EA037031B1 - Imidazopyrazine inhibitors of bruton's tyrosine kinase - Google Patents

Abstract

In some embodiments, the invention relates to the compounds of Formulae (I) and (II) or a pharmaceutically acceptable salt thereof and to their use in therapy. In particular, in some embodiments, the invention relates to the compounds of Formulae (I) and (II) or a pharmaceutically acceptable salt thereof and to their use in the treatment of a hyperproliferative disorder, an inflammatory disorder, an immune disorder, or an autoimmune disorder. The compounds of Formulae (I) and (II) have the following structures:

Description

Technology area

In some embodiments, this invention relates to compounds of formula (I) and (II), to pharmaceutical compositions containing these compounds, and to their use in therapy. In some embodiments, this invention relates to the use of a compound of formula (I) or (II) or a pharmaceutically acceptable salt in the treatment of a hyperproliferative disorder, an inflammatory disorder, an immune disorder, or an autoimmune disorder.

State of the art

Bruton's tyrosine kinase (TKB) is a non-receptor tyrosine kinase of the Tes family, expressed in B cells and myeloid cells. Research results confirm the key role of TCB in regulating antibody formation in autoimmune diseases. Also, inhibition of TCB appears to be particularly relevant to B-cell lymphomas due to chronic active BCR signaling, as described in Davis, et al., Nature, 2010, 463, 88-94.

In many solid tumors, the supporting microenvironment (which can make up the bulk of the tumor mass) is the dynamic force that allows the tumor to survive. A tumor microenvironment is mainly defined as a complex mixture of cells, soluble factors, signaling molecules, extracellular matrices and mechanical signals that promote neoplasm transformation, support tumor growth and invasion, protect the tumor from host immunity, maintain therapeutic resistance and provide niches for the growth of dominant metastases like described in Swartz, et al., Cancer Res., 2012,72, 2473. Although tumors express antigens that must be recognized by T cells, tumor clearance by the immune system is rare due to immune suppression by the microenvironment. Turning to the tumor cells themselves, for example, chemotherapy also proved to be insufficient to overcome the protective effects of the microenvironment. Thus, new approaches are urgently needed to more effectively treat solid tumors that take into account the role of the microenvironment.

The essence of the invention

In one aspect, the TCB inhibitor is a compound of formula (I) having the structure:

(I) or a pharmaceutically acceptable salt thereof.

In another aspect, the TCB inhibitor is a compound of formula (II) having the structure:

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(Ii) or a pharmaceutically acceptable salt thereof.

In another aspect, the invention relates to the use of a compound of formula (I) or (II) in the treatment of a hyperproliferative disorder, an inflammatory disorder, an immune disorder or an autoimmune disorder.

Brief Description of Drawings

The foregoing summary as well as the following detailed description of the invention will be better understood when read in conjunction with the accompanying drawings.

FIG. 1 shows a 1H- ¹³ C two / three-band correlation NMR spectrum of a compound of formula (I).

FIG. 2 shows the effect of a compound of formula (I) on a change in pre-incubation time (0, 30 or 60 min) and ATP concentration (5, 25 or 100 μM) in the IMAP TKB assay.

FIG. 3 shows the observed IC50 of the compound of formula (I) over time for TKB wild type (TKB-DT) and mutant TKB Cys481Ser (TKB-C481S) using the LanthaScreen assay.

FIG. 4 shows the dose-dependent effect of a compound of formula (I) on the occupancy of a TCB target in B Ramos cells.

FIG. 5 shows the primary metabolic pathways of acalabrutinib.

FIG. 6 shows the main oxidative metabolic pathway to M27 from acalabrutinib.

FIG. 7 shows metabolic pathways to M23 from acalabrutinib.

FIG. 8A and FIG. 8B shows the biotransformation pathways of acalabrutinib in humans together.

FIG. 9 shows that acalabrutinib and M27 are covalent inhibitors of TCB. The left figure shows the increase in efficiency over time for TKB-DT due to covalent binding over time. The right figure shows the reversible binding (affinity) of compounds with TCB-C481S and does not change over time. The difference in potency between TKB-DT and TKBC481S shows the effect of covalent binding.

FIG. 10 shows the occupancy of the target (3M TCB) with acalabrutinib and M27 in Ramos cells.

FIG. 11 shows a KINOMEscan profile analysis in a single dose (1 μM) M27 (DiscoveRx scanMAX).

FIG. 12 shows metabolic pathways to a compound of formula (II) from formula (III).

Detailed description of the invention

While preferred embodiments of the invention have been shown and described in the specification, such embodiments are presented by way of example only and do not limit the scope of the invention in any way. Various alternatives to the described embodiments of the invention may be used in the practice of the invention.

Unless otherwise indicated, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications cited are incorporated by reference in their entirety.

The term pharmaceutically acceptable salt refers to salts made from a variety of organic and inorganic counterions known in the art.

Pharmaceutically acceptable acid addition salts can be formed with inorganic and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, ptoluenesulfonic acid, and salicylic acid. Pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum. Organic bases from which salts can be derived include, for example, primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins. Specific examples include isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In selected embodiments, the pharmaceutically acceptable base addition salts are selected from ammonium, potassium, sodium, calcium, and magnesium.

Pharmaceutically acceptable carrier or pharmaceutically acceptable excipient includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents. The use of such media and agents for pharmaceutically active substances is well known in the art. Except where any conventional medium or agent is incompatible with the active ingredient, it is contemplated for its use in therapeutic compositions in accordance with this invention. Additional active ingredients can also be included in the described compositions.

Where ranges of, for example, physical or chemical properties such as molecular weight or chemical formulas are used herein, all combinations and sub-combinations of ranges and their specific variations are included. The use of the term about in relation to a number or numerical range means that the number or numerical range is approximate within the experimental variability (or within the experimental statistical error), and thus, the number or numerical range may vary from, for example, 1% -15 % of the specified number or numeric range. The term comprising (and related terms such as contains or contain, or having or including) include variations such as, for example, a variation of any composition of a substance, method or process that consists of or consists essentially of the described characteristics.

CONNECTIONS

A first embodiment provides a compound of formula (I), also named M27:

or a pharmaceutically acceptable salt thereof; or a compound of formula (II):

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(Ii) or a pharmaceutically acceptable salt thereof.

In one embodiment, the TCB inhibitor is a compound selected from the groups consisting of: 4- (8amino-3 - (4- (but-2-inamido) butanoyl) imidazo [1,5-a] pyrazin-1-yl) - N- (pyridin-2 yl) benzamide and 4- (8-amino-3- (4- (but-2inamido) butanoyl) imidazo [1,5-a] pyrazin-1-yl) -2-methoxy-N- (pyridin-2-yl) benzamide.

In one embodiment, the compound is a compound of formula (I), or a pharmaceutically acceptable salt thereof.

In one embodiment, the compound is a compound of formula (II), or a pharmaceutically acceptable salt thereof.

The compounds and salts of formulas (I) and (II) can exist in solvated forms and non-solvated forms. For example, the solvated form can be a hydrogenated form such as a hemihydrate, monohydrate, dihydrate, trihydrate, or an alternative amount thereof.

Compounds and salts of formulas (I) and (II) also include crystalline and amorphous forms of compounds of formulas (I) and (II), including, for example, polymorphs, pseudopolymorphs, solvates, hydrates, unsolvated polymorphs (including anhydrates), conformational polymorphs and amorphous forms of compounds, as well as mixtures thereof. Crystalline form and polymorph include all crystalline and amorphous forms of the compound, including, for example, polymorphs, pseudopolymorphs, solvates, hydrates, unsolvated polymorphs (including anhydrates), conformational polymorphs and amorphous forms of the compounds, as well as mixtures thereof, unless a specific crystalline or amorphous is indicated. the form.

In one embodiment, there is a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a compound of formula (II), or a pharmaceutically acceptable salt thereof, wherein the compound is in an isolated form.

A compound of formula (I) or a pharmaceutically acceptable salt thereof or of formula (II) or a pharmaceutically acceptable salt thereof in isolated form is one that is substantially free of other components, for example, organic components found in a living organism.

In one embodiment, there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, or a compound of Formula (II), or a pharmaceutically acceptable salt thereof, wherein the compound has a high purity of at least 90% as measured by HPLC.

In one embodiment, there is provided a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a compound of formula (II), or a pharmaceutically acceptable salt thereof, wherein the compound has a high purity of at least 95% as measured by HPLC.

In one embodiment, there is provided a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a compound of formula (II), or a pharmaceutically acceptable salt thereof, wherein the compound has a high purity of at least 96% as measured by HPLC.

In one embodiment, there is provided a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a compound of formula (II), or a pharmaceutically acceptable salt thereof, wherein the compound has a high purity of at least 97% as measured by HPLC.

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In one embodiment, there is provided a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a compound of formula (II), or a pharmaceutically acceptable salt thereof, wherein the compound has a high purity of at least 98% as measured by HPLC.

In one embodiment, there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, or a compound of Formula (II), or a pharmaceutically acceptable salt thereof, wherein the compound has a high purity of at least 99% as measured by HPLC.

In one embodiment, there is provided a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a compound of formula (II), or a pharmaceutically acceptable salt thereof, wherein the compound has a high purity of at least 10 0% as measured by HPLC.

In one embodiment, there is a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a compound of formula (II), or a pharmaceutically acceptable salt thereof, wherein the compound is prepared ex-vivo.

Ex-vivo means outside of a living organism, such as a human being being treated for cancer or other disease.

In one embodiment, there is a compound of Formula (I), or a pharmaceutically acceptable salt thereof, or a compound of Formula (II), or a pharmaceutically acceptable salt thereof, wherein the compound is prepared by organic synthesis. Organic synthetic routes are available to prepare a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, in relatively pure form, for example, 80% or more purity, 90% or more, 95% or more, 96% or more purity, 97 % or more, 98% or more, and 99% or more as measured by HPLC. Recrystallization and other purification methods can be carried out to obtain compounds that are substantially 100% pure. Such synthesis and purification methods are known in the art and are illustrated in a non-limiting manner in the examples that are presented below.

Organic synthesis means carrying out synthetic reactions in a laboratory or industrial equipment to obtain a product.

In one embodiment, a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, is in substantially pure form. Substantially pure means that the compounds are pure enough for FDA approval and are substantially free of impurities or other materials, or alternatively have an impurity level that does not adversely or unacceptably affect the properties of the compounds in terms of safety, efficacy, stability, and other desired properties.

PHARMACEUTICAL COMPOSITIONS

In selected embodiments, the invention provides pharmaceutical compositions for treating solid tumors, lymphomas, and leukemias.

