UA107569C2 - Crystalline Tripeptide Inhibitors of Epoxyketone Protease - Google Patents

Abstract

The invention relates to crystalline tripeptide ketoepoxide compounds, methods for their preparation and related pharmaceutical compositions.

Description

Related applications

This application claims priority to prior US patent application Serial No. MO 61/162196, filed Mar. 20, 2009, and prior US patent application Serial No. MO 61/180561, filed May 22, 2009. The disclosures of the above applications are fully incorporated herein application by reference.

Prerequisites of the invention

In eukaryotes, protein degradation is predominantly mediated by the ubiquitin pathway, in which proteins targeted for degradation are ligated to the 76 amino acid ubiquitin polypeptide. Once targeted, ubiquitinated proteins then serve as substrates for the proteasome 265, a multicatalytic protease that cleaves proteins into short peptides through three major proteolytic activities.

Although proteasome-mediated degradation has a general function in intracellular protein turnover, it also plays a key role in many processes, such as presentation of major histocompatibility complex (MHC) class I antigens, apoptosis, regulation of cell growth, activation

ME-KV (transcription factor K), processing of antigens and transduction of pro-inflammatory signals.

Proteasome 205 is a multicatalytic protease complex with a molecular weight of 700 kDa, consisting of 28 subunits organized into four rings. In yeast and other eukaryotes, 7 different α-subunits form the outer rings, and 7 different B-subunits comprise the inner rings. "-subunits serve as binding sites for regulatory complexes 195 (РА7О0) and 1115 (РА28), as well as a physical barrier for the internal proteolytic chamber formed by two rings of B-subunits. Thus, the proteasome is thought to exist as particle 265 ("proteasome 265"). IP mimo experiments showed that inhibition of the 205 form of the proteasome can be easily correlated with inhibition of the 265 proteasome. Cleavage of the amino-terminal prosequences of the |- subunits during particle formation exposes amino-terminal threonine residues that serve as catalytic nucleophiles. Thus, the subunits responsible for catalytic activity in proteasomes have an amino-terminal nucleophilic residue, and these subunits belong to the M-terminal nucleophile (Mip) hydrolase family (where the nucleophilic M-terminal residue is, for example, Suv, Zeg, TNg and others nucleophilic parts). This family includes, for example, penicillin-(z-acylase (ROSA), penicillin-M-acylase (PMA), glutamine-PAPP-amidotransferase (CAT) and bacterial glycosylparaginase. In addition to ubiquitously expressed β-subunits, higher vertebrates also have three interferon-γ-induced B-subunits (CMP7, HMP2 and MECI) replacing their normal copies, respectively B», B: and pD7, thus changing the catalytic activity of the proteasome. By using different peptide substrates, the three main types of proteolytic activities were determined for 205 eukaryotic proteasomes: a chymotrypsin-like activity (CT-I), which cleaves after large hydrophobic residues; a trypsin-like activity (T-G), which cleaves after basic residues; and a peptidylglutamyl-peptide (POPH) hydrolyzing activity , which cleaves after acidic residues. Two additional less characterized activities have also been attributed to the proteasome: the activity of VgHAAR, which cleaves after branched-chain amino acids; and active is 5MAAR, which cleaves after small neutral amino acids. Different catalytic sites appear to contribute to the major types of proteasome proteolytic activity, as inhibitors, point mutations in B subunits, and exchange of interferon-γ-inducing B subunits alter these types of activity to varying degrees.

Improved compositions and methods of preparation and assembly of proteasome inhibitor(s) are needed.

Brief summary of the essence of the invention

One aspect of the invention relates to crystalline compounds having the structure of formula (I), or pharmaceutically acceptable salts thereof,

M oo U?

From then to Tuesday in!

And where

X represents 0, MH or M-alkyl, preferably 0;

U represents M, 5 or C(P8)2", mainly MH; 2 represents MH, M-alkyl, O, 5 or C(P8)", preferably 5;

B", B: and EZ represent hydrogen;

each of the groups BK", B", NE, B" and KZ is independently selected from hydrogen, C1-alkyl, C1-6hydroxyalkyl, C1-6hydroxyalkyl, aryl and C1-alkyl, each of which is optionally substituted with a group selected from alkyl, amide, amine, carboxylic acid or its pharmaceutically acceptable salt, carboxylic acid ester, thiol and thioether, preferably BH", B? and KU are independently selected from C.-ethioether, C-vhydroxyalkyl and C-varalkyl, and K" represents Si -alkyl, preferably K" and KE? represent C--ethioether, and K" represents C-alkyl.

Brief description of the drawings

In fig. 1 shows the О5С (differential scanning calorimetric) thermogram of crystalline compound 1.

In fig. 2 shows the XRD (powder X-ray diffraction) type of crystalline compound 1.

In fig. C shows the TO (thermogram) of crystalline compound 1.

In fig. 4 shows modulated thermograms of amorphous compound 1, reversed heat flow (lower curve) and non-reversed heat flow (upper curve).

In fig. 5 shows a comparison of О5С thermograms of crystalline compound 1 obtained according to example 2 (middle curve), example C (upper curve) and example 4 (lower curve).

In fig. 6 shows the HCR type of amorphous compound 1 obtained according to example 1 (upper curve) in comparison with the HCR types of crystalline compound 1 obtained according to example 2 (upper curve), example C (2nd curve from the bottom) and example 4 ( 2nd curve from above).

In fig. 7 shows the TO thermogram of amorphous compound 1.

Detailed description of the invention

In certain embodiments, the invention relates to crystalline compounds having the structure of formula (I) or their pharmaceutically acceptable salts,

Ov V! of vk

M kh yosh U y-2 vo vi vo v

And where

X represents 0, MH or M-alkyl, preferably 0;

U represents M, 5 or C(K8)", mainly MH; 2 represents MH, M-alkyl, O, 5 or C(KZ)", preferably 5;

B', B2 and EZ represent hydrogen; each of the groups KU, A», He, B» and KZ is independently selected from hydrogen, C:-alkyl, C:-hydroxyalkyl, C-C-alkyloxyalkyl, aryl and Sivaralkyl, each of which is optionally substituted by a group selected from alkyl, amide, amine, carboxylic acid or its pharmaceutically acceptable salt, carboxylic acid ester, thiol and thioether, preferably NN", B? and KU are independently selected from C1-thioether, C1-hydroxyalkyl and C1-aralkyl, and E/ represents C1-alkyl, preferably KK" and B? represent C1-thioether, and 2" represents C1-alkyl.

In certain embodiments, the invention relates to a crystalline compound of the formula (II) about ОМе о

H (c)

AI, in J o xx No /- OMe

Me (1)

In certain embodiments, the invention relates to a method of obtaining a crystalline compound of formula (I) or (II), which includes: (i) obtaining an amorphous compound, for example, according to US patent application No. 11/595804; (iii) dissolution of the amorphous compound in an organic solvent; (its) bringing the solution to supersaturation; (im) separation of crystals, for example, by filtration of crystals, decantation of liquid from crystals or other suitable method of separation; and (m) crystal washing. In certain embodiments, the preparation further includes inducing crystallization. In certain embodiments, the preparation also includes drying, preferably under reduced pressure, for example under vacuum conditions.

In certain embodiments, the amorphous compound may be dissolved in a solvent selected from acetonitrile, ethyl acetate, heptanes, hexanes, isopropyl acetate, methanol, methyl ethyl ketone, tetrahydrofuran, toluene, and water, or any combination thereof. In certain embodiments, the amorphous compound of formula (II) can be dissolved in an organic solvent selected from acetonitrile, heptanes, hexanes, methanol, tetrahydrofuran and toluene or any combination thereof. In certain preferred embodiments, the organic solvent is toluene, hydrofuran or acetonitrile, preferably acetonitrile or toluene.

In certain embodiments, supersaturating the solution includes slowly adding an antisolvent such as water, heptanes, hexanes, or other polar or non-polar liquid mixed with the organic solvent, allowing the solution to cool (with or without precipitation of the solution), reducing the volume of the solution or any combination thereof. In certain embodiments, bringing the solution to supersaturation includes adding an antisolvent, cooling the solution to ambient temperature or below, and reducing the volume of the solution, for example, by evaporating the solvent from the solution. In certain embodiments, allowing the solution to cool can be passive (eg, allowing the solution to settle at ambient temperature) or active (eg, cooling the solution in an ice bath or freezer).

In certain embodiments, the method further includes inducing precipitation or crystallization. In certain embodiments, induction of precipitation or crystallization includes secondary nucleation, where nucleation occurs in the presence of seed crystals or interactions with the environment (crystallizer walls, mixing impellers, sonication, etc.).

In certain embodiments, washing the crystals includes washing with a liquid selected from an antisolvent, acetonitrile, heptanes, hexanes, methanol, tetrahydrofuran, toluene, water, or a combination thereof. In certain embodiments, the crystals are washed with a combination of an antisolvent and an organic solvent. In certain embodiments, the antisolvent is water, while in other embodiments, it is an alkane solvent, such as hexane or pentane, or an aromatic hydrocarbon solvent, such as benzene, toluene, or xylene.

In certain embodiments, crystal washing includes washing the crystalline compound of formula (II) with a mixture of tetrahydrofuran and an alkane solvent, such as hexanes or heptanes, or with a mixture of acetonitrile and water. In certain embodiments, crystal washing includes washing the crystalline compound of formula (II) with toluene. In preferred such embodiments, the toluene is cooled prior to washing.

In certain embodiments, the crystalline compound of formula (I) is essentially pure. In certain embodiments, the melting point of the crystalline compound of formula (II) is in the range of from about 135 to about 160 °C, from about 140 to about 155 °C, from about 145 to about 150 °C, or even from about 147 to about 149 °C. C, for example approximately 149 "C.

In certain embodiments, ХО5С of the crystalline compound of formula (II) has a sharp endothermic maximum at a temperature of approximately 147 "С, for example, as a result of melting and destruction of the crystalline form, as shown in Fig. 1.

In certain embodiments, the type of powder X-ray diffraction of the crystalline compound of formula (III) is (808-287): 8.94; 9.39; 9.76; 10.60; 11.09; 12.74; 15.27; 17.74; 18.96; 20.58; 20.88; 21.58; 21.78; 22.25; 22.80; 24,25; 24.66; 26.04; 26.44; 28.32; 28.96; 29.65; 30.22; 30.46; 30.78; 32.17; 33.65; 34.49; 35.08; 35.33; 37.85; 38,48, as shown in fig. 2.

