EA044974B1 - COMBINED THERAPY WITH A PHOSPHOINOSITIDE 3-KINASE INHIBITOR WITH A ZINC-BINDING GROUP - Google Patents

Description

Related Application

This application claims priority to US Provisional Application No. 62/729,648, filed September 11, 2018. All teachings of the above application are incorporated herein by reference.

Prior Art

Therapeutic regimens for the treatment of malignancies often include combination therapy with two or more agents. In particular, targeted therapies can be combined to more effectively treat different types of cancer and inhibit the development of treatment-resistant cancer cells. In the field of anticancer drug development, there is a need for particularly effective drug combinations to treat specific types of cancer.

Brief description of the invention

The present invention relates to combination therapy with a compound of formula I

or a pharmaceutically acceptable salt thereof, wherein R represents a hydrogen atom or an acyl group; the acyl group is preferably R ₁ C(O)-, where R ₁ is substituted or unsubstituted C 1 -C ₂₄ alkyl, preferably C ₁ -C ₁₀ alkyl and more preferably C ₁ -C ₆ alkyl; substituted or unsubstituted C ₂ -C ₂₄ -alkenyl, preferably C ₂ -C ₁₀ -alkenyl and more preferably C ₂ -C ₆ -alkenyl; substituted or unsubstituted C ₂ -C ₂₄ -alkynyl, preferably C ₂ -C ₁₀ -alkynyl and more preferably C ₂ -C ₆ -alkynyl; substituted or unsubstituted aryl, preferably substituted or unsubstituted phenyl; either substituted or unsubstituted heteroaryl, and A is optionally substituted phenyl, optionally substituted pyridyl, or optionally substituted pyrimidyl; and an inhibitor of programmed cell death protein 1 (PD-1) signaling for the treatment of cancer. For example, one embodiment of the invention provides a method for preventing or treating cancer in a subject in need thereof. The method comprises administering to a subject a compound of Formula I or a pharmaceutically acceptable salt thereof and an inhibitor of PD-1-mediated signaling, wherein the compound of Formula I and the inhibitor of PD-1-mediated signaling are administered in amounts that in combination are therapeutically effective. Preferably, a compound of Formula I or a salt thereof and an inhibitor of PD-1-mediated signaling are administered to a subject in amounts that provide a synergistic effect.

In addition, this invention relates to pharmaceutical compositions containing a compound of formula I or a pharmaceutically acceptable salt thereof in combination with an inhibitor of PD-1-mediated signal transduction and a pharmaceutically acceptable excipient or carrier.

The compounds of formula I, and in particular the compound of formula I wherein R is a hydrogen atom and A is 2-methoxy-5-pyridyl, also referred to herein as compound 1, have advantageous properties for use as therapeutic agents. such as for the treatment of cancer and other diseases and disorders associated with phosphoinositide 3 (PI3) kinase and/or histone deacetylase (HDAC) activity. Compound 1, for example, has potent inhibitory activity against molecular targets such as phosphoinositide 3-kinase (PI3K) and HDAC, and potent antiproliferative activity against a number of cancer cell lines in vitro. Compound 1 has significant oral bioavailability, as observed in animal models. Following either oral or intravenous administration to mice bearing tumor xenografts, significant uptake of the compound into tumor tissue was demonstrated and pharmacodynamic activity was demonstrated in tumor tissue. Compound 1 also exhibits significant antitumor activity in murine tumor xenograft models following either oral or intravenous administration. In addition, the compound has a favorable safety profile, as demonstrated, for example, by genotoxicity testing using the Ames test.

Brief description of graphic materials

The above and other objects, features and advantages of the present invention will be apparent from the following more specific description of preferred embodiments of the invention, as illustrated in the accompanying drawings.

In fig. 1 is a graph of tumor volume versus time in the CT26.WT xenograft mouse model as described in Example 11.

In fig. 2 is a graph of tumor volume versus time in the A20 xenograft mouse model as described in Example 12.

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Detailed Description of the Invention

The present invention relates to methods and compositions for combination therapy of cancer containing a compound of formula I or a pharmaceutically acceptable salt thereof and an inhibitor of PD-1-mediated signaling. In a preferred embodiment of the compounds of formula I, A is phenyl, pyridyl or pyrimidyl substituted with a methoxy, amino or N-methylamino group. More preferably, A is one of the groups below.

In some embodiments of the compounds of Formula I, A is one of the groups shown above and R is a hydrogen atom.

In a preferred embodiment, the compound of formula I is selected from compounds 1, 2 and 3 below, and their pharmaceutically acceptable salts.

The invention provides a method for preventing or treating cancer in a subject in need thereof. The method includes the step of administering to a subject a compound of formula I or a pharmaceutically acceptable salt thereof and an inhibitor of PD-1-mediated signaling, wherein the compound of formula I or a pharmaceutically acceptable salt thereof and an inhibitor of PD-1-mediated signaling is administered in amounts that in combination are therapeutically effective.

A PD-1-mediated signaling inhibitor can be any compound that inhibits PD-1-mediated signaling. For example, an inhibitor of PD-1-mediated signaling may inhibit PD-1 and/or an activating ligand (L) of PD-1, such as PD-L1 or PD-L2. The PD-1-mediated signaling inhibitor may be a small molecule, a polynucleotide, or a protein such as an antibody. Preferably, the PD-1-mediated signaling inhibitor is a monoclonal antibody, more preferably a humanized or fully human monoclonal antibody. In one embodiment, the inhibitor of PD-1-mediated signaling is an anti-PD-1 monoclonal antibody. In another embodiment, the inhibitor of PD-1-mediated signaling is a monoclonal antibody to PD-L1. In another embodiment, an inhibitor of PD-1-mediated signaling is a mo

- 2 044974 noclonal antibody to PD-L2. Suitable PD-1-mediated signal transmission inhibitors include, but this is not limited to this, inhibitors described in US 8008449, Wo 2006/121168, Wo 2009/114335, US 8354509, US 8609089, US 2010/0028330, US 2012/0114649, Wo 2007/005874, WO 2010/077634, US 7943743, US 2012/0039906 and WO/2011/066342, all of which are incorporated herein in their entirety. Examples of suitable inhibitors of PD-1-mediated signaling include YW243.55.S70, MPDL3280A, MEDI-4736, MSB-0010718C, MDX-1105 and AMP-224. Preferred inhibitors of PD-1-mediated signaling include pembrolizumab (KEYTRUDA™), nivolumab (OPDIVO™), atelolizumab (TECENTRIQ™), avelumab (BAVENCIO™), durvalumab (IMFINZI™) and pidilizumab.

