not necessarily correspond to the same polymorph, but represent different forms having very similar molecular structures. The FT-raman class corresponds to a PXRD cluster in all cases.

b. Suspension equilibrium experiment

Suspension equilibration experiments were performed in one solvent and 11 solvent mixtures (table 13). A suspension of about 100mg PP415-P1 in 0.2-2.0mL of the selected solvent was prepared and shaken at 22 deg.C-24 deg.C for 4-15 days. The solid was recovered and characterized by FT-raman spectroscopy; most solids were also characterized by PXRD.

TABLE 13 suspension equilibration experiments with the amorphous form (PP415-P1) as starting material

Sample (I)	Solvent/mixture	FT-Raman class	PXRD cluster
				PP415-P6	2PrOH	3	3
PP415-P7	1: 2 EtOAc/heptane	2	2
				PP415-P8	1: 2 acetone/hexane	2	2
PP415-P9	1: 3 toluene/DEE	2d	--
				PP415-P10	1∶3MeOH/TBME	2	2
PP415-P11	1: 2 MEK/cyclohexane	2d	--
				PP415-P12	9∶1EtOH/H2Oa	3	3
PP415-P13	7∶3MeCN/H2Ob	4d	4
				PP415-P14	About 1: 1THF/H2Oc	5d	5
PP415-P29	1∶2EtOAc/TEA	2	2
				PP415-P31	9∶1PEG/H2O	1	1
PP415-P35	7∶3MeCN/H2Ob	4d	4

Water activity:^aa (H) at 50 ℃₂O) is about 0.5;^ba (H) at 50 ℃₂O) is about 0.85;^ca (H) at 64 DEG C₂O)＞0.99；

^dThe spectrum contains a solvent signal

c. Crystallization from hot solution

A hot solution of PP415-P1 was prepared in one solvent and a mixture of 4 solvents (Table 14). After slow cooling to 5 ℃ at a rate of about 0.2K/min, precipitation was observed in three cases (-P20, -P21, -P24). In both cases (-P22, -P23), no solid precipitated even after 2 days of storage at 4 ℃ -5 ℃.Here, in N₂The solvent was evaporated at room temperature under reduced pressure. Solids were recovered and characterized by FT-raman spectroscopy, and for those solids with spectra different from the amorphous starting material (FT-raman class 1), also by PXRD.

Table 14: slow Cooling experiment starting from amorphous form (PP415-P1)

^aNo precipitate was formed after slow cooling and stirring at 5 ℃ for 2 days; in N₂Evaporation of solvent at room temperature under stream

^bThe spectrum contains a solvent signal

d. Evaporation/precipitation experiments

Clear solutions of PP415-P1 were prepared in a three solvent mixture (Table 15). The solvent was then slowly evaporated at room temperature under ambient conditions. However, in two of the three experiments (-P15 and-P17), a white solid precipitated before evaporation began. The obtained solid was checked by FT-raman spectroscopy and PXRD.

TABLE 15 Slow Evaporation experiment of the amorphous form (PP415-P1)

Sample (I)	Solvent/mixtureCompound (I)	FT-Raman class	PXRD cluster
				PP415-P15	1∶2DCM/IPE	2a	2
PP415-P16	1: 2 MeOH/toluene	1a	--
				PP415-P17	1: 3 EtOAc/heptane	2a	2

^aThe spectrum contains a solvent signal

e. Drying experiment

At least one sample of each class was dried under vacuum in order to desolvate the solvate and obtain 63415 as an unsolvated crystalline form (table 16). The dried material was further characterized by FT-Raman, PXRD, and TG-FTIR.

TABLE 16 drying experiments on samples obtained from crystallization experiments

^aThe desolvation is successful, the solvent content is obviously reduced, and the sample is mainly amorphous; there are only few broad peaks in PXRD

^bThe samples were less crystalline as indicated by the broader peaks in PXRD

^cThe desolvation is successful, the solvent content is obviously reduced, and the sample is still crystallized; no change in structure

7. Characterization of novel forms (classes)

a. Summary of New Categories

In addition to the amorphous form of 63415, 4 new crystalline forms were obtained in this study (table 17).

Table 17: summary of obtained categories

Class 2: most crystallization experiments produced solid material of class 2. These samples may correspond to isomorphic non-stoichiometric (< 0.5 equivalents) solvates with tightly bound solvent molecules (solvates of heptane, cyclohexane, isopropyl ether, 1-butanol, triethylamine, and possibly hexane and other ethers, etc.). Raman spectra and PXRD patterns within this class are very similar to each other and therefore the structures may be substantially identical with only small differences due to the different solvents incorporated.

Drying experiments on Category 2 samples did not produce crystalline, unsolvated forms, even at high temperatures (80 ℃) and high vacuum (< 1 × 10)^-3Mbar) still do not completely remove tightly bound solvent molecules; always maintain a solvent content of > 2 wt%. The crystallinity of these samples decreased, but neither conversion to a different structure nor significant amorphization was observed.

Class 3: the class 3 solid material was obtained from multiple crystallization experiments. A class 3 sample may be an isomorphic solvate of 2PrOH, EtOH and possibly acetone with tightly bound solvent molecules. They may correspond to a stoichiometric hemisolvate or non-stoichiometric solvate with a solvent content of about 0.5 equivalents. As with class 2, the raman spectra and PXRD patterns within this class are very similar to each other, indicating similar structures incorporating different solvents.

Similar to class 2, drying experiments were also unsuccessful, very tightly bound solvent molecules could only be partially removed (at 1 × 10)^-3About 5.4 wt.% to about 4.8 wt.% was removed after mbar and 80 ℃ for up to 3 days). The PXRD pattern remains unchanged.

Class 4: from 7: 3MeCN/H only₂The O solvent system yields a class 4 material. It most likely corresponds to a crystalline acetonitrile hemisolvate.

By drying (under vacuum or N)₂Flowing down at high temperature), most of the solvent can be removed without changing or destroying the crystal structure (PXRD remains unchanged). Thus, a crystalline unsolvated form (or more precisely, a desolvated solvate) is obtained. It is slightly hygroscopic (about 0.7 wt% increase in mass from 50% relative humidity to 85% relative humidity) and has a possible melting point of 196.1 ℃ (Δ H ═ 29.31J/g).

Class 5: also from only one solvent system (about 1: 1 THF/H)₂O) obtained class 5 and which contains bound THF (and possibly H)₂O). Since the contents of these two components cannot be quantified separately, the exact nature of this crystalline solvate cannot be determined.

Drying category 5 causes complete desolvation and conversion to the amorphous form (category 1). One possible method of preparing the amorphous form from the class 2 material is to convert class 2 to class 5, followed by drying and amorphization.

b. Class 1-amorphous form

The amorphous form of category 1, 63415, was obtained from several crystallization experiments (table 18). Most crystallization experiments yielded crystalline material of class 2, 3, 4 or 5.

The starting material PP415-P1 is amorphous and belongs to class 1. Further experiments were carried out with the aim of preparing the amorphous form only (category 1).

TABLE 18 crystallization experiments to produce solid materials of Category 1

^aNo precipitate after slow cooling and stirring for 2 at 5 ℃; in N₂Evaporation of solvent at room temperature under stream

c. Class 2-isomorphic solvates (e.g. heptane)

Most crystallization experiments yielded solid material of class 2 (table 19). In addition, a batch of the class 2 heptane solvate PP415-P40 was used as starting material (see Table 8).