The pharmaceutical compositions are usually formulated to provide a therapeutically effective amount of a compound of formula (I) or (II) as active ingredients, or a pharmaceutically acceptable salt thereof. If desired, the pharmaceutical compositions contain a pharmaceutically acceptable salt and / or their coordination complex, and one or more pharmaceutically acceptable excipients, carriers including inert solid diluents and excipients, diluents including sterile aqueous solution and various organic solvents, penetration enhancers, solubilizers and adjuvants ...

If desired, other agents can be mixed into a formulation, or both components can be formulated into separate formulations for use in combination, separately or simultaneously.

In selected embodiments, the concentration of each compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, provided in pharmaceutical compositions according to this invention is independently less than, for example, 100%, 90%, 80%, 70%, 60 %, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09% , 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0, 0003%, 0.0002% or 0.0001% w / w, w / v or about./about. in relation to the total weight or volume of the pharmaceutical composition.

In selected embodiments, the concentration of each compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, provided in pharmaceutical compositions according to this invention is independently greater than 90%, 80%, 70%, 60%, 50%, 40 %, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50 %, 17.25% 17%, 16.75%, 16.50%, 16.25% 16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14, 50%, 14.25% 14%, 13.75%, 13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11 , 50%, 11.25% 11%, 10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25% 8%, 7.75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75% , 5.50%, 5.25% 5%, 4.75%, 4.50%, 4.25%,

4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%,

1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07% , 0.06%, 0.05%, 0.04%, 0.03%,

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0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%,

0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w / w, w / v or about./about. in relation to the total weight or volume of the pharmaceutical composition.

In selected embodiments, the concentration of each compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, in accordance with this invention independently ranges from about 0.0001% to about 50%, from about 0.001% to about 40%, from about 0.01% to about 30%, about 0.02% to about 29%, about

0.03% to about 28%, about 0.04% to about 27%, about

0.05% to about 26%, about 0.06% to about 25%, about

0.07% to about 24%, about 0.08% to about 23%, about

0.09% to about 22%, about 0.1% to about 21%, about

0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, or about 1% to about 10% w / w, w ./about. or about./about. in relation to the total weight or volume of the pharmaceutical composition.

In selected embodiments, the concentration of each compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, in accordance with this invention independently ranges from about 0.001% to about 10%, from about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, about 0, 1% to about 0.9% w / w, w / v. or about./about. in relation to the total weight or volume of the pharmaceutical composition.

In selected embodiments, the amount of each compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof according to this invention is independently equal to or less than 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g,

0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007 g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g, 0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g or 0.0001 g.

In selected embodiments, the amount of each compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, according to this invention is independently greater than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g, 0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004 g, 0 , 0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g, 0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07 g, 0.075 g, 0.08 g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25 g, 0.3 g, 0.35 g, 0.4 g , 0.45 g, 0.5 g, 0.55 g, 0.6 g, 0.65 g, 0.7 g, 0.75 g, 0.8 g, 0.85 g, 0.9 g , 0.95 g, 1 g, 1.5 g, 2 g, 2.5 g or 3 g.

Each compound of formula (I) or (II) or pharmaceutically acceptable salt according to this invention is effective over a wide dosage range. For example, in the treatment of an adult, doses independently range from 0.01 to 1000 mg, 0.5 to 100 mg, 1 to 50 mg per day, and 5 to 40 mg per day are examples of dosages used. The exact dosage depends on the route of administration, the form in which the compound is administered, the sex and age of the subject being treated, the body weight of the subject being treated, and the preference and experience of the observing therapist.

Non-limiting pharmaceutical compositions and methods for their preparation are described below.

DOSES AND MODES OF ADMINISTRATION

The amount of a compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof to be administered depends on the mammal being treated, the severity of the disorder or condition, the rate of administration, the location of the compounds, and the discretion of the prescribing physician. However, an effective dose ranges from about 0.001 to about 100 mg per kg of body weight per day, for example, from about 1 to about 35 mg / kg / day, in one or more doses. For a person weighing 70 kg, this amount will be from about 0.05 to 7 days, for example, from about 0.05 to about 2.5 g / day. In some cases, dosage levels below the lower end of the above range may be more than adequate, and in other instances, large doses can be administered without causing harmful side effects - for example, dividing such large doses into several smaller doses for administration throughout the day.

In selected embodiments, a compound of formula (I) or (II) or a pharmaceutically acceptable salt is administered in a single dose. Typically, such administration is by injection, eg, intravenous injection, for rapid administration of agents. However, other routes can be applied as needed. A single dose of a compound of formula (I) or (II) or a pharmaceutically acceptable salt can also be varied for the treatment of an acute condition.

In selected embodiments, the compound of formula (I) or (II) or a pharmaceutically acceptable salt is administered in multiple doses. The introduction can be carried out one, two, three times, four times, five times, six times or more than six times a day. The introduction can be carried out once a month, every two weeks, once a week or every other day. In other embodiments, a compound of formula (I) or (II) or a pharmaceutically acceptable salt is administered from about once a day to about 6 times a day. In another embodiment, administration of a compound of formula (I) or (II) is continued for less than about days. In yet another embodiment, administration is continued for more than about 6, 10, 14, 28 days, two months, six months, or one year. In some cases, continuous administration is achieved and maintained for as long as necessary.

The introduction of agents in accordance with this invention can be continued as long as necessary. In selected embodiments, a compound of formula (I) or (II) or a pharmaceutically acceptable salt is administered for more than 1, 2, 3, 4, 5, 6, 7, 14, or 28 days.

In some embodiments, the compound of formula (I) or (II) or a pharmaceutically acceptable salt is administered over less than 28, 14, 7, 6, 5, 4, 3, 2, or 1 day. In selected embodiments, a compound of formula (I) or (II) or a pharmaceutically acceptable salt is administered chronically or continuously — for example, to treat chronic effects.

An effective amount of a combination of a compound of formula (I) or (II) or a pharmaceutically acceptable salt can be administered in one or more doses by any of the acceptable routes of administration of agents having similar uses, including rectal, buccal, intranasal and transdermal routes, by intra-arterial injection, intravenous, intraperitoneal, parenterally, intramuscularly, subcutaneously, orally, topically or by inhalation.

Based on in vitro studies, acalabrutinib is predominantly metabolized by CYP3A enzymes, and to a lesser extent by glutathione conjugation and amide hydrolysis. The compound of formula (I) is identified as a major plasma metabolite with a geometric mean contact (PIC) approximately 2-3 times higher than the acalabrutinib contact. The main metabolite is approximately 50% less potent than acalabrutinib in relation to TKP inhibition in biochemical analysis.

Akalabrutinib and the main metabolite form a covalent bond with a cysteine residue in the TKB active site, which leads to inhibition of the enzymatic activity of TKB. Binding of Akalabrutinib to the cysteine residue is rapid and irreversible and provides high target occupancy at steady state. Based on clinical studies where subjects received a single 100 mg oral dose of acalabrutinib, the median elimination half-lives (t1 / 2) of acalabrutinib and the major metabolite are 0.9 (range: 0.6-2.8) hours and 6.9 hours, respectively ... The mean apparent total clearance of Akalabrutinib (CL / F) is 159 L / h with similar pharmacokinetics in patients and healthy subjects based on population pharmacokinetic analysis.

It is possible that the more slowly cleared major metabolite, which is available over a longer period of time as serum levels of rapidly purified acalabrutinib decrease, provide additional benefit by inhibiting the newly synthesized TCB enzyme and maintaining a higher effective occupancy of the TCB target during the dosing interval. ...

Acalabrutinib is a weak inhibitor of CYP3A4 / 5, CYP2C8, and CYP2C9, but does not inhibit CYP1A2, CYP2B6, CYP2C19, and CYP2D6. It is a weak inducer of CYP1A2, CYP2B6 and CYP3A4. The main metabolite is a weak inhibitor of CYP2C8, CYP2C9 and CYP2C19, but does not inhibit CYP1A2, CYP2B6, CYP2D6 and CYP3A4 / 5. It is a weak inducer of CYP3A4.

METHODS OF TREATMENT

In one embodiment, the invention relates to a method for treating TKP-mediated disorder in a mammal, which comprises administering to said mammal a therapeutically effective amount of a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof.

In some embodiments, the invention provides a method of treating a hyperproliferative disorder, inflammatory disorder, immune disorder, or autoimmune disorder in a mammal, which comprises administering to said mammal a therapeutically effective amount of a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof.

In some embodiments, the invention relates to a method of treating, with a compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof, a hyperproliferative disorder in a mammal selected from the group consisting of bladder cancer, head and neck cancer, pancreatic ductal adenocarcinoma ( PAW), pancreatic cancer, colon cancer, breast carcinoma, breast cancer, fibrosarcoma, mesothelioma, renal cell carcinoma, lung cancer, thymoma, prostate cancer, rectal and colon cancer, ovarian cancer, acute myeloid leukemia, thymus cancer, brain cancer, plo- 7 037031 100-cell cancer, skin cancer, eye cancer, retinoblastoma, melanoma, intraocular melanoma, oral and oropharyngeal cancer, gastrointestinal cancer, stomach cancer, cervical cancer, head, neck, kidney cancer, kidney cancer, liver cancer, ovarian cancer, prostate cancer, colon cancer, esophageal cancer, testicular cancer, gynecological cancer, thyroid cancer, cancer, related o with acquired immunodeficiency syndrome (AIDS) (for example, lymphomas and Kaposi's sarcoma), viral cancer, glioblastoma, tumors of the esophagus, hematologic neoplasms, primary central nervous system lymphoma, non-small cell lung cancer (NSCLC), chronic myelocytic leukemia, diffuse ICL ), tumors of the esophagus, lymphoma of the center of follicle cells, tumors of the head and neck, viral hepatitis C, hepatocellular carcinoma, Hodgkin's disease, metastatic colon cancer, multiple myeloma, non-Hodgkin's lymphoma, ovarian tumor, pancreatic tumor, renal cell carcinoma, small cell carcinoma lung or stage IV melanoma. In selected embodiments, the invention relates to a method for treating a TCB inhibitor of disorders such as hyperreproliferative disorder, including but not limited to cancer such as acute myeloid leukemia, thymus, brain, lung, squamous cell, skin, eye, retinoblastoma, intraocular melanoma, cancer of the oral cavity and oropharynx, bladder, gastrointestinal tract, stomach, pancreas, bladder, breast, cervix, head, neck, kidneys, kidneys, liver, ovaries, prostate, rectum and colon, esophagus, testicles , gynecological cancer, thyroid cancer, CNS, PNS associated with AIDS (eg, lymphoma and Kaposi's sarcoma), or viral cancer. In some embodiments, the pharmaceutical composition is for the treatment of a non-cancerous hyperproliferative disorder such as benign skin hyperplasia (eg, psoriasis), restenosis, or prostate cancer (eg, benign prostatic hyperplasia (BPH)). In specific embodiments, a method of treating a hyperproliferative disorder comprises administering to a mammal a compound of this invention (eg, a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof). In one embodiment, a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, is directly administered to the mammal, but not administered to the mammal concurrently with another TCB inhibitor. In one embodiment, a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, is directly administered to the mammal, but not administered to the mammal simultaneously with acalabrutinib. In one embodiment, a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, is the only TCB inhibitor directly administered to a mammal.