In particularly preferred embodiments of the methods and compositions of the invention, the compound of formula I is compound 1.

In particularly preferred embodiments of the invention, the PD-1-mediated signaling inhibitor is pembrolizumab or nivolumab.

In some embodiments of the method of the invention, a compound of Formula I, or a pharmaceutically acceptable salt thereof, and an inhibitor of PD-1-mediated signaling are administered to a subject simultaneously as separate compositions. In some embodiments, a compound of Formula I, or a pharmaceutically acceptable salt thereof, and an inhibitor of PD-1-mediated signaling are co-administered to a subject via the same or different routes of administration.

In some embodiments, a compound of Formula I, or a pharmaceutically acceptable salt thereof, and an inhibitor of PD-1-mediated signaling are administered sequentially to a subject as separate compositions. In some embodiments, a compound of Formula I and an inhibitor of PD-1-mediated signaling are administered to a subject sequentially through the same or different routes of administration. In one embodiment, an inhibitor of PD-1-mediated signaling is administered to a subject following administration of a compound of Formula I or a pharmaceutically acceptable salt thereof. In another embodiment, an inhibitor of PD1-mediated signaling is administered to a subject prior to administration of a compound of Formula I or a pharmaceutically acceptable salt thereof to the subject.

In certain embodiments in which a compound of Formula I or a pharmaceutically acceptable salt thereof and an inhibitor of PD-1-mediated signaling are administered as separate compositions, each composition is independently administered transmucosally, orally, rectally, vaginally, sublingually, intravenously, intramuscularly, subcutaneously, buccally , intranasally, intracisternally, intraperitoneally or inside the ear. In some embodiments, one or both of the compositions are included in a suppository or hydrogel. In preferred embodiments, both compositions are administered orally.

In some embodiments, in which a compound of Formula I or a pharmaceutically acceptable salt thereof and an inhibitor of PD-1-mediated signaling are administered as separate compositions, the timing of their administration is selected such that the effects of the pharmacological agents overlap in time, thereby achieving combined therapeutic effect. For example, a compound of Formula I, or a pharmaceutically acceptable salt thereof, and an inhibitor of PD-1-mediated signaling can be administered sequentially, separated by time greater than about 60 minutes. For example, the time interval between sequential administration of a compound of formula I or a pharmaceutically acceptable salt thereof and an inhibitor of PD-1-mediated signaling may be greater than 60 minutes, greater than 2 hours, greater than 5 hours, greater than 10 hours, greater than 1 day, greater than 2 days, more than 3 days or more than 1 week. Optimal administration times will depend on the rates of absorption, distribution, metabolism and/or elimination of the compound of Formula I or a pharmaceutically acceptable salt thereof and the PD-1-mediated signaling inhibitor.

First, either a compound of formula I or a pharmaceutically acceptable salt thereof, or an inhibitor of PD-1-mediated signaling may be administered. For example, an inhibitor of PD-1-mediated signaling can be administered to a subject some time after administration of a compound of Formula I or a pharmaceutically acceptable salt thereof. In this case, it may be desirable to administer the PD-1-mediated signaling inhibitor until about 50% has been metabolized or excreted by the subject (e.g., until about 40% has been metabolized or excreted in the subject, about 30% , about 20%, about 10%, or about 5%) of a compound of Formula I. In another example, a subject is administered a first dose of a compound of Formula I, or a pharmaceutically acceptable salt thereof, followed by a single dose of a PD-1-mediated signaling inhibitor, followed by an additional dose of a compound of formula I or a pharmaceutically acceptable salt thereof.

In some embodiments, a compound of formula I or a pharmaceutically acceptable salt thereof and an inhibitor of PD-1-mediated signaling is administered in a unit dosage form that is administered transmucosally, orally, rectally, vaginally, sublingually, intravenously, intramuscularly, subcutaneously, buccally, intranasally, intracisternally, intraperitoneally or inside the ear. Preferably, the unit dosage form is administered orally.

A compound of formula I can be administered about once a week, about once a day, or

- 3 044974 more than once a day. In one embodiment, a compound of formula I or a pharmaceutically acceptable salt thereof is administered orally. In another embodiment, a compound of formula I or a pharmaceutically acceptable salt thereof is administered parenterally, for example, intravenously. A compound of Formula I or a pharmaceutically acceptable salt thereof can be administered in a daily dose of from about 1 mg to about 1500 mg. For example, a compound of Formula I or a pharmaceutically acceptable salt thereof can be administered at a daily dose of about 200 mg. In one embodiment, a compound of Formula I or a pharmaceutically acceptable salt thereof is administered at a dosage of from about 1 mg to about 250 mg per kg of body weight.

However, it will be appreciated that the frequency of administration and total daily dosage of a compound of Formula I or a pharmaceutically acceptable salt thereof and a PD-1-mediated signaling inhibitor may be determined for an individual patient by one skilled in the art, e.g., a physician points of view. The specific dose(s) for any particular patient will depend on a number of factors, including the disorder being treated and the severity of that disorder; the activity of the particular compound used; the specific composition used; age, body weight, general health, gender and nutrition of the patient; time of administration, route of administration and release rate of the particular compound used; duration of treatment; drugs used in combination or in conjunction with the specific compound used; and similar factors well known in the medical field.