In certain embodiments of TO implementation, the thermogram of the crystalline compound of formula (II) reveals a mass loss of 0.0 to 0.3 95 in the temperature range of 25 to 125 "С, as shown in Fig. 3.

In certain embodiments, the crystalline compound of formula (II) is non-solvated (for example, the crystal lattice does not contain solvent molecules). In certain embodiments, the crystalline compound of formula (II) is solvated.

In certain embodiments, the invention relates to a method of obtaining a crystalline compound of the formula (II)

OMe (6) (6)

Ch is)

M

A Ж ув No хх no /- ОМе

Me (1), which includes (i) the interaction of the compound of the formula (IP) hu ts

NM

(6) (PP) where X represents any suitable counterion, with a compound of formula (IM) in an organic solvent

ОМе (in) H (in)

Makon u-5 Oh /- 7oMe

Me; aM) (i) obtaining a solution of the compound of formula (II) in an organic solvent; (ii) bringing the solution to supersaturation to ensure the possibility of crystal formation; and (im) separating the crystals to obtain a crystalline compound of formula (I), for example, by crystal filtration, decantation or any other suitable separation technique.

In certain embodiments, the compound of formula (II) is not purified by chromatography before obtaining a solution in an organic solvent.

In certain embodiments, the preparation further includes inducing crystallization. In certain embodiments, the preparation further includes washing the crystals, for example, with a solvent or non-solvent liquid. In certain embodiments, the preparation also includes drying, preferably under reduced pressure, for example in a vacuum.

In certain embodiments, X represents a counterion selected from hydrobromide, hydrochloride, sulfate, phosphate, nitrate, acetate, trifluoroacetate, citrate, methanesulfonate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, succinate, tosylate, malonate, maleate, fumarate, tartrate, mesylate, 2-hydroxyethanesulfonate and the like (see, for example, the publication of Vegde ei ai. (1977) "Rpagtaseciisa! Zaive", U. Rpagt. 5si. 66: 1-19). In certain embodiments, X is selected from trifluoroacetate, methanesulfonate, toluenesulfonate, acetate, chloride and bromide, preferably trifluoroacetate.

In certain embodiments, the organic solvent is selected from acetonitrile, ethyl acetate, heptanes, hexanes, isopropyl acetate, methanol, methyl ethyl ketone, tetrahydrofuran, toluene, and water, or any combination thereof. In certain embodiments, the amorphous compound of formula (III) can be dissolved in an organic solvent selected from acetonitrile, heptanes, hexanes, methanol, tetrahydrofuran and toluene or any combination thereof. In certain embodiments, the organic solvent is preferably acetonitrile or toluene.

In certain embodiments, the preparation also includes washing crystals of formula (II). In certain embodiments, washing the crystals includes washing with a liquid selected from an antisolvent, acetonitrile, heptanes, hexanes, methanol, tetrahydrofuran, toluene, water, or a combination thereof. In certain embodiments, the crystals are washed with a combination of an antisolvent and an organic solvent. In certain embodiments, the antisolvent is water, while in other embodiments, it is an alkaline solvent, such as hexane or pentane, or an aromatic hydrocarbon solvent, such as benzene, toluene, or xylene.

In certain embodiments, the preparation also includes drying crystals of both compounds of formula (II), preferably under reduced pressure, for example in a vacuum.

In certain embodiments, the invention relates to a pharmaceutical composition containing a crystalline compound of formula (I) and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition is selected from tablets, capsules and solutions for injections.

Types of application of crystalline tripeptide epoxy ketones

Organized protein degradation plays a critical role in maintaining normal cellular functions, and the proteasome is an integral part of the protein degradation process. The proteasome regulates the levels of proteins that are important for the development of the cell cycle and apoptosis in normal and malignant cells, for example, cyclins, caspases, VSI 2 and ME-KV (Kiptayogi yei ai., Rgos. Mai. Asad. 5si. OBA (1990) 87: 7071-7075; AItopa and I., Heyketia (2002) 16: 433-443). Thus, it is not surprising that inhibition of proteasome activity can be incorporated into therapeutic approaches for the treatment of various pathological conditions, such as malignant, non-malignant and autoimmune diseases, depending on the cells involved.

Both ip miigo and ip mimo models have shown that malignant cells are, in general, susceptible to proteasome inhibition. Indeed, proteasome inhibition has also been substantiated as a therapeutic strategy for the treatment of multiple melanoma. This may be due in part to the dependence of highly proliferative malignant cells on the proteasome system for rapid protein clearance (Roige et al., U. Mo. Med. (1997) 75:5-17; Adatv, Maishte ( 2004) 4: 349-360). Therefore, certain embodiments of the invention relate to a method of treating oncological diseases, which includes administering to an individual in need of such treatment an effective amount of the compound described in this invention, a proteasome inhibitor. As used herein, the term "oncological diseases" includes, without limitation, hematological oncological diseases and solid tumors. Oncological diseases include diseases of the blood, bones, organs, skin tissue and vascular system, including, without limitation, oncological diseases of the bladder, blood, bones, brain, mammary glands, cervix, chest, colon, endometrium, esophagus, eyes , head, kidneys, liver, lungs, lymph nodes, mouth, neck, ovaries, pancreas, prostate, rectum, kidney structures, skin, stomach, testicles, throat and uterus. Specified cancers include, but are not limited to, leukemia (acute lymphocytic leukemia (AHL), acute myeloid leukemia (AMI), chronic lymphocytic leukemia (CI), chronic myeloid leukemia (CML), hairy cell leukemia), mature B-cell neoplasms (small lymphocytic lymphoma, B- prolymphocytic leukemia, lymphoplasmacytic lymphoma (such as Waldenström's macroglobulinemia), splenic marginal zone lymphoma, plasmacytic myeloma, plasmacytoma, monoclonal immunoglobulin deposition disease, heavy chain disease, extranodal marginal zone B-cell lymphoma (MAG T lymphoma), nodular B-cell lymphoma marginal zone (MMA), follicular lymphoma, mantle cell lymphoma, diffuse B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma and Burkitt's lymphoma/leukemia), mature T-cell neoplasms and natural killer (NK) cells (T-cell prolymphocytic leukemia, T-cell large granular lymph oleukosis, aggressive MK-cell leukemia, adult T-cell leukemia/lymphoma, extranodal MK/I-cell lymphoma, enteropathy-type T-cell lymphoma, hepatosplenic T-cell lymphoma, blast MK-cell lymphoma, mycosis fungoides (Sézary syndrome ), primary cutaneous anaplastic large-cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T-cell lymphoma, unspecified T-cell lymphoma and anaplastic large-cell lymphoma), Hodgkin's lymphoma (nodular sclerosis, mixed cellularity, enriched in lymphocytes, with depleted or non-depleted lymphocyte reserve, nodular with lymphocyte predominance), myeloma (multiple myeloma, painless myeloma, indolent myeloma), chronic myeloproliferative disease, myelodysplastic/myeloproliferative disease, myelodysplastic syndromes, lymphoproliferative disorders associated with immunodeficiency, histiocytic and dendritic cell neoplasms, mast sarcomocytosis, chondro , Ewing's sarcoma, fibrosarcoma, malignant g giant cell tumor, myeloma bone disease, osteosarcoma, breast cancer (hormone-dependent, hormone-independent), gynecological oncological diseases (cervix, endometrium, fallopian tubes, gestational trophoblastic disease, ovaries, peritoneum, uterus, vagina and vulva), basal cell carcinoma ( BCC), squamous cell carcinoma (5CC), malignant melanoma, protruding dermatofibrosarcoma, Merkel cell carcinoma, Kaposi's sarcoma, astrocytoma,

pilocytic astrocytoma, dysembryoplastic neuroepithelial tumor, oligodendroglioma, ependymoma, glioblastoma multiforme, mixed glioma, oligoastrocytoma, medulloblastoma, retinoblastoma, neuroblastoma, germinoma, teratoma, malignant mesothelioma (peritoneal mesothelioma, pericardial mesothelioma, pleural mesothelioma), gastroenteropancreatic or gastroenteropancreatic tumor MET), carcinoid, pancreatic endocrine tumor (PET), colorectal adenocarcinoma, colorectal carcinoma, aggressive neuroendocrine tumor, leiomyosarcoma, mucinous adenocarcinoma, signet ring cell adenocarcinoma, hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, hemangioma, hepatic adenoma, focal nodular hyperplasia (hyperplastic, regenerative hamartoma), non-small cell lung carcinoma (M5CI C) (squamous cell lung carcinoma, adenocarcinoma, large cell lung carcinoma), small cell lung carcinoma, lung carcinoma prostate cancer (hormonally refractory, androgen-independent, androgen-dependent, hormone-insensitive), renal cell carcinoma and soft tissue sarcoma (fibrosarcoma, malignant fibrous histiocytoma, dermatofibrosarcoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, hemangiosarcoma, synovial sarcoma, malignant tumor of the perineurium of peripheral nerves/neurofibrosarcoma, extraskeletal osteosarcoma).

Many tumors of hematopoietic and lymphoid tissues are characterized by increased cell proliferation or a specific type of cells. Chronic myeloproliferative diseases (CMDs) are clonal disorders of hematopoietic stem cells, characterized by proliferation in the bone marrow of one or more myeloid differentiation lines, leading to an increased number of granulocytes, erythrocytes and/or platelets in the peripheral blood. In fact, the use of proteasome inhibitors for the treatment of such diseases is attractive and is currently being investigated (Siyopi et al., Nayetajuiodisa (2007) 92: 1124-1229). AML can include chronic myelogenous leukemia, chronic neutrophilic leukemia, chronic eosinophilic leukemia, polycythemia vera, chronic idiopathic myelofibrosis, essential thrombocytopenia, and chronic myeloproliferative disease not classified. One aspect of the invention is a method of treating PMS, which includes administering to an individual in need of such treatment an effective amount of a compound described in this application, which is a proteasome inhibitor.