Although the FT-raman spectra of class 2 are significantly similar to each other (fig. 70), small differences are still shown. They differ significantly from the spectrum of amorphous starting material class 1 (fig. 71) and the spectra of classes 3, 4 and 5 (fig. 72).

The PXRD pattern for class 2 (fig. 73) confirmed the crystallinity of the material. Although the profiles of the samples were very similar to each other, small differences were still shown (fig. 74). The profile of category 2 is significantly different from the profiles of categories 3, 4 and 5 (fig. 75).

The TG-FTIR thermogram of sample PP415-P7 (fig. 76) shows about 7.5 wt% loss of EtOAc and heptane in two steps from about 100 ℃ to 290 ℃ and decomposition at temperature T > 290 ℃. Before the TG-FTIR experiment, the sample was dried briefly (about 5 minutes) under vacuum (10-20 mbar) to remove excess unbound solvent. Loss of both EtOAc and heptane occurred together in the same temperature range; both solvents appear to be tightly bound within the structure. The theoretical EtOAc content (boiling point 76 ℃) of the hemisolvate was 7.4 wt%, and the theoretical heptane content (boiling point 98 ℃) of the hemisolvate was 8.3 wt%. Unfortunately, the contents of the two components cannot be quantified separately.

The TG-FTIR thermogram (fig. 77) of sample PP415-P21 shows about 5.8 wt% loss of cyclohexane in two steps from about 140 ℃ to about 250 ℃ and decomposition at temperatures T > 250 ℃. In the case of cyclohexane having a boiling point of 81 ℃, the solvent appears to be tightly bound within the structure. The theoretical cyclohexane content of the hemisolvate is 7.1% by weight. Thus, samples PP415-P18 likely correspond to a non-stoichiometric cyclohexane solvate (with a solvent content of < 0.5 equivalents).

The TG-FTIR thermogram (fig. 78) of sample PP415-P24 shows a loss of about 16.6 wt.% of 1BuOH in the step from about 50 ℃ to about 160 ℃, a further loss of 1BuOH (6.6 wt.%) in the second step from 160 ℃ to 230 ℃ and decomposition at a temperature T > 230 ℃. At least the solvent of the second step appears to be tightly bound within the structure in the case of 1BuOH having a boiling point of 117 ℃. The theoretical 1BuOH content of the hemisolvate was 6.3% by weight.

The TG-FTIR thermogram of sample PP415-P29 (fig. 79) showed a loss of about 5.1 wt% EtOAc and TEA from about 50 ℃ to about 220 ℃, most of which was lost in the step from 180 ℃ to 210 ℃. The decomposition takes place at a temperature T > 220 ℃. Both EtOAc and TEA losses occur together in the same temperature range; both solvents appear to be tightly bound within the structure (boiling point of EtOAc 77 ℃ and boiling point of TEA 89 ℃).

TG-FTIR thermogram of sample PP415-P47 (fig. 80) shows typical two-step mass loss of class 2 at temperatures up to 240 ℃ (total about 7.9 wt% EtOAc), indicating very tight binding of solvent molecules.

The TG-FTIR thermogram (fig. 81) of sample PP415-P48 shows a mass loss of about 3.5 wt% ethyl formate and water, which occurs gradually at the beginning and then in distinct steps from 180 ℃ to 200 ℃. There may be further loss of ethyl formate accompanied by decomposition at T > 240 ℃.

Thus, samples of class 2 may all correspond to non-stoichiometric (< 0.5 equivalents) isomorphic solvates with tightly bound solvent molecules. Since the raman spectrum and PXRD pattern within this class are very similar to each other, the structures may be substantially identical to each other with only small distortions of the unit cell size or small changes in the atomic positions within the unit cell due to the differences in the size and shape of the solvent molecules being incorporated.

Table 19: crystallization experiments to produce solid materials of class 2

^aStarting materials: PP415-P40, Category 2; in all other experiments, PP415-P1 (Category 1) was used as starting material.

d. Drying experiments on class 2 samples

Various samples of class 2 were dried under vacuum (and some at elevated temperatures) and attempted to desolvate them with the aim of obtaining 63415 in anhydrous form. Details and characterization of the dried samples are provided in table 20 below.

However, even at 80 ℃ and vacuum < 1 × 10^-3Drying for 3 days in mbar does not allow complete removal of the tightly bound solvent molecules; solvent content > 2 wt% was retained (see sample-P32 and sample-P34). PXRD patterns showed that the crystallinity of these samples decreased, but no conversion to a different structure was observed.

TABLE 20 drying experiments on Category 2 samples

^aSlightly less crystalline according to PXRD

Thus, the class 2 solvate appears to have very tightly bound solvent molecules. It is difficult to desolvate or transform/amorphize them.

e.PP415-P7→PP415-P30

The solid material of sample PP415-P7 (Category 2) obtained from the suspension equilibration experiment in 1: 2 EtOAc/heptane was dried (as PP415-P30) under vacuum for several days (1-10 mbar, 50 ℃ -70 ℃).

The FT-raman spectrum of the dried class 2 species (PP415-P30) showed little difference from the original spectrum (sample PP415-P7, fig. 82), but still corresponded to class 2.

The PXRD pattern of the dried class 2 material (PP415-P30) showed a slightly broader peak of lesser intensity (fig. 83), but still corresponded to class 2.

The TG-FTIR thermogram (fig. 84) of the dried sample PP415-P30 shows a loss of about 2.5 wt% heptane (and some EtOAc) in two steps from about 50 ℃ to about 250 ℃ and decomposition at a temperature T > 250 ℃. Two solvent loss steps were maintained compared to the TG-FTIR of samples PP415-P7 (FIG. 76), but the total amount of solvent in the samples was reduced from about 7.5 wt% in PP415-P7 to about 2.5 wt% in PP 415-P30.

Therefore, attempts to desolvate this solvate at high temperatures (50 ℃ C. -70 ℃ C.) and under vacuum of 1-10 mbar caused only a partial loss of solvent.

f.PP415-P15→PP415-P18

The solid material of sample PP415-P15 (Category 2) obtained from the precipitation experiment in 1: 2DCM/IPE was dried (as PP415-P18) under vacuum (ca. 2-20 mbar) at room temperature for about 2 hours.

The FT-Raman spectra of the PP415-P18 were identical to the spectra of the sample PP415-P15 (FIG. 85), both of which correspond to Category 2.

The PXRD pattern of PP415-P18 showed little difference from the pattern of PP415-P15 (FIG. 86). PP415-P18 still corresponds to category 2.

TG-FTIR thermogram (fig. 87) shows about 7.0 wt% IPE loss in two steps from about 140 ℃ to about 250 ℃ and decomposition at temperature T > 250 ℃. In the case of IPE with a boiling point of 67 ℃, the solvent appears to be tightly bound within the structure. The theoretical IPE content of the hemisolvate was 8.4 wt%.

Unfortunately, no TG-FTIR of the material was recorded before the drying step. However, since the solvent appears to be so tightly incorporated into the structure and no changes (or only small changes) are observed in the FT-raman spectra and PXRD patterns, it is assumed that the drying has no significant effect on structure or solvent content.

g.PP415-P17→PP415-P19→PP415-P32

The solid material of sample PP415-P17 (Category 2) obtained from the precipitation experiment in 1: 3 EtOAc/heptane was dried (as PP415-P19) under vacuum (ca. 2-20 mbar) at room temperature for about 2 hours.

The FT-Raman spectra of PP415-P19 were identical to the spectra of sample PP415-P17 (FIG. 88); no change was observed and both correspond to category 2.