The term cancer refers to any type of cancer caused by the proliferation of malignant neoplastic cells, such as tumors, neoplasms, carcinomas, sarcomas, leukemias, lymphomas and the like. For example, types of cancer include, but are not limited to, mesothelioma, leukemias and lymphomas such as cutaneous T-cell lymphomas (CTCL), non-cutaneous peripheral T-cell lymphomas, human T-cell lymphotropic virus (HTLV)-associated lymphomas such as T - adult cell leukemia/lymphoma (ATLL), B-cell lymphoma such as diffuse large B-cell lymphoma (DLBCL), acute non-lymphocytic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, lymphomas and multiple myeloma, non-Hodgkin's lymphoma , acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), Hodgkin's lymphoma, Burkitt's lymphoma, adult T-cell leukemia/lymphoma, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), or hepatocellular carcinoma. Other examples include myelodysplastic syndrome, solid tumors in children such as brain tumors, neuroblastoma, retinoblastoma, Wilms tumor, bone tumors and soft tissue sarcomas, common solid tumors in adults such as cancers of the head and neck region (eg, cavity cancer mouth, larynx, nasopharynx, and esophagus), genitourinary cancers (eg, prostate, bladder, kidney, uterus, ovarian, testicular), lung cancer (eg, small cell and non-small cell), breast cancer, pancreatic cancer, melanoma and other types of skin cancer, stomach cancer, brain tumors, tumors associated with Gorlin syndrome (eg medulloblastoma, meningioma, etc.) and liver cancer. Additional typical forms of cancer that may be treated with the compounds of the invention include, but are not limited to, skeletal or smooth muscle cancer, gastric cancer, small intestinal cancer, rectal carcinoma, salivary gland cancer, endometrial cancer, adrenal cancer, anal cancer, rectal cancer, parathyroid cancer and pituitary cancer.

Additional types of cancer that the compounds described herein can be used to prevent, treat, and study include, for example, colon carcinoma, familial adenomatous polyposis carcinoma, and hereditary nonpolyposis colorectal cancer or melanoma. In addition, cancers include, but are not limited to, lip cancer, laryngeal cancer, hypopharyngeal cancer, tongue cancer, salivary gland cancer, gastric carcinoma, adenocarcinoma, thyroid cancer (medullary and papillary thyroid cancer), renal carcinoma, parenchymal cancer kidney, cervical cancer, uterine body cancer, endometrial cancer, choriocarcinoma, testicular cancer, urinary tract cancer, melanoma, brain tumors such as glioblastoma, astrocytoma, meningioma, medulloblastoma and peripheral neuroectodermal tumors, gallbladder cancer, bronchial cancer, multiple myeloma, basalioma, teratoma, retinoblastoma, choroidal melanoma, seminoma, rhabdomyosarcoma, crane iopharyngioma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, Ewing's sarcoma and plasmacytoma. According to one aspect of the present invention, there is provided the use of one or more compounds of the invention for the preparation of a medicament for the treatment of cancer.

In one embodiment, the cancer being treated is a hematologic malignancy. Hemoblastoses include leukemia, lymphoma and multiple myeloma. Examples include lymphocytic leukemias such as acute lymphocytic leukemia, including B-cell precursor acute lymphoblastic leukemia, T-cell precursor acute lymphoblastic leukemia, Burkitt's leukemia, and acute biphenotypic leukemia; and chronic lymphocytic leukemia, including B-cell prolymphocytic leukemia; and myelogenous leukemias such as acute myelogenous leukemia, including acute promyelocytic leukemia, acute myeloblastic leukemia and acute megakaryoblastic leukemia; and chronic myelogenous leukemia, including chronic monocytic leukemia; acute monocytic

- 4 044974 leukemia. Other leukemias include hairy cell leukemia; T-cell prolymphocytic leukemia;

leukemia of large granular lymphocytes; and adult T-cell leukemia.

Lymphomas include Hodgkin lymphoma and non-Hodgkin lymphoma, including B-cell lymphomas, T-cell lymphomas, natural killer cell (NK) cell lymphomas, and neoplasms of lymphoid progenitor cells. B-cell lymphomas include Burkitt's lymphoma/leukemia, diffuse large B-cell lymphoma, B-cell chronic lymphocytic leukemia/small cell lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma (such as Waldenström's macroglobulinemia), splenic marginal zone cells, hairy cell leukemia, plasma cell tumor, plasma cell myeloma (also known as multiple myeloma), plasmacytoma, monoclonal immunoglobulin deposition, heavy chain disease, extranodal marginal zone cell B-cell lymphoma, also called lymphoid tissue-derived lymphoma, associated with mucous membranes (MALT, from the English lymphoma originating from mucosa-associated lymphoid tissue), nodal B-cell lymphoma from marginal zone cells, follicular lymphoma, primary cutaneous lymphoma from the cells of the center of the follicle, lymphoma from the mantle zone cells, lymphogranulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK (anaplastic lymphoma kinase) plus large B-cell lymphoma, plasmablastic lymphoma, primary serous cavity lymphoma and virus-associated large B-cell lymphoma human herpes type 8 (HHV8) multifocal Castleman disease.

T-cell and NK-cell lymphomas include cutaneous T-cell lymphoma, T-cell prolymphocytic leukemia, large granular lymphocyte T-cell leukemia, aggressive NK-cell leukemia, adult T-cell leukemia/T-cell lymphoma, extranodal NK/T-cell lymphoma nasal type, enteropathy type T-cell lymphoma, hepatolienal T-cell lymphoma, blast NK-cell lymphoma, mycosis fungoides/Sézary syndrome, primary cutaneous CD30 cluster of differentiation 30 (CD30) positive T-cell lymphoproliferative disorders such as primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, lymphoma of cells with a peripheral T-cell immunophenotype, unspecified, angioimmunoblastic T-cell lymphoma and anaplastic large cell lymphoma.

In preferred embodiments, the cancer being treated is non-Hodgkin's lymphoma and, more preferably, B-cell lymphoma. In particularly preferred embodiments, the cancer to be treated is diffuse large B-cell lymphoma (DLBCL), for example, activated B cell (ABC) subtype DLBCL, germinal center cell (GCB) subtype DLBCL, double-hit DLBCL or double-hit lymphoma. expresser lymphoma DLBCL (Quintanilla-Martinez, L, Hematol. Oncol., 2015, 33: 50-55). In some embodiments, the cancer is recurrent or difficult to treat DLBCL.

In one embodiment, the present invention provides the use of a compound of formula I or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of cancer in combination with an inhibitor of PD-1-mediated signaling. Another embodiment of the present invention provides the use of a compound of formula I or a pharmaceutically acceptable salt thereof and an inhibitor of PD-1-mediated signal transduction in the manufacture of a medicament for the treatment of cancer. In a preferred embodiment, the compound of Formula I is Compound 1 or a pharmaceutically acceptable salt thereof, and the PD-1-mediated signaling inhibitor is pembrolizumab or nivolumab.

The invention further covers pharmaceutical compositions containing a compound of Formula I or a pharmaceutically acceptable salt thereof and an inhibitor of PD-1-mediated signal transduction. In one embodiment, the compound of formula I is compound 1, compound 2, or compound 3, and the PD-1-mediated signaling inhibitor is pembrolizumab or nivolumab.