In some embodiments, the invention provides a method for treating an inflammatory, immune, or autoimmune disorder in a mammal with a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof. In particular embodiments, a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, is administered directly to the mammal. In one embodiment, a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, is administered directly to the mammal, but not administered to the mammal concurrently with another TCB inhibitor. In one embodiment, a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, is administered directly to the mammal, but is not administered to the mammal simultaneously with acalabrutinib. In one embodiment, a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, is the only TCB inhibitor directly administered to a mammal. In selected embodiments, the invention also provides a method of treating a disease with a compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof, wherein the disease is selected from the group consisting of tumor angiogenesis, chronic inflammatory disease, rheumatoid arthritis, atherosclerosis, inflammatory bowel disease, skin diseases such as psoriasis, eczema and scleroderma, type 1 diabetes, type 2 diabetes, diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, hemangioma, glioma and melanoma, ulcerative colitis, atopic dermatitis, pauchitis, spondyloarthritis, uveitis, Behcet's disease polymyalgia rheumatica, giant cell arteritis, sarcoidosis, Kawasaki disease, juvenile idiopathic arthritis, purulent hydradenitis, Sorgen's syndrome, psoriasis arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, lupus, lupus nephritis Nom (PPA), autoantibodies, immunotherapy, Addison's disease, autoimmune polyendocrine syndrome type 1 (APS-1), autoimmune polyendocrine syndrome type 2 (APS-2), Graves disease, Hashimoto's thyroiditis, polyendocrine autoimmunity and hypothyroidism, hypothyroidism and lupus nephritis.

Direct administration means that a compound of formula (I) or formula (II), or a pharmaceutically acceptable salt of any of them, is administered to a patient directly, and not indirectly via administration of a precursor molecule. For any option that mentions the administration of a compound of formula (I) or formula (II), or a pharmaceutically acceptable salt of any of them, to a warm-blooded animal in a general sense, another option is provided wherein said compound or salt is administered directly.

In some embodiments, the invention provides a method for treating an inflammatory, immune, or autoimmune disorder selected from the group consisting of rheumatoid arthritis (RA), juvenile RA, juvenile idiopathic arthritis, osteoarthritis, psoriatic arthritis, common psoriasis, pemphigus, bullous pemphigoid, osteoarthritis infectious arthritis, progressive chronic arthritis, polymyalgia rheumatica, deforming arthritis, traumatic arthritis, gouty arthritis, Reiter's syndrome, polychondria, acute synovitis, ankylosing spondylitis, spondylitis, Sorgen's syndrome (SS), systemic lupus erythematosus (SLE), discoid KV), edematous KV, lupus nephritis (LN), antiphospholipidosis, dermatomyositis, polymyositis, autoimmune hematological diseases, thrombocytopenia, idiopathic thrombocytopenia purpura, thrombotic thrombocytopenia purpura, autoimmune) aglutogenous syndrome (cold) coy anemia, cryoglobulinemia, aplastic anemia, neutropenia, autoimmune vasculitis, Behcet's disease, vasculitis associated with antineutrophilic cytoplasmic antibody (ANCA), scleroderma, systemic sclerosis, myasthenia gravis, multiple sclerosis syndrome (PC), chronic hepatitis syndrome chronic fatigue, intolerance to systemic stress, optic neuromyelitis, autoimmune pruritus, conjunctivitis, keratoconjunctivitis, Gweves disease, thyroid-associated ophthalmopathy, chronic thyroiditis, granulomatosis with microscopic polyangitis, granulomatous ulcers, autoimmune diseases Crohn's, graft versus host disease, idiopathic celiac disease, autoimmune hepatitis, active hepatitis (acute and chronic), idiopathic pulmonary fibrosis, bronchitis, pulmonary interstitial fibrosis, chronic inflammatory disease lung disease, sarcoidosis, idiopathic membrane nephropathy, IgA nephropathy, glomerulosclerosis, glomerulonephritis (with or without nephrotic syndrome), pancreatitis, and type 1 or 2 diabetes.

In some embodiments, the invention provides a method for treating an inflammatory, immune, or autoimmune disorder selected from the group consisting of diabetic retinopathy, giant cell arteritis, Kawasaki disease, inflammatory bowel disease, irritable bowel disease, idiopathic celiac disease, enteropathy, postherpetic neuralgia, polymyalgia rheumatica, primary biliary cirrhosis, myasthenia gravis, inflammatory pain, cachexia, periodontal disease, pneumonia, otitis media, pneumoconiosis, mononucleosis, pulmonary emphysema, pulmonary fibrosis, silicosis, chronic inflammatory lung disease, chronic obstructive pulmonary disease, pulmonary insufficiency, pulmonary interstitial fibrosis Whipple disease, benign skin hyperplasia (eg, psoriasis), myalgias caused by infections, cachexia secondary to infections, systemic exercise intolerance, atherosclerosis, granulomatosis, granuloma toosis with microscopic polyangitis, purulent hydradenitis, age-related macular degeneration and amyloidosis.

In some embodiments, the invention provides a method of treating an inflammatory, immune, or autoimmune disorder, wherein the inflammatory, immune, or autoimmune disorder is dermatosis, in which TCB-mediated signals are involved in the recruitment, activation and / or proliferation of cells and the formation of inflammatory mediators and antimicrobial peptides in skin. In some embodiments, the invention provides a method for treating dermatosis, where the dermatosis arises from skin manifestations of systemic diseases, where sensitization, recruitment of lymphocytes, rejection of lymphocytes by antigen-presenting cells, local or in lymph nodes, activation of lymphocytes present or returned to the skin, innate immune perception, keratinocyte antimicrobial reactions, activation of resident or infiltration myeloid dendritic cells, plasmacytoid dendritic cells, macrophages, mast cells, neutrophils and / or Langerance cells, leads to the development of skin lesions. In some embodiments, the invention provides a method for treating dermatosis selected from the group consisting of psoriasis, guttate psoriasis, erythrodermic psoriasis, psoriasis nails, annular pustular psoriasis, pustular psoriasis, inverse psoriasis, psoriasis arthritis, blennorrhagic keratoderma, nodosum, parasitic - plantar hydradenitis, atopic dermatitis, atopic eczema, seborrheic eczema, seborrheic dermatitis, dyshidrosis, rosacea, cutaneous lupus erythematosus, acute cutaneous lupus erythematosus, subacute cutaneous lupus erythematosus, discoid lupus erythematosus, edematous lupus erythematosus (lupus erythematosus) panniculitis, erythema, warts, warty lupus erythematosus, vitiligo, alopecia areata, purigo nodosum, lichen planus, pruritus nodosum, lichen planus, pruritus pigmentosa, pemphigus vulgaris, bullous pemphigoid, pemphigus lupus, erythematosus sarcoidosis, granulomatous dermatitis, scleroderma, systemic sclerosis, cutaneous manifestations of systemic sclerosis, diffuse cutaneous mastocytosis, erythrodermic mastocytosis, annular granuloma,

- 9 037031 chondrodermatitis nodosa, contact dermatitis, drug toxidermia, linear IgA bullous dermatosis, eosinophilic dermatitis, follicular keratosis, lymphomatoid papulosis, acute lymphoid papuloid parapsoriasis (PLEVA), chronical fever leuchoriatic fever (PLEVA) FUMHD), chronic urticaria, rheumatoid neutrophilic dermatitis, cryoglobulinemic purpura, and hyperglobulinemic purpura.

In some embodiments, the invention provides a method for treating a hyperproliferative disorder, wherein the hyperproliferative disorder is a chronic autoimmune and inflammatory bone disorder, wherein TCB signaling in osteoclasts, mast cells, and myeloid cells is involved in osteolysis, osteoclastic processes, tissue imbalance or bone density remodeling bones. Diseases of this nature, which often also have an autoimmune component, include osteoarthritis, bone loss due to metastases, osteolytic lesions, osteoporosis, ankylosing spondylitis, spondyloarthritis, diffuse idiopathic skeletal hyperostosis, gouty arthritis, and bone disorders associated with multiple myeloma. In some embodiments, the invention provides a method for treating a hyperproliferative disorder, wherein the hyperproliferative disorder is selected from the group consisting of osteoarthritis, bone loss due to local stasis, osteolytic lesions, osteoporosis, ankylosing spondylitis, spondyloarthritis, diffuse idiopathic skeletal hyperostosis, and gouty disorders associated with with multiple myeloma.

In some embodiments, the invention provides a method of treating allergic and atopic diseases, in which activated B cells produce IgE antibodies and mast cells degranulate the next involvement of FceR, leading to the release of pro-inflammatory factors and acute activation of local tissue reactions, as well as chronic changes in endothelial cells, neuroreceptors and other proximal structures that control organ functions. Such conditions include atopic dermatitis, contact dermatitis, eczema, atopic eczema, pemphigus vulgaris, pemphigus bullous, pruritus nodosum, Stevens-Johnson syndrome, asthma, airway hypersensitivity, bronchitis, reactive asthma, chronic obstructive pulmonary disease, hypersensitivity 1 type, allergic rhinitis, allergic conjunctivitis and other inflammatory and obstructive respiratory diseases. Allergies that can be treated or prevented include, among others, allergies to food, food additives, insecticides, dust mites, pollen, animal products, metals, and certain drugs.

In one embodiment, the invention provides a method for treating graft versus host disease (GVHD) comprising the step of administering a compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof, wherein GVHD is selected from the group consisting of GVHD associated with a stem cell transplant, GVHD associated with bone marrow transplant, GVHD of the thymus, GVHD of the skin, gastrointestinal GVHD, GVHD of the liver, acute GVHD, and chronic GVHD. In specific embodiments, a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, is directly administered to a mammal. In one embodiment, a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, is directly administered to the mammal, but is not administered to the mammal concurrently with another TCB inhibitor. In one embodiment, a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, is directly administered to the mammal, but is not administered to the mammal simultaneously with acalabrutinib. In one embodiment, a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, is the only TCB inhibitor administered directly to the mammal.