The PXRD pattern of PP415-P19 is slightly different from the pattern of PP415-P17 (FIG. 89), but still corresponds to Category 2.

TG-FTIR thermogram (fig. 90) shows about 7.6 wt% loss of heptane in two steps from about 140 ℃ to about 270 ℃ and decomposition at temperature T > 270 ℃. In the case of heptane having a boiling point of 98 ℃, the solvent appears to be tightly bound in the structure. The theoretical heptane content of the hemisolvate was 8.3 wt%.

Further drying experiments were carried out on the same samples as PP415-P32 (80 ℃ C., < 1 × 10)^-3Mbar, 3 days).

The FT-Raman spectrum remained unchanged (FIG. 88). The PXRD pattern still corresponded to class 2 (fig. 89), but the sample was less crystalline (because the peaks were broader and had a lower S/N ratio).

The TG-FTIR thermogram (fig. 90) shows about 2.2 wt% heptane loss, most of which is lost in the steps from 170 ℃ to 200 ℃, and decomposition at temperatures T > 250 ℃.

Thus, the heptane content was reduced only from 7.6 wt% to 2.2 wt%, confirming the tight binding of the solvent molecules.

h.PP415-P21→PP415-P28→PP415-P34

The solid material from sample PP415-P21 (Category 2) obtained from a slow cooling experiment in about 1: 5 EtOH/cyclohexane was dried (as PP415-P28) under vacuum for several days (2-20 mbar, room temperature to 60 ℃).

The FT-Raman spectrum of the dried class 2 species (PP415-P28) showed little difference from the spectrum of class 2 (sample PP415-P21, FIG. 92), but still corresponded to class 2.

The PXRD pattern of the dried class 2 material (PP415-P28) showed broader, less intense peaks (fig. 93) compared to the pattern of PP415-P21, indicating that the dried sample was less crystalline. However, the map still corresponds to class 2.

The TG-FTIR thermogram (fig. 94) of the dried sample PP415-P28 shows about 3.0 wt.% loss of cyclohexane in two steps from about 140 ℃ to about 250 ℃ and decomposition at a temperature T > 250 ℃. Two solvent loss steps were maintained compared to the TG-FTIR of samples PP415-P21 (fig. 77), but the total amount of solvent in the samples was reduced from about 5.8 wt% in PP415-P21 to about 3.0 wt% in PP 415-P28.

Thus, desolvation of this solvate appears to cause only a partial loss of solvent, similar to a partial loss of crystallinity.

This sample was further dried (< 1 × 10 at 80 ℃^-3Mbar, 3 days) (as PP 415-P34).

The FT-Raman spectrum remained unchanged (FIG. 92). The PXRD pattern still corresponds to class 2 (fig. 93), but the sample is less crystalline (because the peaks are broader and have a lower S/N ratio).

The TG-FTIR thermogram (fig. 95) shows a loss of about 2.3 wt.% cyclohexane in two steps from 25 ℃ to 270 ℃ and decomposition at a temperature T > 270 ℃.

Thus, the cyclohexane content was reduced only from 3.0 wt% to 2.3 wt%, confirming the tight binding of the solvent molecules.

i. Class 3-isomorphic solvates (e.g. ethanol)

Several crystallization experiments yielded a solid material of class 3 and was characterized by FT-raman spectroscopy, PXRD and TG-FTIR (table 21).

The FT-raman spectra of class 3 are clearly similar to each other (fig. 96), but still show small differences (fig. 97). The spectrum of class 3 is significantly different from the spectrum of the amorphous starting material (class 1) (fig. 98) and the spectra of classes 2, 4 and 5 (fig. 72).

The PXRD pattern for class 3 (fig. 99) confirmed the crystallinity of the material. The profiles of the three samples, although similar to each other, still showed a small but significant difference (figure 100). The class 3 pattern is significantly different from the crystalline patterns of classes 2, 4 and 5 (fig. 75).

The TG-FTIR thermogram (plot 100) of sample PP415-P6 showed a loss of about 5.4 wt% 2PrOH from 25 ℃ to 250 ℃, most of which was lost in the step from about 170 ℃ to 190 ℃. Decomposition starts at temperatures T > 250 ℃. Before the TG-FTIR experiment, the sample was dried briefly (for about 5 minutes) under vacuum (10-20 mbar) to remove excess unbound solvent. The theoretical 2PrOH (boiling point 82 ℃) content of the hemisolvate is 5.1% by weight.

The TG-FTIR thermogram (fig. 101) of sample PP415-P12 shows a loss of about 4.9 wt% EtOH (with trace amounts of water) from 25 ℃ to 250 ℃, most of which is lost in the step from about 160 ℃ to 190 ℃. Decomposition starts at temperatures T > 250 ℃. The theoretical EtOH (boiling point. 78 ℃) content of the hemisolvate was 4.0 wt.%.

Thus, the class 3 sample appears to be an isomorphic solvate of 2PrOH, EtOH and possibly acetone with tightly bound solvent content. They may correspond to stoichiometric hemisolvates. However, non-stoichiometric solvates of these forms cannot be excluded.

Since the raman spectrum and PXRD pattern within this class are very similar to each other, the structure may be essentially the same with only a small distortion of the unit cell size or a small change in the atomic position within the unit cell due to incorporation of different solvent molecules.

TABLE 21 crystallization experiments to produce solid materials of Category 3

j. Drying experiments on class 3 samples

One of the samples of class 3 (PP415-P6) obtained from the suspension equilibration experiments performed in 2PrOH was dried (as PP415-P25) under vacuum for several days (2-20 mbar, room temperature to 60 ℃, table 22).

The TG-FTIR thermogram (fig. 102) of this dried class 3 material sample PP415-P25 showed a loss of about 5.4 wt% 2PrOH from 50 ℃ to 250 ℃, most of which was lost in the step from 170 ℃ to 190 ℃, another loss of about 1.0 wt% 2PrOH from 290 ℃ to 320 ℃ and decomposition at a temperature T > 320 ℃. The solvent content did not appear to decrease significantly with a solvent content of about 5.4 wt% of 2PrOH compared to TG-FTIR of original class 3 sample PP415-P6 (figure 103).

Under high vacuum and high temperature (< 1 × 10)^-3This material was further dried (as PP415-P33, table 22) at 80 ℃) for 3 days with the aim of desolvating the solvate and obtaining 63415 as an unsolvated anhydrous form.

The TG-FTIR thermogram (fig. 103) of this further dried class 3 species sample PP415-P33 shows a loss of about 4.2 wt.% 2PrOH from 50 ℃ to 210 ℃, most of which is lost in the step from 160 ℃ to 190 ℃, another loss of about 0.5 wt.% 2PrOH from 210 ℃ to 290 ℃ and decomposition at a temperature T > 290 ℃.

The solvent content was reduced from only about 5.4 wt% to about 4.8 wt% compared to the solvent content of samples PP415-P6 and PP 415-P25.

TABLE 22 drying experiments on samples of Category 3

The FT-raman spectra of class 3 (samples PP415-P6), the dried substance of class 3 (samples PP415-P25) and the further dried substance of class 3 (samples PP415-P33) were identical and showed no change (fig. 104).

The PXRD patterns of class 3 (samples PP415-P6) and class 3 further dried material (samples PP415-P33) did not show any significant difference, but had little small shift and difference from the patterns of class 3 preliminary dried material (samples PP415-P25, fig. 105). All maps correspond to class 3.

Since drying does not have a great influence on the solvent content, it is not surprising that the FT-raman spectrum and PXRD pattern of the dried material show no difference compared to the undried material.