Myelodysplastic/myeloproliferative diseases, such as chronic myelogenous leukemia, atypical chronic myelogenous leukemia, juvenile myelogenous leukemia, and myelodysplastic/myeloproliferative disease unclassified, are characterized by hypercellularity of the bone marrow due to proliferation in one or more of the myeloid lineages of differentiation. Inhibition of the proteasome described in this application by a compound or composition may serve as a treatment for said myelodysplastic/myeloproliferative diseases by administering to an individual in need of such treatment an effective amount of the compound or composition.

Myelodysplastic syndromes (MO5) belong to a group of hematopoietic stem cell disorders characterized by dysplasia and ineffective hematopoiesis in one or more of the main myeloid cell lineages. The effect of a proteasome inhibitor on ME-KV as a target in these hematological malignant diseases causes apoptosis, thereby destroying malignant cells (Vgatsp ei ai. Sei!i Oyeai apa Orinegepiiainop (2006) 13:748-758). Another embodiment of the invention is a method of treating MO5, which includes the introduction of an effective amount of the compound described in this invention to an individual in need of such treatment. MO5 includes refractory anemia with ring-shaped sideroblasts, refractory cytopenia with dysplasia of multiple lines of differentiation, refractory anemia with excess blasts, myelodysplastic syndrome, unclassified (|and myelodysplastic syndrome associated with an isolated chromosome de abnormality|(54).

Mastocytosis is the proliferation of mast cells and their subsequent accumulation in one or more organ systems. Mastocytosis includes, but is not limited to, cutaneous mastocytosis, painless systemic mastocytosis (PSM), systemic mastocytosis with associated clonal hematological, non-mast cell lineage-associated disease (CM-ANMMO), aggressive systemic mastocytosis (ASM), mast cell leukemia (MS), mast cell sarcoma (MO5) and subcutaneous mastocytoma. Another embodiment of the invention is a method of treating mastocytosis, which includes the administration of an effective amount of the compound or composition described in the present invention to an individual diagnosed with mastocytosis.

The proteasome regulates ME-KV, which in turn regulates genes involved in the immune and inflammatory response. For example, ME-KV is required for the expression of the gene K of the light chain of immunoglobulin, the gene of the α-chain of the receptor 1-2, the class I gene of the major histocompatibility complex and a number of cytokine genes encoding, for example, II--2, I-6, a factor stimulating granulocyte colonies, and IEM-B (Raiotrbega ei ai.,

Sow! (1994) 78:773-785). Thus, in certain embodiments, the invention relates to methods of influencing the expression level of I/-2, MNO-Y, I/-6, TMEa, IPM-B, or any of the previously mentioned proteins, each method comprising administering to an individual an effective amount the compound or proteasome inhibitor composition described in this invention. In certain embodiments, the invention includes a method of treating autoimmune diseases in a mammal, which includes administering a therapeutically effective amount of a compound or composition described in this application. As used herein, an "autoimmune disease" is a disease or disorder arising from and directed against an individual's own tissues.

Examples of autoimmune diseases or disorders include, without limitation, inflammatory responses such as inflammatory skin diseases, including psoriasis and dermatitis (eg, atopic dermatitis); systemic scleroderma and sclerosis; reactions associated with inflammatory bowel disease (such as Crohn's disease and ulcerative colitis); respiratory distress syndrome (including respiratory distress syndrome in adults; AKO5B); dermatitis; meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergic conditions such as eczema and asthma and other conditions involving T-cell infiltration and chronic inflammatory responses; atherosclerosis; insufficient adhesion of leukocytes; rheumatoid arthritis; systemic lupus erythematosus (SLE); diabetes (eg type 2 diabetes or insulin-dependent diabetes); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; allergic encephalomyelitis; Sjogren's syndrome; juvenile diabetes; and immune reactions associated with acute and delayed hypersensitivity, mediated by cytokines and T-lymphocytes, are usually found in tuberculosis, sarcoidosis, polymyositis, granulomatosis and vasculitis; pernicious anemia (disease

Edison); diseases involving leukocytic diapedesis; inflammatory disorder of the central nervous system (CNS); multiple organ damage syndrome; hemolytic anemia (including without limitation cryoglobulinemia or Coombs-positive anemia); generalized myasthenia; diseases mediated by the antigen-antibody complex; disease caused by an antibody against the glomerular basement membrane; antiphospholipid syndrome; allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome; bullous pseudobubble; pemphigus; autoimmune polyendocrinopathies; Reiter's disease; muscle stiffness syndrome; Behcet's disease; giant cell arteritis; nephritis caused by immune complexes; nephropathy caused by IdA; polyneuropathies caused by I9M; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia.

The immune system screens for autologous cells infected with viruses, undergoing oncogenic transformation, or presenting unfamiliar peptides on their surface.

Intracellular proteolysis generates small peptides for presentation to T lymphocytes to elicit class-mediated immune responses | Ministry of Emergency Situations Thus, in certain embodiments, the invention relates to a method of using a compound as an immunomodulatory agent to inhibit the alteration or presentation of an antigen in a cell, which includes exposure to a cell (or administration to an individual) of a compound described in this invention. Certain embodiments include a method of treating graft-versus-host disease or graft-versus-host disease in a mammal, which comprises administering a therapeutically effective amount of a compound described herein. As used herein, the term "graft" refers to biological material obtained from a donor for transplantation into a recipient. Grafts include such diverse materials as, for example, isolated cells such as islet cells; tissue such as infant amniotic membrane, bone marrow, hematopoietic progenitor cells, and ocular tissue such as corneal tissue; and organs such as skin, heart, liver, spleen, pancreas, thyroid lobe, kidney, tubular organs (eg, intestine, blood vessels, or esophagus). Tubular organs can be used to replace damaged parts of the esophagus, blood vessels, or bile duct. Skin grafts can be used not only for burns, but also to cover damaged intestines or to close specific defects, such as a diaphragmatic hernia. The source of the graft can be any mammal, including humans, or cadavers or living donors. In some cases, the donor and the recipient are the same mammal. Preferably, the transplant is bone marrow or an organ such as a heart, and the donor of the transplant and the host are matched for class II NHA antigens.

Neoplasms from histiocytic and dendritic cells originate from phagocytes and additional cells that play the main roles in the processing and presentation of antigens to lymphocytes. It was shown that depleting the proteasome reserve in dendritic cells changes their antigen-induced responses (Sparatse et al., Sapseg Vez. (2006) 66:5461-5468). Thus, another embodiment of the invention includes the administration of an effective amount of the compound or composition described in the present invention to an individual with a tumor of histiocytes or dendritic cells. Neoplasms of histiocytes or dendritic cells include histiocytic sarcoma, Langerhans cell histiocytosis, Langerhans cell sarcoma, dendritic cell sarcoma/tumor, and unspecified dendritic cell sarcoma.

Proteasome inhibition has been shown to be beneficial in the treatment of proliferative diseases and immune disorders; thus, one embodiment of the invention includes the treatment of lymphoproliferative diseases (LPDs) associated with primary immune disorders (PIDs), which consists in administering an effective amount of the described compound to an individual in need thereof. The most common clinical forms of immunodeficiency associated with an increased frequency of lymphoproliferative disorders, including B-cell and T-cell neoplasms and lymphomas, are primary immunodeficiency syndromes and other primary immune disorders, infection with the human immunodeficiency virus (HIV, HIV), iatrogenic suppression of immunity in patients who received organ or bone marrow allotransplants, and iatrogenic immunosuppression associated with methotrexate treatment.

Other RIOs commonly associated with IPO include, but are not limited to, ataxia telangiectasia (AT), Wiskott-Aldrich syndrome (M/A5), common variable immunodeficiency (CVID), severe combined immunodeficiency (SCI), X-linked lymphoproliferative disorder (LID), Nijmegen syndrome of chromosomal breaks (MV5), hyper-IAM syndrome and autoimmune lymphoproliferative syndrome (AG RB).

Additional variants of the invention relate to methods of influencing the proteasome-dependent regulation of oncoproteins and methods of treating or inhibiting the growth of a malignant neoplasm, and each method includes the effect on a cell (i.p. mimo, for example, in an individual, or i.p. migo) described in this application with the proteasome inhibitor composition. Proteins derived from HRM-16 and NRMU-18 Eb stimulate ATIF- and ubiquitin-dependent conjugation and degradation of p53 in crude reticulocyte lysates.

It was shown that the recessive oncogene p53 accumulates at a non-permissive temperature in a cell line with mutated thermolabile ET. Increased levels of p5OZ can lead to apoptosis. Examples of proto-oncoproteins degraded by the ubiquitin system include c-Mo5, c-Bo5, and c-Up. In certain embodiments, the invention relates to a method of treating p53-related apoptosis, which includes administering to an individual an effective amount of a proteasome inhibitor composition described in this invention.

Another aspect of the invention relates to the use of the proteasome inhibitor compositions described herein for the treatment of neurodegenerative diseases and conditions, including but not limited to stroke, ischemic nerve injury, nerve injury (e.g., concussion, spinal cord injury, and traumatic nerve injury), multiple sclerosis, and other immune-mediated neuropathies (eg, Guillain-Barre syndrome and its variants, acute motor axonal neuropathy, acute inflammatory demyelinating polyneuropathy, and Fisher syndrome), HIV/AIDS dementia complex, axonomia, diabetic neuropathy, Parkinson's disease,

Huntington's disease, multiple sclerosis, bacterial, parasitic, fungal and viral meningitis, encephalitis, vascular dementia, dementia due to multiple infarcts, dementia with Lewy bodies, dementia associated with frontal lobe lesions such as Pick's disease, subcortical dementias (such such as Huntington's disease or progressive supranuclear palsy), focal cortical atrophy syndromes (such as primary aphasia), metabolic-toxic dementias (such as chronic hypothyroidism or B12 deficiency), and dementias caused by infections (such as syphilis or chronic meningitis).

Alzheimer's disease is characterized by extracellular deposits of B-amyloid protein (B-AR) in senile plaques and brain vessels. B-AP is a peptide fragment of 39-42 amino acids derived from the precursor of amyloid protein (APP). At least three APP isoforms (695, 751 and 770 amino acids) are known. Alternative mRNA splicing generates isoforms; normal processing affects part of the VD-AR sequence, thereby preventing the generation of B-AR. Abnormal protein processing by the proteasome is thought to contribute to the abundance of B-ARs in the brain in disease

Alzheimer's The APP processing enzyme in rats contains approximately 10 different subunits (22-32 kDa).