A compound of Formula I or a pharmaceutically acceptable salt thereof and an inhibitor of PD-1-mediated signaling can be administered by any suitable route, including, without limitation, parenteral, intravenous, intramuscular, subcutaneous, implantable, oral, sublingual, buccal, nasal, pulmonary, transdermal, topical , vaginal, rectal and transmucosal routes of administration or the like. Topical administration may also include the use of transdermal administration, such as transdermal patches or iontophoresis devices. Pharmaceutical preparations include a preparation in solid, semi-solid or liquid form (tablet, pellet, lozenge, capsule, suppository, cream, ointment, aerosol, powder, liquid, emulsion, suspension, syrup, injection, etc.) containing a compound of formula I or a pharmaceutically acceptable salt thereof, an inhibitor of PD-1-mediated signaling, or both, as appropriate for the selected route of administration. In one embodiment, the pharmaceutical compositions are administered orally and are therefore prepared in a form suitable for oral administration, i.e. in the form of a drug in solid or liquid form. Suitable solid oral formulations include tablets, capsules, pills, granules, pellets, sachets and effervescent forms, powders and the like. Suitable liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils and the like. In one embodiment of the present invention, the composition is prepared in capsule form. According to this embodiment, the compositions of the present invention contain, in addition to the active compound and an inert carrier or diluent, a hard gelatin capsule.

Any inert excipient that is commonly used as a carrier or diluent, such as, for example, gum, starch, sugar, cellulose-based material, acrylate, or mixtures thereof, can be used in the compositions of the present invention. The preferred diluent is microcrystalline cellulose. The compositions may further contain a disintegrant (e.g., croscarmellose sodium) and a lubricant (e.g., magnesium stearate), and may further contain one or more excipients selected from a binder, a buffer, a protease inhibitor, a surfactant, a solubilizing agent, a plasticizer, an emulsifier, a stabilizing agent, a viscosity-increasing agent, a sweetener, a film-forming agent, or any combination thereof. In addition, the compositions of the present invention may be in the form of controlled release or immediate release formulations.

For liquid formulations, pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, emulsions or oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol and injectable organic ethers such as ethyl oleate. Aqueous vehicles include water, alcohol/aqueous solutions, emulsions or suspensions, including saline and buffered media. Examples of oils are those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil and fish oil. Solutions or suspensions may also include the following components:

a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate or phosphate; and tonicity adjusting agents such as sodium chloride or dextrose. The pH value can be adjusted using acids or bases such as hydrochloric acid or sodium hydroxide.

In addition, the compositions may further contain binders (e.g., gum arabic, corn starch, gelatin, carbomer, ethylcellulose, guar gum, hydroxypropylcellulose, hydroxypropylmethylcellulose, povidone), disintegrants (e.g., corn starch, potato starch, alginic acid, silica, croscarmellose sodium, crospovidone, guar gum, sodium starch glycolate, primoyel), buffers (e.g. Tris-HCl, acetate, phosphate) with different pH and ionic strength, excipients such as albumin or gelatin to prevent absorption at surfaces, detergents (e.g. Tween 20, Tween 80, Pluronic F68, bile salts), protease inhibitors, surfactants (e.g. sodium lauryl sulfate), penetration enhancer, solubilizing agents (e.g. glycerin, polyethylene glycerol, polyethylene glycol), glidant (e.g. colloidal silica), antioxidants (e.g. ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g. hydroxypropylcellulose, hydroxypropylmethylcellulose), viscosity-increasing agents (e.g. carbomer, colloidal silica, ethylcellulose, guar gum), sweeteners (e.g., sucrose, aspartame, citric acid), flavors (e.g., peppermint, methyl salicylate, or orange flavor), preservatives (e.g., thimerosal, benzyl alcohol, parabens), lubricants (e.g., stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow agents (e.g. colloidal silica), plasticizers (e.g. diethyl phthalate, triethyl citrate), emulsifiers (e.g. carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymer coatings (e.g. poloxamers or poloxamines , coating and film-forming agents (eg, ethylcellulose, acrylates, polymethacrylates) and/or adjuvants.

In one embodiment, active compounds are formulated using carriers that will protect the compound from rapid elimination from the body, such as controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. Methods for preparing such compositions will be apparent to those skilled in the art. These materials may also be obtained from commercial sources Alza Corporation and Nova Pharmaceuticals, Inc. Suspensions of liposomes can also be used as pharmaceutically acceptable carriers. They can be prepared according to methods known to those skilled in the art.

- 6 044974 of the art, for example as described in US patent No. 4522811.

It is particularly advantageous to formulate oral compositions in unit dosage form for ease of administration and uniformity of dosage. The term unit dosage form as used herein refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit containing a predetermined amount of active compound calculated to achieve the desired therapeutic effect, in combination with the required pharmaceutical carrier. The specification of the unit dosage forms of the invention is dictated by and is directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, as well as the limitations inherent in the formulation of such active compound in terms of treatment of individuals.

The compositions of the invention for oral administration may include one or more penetration enhancers, including long chain fatty acids or salts thereof, such as decanoic acid and sodium decanoate.

In one preferred embodiment, this compound can be formulated into an aqueous solution for intravenous injection. In one embodiment, solubilizing agents may be suitably used. Particularly preferred solubilizing agents include cyclodextrins and modified cyclodextrins, such as a sulfonic acid-substituted β-cyclodextrin derivative or a salt thereof, including sulfobutyl modified β-cyclodextrin, such as 7-β-cyclodextrin sulfobutyl ester, which is sold by CyDex, Inc. under the trade name CAPTISOL®.

The pharmaceutical compositions may be contained in a container, package, or dispenser along with instructions for administration.

Daily administration can be repeated continuously over a period of time ranging from several days to several years. Treatment by oral administration can continue for a period of time from one week until the end of the patient's life. Preferably, administration may occur over five consecutive days, after which the patient's condition can be assessed to determine the need for further administration. Administration may be continuous or intermittent, such as treatment over several consecutive days followed by a rest period. The compounds of the present invention can be administered intravenously on the first day of treatment, with oral administration on the second day and all subsequent days thereafter.