In one embodiment, the drug inhibits neurodegenerative diseases that include microglial activation, recruitment and activation of macrophages, infiltration of inflammatory cells, including myeloid cells, which require TCB signaling for activation signals, recognition of integrins on activated endothelial cells, extravasate, or development into cytokine. and / or chemokine-producing cells in situ. Inhibition of TCB by a compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof will inhibit painful activity or disease progression through inhibition of neurodegenerative diseases associated with toxic protein aggregation, such as accumulation of beta-amyloid deposits (amyloid plaques), neurofibrillary tangles, tau aggregation and hyper-phosphorylation, intracytoplasmic viral inclusions, intracytoplasmic paired helical filaments, polyglycosan inclusions, Papp-Lantos bodies, ubiquitin-containing inclusions, and disorders in which inadequate control of protein degradation and / or inability to locate unfolded proteins leads to neurodegeneration. Such diseases include sporadic and hereditary Alzheimer's disease, mild cognitive impairment, cerebral amyloid angiopathy, Lewy body dementia, Lewy body variant of Alzheimer's disease, Down syndrome, Huntington's disease, strianigral degeneration, multiple system atrophy (MCA-R, MCA-C, syndrome Shay-Drager), sporadic or hereditary amyotrophic lateral sclerosis (ABS or Lou Gehrig's disease), primary lateral sclerosis, juvenile primary lateral sclerosis, neurodegenerative taupathies, sporadic or hereditary synucleinopathies, neuronal intranuclear inclusion disease, paroxysmal temporal lobe dementia with parkinsonism associated with chromosome 17 (FTDP-17).

In one embodiment, the invention relates to a method of treating a compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof, a neurodegenerative disorder in a mammal, wherein inhibition of inflammatory processes in glial cells, myeloid cells, Schwann cells, oligodendrocytes and other myeloid-derived cell types located in the central nervous system is achieved through their covalent interaction with TKP and inhibition of signaling through the TKP pathway. Administration of a compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof to prevent or reduce neurodegeneration through inhibition of immune recognition and inflammatory responses towards misfolded and / or accumulated intracellular proteins due to trinucleotide repeat disorders (polyglutamine diseases), Huntington's disease, spinocerebellar ataxia types 1, 2, 3 (Machado-Joseph disease), 6, 7 and 17; spinal and bulbar muscular atrophy, dentato-rubro-pallido-Lewis atrophy, neural waxy lipofuscinosis, frontotemporal dementia (Pick's disease, primary progressive aphasia and semantic dementia), cortico-basal degeneration, and progressive supranuclear palsy. In certain embodiments, the compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, is administered directly to the mammal. In one embodiment, a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, is directly administered to the mammal, but is not administered to the mammal concurrently with another TCB inhibitor. In one embodiment, a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, is directly administered to the mammal, but is not administered to the mammal simultaneously with acalabrutinib. In one embodiment, a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, is the only TCB inhibitor administered directly to the mammal.

In another embodiment, administration of a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, can be used to inhibit TCB in a mammal, thereby facilitating inflammation-mediated neuronal death and other neuroinflammatory effects due to sporadic or hereditary prion disease, prion disorders, such as Creutzfeldt-Jakob disease, Kuru, Gerstmann-Streussler-Scheinker syndrome, and disorders leading to olivopontocerebellar atrophy, sporadic fatal insomnia, fatal familial insomnia. In the case of familial prion disorders, administration of a compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof in mammals can also be used to prevent and / or delay the onset of clinical manifestations of the disease, in addition to reducing the symptoms of the disease and slowing the progression of the disease after the onset of clinical signs. In specific embodiments, a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, is administered directly to the mammal. In one embodiment, a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, is directly administered to the mammal, but is not administered to the mammal concurrently with another TCB inhibitor. In one embodiment, a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, is directly administered to the mammal, but is not administered to the mammal simultaneously with acalabrutinib. In one embodiment, the compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, is the only TCB inhibitor administered directly to the mammal.

In one embodiment, the invention relates to a method for treating a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, for an autoimmune mediated neurodegenerative disorder in the central and / or peripheral nervous system. By inhibiting TCB-mediated antibody formation, a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, can reduce the activation of myeloid-derived cells located in tissues and inhibit transcytosis, hemorrhage and infiltration of myeloid cell circulation, thereby reducing inflammation. In addition, treatment with a compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof can reduce the activation of inflammatory processes at the endothelial-microglial border and interstitial space, where lymphoid aggregates are observed in autoimmune neuropathies, through 1) a change in the cross-talk between microglia and endothelial cells, 2) inhibiting the activation of B lymphocytes and their presentation of the recognized antigen to circulating or infiltrating T cells, and 3) reducing the formation of cytokines and / or chemokines. It is believed that these effects of inhibition of TCB by covalent interaction with a compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof reduce the infiltration of autoimmune T cells into gray matter and white matter, inhibiting B cell activation, cytokine activation and APC function, and altering the developmental and maturational status of professional APCs, including infiltrating monocytes, activated microglia and oligodendrocytes. Thus, a method of treating autoimmunity mediated neurodegenerative disorders with a covalent TCB inhibitor, such as compounds of formula (I) and (II), can worsen disease progression through inhibition of natural immune processes, as well as decrease antibody formation and activation of autoimmune T cells. The invention can slow down the development or cause remission of experimental autoimmune encephalopathy in

- 11 037031 animal models, and in human neuropathies, including opticomyelitis (Devik syndrome), Guillain-Barré syndrome, multiple sclerosis, clinically isolated syndrome, relapsing remitting multiple sclerosis, malignant multiple sclerosis, primary progressive multiple sclerosis, diseases of the spectrum of optic sclerosis , Marburg disease, diffuse myelinoclastic sclerosis, chronic focal encephalitis, Rasmussen's encephalitis, constrained person's syndrome, myasthenia gravis, polyneuropathy associated with anti-MAG IgM monoclonal gammopathy. In specific embodiments, a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, is administered directly to the mammal. In one embodiment, a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, is directly administered to the mammal, but is not administered to the mammal concurrently with another TCB inhibitor. In one embodiment, a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, is directly administered to the mammal, but is not administered to the mammal simultaneously with acalabrutinib. In one embodiment, a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, is the only TCB inhibitor administered directly to the mammal.

In another embodiment, the invention relates to a method of treating a compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof for polyneuropathies arising from infectious or post-infectious neuroinflammation in mammals, including Bannworth syndrome (Lyme disease), chronic encephalomyelitis (Lyme disease); postherpetic neuralgia; HTLV-1 associated myelopathy; progressive multifocal leukoencephalopathy; chronic fatigue syndrome (CFS), systemic exercise intolerance (SFS), myalgic encephalomyelitis (ME), post-viral fatigue syndrome (PFS), chronic fatigue and immune dysfunction syndrome (SFS); Ménière's disease (dizziness - regulation of the endolymph of the inner ear fluid), Guillain-Barré syndrome, amyotrophic lateral sclerosis, progressive bulbar palsy, childhood progressive bulbar palsy (or juvenile progressive bulbar palsy), Bell's palsy, vestibular neuritis, acute disseminated encephalomyelitis or multiforme, recurrent disseminated encephalomyelitis and chronic encephalomyelitis. In certain embodiments, the compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, is administered directly to the mammal. In one embodiment, a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, is directly administered to the mammal, but is not administered to the mammal concurrently with another TCB inhibitor. In one embodiment, a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, is directly administered to the mammal, but is not administered to the mammal simultaneously with acalabrutinib. In one embodiment, the compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, is the only TCB inhibitor administered directly to the mammal.

In some embodiments, the hyperproliferative disorder is a solid tumor selected from the group consisting of bladder cancer, squamous cell carcinoma, head and neck cancer, pancreatic ductal adenocarcinoma (PAD), pancreatic cancer, colon carcinoma, breast carcinoma, breast cancer , fibrosarcoma, mesothelioma, renal cell carcinoma, lung carcinoma, thymoma, prostate cancer, rectal and colon cancer, ovarian cancer, acute myeloid leukemia, thymus cancer, brain cancer, squamous cell carcinoma, skin cancer, eye cancer, retinoblastoma, melanoma, intraocular melanoma, oral cancer, oropharyngeal cancer, gastrointestinal cancer, stomach cancer, cervical cancer, kidney cancer, kidney cancer, liver cancer, ovarian cancer, prostate cancer, rectal and colon cancer, esophageal cancer, testicular cancer, gynecological cancer, cancers of the thyroid gland, cancers associated with acquired immunodeficiency syndrome (AIDS) (eg, lymphomas and Kaposi's sarcoma), viral x cancers such as cervical carcinoma (human papillomavirus), B-cell lymphoproliferative disease, nasopharyngeal carcinoma (Epstein-Barr virus), Kaposi's sarcoma and body cavity lymphomas (herpesvirus Kaposi's sarcoma), hepatocellular carcinoma B and hepatitis viruses C) and T-cell leukemia (human T-cell leukemia virus-1), glioblastoma, esophageal tumors, head and neck tumors, metastatic colon cancer, squamous cell carcinoma of the head and neck, ovarian cancer, pancreatic cancer, renal cell carcinoma , hematological neoplasms, small cell lung cancer, non-small cell lung cancer, stage IV melanoma and glioma.

In some embodiments, the hyperproliferative disorder is B-cell hemoblastosis selected from the group consisting of chronic lymphocytic leukemia (CLL), small cell lymphocytic leukemia (MLL), non-Hodgkin's lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), follicular ), mantle cell lymphoma (MCL), Hodgkin's lymphoma, B-cell acute lymphoblastic leukemia (B-ALL), Burkitt's lymphoma, Waldenstrom's macroglobulinemia (MB), Burkitt's lymphoma, multiple myeloma, myelodysplastic syndromes or myelofibrosis. In one embodiment, the invention relates to a method for treating cancer in a mammal, wherein the cancer is chronic myelocytic leukemia, acute myeloid leukemia, DLBCL (including activated B cell (ABC) and germline B cell (GCB) subtypes), follicular center lymphoma, Hodgkin's disease, multiple myeloma, insensitive non-Hodgkin's lymphoma, and mature B-cell ALL.

- 12 037031

In some embodiments, the hyperproliferative disease is a subtype of CLL. Many subtypes of CLL have been characterized. CLL is often classified by the mutational status of the immunoglobulin heavy chain variable region (IgVH) in leukemic cells. R. N. Damle, et al., Blood 1999.94, 1840-47; T. J. Hamblin et al., Blood 1999, 94, 1848-54. Patients with IgVH mutations generally survive longer than those without IgVH mutations. Expression of ZAP70 (positive or negative) is also used to characterize CLL. L. Z. Rassenti et al., N. Engl. J. Med. 2004,351, 893-901. Methylation of ZAP-70 on CpG3 is also used to characterize CLL, for example, via pyrosequencing. R. Claus et al., J. Clin. Oncol. 2012,30,2483-91; J. A. Woyach, et al., Blood 2014,123, 1810-17. CLL is also classified according to the stage of the disease according to the Binet or Rai criteria. J. L. Binet, et al., Cancer 1977, 40, 855-64; K. R. Rai, T. Han, Hematol. Oncol. Clin. North Am. 1990.4, 447-56. Other common mutations such as 11q deletion, 13q deletion and 17p deletion can be assessed using well known techniques such as fluorescence in situ hybridization (FISH). In one embodiment, the invention relates to a method for treating CLL in a human, wherein CLL is selected from the group consisting of CLL negative with an IgVH mutation, ZAP-70 CLL positive, ZAP-70 CpG3 methylated CLL, CD38 CLL positive, chronic lymphocytic leukemia characterized by 17p13 .1 (17p) deletion and CLL characterized by an 11q22.3 (11q) deletion.