The 25 kDa subunit has the M-terminal sequence X-Cs1Ip-Avp-Rgo-Mei-x-TNg-Stpu-Tyg-Zeg, which is identical to the p-subunit of the human multicatalytic protease (Koiiita, 5. ei aiI.,, Ed. Eig. Viospet. Zos, (1992) 304:57-60). The APP processing enzyme cleaves the SiP'-I uv!b bond: in the presence of a calcium ion, the enzyme also cleaves the Mei"--Avr' bond and the A5p!--AIa?g bond to release the extracellular domain In AR.

Therefore, one aspect of the invention relates to a method of treating Alzheimer's disease, which comprises administering to an individual an effective amount of a proteasome inhibitor compound or composition described herein. Such treatment includes reducing the rate of B-AR processing, reducing the rate of B-AR plaque formation, reducing the rate of generating VD-AR and reducing the clinical signs of the disease

Alzheimer's

Fibrosis is an excessive and persistent formation of fibrous connective tissue that occurs as a result of hyperproliferative growth of fibroblasts and is associated with the activation of the TOE-VD signaling pathway. Fibrosis involves extensive deposition of extracellular matrix and can occur essentially within any tissue or across several different tissues. Of course, the level of intracellular signaling protein (Zta(y), which activates the transcription of target genes after TOE-B stimulation, is regulated by the activity of the proteasome (Khi ei ai!., 2000). However, accelerated degradation of TOE-D signaling components was observed in fibrotic conditions such as cystic fibrosis, injection fibrosis, endomyocardial fibrosis, idiopathic pulmonary fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis. Other conditions often associated with fibrosis include cirrhosis, diffuse parenchymal lung disease, pain syndrome after vasectomy, tuberculosis, sickle cell anemia and rheumatoid arthritis.

One embodiment of the invention is a method of treating a fibrotic or fibrotic-related condition, which includes the administration of an effective amount of the composition described in this invention to an individual in need of such treatment.

Treatment of burn victims is often hindered by fibrosis. Thus, in certain embodiments, the invention relates to local or systemic administration of the subject inhibitor for the treatment of burns. Closure of wounds after surgical interventions is often associated with the formation of deforming scars, which can be prevented by inhibition of fibrosis. Thus, in certain embodiments, the invention relates to a method of preventing or reducing deforming scarring.

It is believed that overproduction of lipopolysaccharide (LPS)-induced cytokines such as

TMEa is a central mechanism in the processes associated with septic shock. In addition, it is generally accepted that the first stage in the activation of GR5 cells is the binding of I RB5 to specific membrane receptors. α- and VD-subunits of the proteasome complex 205 have been identified as I/P5-binding proteins, suggesting that GR5-induced signal transduction may be an important therapeutic target in the treatment or prevention of sepsis (O!tsgezpi, M. ei ai ., U. Ittip (2003) 171: 1515-1525). Therefore, in certain embodiments, the proteasome inhibitor composition can be used to inhibit TMEs to prevent and/or treat septic shock.

Ischemia and reperfusion injury lead to hypoxia, a condition in which there is a shortage of oxygen reaching body tissues. This condition causes an increased decay of Ik-Va, with the help of this, leading to the activation of ME-KV (Koopd ei aiI., 1994). It has been demonstrated that the severity of hypoxia damage can be reduced by administration of a proteasome inhibitor (Bao et al., 2000; Bao et al., 2001; Ruye et al., 2003). Therefore, certain embodiments of the invention relate to a method of treating an ischemic condition or reperfusion injury, which includes administering to an individual in need of such treatment an effective amount of the proteasome inhibitor compound described in this invention.

Examples of such conditions or damage include, without limitation, acute coronary syndrome (vulnerable plaques), arterial occlusive disease (occlusions of the heart, peripheral arteries and other vessels), atherosclerosis (coronary sclerosis, lesions of the coronary arteries), heart attacks, heart failure, pancreatitis, hypertrophy myocardium, stenosis and restenosis.

ME-KV also specifically binds to the HIV enhancer/promoter. Compared to Mei tas239,

The HIV-regulatory protein Mei rri14 differs by two amino acids in the region that regulates protein kinase binding. It is believed that the protein kinase transmits the phosphorylation signals of IKkV, triggering the degradation of IKV through the ubiquitin-proteasome pathway. After decay, ME-KV is released into the nucleus, thus enhancing HIV transcription (Sopep, 9., zZsiepse, (1995) 267:960). In certain embodiments, the invention relates to a method of inhibiting or reducing HIV infection in an individual or a method of reducing the level of viral gene expression, and each method includes administering to the individual an effective amount of the proteasome inhibitor compound or composition described in this application.

Viral infections contribute to the pathology of many diseases. Heart conditions such as ongoing myocarditis and dilated cardiomyopathy have been associated with coxsackievirus B3. In a comparative whole-genome microarray analysis of infected mouse hearts, specific proteasome subunits were uniformly stimulated in the hearts of mice that developed chronic myocarditis (5laiau eyi a At U Raiyo! 168:11542-52, 2006). Some viruses use the ubiquitin-proteasome system at the stage of viral entry, where the virus is released from the endosome into the cytosol. Murine hepatitis virus (MUHV) belongs to the Sogopamygidae family, which also includes severe acute respiratory syndrome (SARS) coronavirus. We et al. (9) MigoY 79:644-648, 2005) demonstrated that treatment of MNM-infected cells with a proteasome inhibitor resulted in decreased viral replication, which correlated with a reduced viral titer, compared to the titer of untreated cells. Hepatitis virus

In humans (NVU/), a member of the Neradpamigidae family of viruses, similarly requires virally encoded envelope proteins for reproduction. Inhibition of the proteasome degradation pathway causes a significant decrease in the number of secreted membrane proteins (bZitvzek ei ai., / Myo! 79:12914-12920, 2005). In addition to HBV, other hepatitis viruses (A, C, O and E) can also use the ubiquin-proteasome degradation pathway for secretion, morphogenesis and pathogenesis. Accordingly, in certain embodiments, the invention relates to a method of treating a viral infection, such as BAS or hepatitis A, B, C, p, and E, which involves contacting a cell with (or administering to an individual) an effective amount of a compound or composition described herein.

In certain embodiments, the described composition can be used to treat a parasitic infection, such as an infection caused by protozoan parasites. It is believed that the proteasome of these parasites is primarily involved in such types of cellular activity as differentiation and replication (Radat ei ai., Tgepab5 Ragavzjoi. 2003, 19(23: 55-59). In addition, it was shown that Entamoeba species lose encystment ability under the influence of proteasome inhibitors (Soplaiez ei ai!., Agsp. Mei. Kev. 1997, 28, res Mo 139-140).In such specified embodiments, administration protocols for proteasome inhibitor compositions are used to treat parasitic infections in humans caused by a protozoan parasite selected from Riaztoaicht species (including R. TaIsiragit, R. mimah, R. Maiagiae and R. omaie, which cause malaria), Tgurapozhota species (including T. sgy7i, which causes Chagas disease, and T. Vgysei, which causes African sleeping sickness), IGeizhptapia species (including /. atalopev, |. dopomapi, Y.. iptapisht, G. tehisapa, etc.),

Rpesthosuzvia sagipii (the most common protozoa known to cause pneumonia in patients with AIDS and other immunosuppression), Tochoriaeta dopai, Epiatoeba ppiziouis, Epiatoeba ipmadepz and Siagaia

Iatbia. In certain embodiments, the described proteasome inhibitor compositions are used for the treatment of parasitic infections in animals and livestock caused by protozoan parasites selected from Riaztoditschst Pegtapi, Sturiob5rogiaiisht species, Espiposossib5 dgapiyo5iv5, Eitegia eepePa,

Zagsosuzii5 pepygopa and Meigozroga sgazza. Other compounds that act as proteasome inhibitors in the treatment of parasitic diseases are described in UMO 98/10779, which is fully incorporated herein by reference.

In certain embodiments, the proteasome inhibitor composition inhibits proteasome activity in the parasite without extracting it from erythrocytes and leukocytes. In such defined embodiments, a long half-life in blood cells can provide long-lasting protection against therapy against recurrent parasite infestation. In certain embodiments, proteasome inhibitor compositions can provide long-lasting chemoprophylaxis protection against future infection.

Prokaryotes have the equivalent of the eukaryotic proteasome particle 205. Although the subunit composition of the prokaryotic particle 205 is simpler than that of eukaryotes, it is capable of hydrolyzing peptide bonds in a similar manner. For example, a nucleophilic attack on a peptide bond occurs through a threonine residue at the M-end of B-subunits. Thus, one embodiment of the present invention relates to a method of treating prokaryotic infections that includes administering to an individual an effective amount of a compound or composition described herein. Prokaryotic infections may include diseases caused by either mycobacteria (such as tuberculosis, leprosy, or ulcer

Buruli), or archaebacteria.

Inhibitors that bind to the proteasome 205 have also been shown to stimulate bone formation in organ bone cultures. In addition, when such inhibitors were administered systemically to mice, the identified proteasome inhibitors increased bone volume and bone formation rate by more than 70 95 (Satei, I.V. Yei Ai., U. Siip. Ipmevi. (2003) 111: 1771-1782), indicating that the ubiquitin-proteasome mechanism regulates the differentiation of osteoblasts and the formation of bone tissue. Therefore, the described compound or proteasome inhibitor composition can be used in the treatment and/or prevention of diseases associated with bone loss, such as osteoporosis.

Thus, in certain embodiments, the invention relates to a method of treating a disease or condition selected from an oncological disease, an autoimmune disease, a transplant-related condition, a neurodegenerative disease, a fibrosis-related condition, an ischemia-related condition, infection (viral, parasitic or prokaryotic) and diseases associated with the loss of bone tissue, which includes the introduction of the compound or composition described in this application.

Introduction of crystalline tripeptide epoxy ketones

The compounds obtained as described in this application can be administered in various forms, depending on the disorder to be treated and the age, condition and body weight of the patient, as is well known in the art.