The method of preparing pharmaceutical compositions that contain the active ingredient is well known in the art, for example methods carried out by mixing, granulating or forming tablets. The active therapeutic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. In the case of oral administration, the active agents are mixed with excipients generally used for this purpose, such as diluents, stabilizers or inert diluents, and converted by conventional methods into forms suitable for administration, such as tablets, film-coated tablets, hard or soft gelatin capsules, aqueous, alcoholic or oily solutions and the like, which are described in detail above.

The amount of compound administered to a patient should be less than the amount that would cause toxicity in the patient. In some embodiments, the amount of the compound that is administered to the patient is less than the amount that results in a concentration of the compound in the patient's blood plasma equal to or greater than the toxicity level of the compound. Preferably, the concentration of the compound in the patient's plasma is maintained at about 10 nM. In one embodiment, the concentration of the compound in the patient's blood plasma is maintained at approximately 25 nM. In one embodiment, the concentration of the compound in the patient's blood plasma is maintained at approximately 50 nM. In one embodiment, the concentration of the compound in the patient's blood plasma is maintained at approximately 100 nM. In one embodiment, the concentration of the compound in the patient's blood plasma is maintained at approximately 500 nM. In one embodiment, the concentration of the compound in the patient's blood plasma is maintained at approximately 1000 nM. In one embodiment, the concentration of the compound in the patient's blood plasma is maintained at approximately 2500 nM. In one embodiment, the concentration of the compound in the patient's plasma is maintained at approximately 5000 nM. The optimal amount of compound to be administered to a patient in the practice of the present invention will depend on the particular compound used and the type of cancer being treated.

Definitions

Listed below are definitions of various terms used to describe this invention. These definitions apply to the terms as they are used throughout this specification and in the claims, unless otherwise limited in special cases, either independently or as part of a larger group.

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The term acyl refers to hydrogen atom, alkyl, partially saturated or fully saturated cycloalkyl, partially saturated or fully saturated heterocycle, aryl- and heteroaryl-substituted carbonyl groups. For example, acyl includes groups such as ( _C1C6 )alkanoyl (for example, formyl, acetyl, propionyl, butyryl, valeryl, caproyl, tert-butylacetyl, etc.), ( _C3 - _C6 )cycloalkylcarbonyl (for example, cyclopropylcarbonyl , cyclobutylcarbonyl, cyclopentylcarbonyl, cyclohexylcarbonyl, etc.), heterocyclic carbonyl (e.g. pyrrolidinylcarbonyl, pyrrolid-2-one-5-carbonyl, piperidinylcarbonyl, piperazinylcarbonyl, tetrahydrofuranylcarbonyl, etc.), aroyl (e.g. benzoyl) and heteroaroyl (e.g. thiophenyl-2-carbonyl, thiophenyl-3-carbonyl, furanyl-2-carbonyl, furanyl-3-carbonyl, 1 H-pyrroyl-2-carbonyl, 1 H-pyrroyl-3-carbonyl, benzo[b]thiophenyl-2 -carbonyl, etc.). In addition, the alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl part of the acyl group may be one of the groups described in the respective definitions. When the term optionally substituted is indicated, the acyl group may be unsubstituted or optionally substituted by one or more substituents (usually one to three substituents) independently selected from the group of substituents listed below in the definition for the term substituted, or alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl the acyl group portion may be substituted as described above in the preferred and more preferred substituent lists, respectively.

The term alkyl includes linear or branched radicals having from one to about twenty carbon atoms, or preferably from one to about twelve carbon atoms. More preferred alkyl radicals are lower alkyl radicals having from one to about ten carbon atoms. Most preferred are lower alkyl radicals having from one to about eight carbon atoms. Examples of such radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl, hexyl and the like.

The term alkenyl includes linear or branched radicals having at least one carbon-carbon double bond of two to about twenty carbon atoms, or preferably two to about twelve carbon atoms. More preferred alkenyl radicals are lower alkenyl radicals having from two to about ten carbon atoms, and more preferably from about two to about eight carbon atoms. Examples of alkenyl radicals include ethenyl, allyl, propenyl, butenyl and 4-methylbutenyl. The terms alkenyl and lower alkenyl include radicals having cis and trans orientations or, alternatively, E and Z'' orientations.

The term alkynyl includes linear or branched radicals having at least one carbon-carbon triple bond of two to about twenty carbon atoms, or preferably two to about twelve carbon atoms. More preferred alkynyl radicals are lower alkynyl radicals having from two to about ten carbon atoms, and more preferably from about two to about eight carbon atoms. Examples of alkynyl radicals include propargyl, 1-propynyl, 2-propynyl, 1-butyn, 2-butynyl and 1-pentynyl.

The term aryl, alone or in combination, means a carbocyclic aromatic system containing one, two or three rings, which rings may be attached to each other in a suspended manner or may be fused. The term aryl includes aromatic radicals such as phenyl, naphthyl, tetrahydronaphthyl, indan and biphenyl.

The term heteroaryl includes unsaturated heterocyclyl radicals. Examples of heteroaryl radicals include an unsaturated 3-6 membered, preferably 5 or 6 membered heteromonocyclic group containing 1-4 nitrogen atoms, for example pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazolyl (for example, 4H -1,2,4triazolyl, 1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, etc.), tetrazolyl (for example, 1H-tetrazolyl, 2Htetrazolyl, etc.) etc. .d.; an unsaturated fused heterocyclyl group containing 1-5 nitrogen atoms, for example, indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl, tetrazolopyridazinyl (for example, tetrazolo[1,5-b]pyridazinyl, etc.) and etc.; an unsaturated 3-6 membered, preferably 5 or 6 membered, heteromonocyclic group containing an oxygen atom, for example, pyranyl, furyl, etc.; an unsaturated 3-6 membered heteromonocyclic group containing a sulfur atom, for example, thienyl, etc.; unsaturated 3-6-membered, preferably 5- or 6-membered, heteromonocyclic group containing 1 or 2 oxygen atoms and 1-3 nitrogen atoms, for example, oxazolyl, isoxazolyl, oxadiazolyl (for example, 1,2,4-oxadiazolyl, 1 ,3,4-oxadiazolyl, 1,2,5-oxadiazolyl, etc.) etc.; an unsaturated fused heterocyclyl group containing 1 or 2 oxygen atoms and 1-3 nitrogen atoms (for example, benzoxazolyl, benzoxadiazolyl, etc.); an unsaturated 3-6 membered, preferably 5 or 6 membered, heteromonocyclic group containing 1 or 2 sulfur atoms and 1-3 nitrogen atoms, for example, thiazolyl, thiadiazolyl (for example, 1,2,4-thiadiazolyl, 1,3 ,4-thiadiazolyl, 1,2,5-thiadiazolyl, etc.) etc.; an unsaturated condensed heterocyclyl group containing 1 or 2 sulfur atoms and 1-3 nitrogen atoms (for example, benzothiazolyl, benzothiadiazolyl, etc.) and the like.