In some embodiments, the hyperproliferative disorder is CLL, where CLL has undergone Richter transformation. Methods for evaluating Richter transformation, which is also known as Richter's syndrome, are described in P. Jain and S. O'Brien, Oncology, 2012,26, 1146-52. Richter's transformation is a subtype of CLL that occurs in 5-10% of patients. It involves the development of aggressive CLL lymphoma and usually has a poor prognosis.

In some embodiments, the hyperproliferative disorder is CLL or MLL in a patient where the patient is susceptible to lymphocytosis. In one embodiment, the invention provides a method for treating CLL or MLL in a patient, wherein the patient exhibits lymphocytosis due to a disorder selected from the group consisting of a viral infection, a bacterial infection, a protozoal infection, or a post-splenectomy condition. In one embodiment, the viral infection in any of the above embodiments is selected from the group consisting of infectious mononucleosis, hepatitis, and cytomegalovirus. In one embodiment, the bacterial infection in any of the above embodiments is selected from the group consisting of cough, tuberculosis, and brucellosis.

In some embodiments, the hyperproliferative disorder is selected from the group consisting of myeloproliferative disorders (MPDs), myeloproliferative neoplasms, polycythemia vera (PI), essential thrombocythemia (ET), primary myelofibrosis (PMF), myelodysplastic BCR-positive syndrome (BCR) ), chronic neurophilic leukemia, chronic eosinophilic leukemia or mastocytosis.

In one embodiment, there is provided a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a compound of formula (II), or a pharmaceutically acceptable salt thereof, for use in therapy.

In one embodiment, there is provided the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a compound of formula (II), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament.

In one embodiment, there is provided the use of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, or a compound of Formula (II), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament, wherein the medicament is produced ex-vivo.

In any embodiment where the manufacture of the drug is indicated in a general sense, there is another embodiment where the drug is produced ex-vivo.

In one embodiment, there is provided a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a compound of formula (II), or a pharmaceutically acceptable salt thereof, for use in the treatment of a hyperproliferative disorder, an inflammatory disorder, an immune disorder, or an autoimmune disorder in a mammal.

In one embodiment, there is provided a compound of formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of a TCP mediated disease, wherein the disease is a hyperproliferative disease. The hyperproliferative disease can be any hyperproliferative disease referred to herein.

Examples of

The options discussed here are further described with reference to the following examples. These examples are provided for purposes of illustration only, and the description herein is in no way to be construed as being limited to these examples, but rather should be construed to cover any and all variations that become apparent as a result of the ideas presented herein. The reagents described in these examples are commercially available or can be obtained by methods described in the literature.

Example 1. Analytical Methods

The following liquid chromatography (LC) and mass spectrometry (MS) methods can

- 13 037031 be used to characterize the compounds included in this invention.

Method A.

LC-MS spectrometer (Agilent)

Sensor: DAD (210, 254 and 280 nm)

Mass sensor: API-ES (10-2000 amu, pole / negative ionization mode)

Eluents (mobile phase):

A: 0.1% formic acid in MilliQ water,

B: acetonitrile

Column: Waters XTerra C18 MS, 50x4.6 mm ID, 2.5 μm

Flow rate: 0.5 ml / min

Elution gradient program:

Time (min) AND (%) IN (% 0, 0 90 ten 7, 0 ten 90 7, 1 0 100 one hundred 90 ten

Method B:

HPLC: Gilson Analytical HPLC System

Column: Phenomenex Luna C18 (2) (100x2.00 mm, 5 μm)

Sensor: UV / Vis (210/240 nm)

Flow rate: 1 ml / min

Eluents (mobile phase): A: acetonitrile, B: acetonitrile / MilliQ) -water = 1/9 (v / v), C: 0.1% TFA in MilliQ-water.

Elution gradient program:

Time (min)

0, 0

11.90

14.40

15, 40

Example 2. Synthesis of a compound of formula (I)

Preparation of 4- [8-amino-3- (pyrrolidin-2-yl) imidazo [1,5-a] pyrazin-1-yl] ^ - pyridin-2-ylbenzamide.

This compound is essentially prepared by the methods described in WO 2013/010868.

Preparation of 4- [8-amino-3- (3,4-dihydro-2H-pyrrol-5-yl) imidazo [1,5-a] pyrazin-1-yl] -X-pyridin-2ylbenzamide.

4- [8-amino-3- [(2S) -pyrrolidin-2-yl] imidazo [1,5-a] pyrazin-1-yl] -X- (2-pyridyl) benzamide (1.13 g, 2 , 83 mmol) is suspended in DCM (50 ml). N-chlorosuccinimide (416 mg, 3.11 mmol) was added and the mixture was stirred at 21 ° C. After 10 minutes, the reaction mixture turns into a clear, pale yellow solution. Triethylamine (868 μl, 6.23 mmol) was added and the mixture was continued to stir at 21 ° C. After 15 minutes of stirring, a white precipitate forms. The precipitate is collected on a filter, washed with acetonitrile (20 ml) and air-dried. This gives the title compound as a white solid (800 mg, 70%). MS (ESI +) m / z 398.2 (M + H) +;

¹ H NMR (400 MHz, DMSO-d ₆ , 300K): δ = 7.72 (1H, d, J = 5.0 Hz), 6.98 (1H, d, J = 5.0 Hz), 4 , 45 (1H, t, J = 6.9 Hz), 2.99 (1H, bs), 2.77-2.90 (2H, m), 2.09-2.20 (1H, m), 1.99-2.09 (1H, m), 1.78-1.89 (1H, m), 1.66-1.78 (1H, m).

Preparation of 4- (8-amino-3- (4- (but-2-inamido) butanoyl) imidazo [1,5-a] pyrazin-1-yl) -N- (pyridin-2yl) benzamide.

4- [8-amino-3- (3,4-dihydro-2H-pyrrol-5-yl) imidazo [1,5-a] pyrazin-1-yl] -N- (2-pyridyl) benzamide

- 14 037031 (167 mg, 1.69 mmol) is dispersed in methanol (26.8 ml). Concentrated hydrochloric acid (12 M, 670 μL, 8.04 mmol) was added with stirring. After 30 minutes, a white precipitate forms. All solvent was evaporated, washed with toluene (10 ml) and evaporated again. The resulting white solid is dispersed in acetone (70 ml) and triethylamine (705 μl, 5.06 mmol) is added. A solution of butionyl chloride (207.4 mg, 2.02 mmol) in acetone (2 ml) was added dropwise and the white solid was gradually dissolved. The volatiles were removed in vacuo and the residue was taken up in chloroform (200 ml), washed with water (200 ml) and brine. The aqueous phases are extracted with chloroform (2x50 ml). The combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated by rotary evaporation to give 657 mg of a pale yellow solid. The crude product was purified using chromatography on silica gel (90 g), eluting with 2-5% MeOH (containing 10% ammonium hydroxide) in DCM. The pure product fractions are combined and concentrated in vacuo to give 228 mg (27%) of a pale yellow solid. LCMS (Method A) By: 3.76 min; m / z 482.1 (M + H) +; HPLC (Method B) By: 5.79 min; purity 99.8%;

Ή NMR (400 MHz, DMSO-d6, 300K): δ = 10.91 (1H, s), 8.72 (1H, d, J = 4.8 Hz), 8.54 (1H, t, J = 6.0 Hz), 8.42 (1H, d, J = 4.9 Hz), 8.22 (3H, t, J = 8.5 Hz), 7.87 (1H, dt, J1 = 1, 9 Hz), 7.82 (2H, d, J = 8.5 Hz), 7.51 (1H, d, J = 4.8 Hz), 7.19 (1H, dd, J1 = 0.9 Hz , J2 = 4.8 Hz), 6.47 (2H, s), 3.11-3.26 (4H, m), 1.93 (3H, s), 1.83 (2H, m).

Example 3. Synthesis of a compound of formula (II)

To a solution of 4-bromo-2-methoxybenzoic acid (15.3 g, 66.2 mmol) in dichloromethane (250 ml) were added pyridin-2-amine (6.9 g, 72.8 mmol) and DIPEA (34.6 ml, 198.7 mmol). GATU (32.7 g, 86.1 mmol) was added and the mixture was stirred at room temperature overnight. Water (200 ml) was added and the reaction mixture was stirred for 1 hour. The organic layer was concentrated under reduced pressure. DCM (50 ml) was added and the solution crystallized over the weekend. The solids were filtered, washed twice with diethyl ether (10 ml) and dried under reduced pressure to give 4-bromo-2-methoxy-N- (2-pyridyl) benzamide (14.4 g, 66.8%) as light brown crystals ... LCMS (Method A) By: 6.05 min; m / z 307.0 + 309.0 (1: 1) (M + H) ⁺ .

To a solution of 4-bromo-2-methoxy-N- (2-pyridyl) benzamide (14.4 g, 46.9 mmol) in 1,4-dioxane (175 ml) was added bis (pinacolato) diborone (14.3 g , 56.3 mmol) and potassium acetate (9.2 g, 93.8 mmol). PdCl2 (dppf). DCM (1.9 g, 2.3 mmol) was added and the mixture was stirred at 100 ° C for 5 h. The reaction mixture was diluted with water (150 ml) and extracted twice with ethyl acetate (150 ml). Dry the combined organic layers over sodium sulfate, filter, and concentrate under reduced pressure. The residue was purified by flash column chromatography (0-50% ethyl acetate in heptane). The fractions containing the product are concentrated under reduced pressure. The residue is suspended in heptane (150 ml) and stirred for 30 minutes. The solids were filtered and washed twice with heptane (15 ml) to give 2-methoxy-N- (2-pyridyl) -4- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) benzamide (10.4 g, 62.6%) as a white solid. LCMS (Method A) By: 6.86 min; m / z 355.2 (M + H) ⁺ .

Ή NMR (400 MHz, DMSO-d ₆ , 300 K): δ = 10.51 (1H, s), 8.36 (1H, m), 8.26 (1H, d, J = 8.4 Hz) , 7.89 (1H, d, J = 7.6 Hz), 7.85 (1H, m), 7.41 (1H, dd, J1 = 7.6 Hz, J2 = 0.9 Hz), 7 , 38 (1H, s), 7.17 (1H, m), 4.01 (3H, s), 1.33 (12H, s).