For example, when the compounds are to be administered orally, they may be in the form of a tablet, capsule, granule, powder or syrup; or for parenteral administration, they can be in the form of solutions for injections (intravenous, intramuscular or subcutaneous), preparations for drip infusion or suppositories. For intraocular administration, the compounds may be in the form of eye drops or eye ointments. Said formulations may be prepared by conventional means and, if desired, the active ingredient may be mixed with any conventional additive or excipient, such as a binding agent, disintegrant, lubricant, corrector, solubilizing agent, suspending agent, emulsifying agent, coating, cyclodextrin and/or buffer. Although the dosage will vary depending on the symptoms, the age and weight of the patient, the nature and severity of the disorder to be treated or prevented, the route of administration and the form of the drug; in general, the recommended daily dose is from 0.01 to 2000 mg of the compound for an adult human patient and can be administered as a single dose or in divided doses.

The amount of the active ingredient that can be combined with the carrier material to produce one dosage form is generally the amount of the compound that provides the therapeutic effect.

The exact time of administration and/or the amount of the composition that will give the most effective results in terms of the effectiveness of the treatment in a given patient will depend on the activity, pharmacokinetics and bioavailability of a particular compound, the physiological state of the patient (including age, sex, type and stage of the disease, general physical condition , reactivity to a given dosage and type of drug therapy), route of administration, etc. However, the above recommendations can be used as a basis for careful selection of treatment, for example, determining the optimal time of administration and/or the amount to be administered, which will require no more than conventional experimentation consisting of monitoring the individual and selecting the dosage and/or time of administration.

The phrase "pharmaceutically acceptable" is used herein to refer to those ligands, materials, compositions and/or dosage forms which, within the scope of sound medical judgment,

suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reaction or other problem or complication, according to a reasonable benefit/risk ratio.

As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or excipient, such as a liquid or solid excipient, diluent, excipient, diluent, or encapsulating material. Each carrier must be "acceptable" in the sense of compatibility with other ingredients of the formulation and harmless to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch, potato starch, and substituted and unsubstituted β-cyclodextrin; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (b) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline solution; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic excipients used in pharmaceutical formulations. In certain embodiments, the pharmaceutical compositions of the present invention are non-pyrogenic, that is, they do not cause significant temperature increases when administered to a patient.

The term "pharmaceutically acceptable salt" refers to relatively non-toxic addition salts of inorganic and organic acids of the inhibitor(s). These salts can be obtained separately during the final isolation and purification of the inhibitor(s) or by separate interaction of the purified inhibitor(s) in its free base form with a suitable organic or inorganic acid and isolation of the salt thus formed. Representative salts include hydrobromides, hydrochlorides, sulfates, bisulfates, phosphates, nitrates, acetates, valerates, oleates, palmitates, stearates, laurates, benzoates, lactates, phosphates, tosylates, citrates, maleates, fumarates, succinates, tartrates, naphthylates, mesylates, glucoheptonates , lactobionates, lauryl sulfonates and salts of amino acids, and the like (see, for example,

Vegaoe vie a). (1977) "Rpattasetsyisai! Zav", U. Rnapt. axis 66: 1-19).

In other cases, the inhibitors that can be used in the methods of the present invention can contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term "pharmaceutically acceptable salts" in these cases refers to relatively non-toxic additive salts of the inorganic and organic bases of the inhibitor(s). These salts may similarly be prepared during the final isolation and purification of the inhibitor(s) or by separate interaction of the purified inhibitor(s) in its free acid form with a suitable base such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation with ammonia or with a pharmaceutically acceptable organic primary, secondary or tertiary amine. Representative alkali and alkaline earth salts include lithium, sodium, potassium, calcium, magnesium and aluminum salts and the like. Representative organic amines used to form basic addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like (see, for example, Wegde et al., supra).

Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweeteners, flavoring and deodorizing agents, preservatives and antioxidants may also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal ion chelating agents such as citric acid, ethylenediaminetetraacetic acid (EOTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Dosage forms suitable for oral administration may be presented as capsules, wafers, pills, tablets, cakes (using a flavored base, usually sucrose and acacia or tragacanth), powders, granules, or a solution or suspension in an aqueous or non-aqueous liquid, or in the form of an oil-in-water or water-in-oil emulsion, or in the form of an elixir or syrup, or in the form of lozenges (using an inert matrix such as gelatin and glycerin or sucrose and acacia) and Mabo in the form of mouthwashes and the like, and each form contains a given amount of inhibitor(s) as an active ingredient. The composition can also be administered in the form of a bolus or paste of various consistencies.

In solid dosage forms for oral administration (capsules, tablets, pills, dragees,

powders, granules and the like) the active ingredient may be mixed with one or more pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate and/or one of the following: (1) fillers or bulking agents such as starches, cyclodextrins, lactose, sucrose, glucose, mannitol and/or silicic acid; (2) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants such as glycerin; (4) leavening agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain salicylates, and sodium carbonate; (5) dissolution retarding agents such as paraffin; (b) absorption accelerators such as quaternary ammonium compounds; (7) wetting agents such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents such as kaolin and bentonite clay; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and their mixtures; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also contain buffering agents. Solid compositions of a similar type can also be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like. In certain embodiments, the crystalline tripeptide epoxyketone is administered to the mammal in the form of a capsule. In another embodiment, the crystalline tripeptide epoxyketone is a compound of formula (I). In a more preferred embodiment, the crystalline tripeptide epoxyketone is a compound of formula (II).

The tablet may be obtained by compression or molding, possibly with one or more additional ingredients. Compressed tablets may be prepared using a binder (eg, gelatin or hydroxypropyl methylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (eg, sodium starch glycolate or cross-linked sodium carboxymethylcellulose), a surfactant, or a dispersing agent. Molded tablets may be obtained by molding in a suitable machine a mixture of powdered inhibitor(s) moistened with an inert liquid diluent.

Tablets and other solid dosage forms such as dragees, capsules, pills and granules may be scored or may be provided with coatings and shells such as enteric coatings and other coatings well known in the art of pharmaceutical formulation. They can also be formulated to provide a slow or controlled release of the active ingredient contained therein, using, for example, hydroxypropyl methylcellulose in different proportions to provide the desired release profile, other polymeric matrices, liposomes and/or microspheres. They can be sterilized, for example, by filtering through a bacteria-retaining filter or by including sterilizing agents in the form of a sterile injected medium immediately before use. It is also possible to contain contrast agents in said compositions, and they may also have a composition that releases only the active ingredient(s) or, preferably, in a specific part of the gastrointestinal tract, possibly in a delayed manner. Examples of potting compositions that can be used include polymeric substances and waxes. The active ingredient may also be presented in microencapsulated form, if appropriate, with one or more of the excipients described above.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents, and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (including cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycols, and sorbitan fatty acid esters and their mixtures.

In addition to inert diluents, oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweeteners, flavors, colors, odorants, and preservatives.

Suspensions, in addition to the active ingredient(s), may contain suspending agents, such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth and their mixtures.

Formulations for rectal or vaginal administration may be in the form of a suppository, which may be prepared by mixing one or more inhibitors with one or more suitable non-irritating excipients or carriers, including, for example, cocoa butter, polyethylene glycol, suppository wax, or salicylate, which is solid at room temperature but liquid at body temperature and therefore melts in the rectum or vaginal cavity and releases the active agent.

Formulations suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams, or aerosol formulations containing such carriers as are appropriate in the art.

Dosage forms for topical or transdermal administration of the inhibitor(s) include powders, aerosols, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active ingredient may be admixed under sterile conditions with a pharmaceutically acceptable carrier and with any preservatives, buffers or propellants that may be required.

Ointments, pastes, creams and gels may contain, in addition to the inhibitor(s), excipients such as animal or vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide or their mixtures.

Powders and aerosols may contain, in addition to the inhibitor(s), excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder or mixtures of these substances. Aerosols may additionally contain custom displacer gases such as chlorofluorocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

The inhibitor(s) may alternatively be administered by aerosol. This is done by obtaining an aqueous aerosol, a liposomal preparation or solid particles containing the composition. A non-aqueous suspension (for example, fluorocarbon displacer gas) can be used. Ultrasonic atomizers are preferred because they minimize exposure to the shearing agent that can cause compound disintegration.

Of course, an aqueous aerosol is obtained by including the agent in an aqueous solution or suspension along with conventional pharmaceutically acceptable carriers and stabilizers. Carriers and stabilizers can vary depending on the requirements for a specific composition, but usually include nonionic surfactants (Tmeepv, Rigopis5, sorbitan esters, lecithin,

Cremophores), pharmaceutically acceptable co-solvents such as polyethylene glycol, harmless proteins such as serum albumin, oleic acid, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols are generally obtained from isotonic solutions.

Transdermal patches or systems have the added advantage of providing controlled delivery of the inhibitor(s) into the body. Such dosage forms can be obtained by dissolving or dispersing the agent in a suitable medium. Absorption enhancers may also be used to increase the flux of the inhibitor(s) through the skin. The rate of such flow can be controlled either by providing a regulating frequency of the membrane or by dispersing the inhibitor(s) in the polymer matrix or gel.

Pharmaceutical compositions of the present invention suitable for parenteral administration contain one or more inhibitors in combination with one or more pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders, the moisture content of which can be reconstituted with sterile injectable solutions or dispersions immediately before use, which may contain antioxidants, buffers, bacteriostatic agents, solutes that make the preparations isotonic with the blood of the intended recipient, or suspending agents or thickeners.

Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like) and suitable mixtures thereof, vegetable oils such as olive oil, etc. jected organic esters such as ethyl acetate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be provided by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenolsorbic acid and the like. It may also be desirable to include tonicity-adjusting agents such as sugars, sodium chloride, and similar compounds in the composition. In addition, long-term absorption of the injected pharmaceutical form can be ensured by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

In some cases, to prolong the effect of the drug, it is desirable to ensure the possibility of absorption of the drug as a result of subcutaneous or intramuscular injection. For example, delayed absorption of a parenterally administered dosage form is carried out by dissolving or suspending the drug in an oil carrier.

Injectable depot forms are obtained by forming microcapsule matrices of the inhibitor(s) in biodegradable polymers such as polylactide-polyglycoside. Depending on the ratio of the drug to the polymer and the nature of the specific polymer used, the rate of release of the drug can be adjusted. Examples of other biodegradable polymers include poly(orthoethers) and poly(anhydrides). Injectable preparative depot forms are also obtained by placing the drug in liposomes or microemulsions compatible with body tissues.