Thus, class 3 is a class of isomorphic solvates (2PrOH, EtOH and possibly acetone) with very tightly bound solvent molecules, by the drying conditions applied here (at 1 × 10)^-3Mbar and 80 ℃ for up to 3 days) can only partially (about 5.4% to about 4.8% by weight) remove the solvent molecules.

k. Class 4-acetonitrile solvates

From 7: 3MeCN/H only₂The O solvent mixture yielded category 4 (table 23). The experiment that produced category 4(PP415-P13) (as PP415-P35) was repeated to prepare more material for further drying studies.

The FT-raman spectra (fig. 72) and PXRD spectra (fig. 75) of category 4 (samples PP415-P13) are significantly different from the spectra and spectra of categories 2, 3, and 5.

The TG-FTIR thermogram of category 4 (sample PP415-P13, fig. 106) shows a loss of MeCN (with trace amounts of water) of about 3.4 wt% from 25 ℃ to 270 ℃, most of which is lost in the step from about 180 ℃ to 210 ℃. Decomposition begins at temperatures T > 270 ℃. Before the TG-FTIR experiment, the sample was dried briefly (for about 5 minutes) under vacuum (10-20 mbar) to remove excess unbound solvent. The theoretical MeCN (boiling point 81 ℃) content of the hemisolvate was 3.6 wt%.

TABLE 23 crystallization experiments to produce solid materials of Category 4

Drying experiments on Category 4

Under vacuum or in N₂The flow-down will be from about 7: 3MeCN/H₂Samples of category 4 obtained from suspension equilibration experiments performed in O were dried for several days (table 24).

TABLE 24 drying experiments on samples of Category 4

^aThe solvent content may be MeCN and H₂O, but is difficult to measure because of its small amount

The FT-Raman spectrum of the dried class 4 material (PP415-P26) was identical to that of class 4(PP415-P13, FIG. 107).

The PXRD pattern of the dried class 4 material (PP415-P26) showed only very small differences from the pattern of the class 4 sample PP415-P13 (fig. 108). Some peaks appear to be better resolved and the peak intensities have changed. No amorphization was observed. The map of PP415-P26 corresponds to Category 4.

The TG-FTIR thermogram (fig. 109) of the dried class 4 material sample PP415-P26 shows MeCN loss from 170 ℃ to 250 ℃ of about 2.8 wt% and decomposition at temperature T > 300 ℃. The solvent content of the sample was reduced from 3.4 wt% to 2.8 wt% compared to TG-FTIR of sample PP415-P13 (fig. 106).

Thus, the sample appears to be a partially desolvated solvate. Experiments PP415-P13 were repeated (as PP415-P35) as not enough material was retained for the second drying experiment and subsequent characterization. The class 4 material was then prepared and two drying experiments were performed using this newly prepared material:

● PP415-P36 under vacuum (< 1 × 10)^-3Mbar) at 80 ℃ for 3 days

● PP 415-P37: in N₂Dried under reduced pressure at 80 ℃ for 3 days.

The FT-Raman spectra of these dried class 4 samples (PP415-P36 and PP415-P37) correspond to the spectrum of class 4 (i.e., PP415-P35, FIG. 110).

The PXRD patterns (FIG. 111) for class 4 material (samples PP415-P35) and class 4 dried samples (samples PP415-P36 and PP415-P37) were identical. The dried sample was crystalline.

TG-FTIR thermograms of these dried samples of class 4(PP 415-P36 is FIG. 112, and PP415-P37 is FIG. 113) show that PP415-P36 and PP415-P37 have only small solvent contents (MeCN and/or H) of about 0.6 wt% and about 0.9 wt%, respectively, in two steps from 25 ℃ to 280 ℃. (MeCN and/or H)₂O). The solvent content may be MeCN and H₂O, but the amount is small, and thus it is difficult to measure. Decomposition begins at temperatures T > 280 ℃.

Thus, most of the solvent of this solvate can be removed without destroying the crystal structure. Crystalline unsolvated forms (or more precisely, desolvated solvates) are obtained.

Further characterization of dried and desolvated class 4

Drying class 4(MeCN solvate) produced a desolvated solvate with a reduced solvent content to < 1 wt.% (TG-FTIR).

The structure did not change after desolvation (FT-Raman method and PXRD). No significant loss of crystallinity was observed.

Thus, 63415 was obtained as the only unsolvated crystalline form known to date.

This desolvated class 4 material was further characterized by DVS and DSC.

The DVS isotherm (fig. 114) shows that a mass increase of about 0.4 wt% occurs during the initial equilibration time at 50% relative humidity. During the measurement, after the relative humidity was reduced from 50% relative humidity to 0% relative humidity, a gradual, reversible mass loss of about 1.3% by weight occurred. Equilibrium is reached. After increasing the relative humidity to 95% relative humidity, a gradual mass increase of about 0.8% by weight (relative to the equilibrium mass at 50% relative humidity) was observed. Equilibrium is reached. After lowering the relative humidity to 50% relative humidity, the final mass was still 0.1 wt% lower than the equilibrium starting mass. A mass increase of about 0.7 wt% after increasing the relative humidity from 50% to 85% relative humidity classified the sample as slightly hygroscopic.

The PXRD pattern of the sample after measurement was unchanged from the pattern before measurement (fig. 115).

The DSC thermogram (fig. 116) of a sample of desolvated class 4 material showed no glass transition attributable to the amorphous form that would otherwise be expected to occur at about 150 ℃, but rather an endothermic peak with a maximum at T196.1 ℃ (Δ H29.31J/g), which may correspond to melting, and showed no decomposition up to 270 ℃.

In addition, DSC experiments using an approximately 1: 1 mixture of amorphous material (class 1) and desolvated class 4 material to investigate whether the amorphous material would convert and crystallize into desolvated class 4 would be expected to be at a higher glass transition temperature (T) than the amorphous form_gAbout 150 ℃ and below the melting point (T) of desolvated class 4_mAbout 196 c, if any.

The DSC thermogram (fig. 117) of the mixture shows an endotherm with a peak at 156.7 ℃ (Δ H ═ 1.47J/g) and a second endotherm with a peak at 197.0 ℃ (Δ H ═ 14.1J/g). The first phenomenon can be attributed to the amorphous material (at T)_gGlass transition at about 150 ℃). A second phenomenon may correspond to a T of desolvated class 4 at about 196 deg.C_mMelting. The heat of fusion of the mixture (Δ H ═ 14.1J/g) correlates well with half the heat of fusion of pure desolvated class 4(Δ H ═ 29.3J/g).

No exothermic phenomena corresponding to possible crystallization of the amorphous material are observed in the temperature range from the glass transition to the melting. Thus, no conversion of the amorphous form to the desolvated class 4 form appears to occur on this scale.

In yet another DSC experiment using an approximately 1: 1 mixture of amorphous material (class 1) and desolvated class 4 material, heating was stopped at 173 ℃ (between glass transition and melting) to allow time for possible crystallization.

A DSC thermogram (fig. 118) of the mixture shows an endotherm with a peak at T161.4 ℃ (Δ H0.31J/g) and a second endotherm with a peak at 201.4 ℃ (Δ H11.4J/g). As in the first experiment, the heat of fusion of the second peak was not increased; no evidence was seen of the conversion of the amorphous form to the desolvated class 4 form.

The curved baseline (-50 ℃ to 150 ℃) is most likely an artifact (artifact) (due to the curved sample holder cover).

n. class 5-THF solvates

From 1: 1THF/H only₂The O solvent mixture gave category 5 (table 25).