- 15 037031

Preparation of 1-bromo-3 - [(2S) -pyrrolidin-2-yl] imidazo [1,5-a] pyrazin-8-amine.

A 2000 ml round bottom flask equipped with a magnetic stirrer was charged with 37% hydrogen chloride (660 ml, 7971 mmol). Benzyl (2S) -2- (8-amino-1-bromo-imidazo [1,5-a] pyrazin-3-yl) pyrrolidine-1-carboxylate sulfuric acid (205 g, 399 mmol) was added in portions (approx. 30 min) and the reaction mixture was stirred for 8 h at 50 ° C. The reaction mixture was cooled to room temperature over 8 hours. The reaction mixture was washed with MTBE (3 x 1200 ml). 33% sodium hydroxide in water (-600 ml) is added dropwise to the aqueous phase until a pH of approx. 14, maintaining a temperature of 2030 ° C (MTBE layers appear). After the addition, the aqueous phase is stirred for 1 hour and extracted with dichloromethane (2x1500 ml). Activated carbon (10 g) was added to the combined layers of DCM and the mixture was stirred for 1 hour at 40 ° C. The solids are removed by filtration over dicalite and the filtrate is concentrated under reduced pressure to give 1-bromo-3 - [(2S) -pyrrolidine-2yl] imidazo [1,5-a] pyrazine-8-amine (112.3 g, 397 , 9 mmol, 99.8% yield) as an off-white solid. LCMS (Method A) By: 0.673 min; m / z 282.0 + 284.0 (1: 1) (M + H) ⁺ ;

¹ H NMR (400 MHz, DMSO-6, 300K): δ = 7.72 (1H, d, J = 5.0 Hz), 6.98 (1H, d, J = 5.0 Hz), 4, 45 (1H, t, J = 6.9 Hz), 2.99 (1H, br s), 2.77-2.90 (2H, m), 2.09-2.20 (1H, m), 1 , 99-2.09 (1H, m), 1.78-1.89 (1H, m), 1.66-1.78 (1H, m).

Preparation of 4- [8-amino-3 - [(2S) pyrrolidin-2-yl] imidazo [1,5-a] pyrazin-1-yl] -2-methoxy-N- (2-pyridyl) benzamide.

1-Bromo-3 - [(2S) pyrrolidin-2-yl] imidazo [1,5-a] pyrazin-8-amine (112.3 g, 398.03 mmol), 2-methoxyN- (2-pyridyl) -4- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) benzamide (148.04 g, 417.93 mmol) and potassium iodide (19.82 g, 119.41 mmol) is loaded into a three-necked 3 L flask. 2-Butanol (550 ml) and water (880 ml) were added and the resulting suspension was stirred while bubbling nitrogen gas. Triethylamine (165.97 ml, 1194.1 mmol) was added and the suspension was slowly dissolved. Bis (tert-butyldicyclohexylphosphine) dichloro palladium (II) (Pd-166, 1.37 g, 1.99 mmol) was added and the reaction mixture was reconstituted for 10 min and stirred at 82 ° C overnight to give a tan slurry. The reaction mixture is cooled at room temperature. The mixture was then warmed to 40 ° C. and water (1800 ml) was added and after addition was cooled back to room temperature. The mixture was filtered and the cake washed with water (500 ml) and heptane (300 ml). The solid was suspended in heptane (500 ml) and co-evaporated. The solid was again co-evaporated with heptane (500 ml) and dried under reduced pressure at 50 ° C overnight to give 4- [8amino-3- [(2S) pyrrolidin-2-yl] imidazo [1,5-a] pyrazin-1-yl] -2-methoxy-N- (2-pyridyl) benzamide (142.11 g, 330.9 mmol, 83.1% yield) as a light yellow solid. LCMS (Method A) By: 2.885 min; m / z 430.1 (M + H) +; HPLC (Method B) By: 1.483 min; purity 98.1%;

1H NMR (400 MHz, DMSO-d6, 300K): δ = 10.49 (1H, s), 8.37 (1H, m), 8.30 (1H, d, J = 8.3Hz), 8 , 04 (1H, d, J = 8.0 Hz), 7.87 (1H, dt, J1 = 1.9 Hz, J2 = 7.8 Hz), 7.79 (1H, d, J = 5, 0 Hz), 7.43 (1H, s), 7.39 (1H, dd, J1 = 1.4 Hz, J2 = 8.0 Hz), 7.18 (1H, dd, J1 = 1.0 Hz , J2 = 8.0 Hz), 7.09 (1H, d, J = 4.9 Hz), 6.20 (2H, s), 4.55 (1H, t, J = 7.5 Hz), 4.07 (3H, s), 2.90 (2H, t, J = 7.2 Hz), 2.25 (1H, m), 2.12 (1H, m), 1.89 (1H, m ), 1.78 (1H, m).

- 16 037031

Preparation of 4- [8-amino-3- (3,4-dihydro-2H-pyrrol-5-yl) imidazo [1,5-a] pyrazin-1-yl] -2-methoxy-N (2-pyridyl) benzamide.

4- [8-amino-3 - [(2S) -pyrrolidin-2-yl] imidazo [1,5-a] pyrazin-1-yl] -2-methoxy-N- (2-pyridyl) benzamide (250 mg , 0.58 mmol) is taken and partially dissolved in DCM (20 ml). N-chlorosuccinimide (85.1 mg, 0.64 mmol) was added and the mixture was stirred at 21 ° C. After 10 minutes, the reaction mixture turns into a clear yellow solution. Triethylamine (177.1 μl, 1.27 mmol) was added and the mixture was continued to stir at 21 ° C. After 30 minutes of stirring, a precipitate forms. The precipitate was isolated using a centrifuge to give the title compound as an off-white solid (187.7 mg, 75.8%). HPLC (chlorine intermediate) (method B) By: 5.596 min.

Preparation of 4- [8-amino-3- (4-aminobutanoyl) imidazo [1,5-a] pyrazin-1-yl] -2methoxy-N- (2-pyridyl) benzamide trihydrochloride.

4- [8-amino-3- (3,4-dihydro-2H-pyrrol-5-yl) imidazo [1,5-a] pyrazin-1-yl] -2-methoxy-N- (2-pyridyl) benzamide (187.7 mg, 0.44 mmol) is taken up in methanol (8 ml). Concentrated hydrochloric acid (174.5 μl, 2.0-9 mmol) is added. The mixture was stirred for 1.5 hours. A solid was obtained by filtration to give the title compound as a light yellow solid in quantitative yield (269 mg). HPLC (Method B) By: 3.790 min.

Preparation of 4- (8-amino-3 - (4- (but-2-inamido) butanoyl) imidazo [1,5-a] pyrazin-1-yl) -2-methoxy-N (pyridin-2-yl) benzamide ...

4- [8-amino-3- (4-aminobutanoyl) imidazo [1,5-a] pyrazin-1-yl] -2-methoxy-N- (2pyridyl) benzamide trihydrochloride (250 mg, 0.451 mmol) is suspended in DCM (14 ml) with acetone (2 ml). Add GATU (289.7 mg, 0.762 mmol), triethylamine (254.7 μl, 1.833 mmol) and 2-butic acid (64.1 mg, 0.762 mmol). The mixture is stirred overnight at 21 ° C. The reaction mixture is concentrated in vacuo. The crude product is purified using flash chromatography (0-7% methanol in DCM). The product fractions are combined and concentrated in vacuo. The residue was suspended in methanol (2 ml) and the solvent was removed using a centrifuge. The solids were washed with diethyl ether (2 ml) and dried in vacuo to give the title compound as an off-white solid (76.2 mg, 33.1%). LCMS (Method A) By: 4,300 min; m / z 512.2 (M + H) +; HPLC (Method B) By: 6.499 min; purity 98.8%;

1H NMR (400 MHz, DMSO-dg, 300K): δ = 10.51 (1H, s), 8.73 (1H, d, J = 4.8 Hz), 8.55 (1H, t, J = 6.3 Hz), 8.38 (1H, m), 8.30 (1H, d, J = 8.5 Hz), 8.06 (1H, d, J = 7.9 Hz), 7.88 (1H, dt, J1 = 1.9 Hz, J2 = 4.3 Hz), 7.52 (1H, d, J = 4.8 Hz), 7.50 (1H, d, J = 1.3 Hz ), 7.44 (1H, dd, J1 = 1.5 Hz, J2 = 8.0 Hz), 7.19 (1H, m), 6.55 (2H, s), 4.08 (3H, s ), 3.20 (2H, t, J = 7.2 Hz), 3.16 (2H, q, J = 6.0 Hz), 1.93 (3H, s), 1.83 (2H, t , J = 7.9 Hz).

Example 4. Measurement of the kinase activity of TKB and other kinases with cysteine in the same position as Cys481 in TKB

Table 1

Kinase Method Formula I IC ₅₀ (nM) Formula II IC ₅₀ (nM) TKB IMAP 5.0 + 1.0 9, 3 TEC LanthaScreen 345 + 34 ITK IMAP > 10000 > 10000 THK Z'-LYTE 567 + 174 59 BMX Z'-LYTE 15 + 2 EGFFR Z'-LYTE > 10000 ERBB2 Z'-LYTE 552 + 166 ERBB4 Z'-LYTE 343 + 23 BLK Z'-LYTE 6170 + 3348 JAK3 Z'-LYTE > 10000

TKP enzymatic activity is measured using an IMAP (immobilized metal affinity fluorescence polarization) assay as described below.

Enzyme TKB (His-TKB (Millipore cat # 14-552)) is diluted to 0.4 U / ml in KR buffer (10 mM Tris-HCl, 10 mM MgCl2, 0.01% Tween-20, 0.05% NaN ₃ , 1 mM DTT, 2 mM MnCl ₂ , pH 7.2).

Serial log10 dilutions from 2 mM to 63.2 nM test compounds are prepared in 100% DMSO. DMSO dilutions are then diluted 50-fold in KR buffer. The final concentration range of the compound in the assay is from 10 μM to 0.316 nM.