The term substituted refers to the replacement of one or more hydrogen radicals in a given structure with a radical of a specific substituent, including, but not limited to: halogen, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio, arylthio, alkylthioalkyl, aryltioalkyl, alkylsulfonyl, alkylsulfonylalkyl , arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyal kyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl , aralkoxycarbonyl, carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl, heteroaryl, heterocyclic and aliphatic radical. It will be appreciated that the substituent may be further substituted.

The term inhibition in the context of neoplasia, tumor growth or proliferation of tumor cells can mean the assessment of delaying the appearance of primary or secondary tumors, slowing the development of primary or secondary tumors, reducing the occurrence of primary or secondary tumors, delaying the appearance or reducing the severity of secondary effects of the disease, stopping tumor growth and tumor regression, among other things. In an exceptional situation, complete inhibition is referred to herein as prophylaxis or chemoprevention.

The term metastasis as used herein refers to the migration of cancer cells from the original tumor site through the blood and lymphatic vessels to form cancerous tumors in other tissues. Metastasis is also a term used to refer to a secondary cancer that develops at a distant site.

The term neoplasm as used herein refers to a mass of tissue that results from excessive cell division. The growths can be benign (noncancerous) or malignant (cancerous) and may also be called a tumor. The term neoplasia represents a pathological process that leads to the formation of a tumor.

As used herein, the term precancerous refers to a condition that is not cancerous but may become cancerous if left untreated.

The term proliferation refers to cells undergoing mitosis.

The term treatment refers to any process, act, application, any therapy or the like in which a mammal, including a human, receives medical care for the purpose of improving the condition of the mammal, directly or indirectly.

As used herein, the term pharmaceutically acceptable salt refers to those salts that are medically suitable for use in contact with tissues of humans and lower animals without undue toxicity, irritation, allergic reaction, or the like, and within a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S.M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). The salts can be prepared in situ during the final isolation and final purification of the compounds of the invention or separately by reacting the free base functionality with a suitable organic acid or inorganic acid. Examples of pharmaceutically acceptable non-toxic acid addition salts include, but are not limited to, amino salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid, lactobionic acid or malonic acid, or using other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentane propionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate , gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalene sulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate , 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, salt valerate and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium and the like salts. Other pharmaceutically acceptable salts include, as appropriate, salts with non-toxic ammonium, quaternary ammonium and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl sulfonate having 1 to 6 carbon atoms, sulfonate and arylsulfonate. Certain salts, such as the basic sodium, potassium and choline salts, as well as salts with acids, such as sulfate and methanesulfonate salts, have been found to improve the solubility of compounds of formula I in pharmaceutically acceptable aqueous media. In one embodiment, the pharmaceutically acceptable salt of Compound 1 is a choline salt. Preferred salts of Compound 1 include sodium salt and potassium salt. Other

- 9 044974 preferred salts include sulfate and methanesulfonate salts. In particular, the preferred salts of Compound 1 are the methanesulfonate and benzenesulfonate salts. A particularly preferred salt of compound 2 is the hydrochloride salt.

As used herein, the term pharmaceutically acceptable carrier is intended to include any conceivable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption retarding agents, and the like compatible with pharmaceutical administration, such as sterile pyrogen-free water. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, an authoritative reference in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin solution. In addition, liposomes and non-aqueous diluents such as fixed oils can be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except in cases where any conventional medium or any conventional agent is incompatible with the active compound, their use in the compositions is proposed. Additional active compounds may also be included in the compositions.

As used herein, the term precancerous refers to a condition that is not cancerous but may become malignant if left untreated.

The term subject as used herein refers to an animal. Preferably the animal is a mammal. More preferably, the mammal is a human. The term subject also refers, for example, to dogs, cats, horses, cattle, pigs, guinea pigs, fish, birds and the like.

The compounds of this invention can be modified by adding appropriate functional groups to enhance the biological properties with respect to selectivity. Such modifications are known in the art and may include modifications that increase the penetration of a biological substance into a given biological system (eg, blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, modify the process metabolism and change the rate of excretion.

Pharmaceutical compositions

The pharmaceutical compositions of the present invention contain a therapeutically effective amount of a compound of Formula I, such as Compound 1 or a pharmaceutically acceptable salt thereof, in combination with an inhibitor of PD-1-mediated signaling, formulated together with one or more pharmaceutically acceptable carriers or excipients.

Preferably, the pharmaceutically acceptable carrier or excipient is a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or excipient for any type of formulation. Some examples of materials that can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose and sucrose; cyclodextrins such as alpha(a), beta(b) and gamma(y) cyclodextrins; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline solution; Ringer's solution; ethyl alcohol and phosphate buffer solutions, and in addition, according to the judgment of the formulator, other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, lubricants, coating agents, sweeteners, flavorings and fragrances, preservatives and antioxidants.

The pharmaceutical compositions of this invention can be administered orally, parenterally, by nebulization, topically, rectally, nasally, buccally, vaginally or by implanted reservoir, preferably by oral administration or injection. The pharmaceutical compositions of this invention may contain any conventional non-toxic pharmaceutically acceptable carriers, adjuvants or diluents. In some cases, the pH of the formulation may be adjusted using pharmaceutically acceptable acids, bases, or buffers to enhance the stability of the compound or its delivery form. The term parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal injection or infusion methods, intralesional and intracranial injection or infusion methods.

Liquid dosage forms for oral administration include pharmaceutically acceptable

- 10 044974 emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed, peanut, corn, sprout, olive, castor and sesame oils), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycols and sorbitan fatty acid esters and mixtures thereof. In addition to inert diluents, oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents and flavoring agents.