The FT-raman spectra (fig. 71) and PXRD patterns (fig. 75) for category 5 are significantly different from those for categories 2, 3 and 4.

TG-FTIR thermograms of class 5 (samples PP415-P14, FIG. 119) show about 36.1 wt.% THF and H from 25 ℃ to 200 ℃₂O is lost, most of which is lost in the step from about 100 ℃ to 130 ℃. Before the TG-FTIR experiment, the sample was dried briefly (for about 5 minutes) under vacuum (10-20 mbar) to remove excess unbound solvent. THF and H₂The loss of both O occurs together in the same temperature range. Decomposition starts at temperatures T > 300 ℃. The theoretical THF (boiling point 66 ℃) content of the trisolvent (trisolvate) was 28.1% by weight. Unfortunately, these two components cannot be used separately due to their contentIs quantified and therefore the exact solvation state cannot be determined.

Details regarding the experiments and characterization of samples PP415-P41 and PP415-P45 are provided.

TABLE 25 crystallization experiments to produce solid materials of Category 5

^bStarting materials: PP415-P40, Category 2; in all other experiments in this table, PP415-P1 (Category 1) was used as the starting material

^c3g Scale experiment, not 100mg Scale

Drying experiments on samples of class 5

Under vacuum will be from about 1: 1THF/H₂Suspension equilibration experiments in O samples of category 5(PP 415-P14) obtained were dried (as PP415-P27) for several days (2-20 mbar, room temperature to 60 ℃, Table 26).

TABLE 26 drying experiments for samples of Category 5

^aMainly amorphous with only a few broad peaks with low S/N ratio

The FT-raman spectrum of the dried material (PP415-P27) is different from the spectrum of class 5(PP415-P14, fig. 120) and in case of its broadened peaks, more similar to the spectrum of the class 1 amorphous starting material PP 415-P1.

The PXRD pattern of the dried class 5 material (PP415-P27) showed only a few broad low intensity peaks with low S/N ratios, indicating poor crystallinity of the sample (fig. 121). Some of the peaks may correspond to class 5 while others (i.e., peaks at 7.35 ° 2 θ) are new or shifted.

The TG-FTIR thermogram (fig. 122) of the dried class 5 material shows mass loss from 25 ℃ to 290 ℃ of about 0.3 wt% and decomposition at temperature T > 290 ℃. The sample is anhydrous.

Thus, by drying under vacuum, the material loses its solvent content as well as most of its crystallinity.

8. Experiment for preparing amorphous form

Experiments aimed at the preparation of the amorphous form (category 1) were carried out using the category 2 material (PP415-P40, table 8) as starting material. Various strategies and methods were tried:

● converting class 2 to class 5, followed by drying class 5 to obtain the amorphous form (class 1).

● amorphous form (class 1) was prepared directly from class 2, using ICH class 3 solvent, if possible.

The predominantly amorphous material was prepared in a two-step process via class 5 on a scale of 100mg and 3g starting from class 2 material.

Further experiments were performed with the aim of simplifying the procedure to a one-step process to avoid the ICH class 2 solvent THF and to obtain a completely amorphous material. The most promising method was found to be precipitation from acetone solution in a cold water bath. This direct process gives much better results than the two-step process via class 5.

a. Preparation of the amorphous form via class 5

Crystallization experiments using PP415-P40 of class 2 as starting material were performed with the aim of converting this heptane solvate into class 5 (possibly THF solvate) followed by drying of class 5 to obtain the amorphous material (table 27).

Category 5 is considered a good intermediate step because it is more easily desolvated and amorphized than either category 2 or 3.

TABLE 27 summary of experiments aimed at preparing amorphous form (Category 1) via Category 5 materials

Step (ii) of	Sample (I)	Method of producing a composite material	Condition	Results
					1	PP415-P41	Balancing of the suspension	1∶1THF/H2O, 24 ℃,3 days	Class 5
“	PP415-P45	Balancing of the suspension	1∶1THF/H2O, room temperature, 1 day	Class 5
					2	PP415-P44a	Drying	100 mbar, 80 ℃, 2 days	Class 1a(ii) a 0.9% by weight of THF

“

PP415-P46a

Drying

100 mbar, 80 ℃,4 days

Class 1a(ii) a 0.4 wt.% H2O

^aMainly amorphous with only a few broad peaks with low S/N ratio

b. Step 1: conversion of class 2 to class 5

By suspending the PP415-P40 (heptane solvate) material in (1: 1) THF/H₂O mixture and allowing the suspension to equilibrate at room temperature to successfully convert the heptane solvate (category 2) to THF solvate (category 5) (PP415-P41, 100mg scale). The resulting solid material corresponded to THF solvate, class 5 (fig. 123).

A first scale-up experiment from milligram-scale to gram-scale (× 30, i.e., 3 g-scale) was performed analogously to PP415-P41 by dissolving the class 2 heptane solvate starting material (PP415-P40) in THF/H₂O (1: 1) was equilibrated for one day and successfully converted to class 5, THF solvate (PP415-P45, FIG. 124).

c. Step 2: amorphization of class 5 materials by drying

Class 5 materials (THF solvates) were dried under vacuum (about 100 mbar) at elevated temperature (80 ℃) in view of the conditions that could be used at the API MFG site.

After drying 100mg of the experimental material PP415-P41 at 80 ℃ and 100 mbar for one day, it was converted into a predominantly amorphous material (PP415-P44, FIG. 125). The PXRD pattern shows only a few broad peaks with low intensity. After drying (80 ℃, 100 mbar) overnight, the intensity of these broad peaks was further reduced (PP415-P44 a). TG-FTIR of this material showed gradual loss of THF (with trace amounts of water) from 25 ℃ to 280 ℃ of about 0.9 wt% and decomposition at temperatures T > 300 ℃ (FIG. 126).

3g of the experimental material PP415-P45 were also dried at 80 ℃ and 100 mbar (as PP 415-P46). It converted overnight to a predominantly amorphous material with only a few broad peaks of low intensity (figure 127). After a total of 4 days of drying (80 ℃, 100 mbar), these broad peaks still remain (-P46a, fig. 128). The TG-FTIR of this material did not show THF content, but showed a gradual loss of water from 25 ℃ to 250 ℃ of about 0.4 wt% and decomposition at temperatures T > 250 ℃ (fig. 129).

d. Directly obtaining the amorphous form

The preparation of amorphous form via class 5 in a two-step process starting from class 2 material is basically, but not completely, successful. Therefore, further experiments were carried out with the aim of simplifying the procedure into a one-step process to avoid the use of the ICH class 2 solvent THF and to obtain a completely amorphous material (table 28).

In evaporation experiments on THF solutions of class 2 in N₂Amorphous form (category 1) was prepared directly from category 2 material by flow down (PP415-P42, fig. 129).

To simulate an incompletely dried heptane/hexane solvate with a large amount of remaining solvent, the class 2 solution was evaporated in an 8: 2 THF/hexane solution (hexane was used instead of heptane to have similar boiling points in the solvent mixture). However, the resulting solid corresponded to class 2, the class of isomorphic solvates, rather than class 5(PP415-P43, fig. 130).

To avoid the ICH class 2 solvent THF, evaporation experiments were performed in the ICH class 3 solvent.

In N₂Evaporation of class 2EtOAc solution down stream yielded a crystalline material with PXRD pattern corresponding to class 2 (PP415-P47, figure 130). TG-FTIR (figure 80) shows typical two-step mass loss of class 2 at temperatures up to 240 ℃ (total about 7.9 wt% EtOAc), indicating very tightly bound solvent molecules.