The assay is performed as follows: 5 μl / well of the test compound in KR buffer (the final concentration of DMSO in the assay is 1%) is mixed with 5 μl / well of 0.4 U / ml of TKB enzyme

- 17 037031 (final concentration in the assay is 0.1 U / ml). The test compound and TKB enzyme are preincubated for 60 min at room temperature, then 5 μl / well of 200 nM Fluorescin-labeled substrate peptide (Blk / Lyntide substrate, eg # R7188 / # R7233, Molecular Devices) is added in KR buffer. The final concentration of the peptide substrate in the assay is 50 nM. The kinase assay is started by adding 5 μl / well of 20 μM ATP in KR buffer (final concentration of ATP is 5 μM ATP, Km ATP in TCB IMAP assay). After incubation for 2 hours at room temperature, the enzyme reaction is stopped by adding 40 μl / well of IMAP Progressive Binding Solution (according to the manufacturer's protocol (Molecular Devices) using 75% 1x Buffer A and 25% 1x Buffer B with 1: 600 Progressive Binding Solution ). After 60 min incubation at room temperature in the dark, the FP signal is read. Fluorescence at 535 nm is measured using parallel and perpendicular filters to determine rotation differences due to binding of the phosphorylated substrate peptide to the beads. Values are calculated as the percentage of difference in reading (Δ mPi) between control samples with and without ATP. IC ₅₀ values are determined by fitting the curve of the experimental results in Dotmatics. The results are presented in table. one.

The activity of the ITK enzyme is measured using IMAP (immobilized metal affinity fluorescence polarization) as described below.

ITK enzyme (Millipore # 14-660M) is diluted to 0.2 U / ml in KR buffer (10 mM Tris-HCl, 10 mM MgCl ₂ , 0.01% Tween-20, 0.1% NaN ₃ , 1 mM DTT , 2 mM MnCl ₂ , pH 7.5)

Serial log10 dilutions from 2 mM to 63.2 nM test compounds are performed in 100% DMSO. The DMSO dilutions are then diluted 50-fold in KR buffer. The final concentration range of the compound in the assay is from 10 μM to 0.316 nM.

The assay is performed as follows: 5 μl / well of the test compound in KR buffer (final concentration of DMSO in the assay is 1%) is mixed with 5 μl / well of 0.2 U / ml of ITK enzyme (final concentration in the assay is 0.05 U / ml) (8.4 nM)). Test compound and ITK enzyme are preincubated for 60 minutes at room temperature, then 5 μl / well of 200 nM Fluorescin-labeled substrate peptide (Blk / Lyntide substrate, # R8124, Molecular Devices) is added in KR buffer. The final concentration of the peptide substrate in the assay is 50 nM. The kinase assay is started by adding 5 μl / well of 20 μM ATP in KR buffer (final concentration of ATP is 5 μM ATP, Km ATP in ITK IMAP assay). After incubation for 2 hours at room temperature, the enzyme reaction is stopped by the addition of 40 μl / well of IMAP Progressive Binding Solution (according to manufacturer's protocol (Molecular Devices) using 60% 1x Buffer A and 40% 1x Buffer B with 800x diluted beads (Progressive Binding System, Molecular Devices # R8124) After 60 min incubation at room temperature in the dark, the FP signal is read The fluorescence at 535 nm is measured using parallel and perpendicular filters to determine rotation differences due to binding of the phosphorylated substrate peptide to the beads. percent difference in reading (Δ mPi) of control samples with and without ATP The IC ₅₀ values are determined by fitting a curve of the experimental results in Dotmatics.

TEC enzyme activity is measured using the LanthaScreen assay from ThermoFisher as described below.

TEC enzyme (LifeTech # PV3269) and Eu-anti-HIS antibody (Invitrogen # PV5596) are mixed and diluted in kinase buffer (50 mM Hepes pH 7.5 + 10 mM MgCl ₂ +1 mM EGTA + 0.01% Brij-35 ) up to 3 and 6 nM, respectively. The final concentration in the assay for enzyme and antibody is 1 and 2 nM, respectively.

The label (Kinase tag 178, Invitrogen # PV5593) is diluted in kinase buffer to 3 nM. The final concentration in the assay is 1 nM.

Serial log10 dilutions from 1 mM to 3.16 nM test compounds are carried out in 100% DMSO. Then the dilutions in DMSO are diluted 33-fold in kinase buffer (50 mM Hepes pH 7.5 + 10 mM MgCl2 + 1 mM EGTA + 0.01% Brij-35).

The assay is performed as follows: 5 μl / well of the TEC enzyme dilution and EU-anti-His antibody are mixed with 5 μl / well of the label dilution and 5 μl / well of the compound dilution in kinase buffer. The final concentration of the compound in the assay is 10 μM to 0.316 nm, with a 1% DMSO final concentration in the assay. After 2 h incubation at room temperature, the TRFRET signal is read at 615 and 665 nm. The 665/615 ratio is used to calculate the values, expressed as a percentage of the difference in reading (S / N) of the labeled and unlabeled controls. IC50 values are determined by fitting the curve of the experimental results in Dotmatics.

BMX, TXK, EGFR, ERBB2, ERBB4, JAK3, BLK kinase activities were measured using the Z'LYTE assay on Thermo Fisher. A 10-point dose-response effect (the final concentration in the assay ranges from 10 μM to 0.5 nM in 3-fold dilution per dilution step) is constructed for 1 h of incubation of the test compound with kinase prior to initiation of the kinase reaction by addition of ATP. The ATP concentration in the assay is Km ATP for the final kinases. IC50 values are determined by fitting the curve of the experimental results at Thermo Fisher.

- 18 037031

Example 5. TKB IMAP with ATP competition for the study of covalent binding of compounds

The activity of the TKB enzyme with ATP competition is measured using an IMAP (immobilized metal affinity based fluorescence polarization) assay.

TKB enzyme (Millipore) is diluted to 16 nM, respectively, in a kinase reaction (KR) buffer (10 mM Tris-HCl, 10 mM MgCl2, 0.01% Tween-20, 0.1% NaN3, 1 mM DTT, 2 mM MnCl2, pH 7.5).

Serial log10 dilutions from 1 mM to 31.6 nM test compounds are prepared in 100% DMSO. DMSO dilutions are then diluted 25-fold in KR buffer. Final compound concentrations range from 10 μM to 0.316 nM.

The assay is performed as follows: 5 μl / well of the test compound in KR buffer (final concentration of DMSO in the assay is 1%) is mixed with 5 μl / well of TCB or ITK enzyme (final concentration in the assay is 4 and 8 nM for TCB and ITK, respectively ). Test compounds and kinase enzyme are preincubated for 0, 30 or 60 minutes, then 5 μl / well of 200 nM Fluorescein-labeled substrate peptide (Blk / Lyntide substrate, Molecular Devices) in KR buffer is added. The final concentration of the peptide substrate is 50 nM. The kinase assay is started by adding 5 μl / well of 20, 100 or 400 μM ATP in KR buffer (final ATP concentration is 5.25 or 100 μM ATP). After incubation for 2 hours at room temperature, the enzyme reaction is stopped by adding 40 μl / well of IMAP Progressive Binding Solution (Molecular Devices) according to the manufacturer's instructions, using 60% 1x buffer A and 40% 1x buffer B with 800x diluted beads). After 60 min incubation at room temperature in the dark, the FP signal is read. Fluorescence at 535 nm is measured using parallel and perpendicular filters to determine rotation differences due to binding of the phosphorylated substrate peptide to the beads. Values are calculated as the percentage of difference in reading (Δ mPi) between control samples with and without ATP. IC ₅₀ values are determined by fitting the curve of the experimental results in Dotmatics.

Although standard IMAP analysis has shown the metabolite M27 to be an inhibitor of TCB, further testing is required to determine if M27 is a covalent inhibitor. M27 is tested in TKB IMAP ATP competitive assays with different pre-incubation times (0, 30 and 60 min) and ATP concentrations (5, 25 and 100 μM). The results are in table. 2 and in FIG. 2 confirm that M27 is a covalent TCB inhibitor. Increasing the pre-incubation time gives a shift in the efficiency of M27. Moreover, there is a loss of ATP competition after the preincubation of TCB with M27, a result that is characteristic of compounds that bind covalently.

Example 6. TKB-DT and TKB-C481S LanthaScreen to study the covalent binding of compounds

The inhibitory effect on wild-type TKB (TKB-DT) and the mutant TKB Cys481Ser (TKB-C481S) was measured using ThermoFisher LanthaScreen assay according to the manufacturer's protocol.

TKB-DT or TKB-C481S (Genscript) are mixed and diluted with Eu-anti-GST antibody (Invitrogen) in kinase buffer (50 mM Hepes pH 7.5 + 10 mM MgCl2 + 1 mM EGTK + 0.01% Brij-35 ) up to 15 and 6 nM, respectively. The final concentration in the assay for enzyme and antibody is 5 and 2 nM, respectively.

The label (kinase tag 236, Invitrogen) is diluted in kinase buffer to 90 nM. The final concentration in the assay is 30 nM.

Serial log10 dilutions from 1 mM to 3.16 nM test compounds are made in 100% DMSO. The DMSO dilutions are then diluted 33-fold in kinase buffer (50 mM Hepes pH 7.5 + 10 mM MgCl2 + 1 mM EGTA + 0.01% Brij-35).

The assay is performed as follows: 5 µl / well dilution of TKB-DT or TKB-C481S enzyme and Eu-anti-GST antibodies are mixed with 5 µl / well dilution of the label and 5 µl / well dilution of the compound in kinase buffer. The final concentration of the compound in the assay ranges from 10 μM to 0.316 nM, with 1% DMSO final concentration in the assay. The mixture is incubated at room temperature in the dark and with different incubation times (5 10, 20, 30, 40, 60, 90, 120, 180 and 300 min), the TR-FRET signal is read at 615 and 665 nm. The 665/615 ratio is used to calculate the values, expressed as the percentage of difference in reads (S / N) of the labeled and unlabeled controls. IC ₅₀ values for each time point are determined by fitting a curve of the experimental results in Dotmatics.

To confirm the covalent inhibition of M27, the inhibitory effect of M27 was tested on TKB-DT and the mutant TKB Cys481Ser (TKB C481S) using ThermoFisher LanthaScreen assay according to the manufacturer's protocol. IC50 measurements are taken at different time points after incubation of compounds with TKB and TKB-C481S. The results depicted in FIG. 9, confirm

- 19 037031 covalent binding of M27 with TKB. The difference in efficiency between TKB-DT and TKB-C4 81S shows the effect of covalent bonding of compounds with TKB. The IC50s obtained using TCB-C481S reflect reversible inhibition and do not change or change slightly over time. The increase in efficiency observed for TKB-DT arises from the ability of M27 to covalently bind to C481 in the ATP pocket of TKB. The kinetics in covalent binding is determined by the affinity of the compound for TCB and the reactivity of the electrophile.

Using data for M27 from the LanthaScreen TKB-DT assay, inhibition constants can be calculated for the parent compound and metabolite. To determine the inhibition constants more accurately, the LanthaScreen experiments are repeated to include additional earlier times after the start of incubation. The results of these experiments are summarized in table. 3. Ki and enzyme inactivation rate parameters are determined from IC50 values over time according to the method of Krippendorff et al. (J Biomol Screen. 2009, 14 (8): 913-23) with a measured Km = 102 nM for the label used in the assay.