Drugs can be administered orally, parenterally, topically or rectally. They are, of course, administered in forms suitable for each route of administration. For example, they are administered in the form of tablets or in the form of capsules, injections, inhalations, eye lotion, ointment, suppository, infusion, locally in the form of lotion or ointment, and rectally with a suppository. Oral administration is preferred.

The phrases "parenteral administration" and "parenterally administered" as used herein mean a route of administration other than enteral and local administration, usually by injection, and include without limitation intravenous, intramuscular, intraarterial, intratracheal, intracapsular, intraorbital , intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

As used herein, the phrases "systemic administration," "administered systemically," "peripherally administered," and "peripherally administered" refer to the administration of a ligand, drug, or other material by a route other than direct administration to the central nervous system such that it enters the system of the patient and thus subjected to metabolism and other similar processes, such as subcutaneous administration.

Said inhibitors) may be administered for the treatment of humans and other animals by any suitable route of administration, including administration orally, intranasally, for example in the form of an aerosol, rectally, intravaginally, parenterally, intracisternally and topically, for example in the form of powders, ointments or drops, including buccal and sublingual administration.

Regardless of the chosen route of administration, the inhibitor(s), which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

Effective dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response in a particular patient, composition and route of administration without toxicity to the patient.

The concentration of the described compound in a pharmaceutically acceptable mixture will vary depending on several factors, including the dosage of the compound to be administered, the pharmacokinetic characteristics of the compound used, and the route of administration. In general, the compositions of the present invention can be provided in an aqueous solution containing approximately 0.1-10 95 wt./vol. the compound described in this invention, among other substances, for parenteral administration. Typical dosage ranges are from about 0.01 to about 50 mg/kg body weight per day administered in 1-4 divided doses. Each fractional dose may contain some or different compounds of the invention. The dosage will be an effective amount that depends on several factors, including the general health of the patient, and the formulation and route of administration of the selected compound.

Definition

The term "schualalkyl" refers to substituted or unsubstituted saturated hydrocarbon groups, including straight-chain alkyl and branched-chain alkyl groups containing from x to y carbon atoms in the chain, including haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl, etc. d. Subalkyl indicates a hydrocarbon where the group is in the terminal position, the bond if internal. The terms "coalkenyl" and "coalkenyl" refer to substituted or unsubstituted unsaturated aliphatic groups similar in length and optional substitution to the alkyls described above, but which contain at least one double or triple bond, respectively.

"Alkoxy" refers to an alkyl group that has oxygen attached to it. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy, and the like. A "simple ether" is two hydrocarbons covalently linked by oxygen. Accordingly, the alkyl substituent, which makes the specified alkyl ether, is or resembles alkoxy.

The term "C:-alkoxyalkyl" refers to a C:-alkyl group substituted with an alkoxy group, thereby forming a simple ether.

As used herein, the term "C 1 -varalkyl" refers to a C 1 -alkyl group substituted with an aryl group.

The terms "amine" and "amino" are recognized in this field and refer to both unsubstituted and substituted amines and their salts, for example, a moiety that can be represented by the general formulas: vo vo / I -M -M- '

Кк or в ; where each of the groups RU, Vo and "E independently represents hydrogen, alkyl, alkenyl, -(CHg)t-NU, or KZ and Co,

taken together with the M atom to which they are attached, complete a heterocycle having from 4 to 8 atoms in the ring structure; EZ represents aryl, cycloalkyl, cycloalkenyl, heterocyclyl or polycyclyl; and t is 0 or an integer from 1 to 8. In preferred embodiments, only one of the PE groups? or

IN? can represent a carbonyl, for example RU, VO and nitrogen together do not form an imide. In even more preferred embodiments, CU and CU (and possibly CU) each independently represents hydrogen, alkyl, alkenyl, or -(CHg)t-H8. In certain embodiments, the amino group is basic, meaning that the protonated form has a pKa of 7.00.

The terms "amide" and "amino" are recognized in the art as amino-substituted carbonyl and include a moiety that can be represented by the general formula: (c)

Hv ro de KE", B" are as defined above. Preferred amide embodiments should not include imides, which can be unstable.

As used herein, the term "aryl" includes 5-, 6-, and 7-membered substituted or unsubstituted monocyclic aromatic groups in which each ring atom represents carbon. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbon atoms are common to two adjacent rings, where at least one of the rings is aromatic, for example, other cyclic rings may be cycloalkyl, cycloalkenyl , cycloalkynyl, aryl, heteroaryl and/or heterocyclyl. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.

As used herein, the terms "carbocycle" and "carbocyclyl" refer to non-aromatic substituted or unsubstituted rings in which each ring atom represents carbon. The terms "carbocycle" and "carbocyclyl" also include polycyclic ring systems having two or more cyclic rings in which two or more carbon atoms are common to two adjacent rings, where at least one of the rings is carbocyclic, for example, other cyclic rings may represent cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls and/or heterocyclyls.

The term "carbonyl" is recognized in the art and includes the following moieties, which can be represented by the general formula: (c) (c)

And Yap ZhK or ; where X represents a bond or represents oxygen or sulfur, and B' represents hydrogen, alkyl, alkenyl, -"CHg)t-BZ or a pharmaceutically acceptable salt, K"" represents hydrogen, alkyl, alkenyl or -(CHg)t- AAU, where t and KU are as defined above. When X represents oxygen and B" or KK" does not represent hydrogen, the formula represents an "ester". When X represents oxygen and B" represents hydrogen, the formula represents "carboxylic acid".

The term "heteroaryl" includes substituted or unsubstituted aromatic 5- to 7-membered ring structures, preferably 5- to b-membered rings, the ring structures of which include from one to four heteroatoms. The term "heteroaryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbon atoms are common to two adjacent rings, where at least one of the rings is heteroaromatic, for example, other cyclic rings may be cycloalkyl, cycloalkenyl , cycloalkynyl, aryl, heteroaryl and/or heterocyclyl. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, isoxazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.

As used herein, the term "heteroatom" means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, phosphorus and sulfur.

The term "heterocyclyl" or "heterocyclyl group" refers to substituted or unsubstituted non-aromatic 3- to 10-membered ring structures, preferably 3- to 7-membered rings, the ring structures of which include from one to four heteroatoms. The term "heterocyclyl" or "heterocyclic group" also includes polycyclic ring systems having two or more rings in which two or more carbon atoms are common to two adjacent rings, where at least one of the rings is heterocyclic, for example, other cyclic rings may represent cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls and/or heterocyclyls. Heterocyclic groups include, for example, tetrahydrofuran, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams and similar compounds.

As used herein, the term "C:-heterocycloalkyl" refers to a C1-alkyl group substituted with a heterocyclyl group.

The term "C 1 -hydroxyalkyl" refers to a C 1 -alkyl group substituted by a hydroxy group.

As used herein, the term "inhibitor" is intended to describe a compound that blocks or reduces enzyme activity (eg, inhibition of proteolytic cleavage of standard fluorogenic peptide substrates such as zys-G and MM-AMC, Vox-GI B-AMC and 2-1 E-AMP, inhibition of various types of catalytic activity of the proteasome 205). An inhibitor can act by competitive or non-competitive inhibition. An inhibitor can bind reversibly or irreversibly, and therefore the term includes compounds that are suicidal substrates of the enzyme. The inhibitor can modify one or more sites in or near the active site of the enzyme or it can cause a conformational change in other regions of the enzyme.

As used herein, the term "orally bioavailable" is intended to describe a compound administered to a mouse at a dose of 40 mg/kg or less, 20 mg/kg or less, or even 10 mg/kg or less, where one hour after oral administration, such compound exhibits inhibition of at least about 50 95, at least about 75 95, or even at least about 90 95 of the activity of the proteasome ST-Y in the blood.

As used herein, the term "peptide" includes not only the standard amide bond with standard α-substituents, but commonly used peptidomimetics, other modified bonds, non-naturally occurring side chains, and side chain modifications, as detailed below .

The term "polycyclyl" or "polycyclic" refers to two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and/or heterocyclyl) in which two or more carbon atoms are shared by two adjacent rings, e.g., the rings are "condensed rings". Each of the rings of the polycycle may be substituted or unsubstituted.

As used herein, the term "proteasome" is intended to include immuno- and constitutive proteasomes.

As used herein, the term "substantially pure" refers to a crystalline polymorph whose purity is greater than 90 95 , meaning that it contains less than 10 95 of any other compound, including the corresponding amorphous compound. Preferably, the crystalline polymorph has a purity greater than 95 95 or even a purity greater than 98 95.

The term "prevention" is also recognized in the art when used in relation to a condition such as a local recurrence (eg, pain), a disease such as cancer, a syndrome complex such as heart failure, or any other medical condition well understood in the art and may include administering a composition that reduces the frequency or delays the onset of symptoms of a medical condition in an individual relative to an individual not receiving the composition. Thus, preventing cancer includes, for example, reducing the incidence of detectable malignancy in a prophylactically treated patient population relative to an untreated control population, and/or delaying the occurrence of detectable malignant growth in a treated patient population , compared to an untreated control population, for example, by a statistically and/or clinically significant amount. Prevention of infection includes, for example, a reduction in the number of diagnoses of infection in a treated population compared to a non-treated control population and/or a delay in the onset of symptoms of infection in a treated population compared to a non-treated control population received treatment. Prevention of pain includes, for example, reducing the severity or delay in the onset of pain experienced by individuals in a treated population compared to an untreated control population.

The term "prodrugs" includes compounds that under physiological conditions are converted into therapeutically active agents. A common way to prepare prodrugs is to incorporate selected moieties that hydrolyze under physiological conditions to identify the desired molecule. In other embodiments, the prodrugs are converted by the enzymatic activity of the host animal.

The term "prophylactic or therapeutic" treatment is recognized in this field and includes the introduction into the body of the host of one or more described compositions. If the composition is administered before the clinical manifestation of the undesirable condition (eg, a disease or other undesirable condition in the host animal), then the treatment is prophylactic (that is, it protects the host organism from the development of the undesirable condition), and if it is administered after the manifestation of the undesirable condition, then the treatment is therapeutic (ie intended to reduce, relieve or stabilize an existing unwanted condition or its side effects).