Injectable preparations, such as sterile aqueous or oleaginous injectable suspensions, can be prepared according to methods known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a non-toxic parenterally acceptable diluent or solvent, for example, a solution in 1,3-butanediol. Among the acceptable diluents and solvents that may be used are water, Ringer's solution as mentioned in the United States Pharmacopoeia (U.S.P.), and isotonic sodium chloride solution. In addition, sterile non-volatile oils are traditionally used as a solvent or suspending medium. For this purpose, any low-viscosity fixed oil can be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injections.

Injectable formulations may be sterilized, for example, by filtration through a bacteria-retaining filter or by administering sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injection medium prior to use.

To prolong the effect of a drug, it is often desirable to slow the absorption of a drug administered by subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of a crystalline or amorphous substance having poor solubility in water. Then the rate of absorption of the drug will depend on the rate of its dissolution, which, in turn, may depend on the size of the crystals and the crystalline form. Alternatively, delayed absorption of a parenterally administered dosage form is achieved by dissolving or suspending the drug in an oily diluent. Injectable depot forms are prepared by forming matrices to microencapsulate the drug in biodegradable polymers such as polyglycolide polyglycolide. Depending on the drug/polymer ratio and the nature of the particular polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Additionally, depot forms of injectable formulations are prepared by incorporating the drug into liposomes or microemulsions that are compatible with body tissue.

Compositions for rectal or vaginal administration are preferably suppositories, which can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers, such as cocoa butter, polyethylene glycol or suppository wax, which are solid at ambient temperature but liquid at body temperature , and therefore melt in the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert pharmaceutically acceptable excipient or carrier, such as sodium citrate or dibasic calcium phosphate, and/or with: a) excipients or dry diluents, such as starches, lactose, sucrose, glucose , mannitol and silicic acid; b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; c) wetting agents such as glycerin; d) disintegrants such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, some silicates and sodium carbonate; e) dissolution retarders such as paraffin; f) absorption accelerators such as quaternary ammonium compounds; g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate; h) absorbents such as kaolin and bentonite clay; and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also contain buffering agents.

Solid compositions of this type can also be used as fillers in soft and hard gelatin capsules using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like.

- 11 044974

Solid dosage forms in the form of tablets, dragees, capsules, pills and granules can be formulated with coatings and coatings, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They may optionally contain opacifying agents and may be formulated such that they will release the active ingredient(s) only, or preferentially, in a specific part of the gastrointestinal tract, possibly delayed. Examples of sealing compositions that can be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and patches. The active ingredient is mixed under sterile conditions with a pharmaceutically acceptable carrier and any necessary preservatives or buffering agents that may be required. Ophthalmic composition, ear drops, ophthalmic ointments, powders and solutions are also covered as they fall within the scope of this invention.

Ointments, pastes, creams and gels may contain, in addition to the active compound of this invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide or mixtures thereof.

Powders and sprays may contain, in addition to the compounds of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and powdered polyamide or mixtures of these substances. Sprays may additionally contain conventional propellants such as chlorofluorocarbons.

Transdermal patches have the added benefit of providing controlled delivery of the compound to the body. Such dosage forms can be prepared by dissolving or dispersing the compound in a suitable medium. In addition, absorption enhancers can be used to increase the delivery of the compound through the skin. The rate can be controlled either by providing a rate-regulating membrane or by dispersing the active compound in a polymer matrix or gel.

For delivery to the lungs, the therapeutic composition of the invention is prepared and administered to the patient in particulate form in solid or liquid form by direct administration (eg, by inhalation into the respiratory system). The active compound-containing particles in solid or liquid form prepared for the practice of the present invention include respirable size particles: that is, particles small enough to pass through the mouth and larynx when inhaled into the bronchi and alveoli of the lungs. Delivery of aerosolized therapeutics, especially aerosolized antibiotics, is known in the art (see, for example, US Pat. No. 5,767,068 to Van Devanter et al., US Pat. No. 5,508,269 to Smith et al., and WO 98/43650 filed by Montgomery, all of which incorporated herein by reference). A discussion of pulmonary delivery of antibiotics can also be found in US Pat. No. 6,014,969, which is incorporated herein by reference.

By a therapeutically effective amount of a combination of a compound of Formula I and an inhibitor of PD-1-mediated signaling is meant an amount of each compound that, in combination, produces a therapeutic effect in the subject being treated with a reasonable benefit/risk ratio acceptable for any therapeutic treatment. A therapeutic effect can be assessed as objective (i.e., something that can be measured using some test or marker) or subjective (i.e., where the subject indicates or perceives an effect). In addition, effective doses will vary depending on the route of administration, as well as the possibility of coadministration with other agents. However, it will be understood that the total daily intake of the compounds and compositions of the present invention will be determined by the attending physician from a medical point of view. The specific therapeutically effective dosage level for any particular patient will depend on a number of factors, including the disorder being treated and the severity of that disorder; the activity of the particular compound used; the specific composition used; age, body weight, general health, gender and nutrition of the patient; the duration of administration, route of administration, and release rate of the particular compound used; duration of treatment; drugs used in combination or duration of treatment; drugs used in combination or concurrently with the specific compound used; and similar factors well known in the medical field. In preferred embodiments, a therapeutically effective amount of the combination of a compound of Formula I, or a pharmaceutically acceptable salt thereof, and an inhibitor of PD-1-mediated signaling is synergistic for the type of cancer being treated.

The total daily dose of each compound in the combination therapy of the present invention, administered to a human or other animal in single or divided doses, may be, for example, from 0.01 to 50 mg/kg body weight, or more typically from 0 ,1 to 25 mg/kg body weight. Single dose compositions may contain amounts or amounts thereof in fractions that constitute a daily dose. In general, the treatment regimens of the present invention comprise administering to a patient in need of such treatment from about 10 to about 1000 mg of the compound(s) of the present invention per day in single or multiple doses.

Each compound in the combination therapy of the invention can, for example, be administered by injection, intravenously, intraarterially, subdermally, intraperitoneally, intramuscularly or subcutaneously; or orally, buccally, nasally, transmucosally, topically, as part of an ophthalmic preparation, or by inhalation, dosages ranging from about 0.1 to about 500 mg/kg body weight, alternatively, dosages from 1 to 1000 mg/dose, every 4 to 120 hours or as directed for the specific drug. The methods of this invention involve administering an effective amount of a compound or a composition containing a compound to achieve the desired or specified effect. Typically, the pharmaceutical compositions of this invention will be administered from about 1 to about 6 times per day or. alternatively, as a continuous infusion. This mode of administration can be used to treat chronic or acute conditions. The amount of active ingredient that can be combined with pharmaceutically acceptable excipients or carriers to formulate a unit dosage form will vary depending on the recipient being treated and the particular route of administration. A typical formulation will contain from about 5 to about 95% active compound (w/w). Alternatively, such preparations may contain from about 20 to about 80% active compound.