Evaporation in ethyl formate also yielded a crystalline class 2 material, not an amorphous form (PP415-P48, FIG. 131). TG-FTIR (fig. 78) showed a mass loss of about 3.5 wt% of ethyl formate, initially gradually lost, then lost in distinct steps from 180 ℃ to 200 ℃. There may be further loss of ethyl formate accompanied by decomposition at T > 240 ℃.

However, successfully class 2 material was converted to the amorphous form, class 1, by adding an acetone solution to a cold (5 ℃) water bath (PP415-P49, fig. 132).

This direct process for preparing the amorphous form gives better results and is a more promising process than the two-step process.

TABLE 28 summary of experiments aimed at obtaining the amorphous form directly from the class 2 starting material

Sample (I)	Method of producing a composite material	Solvent(s)	Condition	Results
					PP415-P42	Evaporation of	THF	N2Flow, 1 day	Class 1
PP415-P43	Evaporation of	8: 2 THF/hexane	N2Flow, 1 day	Class 2
					PP415-P47	Evaporation of	EtOAc	N2Flow, 1 day	Class 2
PP415-P48	Evaporation of	Formic acid ethyl ester	N2Flow, 1 day	Class 2
					PP415-P49	Precipitation of	Acetone (II)	5℃H2O bath	Class 1

9. Instrument-typical measurement conditions

FT-Raman spectroscopy: bruker RFS100 with OPUS 6.5 software; nd: YAG 1064nm excitation, Ge detector, 3500-^-1A range; typical measurement conditions are: 100-300mW rated laser power, 64-128 scans, 2cm^-1Resolution.

PXRD: stoe Stadi P; a Mythen1K detector; Cu-Kalpha radiation; standard measurement conditions: transmitting; 40kV and 40mA tube power; a curved Ge monochromator; a step size of 0.02 degrees 2 theta, a step time of 12 seconds or 60 seconds, a scanning range of 1.5 degrees to 50.5 degrees 2 theta or 1.0 degrees to 55 degrees 2 theta; detector mode: step scanning; 1 ° 2 θ or 6 ° 2 θ detector step size; preparation of a standard sample: placing 10mg to 20mg of the sample between two acetate foils; sample support: stoe transmission sample holder; the sample is rotated during the measurement.

TG-FTIR: netzsch Thermo-microbalanceTG 209 with Bruker FT-IR spectrometer Vector 22; aluminum crucible (with micropores), N₂Atmosphere, heating rate of 10K/min, 25-250 deg.C or 25-350 deg.C.

DSC: perkin Elmer DSC 7; gold crucibles (closed or with micro-holes) in N₂The environment was filled with samples, heating rate of 10K/min, range from-50 ℃ to 250 ℃ or 350 ℃, quenching (at-200K/min) to-50 ℃ from time to time between scans.

DVS: projekt Messtechnik Sorptions Pr ufsystem SPS 11-100n or Surface Measurement Systems (Surface Measurement Systems) DVS-1. The sample is placed on an aluminum or platinum holder on top of the microbalance and equilibrated at 50% relative humidity for 2 hours before starting the predetermined humidity program:

(1) 50% → 0% relative humidity (5%/hour); at 0% relative humidity for 5 hours

(2) 0% → 95% relative humidity (5%/hour); 5 hours at 95% relative humidity

(3) 95% → 50% relative humidity (5%/hour); at 50% relative humidity for 2 hours

Hygroscopicity was classified based on the mass increase relative to the initial mass at 85% relative humidity as follows: deliquescence (adsorbing enough water to form a liquid), high hygroscopicity (mass increase > 15%), hygroscopicity (mass increase < 15% but > 2%), slightly hygroscopicity (mass increase < 2% but > 0.2%) or non-hygroscopicity (mass increase < 0.2%).

Solvent: for all experiments, Fluka, Merck or ABCR analytical grade solvents were used.

Approximate solubility determination: the approximate solubility was determined by stepwise dilution of a suspension of about 10mg of the substance in 0.05mL of solvent. Solubility is shown as < 1mg/mL if the material is not dissolved by adding a total of > 10mL of solvent. Due to experimental error inherent in this approach, these solubility values are intended to be considered rough estimates and are only used for the design of crystallization experiments.

And (3) chemical stability determination: 4 samples of 1.0mg of PP415-P1 substance were prepared in 1.0mL of the corresponding solvent. The resulting suspension/solution was equilibrated at 25 ℃ in a temperature controlled Eppendorf Thermomixer Comfort shaker at a shaking rate of 500rpm for 7 days, 2 days, 24 hours and 6 hours. If necessary, the solid phase was separated by filtration centrifugation (0.5 μm PVDF membrane). The filtrate was diluted in diluent (MeCN with 0.1% formic acid) to a concentration of ≦ 0.2mg/mL (unknown and possibly lower for the suspension) and checked using the HPLC method given in Table 29. For reference, substance PP415-P1 was diluted in a diluent to a concentration of 0.25mg/mL and added to the beginning and end of the HPLC sequence.

HPLC results

TABLE 29 HPLC methods for chemical stability determination

10. Abbreviations

The method comprises the following steps:

integral analysis under AUC curve

DSC differential scanning calorimetry

DVS dynamic vapor adsorption

FT Raman Fourier transform (Fourier-transform) Raman spectroscopy

¹H-NMR proton nuclear magnetic resonance spectroscopy

HPLC high performance liquid chromatography

PXRD powder X-ray diffraction

TG-FTIR thermogravimetric analysis-Fourier transform infrared spectroscopy combination

Chemical products:

1BuOH 1-Butanol

CTAB cetyl trimethyl ammonium Bromide

DCM dichloromethane

DEE Ether

DMF N, N-dimethylformamide

EtOAc ethyl acetate

EtOH ethanol

IPE isopropyl ether

MeCN acetonitrile

MEK methyl Ethyl Ketone

MeOH methanol

PEG propylene glycol

PTFE Polytetrafluoroethylene, Teflon (Teflon)

2PrOH 2-propanol and isopropanol

SDS sodium dodecyl sulfate

TBME Tert-butyl methyl Ether

TEA Triethylamine

THF tetrahydrofuran

Tween 80 polyoxyethylene (80) sorbitan monooleate or polysorbate 80

Genes, proteins and biological parameters:

AIM antioxidant inflammation modulators

Akr1c1 aldehyde ketoreductase family 1, member c1

ALP alkaline phosphatase

ALT alanine aminotransferase

ARE anti-oxidation reaction element

AST aspartate aminotransferase

Area under AUC curve

BAL bronchoalveolar lavage

BALF bronchoalveolar lavage fluid

Bil bilirubin

BUN blood urea nitrogen

COPD chronic obstructive pulmonary disease

COX-2 cyclooxygenase-2

Cr creatine

CYP450 cytochrome P450

Eh-1 epoxide hydrolase 1

G6PD glucose-6 phosphate dehydrogenase

Gclc glutamate-cysteine ligase, a catalytic subunit

Gclm glutamate-cysteine ligase, modifying subunit

Ggt1 gamma-glutamyl transferase

Glrx glutaredoxin-1

Glu glucose

GOT glutamate-oxaloacetate transaminase

GPT1 glutamate-pyruvate transaminase

Gpx3 glutathione peroxidase 3

GSH glutathione

GSR glutathione reductase

GSs glutathione synthetase

GST glutathione S-transferase

GSTA1 glutathione S-transferase alpha 1

GSTp1 glutathione S-transferase pi 1

Gy Gray (Gray)

H6PD hexose-6-phosphate dehydrogenase

hERG human ether a-go-go related gene

HMOX1 heme oxygenase (decyclization) 1

HO-1 heme oxygenase

IFN gamma interferon-gamma

IL interleukins

iNOS inducible nitric oxide synthase

I kappa B alpha B cell kappa light chain polypeptide gene enhancer nuclear factor inhibitor alpha

KC mouse IL-8 related protein

Keap1 Kelch-like ECH-related protein-1

LPS lipopolysaccharide

ME1 malic enzyme 1

MPCE micronucleus pleochromatic erythrocyte

Mrp metG related protein

Mrps multidrug resistance associated protein

NADPH reduced nicotinamide adenine dinucleotide phosphate

Kappa light chain enhancer nuclear factor of NF kappa B activated B cells

NO nitric oxide

NQO1 NAD (P) H quinone oxidoreductase 1

Nrf2 nuclear factor (erythrocyte derived) -like 2

phosphorylation of I.kappa.B.alpha.by p-I.kappa.B.alpha.