As used herein, the term "proteasome" is intended to include immuno- and constitutive proteasomes.

The term "substituted" refers to moieties having substituents replacing hydrogen on one or more carbon atoms of the main chain. It should be understood that "substitution" or "substituted by" includes the assumption of the condition that such substitution occurs according to the allowed valence of the substituted atom and the substituent, and that the substitution results in a stable compound, for example, which does not undergo spontaneous transformation, e.g. , rearrangement, cyclization, elimination, etc. As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In a broad aspect, acceptable substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. Acceptable substituents may be single or multiple and may be the same or different for the respective organic compounds. For the purposes of this invention, heteroatoms, such as nitrogen, may have hydrogen substituents and/or any acceptable substituents of the organic compounds described in this invention, corresponding to the valence values of the heteroatoms. Substituents may include, for example, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl), thiocarbonyl (such as a thioether), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano , nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety. One of ordinary skill in the art will appreciate that parts substituted on the hydrocarbon chain may themselves be substituted if appropriate. A "therapeutically effective amount" of a compound with respect to a method of treatment under discussion refers to an amount of the compound(s) in the preparation that, when administered as part of a desired dosage regimen (to a mammal, preferably a human), alleviates a symptom, ameliorates a condition, or delays the onset of a pathological condition in accordance with clinically acceptable standards for of a treatable disorder or condition, or for a cosmetic purpose, for example, with an appropriate benefit/risk balance applicable to any medical treatment.

The term "simple thioether" refers to an alkyl group, as defined above, having a sulfur moiety attached to it. In a preferred embodiment, the "simple thioether" is represented by -5-alkyl.

Representative simple thioester groups include methylthio, ethylthio, and the like.

As used herein, the term "treatment" includes the reversal, reduction, or cessation of symptoms, clinical signs, and the underlying condition in order to improve or stabilize the individual's condition.

EXAMPLES

Example 1

Synthesis of Compound 1

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Me 1

Synthesis of Compound (A)

To a solution of M-Boc-serine methyl ether (43.8 g, 200 mmol), triethylamine (26.5 g, 260 mmol) and 4-(dimethylamino)pyridine in dichloromethane (1.2 L) at 0"C was added a solution of benzyl chloroformate (41 g, 240 mmol) in dichloromethane (250 mL) for 30 min. The resulting mixture was stirred at the same temperature for another 3 h. Saturated aqueous sodium bicarbonate (200 mL) was added, and the organic layer was separated, the residual mixture was extracted dichloromethane (2 x 400 mL).The combined organic layers were washed with saturated aqueous sodium bicarbonate (200 mL) and brine (200 mL), dried over sodium sulfate, and filtered through Celite-545.

The solvents were removed under reduced pressure and the residue was purified by flash chromatography (silica gel, hexane and ethyl acetate). Compound (A) (54 g) was isolated and characterized by IS/M5 (liquid chromatography/mass spectrometry) (M KM5 (MH) mp/7: 310.16).

Synthesis of Compound (B)

Trifluoroacetic acid (200 ml) was added to a solution of Compound (A) (54 g) in dichloromethane (200 ml) at 0 °C for 10 minutes, and the resulting mixture was stirred at the same temperature for another 3 hours. The solvents were removed under reduced pressure, and the residue was placed under high vacuum overnight, obtaining the TEA (trifluoroacetic acid) salt of Compound (B), which was characterized by I C/M5 (I KM5 (MH) t/2: 210.11).

Synthesis of Compound (C)

To a solution of Compound (B) (43.8 g, 200 mmol), M-Voc-serine methyl ether (36.7 g, 167 mmol), NOVT (hydroxybenzotriazole) (27 g, 200 mmol) and NVTI (1H- benzotriazolium) (71.4 g, 200 mmol) in tetrahydrofuran (1.2 L) at 0 "С, a solution of M, M-diethylisopropylamine (75 g, 600 mmol) in tetrahydrofuran (250 ml) was added over 10 minutes, and the pH of the resulting of the mixture amounted to "8. The mixture was stirred at room temperature for another 5 hours. Most of the solvent was removed under reduced pressure at room temperature and diluted with a saturated aqueous solution of sodium bicarbonate (400 ml). Then it was extracted with ethyl acetate (3x400 ml), washed with sodium bicarbonate ( 100 mL) and brine (100 mL). The combined organic layers were dried over sodium sulfate and filtered through Celite-545. The solvents were removed under reduced pressure. The residue was purified by flash chromatography (silica gel, hexane, and ethyl acetate). Compound (C) (65 g) was isolated and characterized

I C/mM5 (I AM5 (MH) t/: 411.21).

Synthesis of Compounds (0)

Trifluoroacetic acid (80 ml) was added to a solution of Compound (C) (18 g) in dichloromethane (100 ml) at 0 °C for 5 minutes, and the resulting mixture was stirred at the same temperature for another 3 hours. The solvents were removed under reduced pressure, and the residue was placed under high vacuum overnight, obtaining the TEA salt of intermediate compound (0), which was characterized by I C/M5 (I KM5 (MN) t/7: 311.15).

Synthesis of Compound (E)

An aqueous solution of sodium hydroxide (5M, 50 ml) was added to a solution of ethyl 2-methylthiazole-5-carboxylate (15 g, 88 mmol) in tetrahydrofuran (50 ml) at 0 °C for 10 minutes, and the resulting solution was stirred at room temperature for another 2 hours. It was then acidified with hydrochloric acid (2M) to pH-1 and extracted with tetrahydrofuran (3x100 mL). The combined organic layers were washed with brine (30 mL) and dried over sodium sulfate. Most of the solvents were removed under reduced pressure and the residue was lyophilized to obtain Compound (E) (14 g).

Compound Synthesis (BE)

To a solution of Compound (0) (41 mmol) and 2-methylthiazole-5-carboxylic acid (E) (6.0 g, 42 mmol),

NOVT (7.9 g, 50 mmol) and NVTIi (18.0 g, 50 mmol) in tetrahydrofuran (800 ml) at 0 "С was added to a solution of M, M-diethylisopropylamine (50 g) in tetrahydrofuran (200 ml) for 5 minutes until its pH reached about 8.5. The resulting mixture was stirred at the same temperature overnight. It was then quenched with saturated aqueous sodium bicarbonate solution (200 mL), and most of the solvents were removed under reduced pressure. The residual mixture was extracted with ethyl acetate (3 x 400 mL). The combined organic layers were washed with saturated aqueous sodium bicarbonate solution (200 mL) and brine (100 mL), dried over sodium sulfate, and filtered through Celite 545. The solvents were removed under reduced pressure, and the residue was purified by flash chromatography (silica gel, ethyl acetate with 2% methanol).Compound (E) (17.1 g) was isolated and characterized by I. C/M5 (I KM5 (MN) n/7: 436.15).

Synthesis of Compounds (0)

10 95 Ra/C (3 g) was added to a solution of Compound (BE) (17.1 g, 95 mmol) in methanol (300 ml). The resulting mixture was allowed to mix at a pressure of 1 atm. hydrogen for 48 hours. The mixture was filtered through Celite-545 and the filter cake was washed with methanol (2200 mL). The organic layers were concentrated under reduced pressure, and the residue was placed under high vacuum to obtain Compound (C), which was characterized by I C/M5 (I KM5 (MN) ip/7: 346.1).

Synthesis of Compound (H)

M-Boc-phenylalanine ketoepoxide (140 mg, 0.46 mmol) was diluted with OSM (dichloromethane) (2 mL) and cooled to 0 °C. Trifluoroacetic acid (6 mL) was added to this solution. The cooling bath was removed and the reaction mixture was stirred for 1 hours, and at this time TLC (thin layer chromatography) showed complete consumption of the starting material.The resulting solution was concentrated under reduced pressure and placed under high vacuum to obtain the TEA salt of Compound (H).

Synthesis of Compound 1

To a solution of the above Compound (H) (131 mg, 0.38 mmol) and (3) (0.46 mmol), NOVT (75 mg, 0.48 mmol) and NVTI (171 mg, 0.48 mmol) in tetrahydrofuran (20 ml) and M, M-dimethylformamide (10 ml) at

M, M-diethylisopropylamine (1 ml) was added dropwise to the solution. The mixture was stirred at the same temperature for another 5 hours. It was then quenched with a saturated aqueous solution of sodium bicarbonate (20 mL), and most of the solvents were removed under reduced pressure. Then the residual mixture was extracted with ethyl acetate (3x40 ml). The combined organic layers were washed with saturated aqueous sodium bicarbonate solution (20 mL) and brine (10 mL), dried over sodium sulfate, and filtered through Celite-545. The solvents were removed under reduced pressure and the residue was purified by HPLC (0.02 M aqueous ammonium acetate and acetonitrile (66/34)) to give Compound 1 (92 mg), which was lyophilized and characterized by I C/M5 (I KM5 (MH ) t/7: 533.2).

Example 2

Amorphous Compound 1 (50 mg) was dissolved in acetonitrile (1 mL), then deionized water (2 mL) was added, and the solution was supersaturated by slow evaporation of 1 mL over approximately 1-2 weeks. The obtained crystals were filtered, washed with 1 ml of acetonitrile-water solution in a ratio of 1:2 and dried in a vacuum for 12 hours to obtain the crystalline polymorph of Compound 1 (25 mu) with a melting point of 148 °C. Ipzigitepich UOShegepia! zZsappipud SaiYogiteseg 2920 at the heating rate "S/minute, shown in fig. 1.

Example C

Amorphous Compound 1 (611 mg) was dissolved in tetrahydrofuran (5 mL) followed by the addition of hexanes (5 mL), and a seed of the crystalline polymorph of Compound 1 obtained in Example 2 was added to the solution, and the solution was brought to supersaturation by slow evaporation of 5 mL for approximately 17 hours The obtained crystals were filtered, washed with 1 ml of tetrahydrofuran-hexanes solution. Chu ratios of 1:11 and dried in a vacuum for 12 hours to obtain a crystalline polymorph

Compound 1 (150 mg) with a melting point of 147 °C.

Example 4

Amorphous Compound 1 (176 mg) was dissolved in tetrahydrofuran (5 mL), then toluene (25 mL) was added.