Table 3

Compound Ki (nM) enzyme inactivation rate (s ^-1 ) Enzyme inactivation rate / Ki (s- ^ m- ¹ ) * M2 7 188 + 9 0.0031 + 0.0003 1.65E + 04 + 7.77E + 02

Example 7. TKB employment of target in B cells of Ramos

Ramos cells (ATCC, cat # CRL-1923) are plated in 24-well culture plates at 2x10 ⁶ cells per well in a total volume of 900 μl DMEMF12 + 10% FCS + 2 mM L-Glutamine + Pen / Strep. The cells are incubated for 1 h at 5-7% CO2 and 37 ° C.

Serial log10 dilutions from 10 mM to 316 nM test compounds are prepared in 100% DMSO, then diluted 100-fold in culture medium.

For each well, 100 μl is transferred to was then transferred to the well of a plate containing 900 μl of Ramos B cells. The final concentration range of the compound in the assay ranges from 10 μM to 0.316 nM, with a final DMSO concentration of 0.1% and incubated at 5-7% CO ₂ and 37 ° C for 2 hours. Cells are then harvested to measure target occupancy with TCB using ELISA employment targets TKB as described below.

The percentage of drug bound TKB in Ramos B cell samples was determined by ELISA method as follows: 96-well OptiPlate (Perkin Elmer) coated 125 ng / well anti-TKB Ab (BD Biosciences) and blocked with ABS (Sigma-Aldrich). Samples containing Ramos B cells are lysed in ice-cold lysis buffer containing 50 mM Tris-HCl pH 7.5, 250 mM sucrose, 5 mM MgCl ₂ , 1 mM dithiothreitol (DTT), 0.05% digitonin, and a mixture of protease inhibitors ( SigmaAldrich). The cell lysates are then incubated for 1 hour in the absence or presence of 1 μM acalabrutinib, a saturating concentration that gives full occupancy of the TCB. The final amount of cell lysate used per well in the target TKB target employment ELISA is typical of 2x10 ⁵ B Ramos cells. The difference with the signal of cell lysates not incubated with an excess of acalabrutinib is free TKB (not occupied by the TKB inhibitor). Samples are incubated for 1 hour with a biotin-labeled compound of formula (II) (100 nM). This probe is covalently bound to Cys481 in the ATP pocket in TKB when the ATP pocket is not occupied by the covalent inhibitor of TKP. Each sample is then added twice to the resulting Optiplate and incubated for 2 hours at ambient temperature. Plates are washed with PBS + 0.05% Tween20 four times. Streptavidin-HRP (Invitrogen; ELISA grade) is added at 100 μl / well (120 ng / ml) and incubated for 1 hour at room temperature. Plates are washed with PBS + 0.05% Tween20 three times and then washed with PBS (no Tween 20) twice. One hundred μl / well of SuperSignal ELISA Femto Substrate (ThermoFisher Scientific) is added and then chemiluminescence is measured after 1 min (Envision® plate reader; PerkinElmer). The percentage of TCB occupancy for each sample is calculated relative to the control vehicle. The signal from the control vehicle without exogenous acalabrutinib represents 100% free TKP (or 0% occupied TKP), and the signal from the control vehicle with exogenous acalabrutinib represents 0% free TKP (or 100% occupied TKP). Incubation of each cell lysate with 1 μM acalabrutinib is used to correct the background signal not associated with free TCB:

% free TCB sample X = (Sample X - Sample X + drug [1 μM]) / (1 day before administration - one day before administration + drug [1 μM]) x 100%% occupied TCB = 100% -% free TKB

The binding of M27 to TKB in cells is carried out using a Ramos cell colony (Burkitt's lymphoma). Ramos cells are incubated with an M27 dose interval and TKP target occupancy as determined by ELISA. The results are shown in FIG. 10 and in table. 4. These data also confirm that M27 binds covalently to TCBs in Ramos cells, consistent with the target ELISA setting for busy detection.

- 20 037031 STB, the reversible inhibitor will be washed out during analysis.

Table 4

Parameter M27 1C ₅₀ (nm) TKB target employment 39

Example 8. Analysis of human peripheral blood mononuclear cell (PBMC) CD69 and analysis in CK

Whole blood is collected in heparin-coated Vacutainer tubes (BD Biosciences, San Jose, Calif.) And used for the isolation of PBMCs using Ficoll-Hypaque (Pharmacia, Uppsala, Sweden). The isolated PBMCs are cryopreserved in 90% FCS / 10% DMSO prior to use.

Cells after cryogenic storage are thawed in a 37 ° C water bath, diluted with RPMI / 1% FCS, washed 2 times and then visited at 2x10 ⁵ cells per well in RPMI / 10% FCS in 96-well plates.

Serial log10 dilutions from 10 mM to 316 nM test compounds are prepared in 100% DMSO, then diluted 100-fold in RPMI / 1% FCS. For each well, 10 μl is then transferred to a deep well plate containing 90 μl of PBMC cells. The range for the final concentration of the compound in the assay is 10 μM to 0.316 nM, with a final concentration of 0.1% DMSO. PBMC are then incubated for 2 h at 37 ° C in the presence or absence of test compounds, prior to stimulation with goat F (ab ') 2 anti-IgM (Southern Biotech, # 2022-14, final assay concentration 5 μg / ml) for 18 hours

After stimulation with anti-IgM, PBMCs are incubated on ice for 30 min with anti-CD69-FITC, anti-CD19-BV421 (BD Biosciences # 555530 and # 562440, respectively) and 7AAD (Life Technologies # A1310). Flow cytometry is performed and fluorescence values are obtained from the CD69-FITC channel in CD19 + gated vital B cells. EC50 values are determined by fitting a curve to the experimental results using the GraphPad Prism.

MCPC analysis:

Cryopreserved PBMCs are thawed, washed and suspended in 2x10 ⁵ cells / well in RPMI + 10% FCS in 96-well plates. Test compounds are added using _^half log dose titration (final concentration of 10 uM to 0.316 nM) and incubated for 2 hours incubation at 37 ° C, 5% CO _2. The final concentration of DMSO in the assay is 0.1%. For the washing portion of the experiment, PBMCs are centrifuged and the cell pellet is resuspended in culture medium without test compound. This is repeated twice. Goat arhuman IgM F (ab ') 2 antibody (Southern Biotech) (final concentration 5 μg / ml) is added to PBMCs with and without washing, and cells are incubated for an additional 18 hours. Cells are then labeled with CD69-FITC and CD19-BV421 antibodies (BD Biosciences) for 30 min at 4 ° C. After washing unbound antibody, 7-AAD is added as a measure of survival, followed by flow cytometry using the FACSVerse instrument (BD Biosciences). The percentage of CD69 positive cells was obtained from the CD19 + B lymphocyte gate using the FCSExpress analysis software (De Novo Software). EC50 values are determined by fitting the curve of the experimental results using Dotmatics.

CC analysis:

Forty-five µl of blood is diluted 1: 1 in RPMI + 1% FCS and incubated with test compound as described above. Blood cells are stimulated with 10 μg / ml mouse anti-human anti-IgD antibody (BD Biosciences, final assay concentration 10 μg / ml) and incubated for 18 hours. Cells are labeled with CD69-FITC, CD86-PE and CD19-BV421 (BD Biosciences) for 15 min at room temperature, then lysis of CCC is carried out with FACS Lysing Solution (BD Biosciences). Cells are washed 3 times with 1 ml / well PBS + 0.5% ABS, followed by flow cytometric analysis. Average fluorescence values for CD69 are obtained from the CD19 + B lymphocyte gate using the FCSExpress analytical software (De Novo Software). EC50 values are determined by fitting the curve of the experimental results using Dotmatics.

The efficacy of TCB inhibitors in primary B cells can be evaluated in assays that assess functional changes following BCR activation in the presence of the inhibitor. Inhibition of specific phosphorylated epitopes on signaling proteins and more distant measures of BCR activation such as increased expression of CD86 (B7-2) and CD69 on the cell surface can be measured by flow cytometry (see Report R2013003A). This study evaluates the effect of M27 on CD69 expression. The results of CD69 activation in preparations of human PBMC and CK are summarized in table. five.

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Table 5

Analysis M27 EC ₅₀ (nM) Formula (II) EC ₅₀ (nM) hMCC: anti-IgM-induced CD69 2 6 ± 16 19 ± 6 hCC: anti-IgD induced CD69 64 + 6 137 + 36

The data (Table 5) support the findings that M27 is a covalent inhibitor of TCB, and M27 covalently binds to and completely occupies TCB in B Ramos cells.

Example 9. Analysis of the profile of the kinoma M27

Kinase profiling analysis was performed with M27 on DiscoveRx at a single dose of 1 μM for all available kinases (KINOMEscan). A general assessment of the selectivity of kinases is presented in table. 6. The results show the overall kinase selectivity profile for M27 at 1 μM. Kinases inhibited> 65% at 1 μM for M27 have a dose-dependent effect on DiscoveRx. The Kd values determined in this experiment are listed in table. 7. M27 has IC50 values <1 μM for practically the same set of kinases, except for TCA, but with additional inhibition of BRK (PTK6).

Table 6. Results of selectivity assessment from KINOMEscan profile analysis (DiscoveRx scanMAX) for М27

Kinase Selectivity assessment M2 7 S (35) 0.013 S (10) 0.05 3 (1) 0

Table 7. Kd values of kinases with> 65% inhibition at 1 μM for M27 (HT = not tested)

Claims (9)

1. A compound of formula (I) or formula (II) having structures or a pharmaceutically acceptable salt thereof. 2. A compound according to claim 1, wherein the compound is a compound of formula (I), or a pharmaceutically acceptable salt thereof. 3. A compound according to claim 1, wherein the compound is a compound of formula (II), or a pharmaceutically acceptable salt thereof. 4. Use of a compound according to claim 2, or a pharmaceutically acceptable salt thereof, for the treatment of a hyperproliferative disorder selected from the group consisting of chronic lymphocytic - 22 037031 leukemia, small cell lymphocytic leukemia, non-Hodgkin's lymphoma, diffuse B large cell lymphoma, follicular lymphoma, mantle cell lymphoma, Hodgkin's lymphoma, B-cell acute lymphoblastic leukemia, Burkitt's lymphoma, macroglobulinemia, myelodibelastromiasis syndrome. 5. Use according to claim 4, wherein the proliferative disorder is chronic lymphocytic leukemia. 6. Use according to claim 4, wherein the proliferative disorder is small cell lymphocytic leukemia. 7. Use according to claim 4, wherein the proliferative disorder is mantle cell lymphoma. 8. Use according to claim 4, wherein the proliferative disorder is diffuse large B cell lymphoma. 9. Use according to claim 4, wherein the proliferative disorder is Waldenstrom's macroglobulinemia.