PBMC peripheral blood mononuclear cells

PCE pleochromocyte

PGD phosphogluconate dehydrogenase

PMN polymorphonuclear

RANTES regulates normal T cell expression and secretion of factors

SOD1 superoxide dismutase 1

SRXN1 thioredoxin-1

TG Total glycerides

TKT transketolase

TNF alpha tumor necrosis factor alpha

Txn thioredoxin

TXNRD1 thioredoxin reductase 1

xCT solute Carrier family 7, Member 11

Miscellaneous items:

API active pharmaceutical ingredient

aq. aqueous phase

b.p. boiling point

crystal of crystal

decomp

d days

eq. equivalent

equilibria

Evaporation by evap

h hours

matter mat

min for

m.p. melting Point

MS molecular sieve

part of part

precipitation of Precip

r.h. relative humidity

rpm rotation/min

Room temperature (about 25 deg.C)

S/N signal-to-noise ratio

solvent (iv)

susp suspension

T temperature

T_gGlass transition temperature

theory of the world

vis

w weeks

wt. -% of weight percent

All of the compounds, polymorphs, formulations and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compounds, polymorphs, formulations and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compounds, polymorphs, formulations and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Reference to the literature

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Claims (99)

1. A compound having the formula:

or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein the compound is not in the form of a salt.

3. A polymorph of a compound having the formula:

wherein the polymorph has an X-ray powder diffraction pattern irradiated with Cu-Ka substantially as shown in figure 59.

4. The polymorph of claim 3, further having a T of 150 ℃ to 155 ℃_g。

5. The polymorph of claim 4, further having a T of about 153 ℃_g。

6. The polymorph of claim 3, further having a differential scanning calorimetry curve comprising an endotherm centered at 150 ℃ to 155 ℃.

7. The polymorph of claim 6, wherein said endotherm is centered at about 153 ℃.

8. The polymorph of claim 3 having a differential scanning calorimetry curve substantially as shown in figure 62.

9. A polymorph of a compound having the formula:

wherein the polymorph is a non-stoichiometric solvate with <0.5 equivalents of heptane, cyclohexane, isopropyl ether, 1-butanol, or triethylamine having an X-ray powder diffraction pattern of radiation with Cu-Ka comprising peaks at about 5.6 ° 2 θ, 7.0 ° 2 θ, 10.6 ° 2 θ, 12.7 ° 2 θ, and 14.6 ° 2 θ.

10. The polymorph of claim 9, wherein the X-ray powder diffraction pattern of radiation with Cu-ka is substantially as shown in the top pattern in figure 75.

11. A polymorph of a compound having the formula:

wherein the polymorph is a solvate of ethanol, isopropanol, or acetone having about 0.5 equivalents, having an X-ray powder diffraction pattern with Cu-ka radiation comprising peaks at about 7.0 ° 2 Θ, 7.8 ° 2 Θ, 8.6 ° 2 Θ, 11.9 ° 2 Θ, 13.9 ° 2 Θ, 14.2 ° 2 Θ, and 16.0 ° 2 Θ, wherein the peak at about 13.9 ° 2 Θ is bimodal.

12. The polymorph of claim 11, wherein the X-ray powder diffraction pattern of radiation with Cu-ka is substantially as shown in the second pattern from the top in figure 75.

13. A polymorph of a compound having the formula:

wherein the polymorph is an acetonitrile hemisolvate having an X-ray powder diffraction pattern with Cu-Ka radiation comprising peaks at about 7.5 ° 2 θ, 11.4 ° 2 θ, 15.6 ° 2 θ, and 16.6 ° 2 θ.

14. The polymorph of claim 13, wherein the X-ray powder diffraction pattern of radiation with Cu-ka is substantially as shown in the second pattern from the bottom of figure 75.

15. Root of herbaceous plantThe polymorph of claim 13, further having a T of about 196 ℃_g。

16. The polymorph of claim 13, further having a differential scanning calorimetry curve comprising an endotherm centered at about 196 ℃.

17. The polymorph of claim 13 having a differential scanning calorimetry curve substantially as shown in figure 116.

18. A polymorph of a compound having the formula:

wherein the polymorph is a tetrahydrofuran solvate having an X-ray powder diffraction pattern with Cu-Ka radiation comprising peaks at about 6.8 ° 2 θ, 9.3 ° 2 θ, 9.5 ° 2 θ, 10.5 ° 2 θ, 13.6 ° 2 θ, and 15.6 ° 2 θ.

19. The polymorph of claim 18, wherein the X-ray powder diffraction pattern of radiation with Cu-ka is substantially as shown in figure 75 at the bottom.

20. A pharmaceutical composition comprising:

an active ingredient consisting of a compound according to any one of claims 1 to 2 or a polymorph according to any one of claims 3 to 19, and

a pharmaceutically acceptable carrier.

21. The pharmaceutical composition of claim 20, wherein the pharmaceutical composition is formulated for administration in a manner that: oral, intralipidic, intraarterial, intraarticular, intracranial, intradermal, intralesional, intramuscular, intranasal, intraocular, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrarectal, intrathecal, intratracheal, intratumoral, intraumbilical, intravaginal, intravenous, intravesical, intravitreal, liposomal, transmucosal, parenteral, rectal, subconjunctival, subcutaneous, sublingual, topical, buccal, transdermal, vaginal, in cream form, in liquid composition form, via catheter, via lavage, via continuous infusion, via inhalation, via injection, via local delivery, or via local infusion.

22. The pharmaceutical composition of claim 21, wherein the pharmaceutical composition is formulated for oral, intra-arterial, intravenous, or topical administration.

23. The pharmaceutical composition of claim 21, wherein the pharmaceutical composition is formulated for oral administration.

24. The pharmaceutical composition of claim 20, wherein the pharmaceutical composition is formulated as a hard or soft capsule, a tablet, a syrup, a suspension, an emulsion, a solution, a solid dispersion, a wafer, or an elixir.

25. The pharmaceutical composition of claim 20, further comprising an agent that enhances solubility and dispersibility.

26. The pharmaceutical composition of claim 20, wherein the compound or polymorph is suspended in sesame oil.

27. The pharmaceutical composition of claim 20, wherein the pharmaceutical composition is formulated for topical administration.

28. The pharmaceutical composition of claim 27, wherein the pharmaceutical composition is formulated as a lotion, cream, gel, oil, ointment, salve, emulsion, solution, or suspension.