A seed of the crystalline polymorph of Compound 1 obtained in Example 2 was added to the solution, and the solution was brought to supersaturation by slow evaporation of 20 ml for about 2 days. The obtained crystals were filtered, washed with 15 ml of toluene and dried under vacuum for 12 hours to obtain the crystalline polymorph of Compound 1 (88 mg) with a melting point of 149 °C.

Example 5

Amorphous Compound 1 (312 mg) was dissolved in toluene (50 mL) heated to about 100°C to complete dissolution, then hexanes (50 mL) were added, and the crystalline polymorph of Compound 1 obtained in Example 2 was seeded and the solution was adjusted to supersaturation by slow evaporation of 60 ml for about 2 days. The obtained crystals were filtered, washed with 10 ml of toluene and dried under vacuum for 12 hours to obtain the crystalline polymorph of Compound 1 (156 mg) with a melting point of 149 °C.

Example 6

Amorphous Compound 1 (1.4 g) was dissolved in toluene (25 mL) heated to 502C to complete dissolution, then the solution was brought to supersaturation by cooling to 222C and allowing the compound to crystallize for 12 hours. The obtained crystals were filtered, washed with 5 ml of hexanes and dried under vacuum for 12 hours to obtain the crystalline polymorph of Compound 1 (0.94 g) with a melting point of 149 "70.

Example 7

Synthesis of Compound 1

Synthesis of Compound (H)

M-Boc-phenylalanine ketoepoxide (1.0 equivalents) was dissolved in OSM (3 L/kg M-Boc-phenylalanine ketoepoxide) in a 3-neck round-bottom flask under an inert atmosphere, and the solution was cooled in an ice bath. TEA (5.0 equivalents) was then added at a rate to maintain the internal temperature below 10°C. The reaction mixture was then heated to approximately 207°C and stirred for 1-3 hours. Then MTBE (simple methyl tert-butyl ether) (3.6 l/kg of M-Voc-phenylalanine ketoepoxide) was added to the reaction mixture while maintaining the temperature of the mixture below 25"C. Then heptane (26.4 l/kg of M-Voc) was added -phenylalanine ketoepoxide), the reaction mixture was cooled to a temperature from -5 to 0 C for 2-3 hours to ensure the possibility of crystallization of Compound (H). The white solid was filtered and washed with heptane (3 l/kg of M-Voc-phenylalanine ketoepoxide). The white solid the substance was then dried under vacuum for 12 hours at 22 76.

The obtained yield was 86 95, with HPLC purity of 99.4 parts.

Synthesis of Compound 1

Compound (H) (1.2 equivalents), Compound (C) (1.0 equivalents), NVTi (1.2 equivalents), NOVT (1.2 equivalents) and M-methylpyrrolidinone (8 l/kg Compound (0) ) was added to a dry flask at an inert temperature, and the mixture was stirred at 23 °C to complete dissolution. The reaction mixture was then cooled to -5 to 0 °C, and isopropylethylamine (2.1 equivalents) was added over 15 minutes, in the same time, maintaining the internal temperature below 0 "C. The reaction mixture was stirred at 0 "C for 12 hours.

Crude Compound 1 was precipitated by pouring the reaction mixture into 8 95 sodium bicarbonate (40 l/kg

Compounds (C)) and a suspension of crude Compound 1 were stirred for 12 hours at 20-25 C, followed by stirring at 0-57 C for 1 hour. The white solid was filtered and washed with water (5 l/kg Compound (0)). Then the white solid was resuspended in water (15 l/kg) for 3 hours at 20-25"7C, filtered and washed with water (5 l/kg of Compound (5)) and isopropyl acetate (2 x 2 l/kg of Compound ()). White the solid substance was dried in a vacuum at 45 "C to a constant weight. The yield of crude Compound 1 was 65 95, with an HPLC purity of 97.2 Vol.

Crude Compound 1 was completely dissolved in isopropyl acetate (20 L/kg crude Compound 1) with stirring and heating at 85 °C. The solution was then filtered while hot to remove any particulate material, and the solution was reheated to 85 °C to give clear solution. The clear solution was allowed to cool at a rate of 10 °C per hour to 20 °C, when significant crystallization of Compound 1 occurred. The suspension was stirred at 20 °C for 6 hours, followed by stirring at 0-5 °C for a minimum of 2 hours and filtering and washing with isopropyl acetate (1 L/kg crude Compound 1).Purified Compound 1 was dried under vacuum at 45" for a minimum of 24 hours to constant weight. The yield of Compound 1 was 87 95, with an HPLC purity of 97.2 95.

Example 8

Synthesis of Compound 1

Compound (H) (1.1 equivalents), Compound (C) (1.0 equivalents), NVTi (1.5 equivalents), NOVT (1.5 equivalents) and UOME (8 l/kg of Compound (5)) were added into a dry flask under an inert atmosphere, and the mixture was stirred at 23 C to complete dissolution. The reaction mixture was then cooled to -5 to 0°C, and diisopropylethylamine (2.1 equivalents) was added over 15 minutes while maintaining the internal reaction temperature below 0°C. Then the reaction mixture was stirred at 0 "C for 3 hours.

The reaction mixture was quenched by adding pre-cooled saturated sodium bicarbonate solution (94 L/kg of Compound (5)), while maintaining the internal temperature at less than 10 °C.

The contents were then transferred to a dividing funnel. The mixture was extracted with ethyl acetate (24 L/kg of Compound (5)), and the organic layer was washed with a saturated solution of sodium bicarbonate (12 L/kg of Compound (0)) and a saturated solution of sodium chloride (12 L/kg of Compound (05)).

The organic layer was concentrated under reduced pressure at a bath temperature of less than 30 "C to 15 l/kg of Compound (0), followed by co-distillation with isopropyl acetate (2x24 l/kg RK-022). The final volume was brought to 82 l/kg Compound (0) with isopropyl acetate before heating to 60 "C to obtain a clear solution. The clear solution mixture was allowed to cool to 50°C before adding the seed crystals. The solution was allowed to cool to 20°C when significant crystallization of Compound 1 occurred. The suspension was stirred at 0°C for 12 hours before filtration and washing with isopropyl acetate (2 L/kg of Compound 1). Compound 1 was dried in a vacuum at "C for 12 hours to a constant mass. The yield of Compound 1 was 48 95, with HPLC purity of 97.4 95.

Equivalents

Those skilled in the art will appreciate or be able to determine by no more than routine experimentation the numerous equivalents of the compounds and their uses described in this application.

It is believed that such equivalents are within the scope of this invention and are covered by the following claims.

All of the above references and publications are incorporated herein by reference. and

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Claims (23)

1. The method of obtaining a crystalline compound of the formula (IP) ОоМе ие в) |c) more 9) M s M t M что : Н З в) х - Томе и Ме ХХО in which the following are carried out: (1) obtaining a solution of the compound of the formula (PI ) in an organic solvent; (11) supersaturating the solution to induce crystal formation; 1 (111) allocation of crystals. 2. The method according to claim 1, where the organic solvent is selected from acetonitrile, ethyl acetate, heptanes, hexanes, isopropyl acetate, methanol, methyl ethyl ketone, tetrahydrofuran, toluene, water or any combination thereof. 3. The method according to claim 2, where the organic solvent is selected from acetonitrile, heptanes, hexanes, methanol, tetrahydrofuran, 1 toluene. 4. The method according to claim 3, where the organic solvent is selected from hexanes, tetrahydrofuran and toluene. 5. The method according to any of claims 1-4, where bringing the solution to supersaturation is carried out by adding an antisolvent, allowing the solution to cool, reducing the volume of the solution, or any combination thereof. b. The method according to claim 5, where bringing the solution to supersaturation is carried out by adding an antisolvent, cooling the solution to ambient temperature 1 and reducing the volume of the solution. 7. The method according to claim 5, where the antisolvent is added slowly. 8. The method according to claim 5, where the antisolvent is selected from hexanes, toluene and water. 9. The method according to claim 5, where the volume reduction is carried out by evaporation. 10. The method according to any of claims 1-9, which additionally includes introducing the seed into the solution. 11. The method according to claim 10, which additionally includes washing the crystals. 12. The method according to item 11, wherein washing includes washing with a liquid selected from an antisolvent, acetonitrile, heptanes, hexanes, methanol, tetrahydrofuran, toluene, water, or a combination thereof. 13. The method according to claim 12, wherein washing includes washing with a combination of an anti-solvent and an organic solvent. 14. The method according to claim 13, where the antisolvent is hexanes or heptanes. 15. The method according to any one of claims 1-14, where the selection of crystals is carried out by filtration of crystals. 16. The method according to any of claims 1-15, in which crystals are additionally dried under reduced pressure. 17. A crystalline compound having the structure of the formula (P) ОМе (c) (c) MAH y M lo : MM х n И n (c) х (c) )- Tome Me «В 18. The crystalline compound according to claim 17, which has a ХО5С thermogram essentially as shown in FIG. 1. 19. The crystalline compound of claim 17 having a melting point of from about 140 to about 155 2C. 20. The crystalline compound of claim 17 having a melting point of from about 145 to about 150 2C. 21. The crystalline compound according to claim 17, which has the HECRI type, essentially as shown in FIG. 2. 22. The crystalline compound according to claim 17, which has values of 29 8.94; 9.39; 9.76; 10.60; 11.09; 12.74; 15.27; 17.74; 18.96; 20.58; 20.88; 21.58; 21.78; 22.25; 22.80; 24,25; 24.66; 26.04; 26.44; 28.32; 28.96; 29.65; 30.22; 30.46; 30.78; 32.17; 33.65; 34.49; 35.08; 35.33; 37.85; 38,48. 23. The method of obtaining a crystalline compound of the formula (P) ОМе (c) (c) МАХ y M lo : М М х н И н 5 (c) х (c) )- Tome Me ; (p) in which (1) the interaction of the compound of the formula (Ш) - Хх но НМ о is carried out; OP) where X represents any suitable protpion, with the compound of formula (II) in the second organic solvent ОМе (c) (c) ЖМ и : он х 5 (c) : )- Томе Ме ; M) (11) obtaining a solution of the compound of formula (PI) in the second organic solvent; (11) bringing the solution to supersaturation to ensure the possibility of crystal formation; 1 (y) separation of crystals to obtain a crystalline compound of formula (P).