29. The pharmaceutical composition of claim 28, wherein the pharmaceutical composition is formulated as a lotion.

30. The pharmaceutical composition of claim 28, wherein the pharmaceutical composition is formulated as a cream.

31. The pharmaceutical composition of claim 28, wherein the pharmaceutical composition is formulated as a gel.

32. The pharmaceutical composition according to any one of claims 20 to 30, wherein the amount of the active ingredient is 0.01 to 5% by weight.

33. The pharmaceutical composition of claim 32, wherein the amount of the active ingredient is 0.01 to 3% by weight.

34. The pharmaceutical composition of claim 33, wherein the amount of the active ingredient is about 0.01% by weight.

35. The pharmaceutical composition of claim 33, wherein the amount of the active ingredient is about 0.1% by weight.

36. The pharmaceutical composition of claim 33, wherein the amount of the active ingredient is about 1% by weight.

37. The pharmaceutical composition of claim 33, wherein the amount of the active ingredient is about 3% by weight.

38. Use of a compound according to any one of claims 1 to 2 or a polymorph according to any one of claims 3 to 19 for the manufacture of a medicament for the treatment or prevention of a condition associated with inflammation or oxidative stress in a patient.

39. The use of claim 38, wherein the condition is inflammation.

40. The use of claim 38, wherein the condition is associated with oxidative stress.

41. The use of claim 38, wherein the condition is a skin disease or disorder, sepsis, osteoarthritis, cancer, an autoimmune disease, inflammatory bowel disease, complications from local or systemic exposure to ionizing radiation, mucositis, acute or chronic organ failure, liver disease, pancreatitis, an ocular disorder, lung disease, or diabetes.

42. The use of claim 41, wherein the condition is a skin disease or disorder.

43. The use of claim 42, wherein the skin disease or disorder is dermatitis, thermal or chemical burns, chronic wounds, acne, alopecia, other follicular disorders, epidermolysis bullosa, sunburn complications, skin pigmentation disorders, aging-related skin conditions, post-operative wounds, scars resulting from skin injury or burns, psoriasis, autoimmune diseases or skin manifestations of graft versus host disease, skin cancer, or a disorder involving hyperproliferation of skin cells.

44. The use of claim 43, wherein the skin disease or disorder is dermatitis.

45. The use of claim 44, wherein the dermatitis is allergic dermatitis, atopic dermatitis, dermatitis due to chemical exposure, or radiation-induced dermatitis.

46. The use of claim 43, wherein the skin disease or condition is chronic trauma.

47. The use of claim 46, wherein the chronic wound is a diabetic ulcer, a decubitus ulcer, or a venous ulcer.

48. The use of claim 43, wherein the skin disease or disorder is alopecia.

49. The use of claim 48, wherein the hair loss is alopecia or drug-induced hair loss.

50. The use of claim 43, wherein the skin disease or disorder is a skin pigmentation disorder.

51. The use according to claim 50, wherein the skin pigmentation disorder is vitiligo.

52. The use of claim 43, wherein the skin disease or disorder is a disorder involving hyperproliferation of skin cells.

53. The use of claim 52, wherein the disorder involving hyperproliferation of skin cells is hyperkeratosis.

54. The use of claim 41, wherein the condition is an autoimmune disease.

55. The use of claim 54, wherein the autoimmune disease is rheumatoid arthritis, lupus, Crohn's disease, or psoriasis.

56. The use of claim 41, wherein the condition is a liver disease.

57. The use according to claim 56 wherein the liver disease is fatty liver disease or hepatitis.

58. The use of claim 41, wherein the condition is an ocular disorder.

59. The use of claim 58, wherein the ocular disorder is uveitis, macular degeneration, glaucoma, diabetic macular edema, blepharitis, diabetic retinopathy, a corneal endothelial disease or disorder, dry eye, or conjunctivitis.

60. The use of claim 59, wherein the ocular disorder is macular degeneration.

61. The use of claim 60, wherein the macular degeneration is in the dry form.

62. The use of claim 60, wherein the macular degeneration is the wet form.

63. The use of claim 59, wherein the corneal endothelial disease or disorder is Foss's corneal endothelial dystrophy.

64. The use of claim 41, wherein the condition is a pulmonary disease.

65. The use of claim 64, wherein the lung disease is pulmonary inflammation, pulmonary fibrosis, COPD, asthma, cystic fibrosis, or idiopathic pulmonary fibrosis.

66. The use of claim 65, wherein the lung disease is pulmonary inflammation.

67. The use of claim 65, wherein the lung disease is pulmonary fibrosis.

68. The use of claim 65, wherein the lung disease is COPD.

69. The use according to claim 68, wherein said COPD is induced by cigarette smoke.

70. The use of claim 65, wherein the pulmonary disease is asthma.

71. The use of claim 41, wherein the condition is sepsis.

72. The use of claim 41, wherein the condition is mucositis due to radiation therapy or chemotherapy.

73. The use of claim 72, wherein the mucositis is oral mucositis.

74. The use of claim 41, wherein the condition is a complication from local or systemic exposure to ionizing radiation.

75. The use of claim 74, wherein said exposure to ionizing radiation results in dermatitis.

76. The use of claim 74, wherein said exposure to ionizing radiation is acute.

77. The use of claim 74, wherein said exposure to ionizing radiation is fractionated.

78. The use of claim 41, wherein the condition is cancer.

79. The use of claim 78, wherein the cancer is carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma, or seminoma.

80. The use of claim 78, wherein the cancer is bladder cancer, blood cancer, bone cancer, brain cancer, breast cancer, central nervous system cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, gall bladder cancer, genital cancer, genitourinary tract cancer, head cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, muscle tissue cancer, neck cancer, oral or nasal mucosa cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, spleen cancer, small intestine cancer, large intestine cancer, stomach cancer, testicular cancer, or thyroid cancer.

81. The use of claim 38, wherein the compound or the pharmaceutical composition is administered in a single dose per day.

82. The use of claim 38, wherein the compound or the pharmaceutical composition is administered in more than one dose per day.

83. The use of claim 38, wherein the pharmaceutical composition is administered in a unit dose of 1mg/kg to 2000 mg/kg.

84. The use of claim 83, wherein the dose is 3mg/kg to 100 mg/kg.

85. The use of claim 83, wherein the dose is about 3 mg/kg.

86. The use of claim 84, wherein the dose is about 10 mg/kg.

87. The use of claim 84, wherein the dose is about 30 mg/kg.

88. The use of claim 83, wherein the dose is about 100 mg/kg.

89. The use of claim 38, wherein the medicament is administered topically.

90. The use according to claim 89, wherein the topical application is to the skin.

91. The use according to claim 89, wherein the topical application is to the eye.

92. The use of claim 38, wherein the medicament is administered orally.

93. The use of claim 38, wherein the medicament is administered intraocularly.

94. The use of claim 38, wherein the medicament is administered prior to or immediately after treating the patient with radiation therapy or chemotherapy, wherein the chemotherapy does not comprise an active ingredient as in claim 20.

95. The use of claim 38, wherein the medicament is administered prior to and immediately after treating the patient with the radiation therapy or the chemotherapy, wherein the chemotherapy does not comprise an active ingredient as in claim 20.

96. The use of claim 95, wherein said treatment reduces side effects of said radiation therapy or said chemotherapy.

97. The use of claim 96, wherein the side effects are mucositis and dermatitis.

98. The use of claim 94, wherein the treatment increases the efficacy of the radiation therapy or the chemotherapy.

99. The use of claim 94, wherein the chemotherapy comprises administering to the patient a therapeutically effective amount of 5-fluorouracil or docetaxel.