Lower or higher doses than those listed above may be required. Specific dosage and treatment regimens for any particular patient will depend on a number of factors, including the potency of the specific compound used, age, body weight, general health status, sex, nutrition, time of administration, rate of elimination, drug combination, severity and the course of the disease, condition or symptoms, the patient's predisposition to the disease, condition or symptoms and the judgment of the attending physician.

If the patient's condition improves, a maintenance dose of a compound, composition, or combination of this invention may be administered if necessary. Subsequently, the dosage or frequency of administration, or both, can be reduced depending on the symptoms to a level where the improved condition is maintained when the symptoms are reduced to the desired level. However, if symptoms recur, patients may require periodic treatment on a long-term basis.

Examples

The compounds and methods of the present invention will be better understood in conjunction with the following examples, which are intended only to illustrate and not to limit the scope of the present invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art, and such changes and modifications, including, without limitation, those relating to the chemical structures, substituents, derivatives, compositions and/or methods of the invention, can be made without deviating from the essence of the invention and the scope of the attached claims.

The synthesis of compound 1 and its methanesulfonate, sodium, potassium and choline salts is illustrated in the schemes below.

- 13 044974 are lustrated below:

Intermediate 107-1 or 107-2 can be prepared by reacting compound 106 with either R-2-1 or R-2-2, respectively. Synthesis schemes for the preparation of R-2-1 and R-2-2 proyl-

or using an alternative method:

- 14 044974

Intermediate 108-1 and 108-2 can be prepared by coupling 107-1 or 107-2 with either R-3-1 or R-3-2, wherein R-3-1 and R-3 -2 can be obtained according to the following scheme:

Example 1. Preparation of N-hydroxy-2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidin- 5-carboxamide (compound 1)

Step a: (2)-ethyl-2-(ethoxymethyl)-3-methoxyacrylate (compound 202)

Sodium (40.9 g; 1.78 mol) was carefully added portionwise to ethanol (750 ml) and the solution was concentrated to give NaOEt powder after all sodium metal had disappeared. Hexane (1.0 L) was added while stirring and the mixture was cooled using an ice water bath. A mixture of compound 201 (130 g, 0.89 mol) and ethyl formate (131 g, 1.78 mol) was added dropwise at 0-5 °C.

The reaction mixture was stirred at room temperature overnight. Dimethyl sulfate (224 g; 1.78 mol) was added dropwise while cooling in an ice water bath. The resulting mixture was heated at 50°C for 2 hours. Triethyl ammonium chloride (122 g) and sodium hydroxide (20 g) were added to the mixture. The mixture was then stirred at room temperature for 4 hours and filtered. The filtrate was washed with water and dried over Na ₂ SO ₄ . It was concentrated to give the title compound (140 g; 37%) as a colorless oil, which was used in the next step without further purification.

Step b: ethyl 2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (compound 203)

A mixture of compound 202 (140 g; 0.745 mol), urea (40.0 g; 0.697 mol) and concentrated hydrochloric acid (34 ml) in ethanol (500 ml) was heated at reflux overnight. After the reaction mixture was evaporated to approximately 50% volume, the resulting suspension was filtered, washed with a small amount of ethanol and dried to give compound 203 (47 g; 37%) as a white solid. LCMS (liquid chromatography coupled with mass spectrometry): 171 [M+1]+.

^1H NMR (nuclear magnetic resonance) (400 MHz, CDCl ₃ ): δ 1.19 (t, J = 7.2 Hz, 3H), 3.92 (s, 2H), 4.08 (q, J = 7.2 Hz, 2H ), 7.0 (s, 1H), 7.08 (d, J = 6.0 Hz, 1H), 8.83 (d, br, J = 4.8 Hz, 1H).

Step c: ethyl 2-oxo-1,2-dihydropyrimidine-5-carboxylate (compound 204)

Bromine (49.0 g, 307 mmol) was added to a solution of compound 203 (47 g, 280 mmol) in acetic acid (500 mL). The mixture was heated at reflux for 2 hours, cooled to room temperature, further cooled at 0-5°C and filtered to obtain the title compound 204 as a yellow solid (38 g; 54%). LCMS: 169 [M+1] ⁺ .

1H NMR (400 MHz, D2O): δ 1.28 (t, J = 7.2 Hz, 3H), 4.32 (q, J = 7.2 Hz, 2H), 9.00 (br, s, 2H).

Step d: ethyl 2-chloropyrimidine-5-carboxylate (compound R-2-1)

A mixture of compound 204 (38.0 g; 153 mmol), phosphoryl trichloride (300 ml) and N,N-dimethylaniline (3 ml) was heated at reflux for 2 hours, cooled to room temperature and concentrated. The residue was carefully quenched with ice water, the pH was adjusted to 7-8 using sodium carbonate, and extracted with EtOAc. The combined organic extracts were washed with ice water and brine, dried over Na ₂ SO ₄ , evaporated and purified by column chromatography (eluted with EtOAc/hexanes, 10%), giving compound R-2-1 (15 g; 52%) as a white solid substances.

- 15 044974

LCMS: 187 [M+1]+.

1H NMR (400 MHz, CDCl ₃ ): δ 1.36 (t, J = 7.5 Hz, 3H), 4.39 (q, J = 7.5 Hz, 2H), 9.08 (s, 2H).

Step e: sodium (Z)-2-(dimethoxymethyl)-3-methoxy-3-oxoprop-1-ene-1-olate (compound 206)

A mixture of NaH (27 g; 60% in mineral oil; 0.675 mol) in anhydrous 1,2-dimethoxyethane (300 ml) was heated to 40-50°C and methyl 3,3-dimethoxypropionate (205) (100 g) was added dropwise ; 0.675 mol). The resulting mixture was stirred for 0.5 h and anhydrous methyl formate (81 g; 1.35 mol) was added dropwise at 40-50°C. The resulting mixture was stirred at 40-50°C (internal temperature) for 2 hours, after which it was cooled to 0°C. The reaction mixture was left to warm slowly to 25°C and stirred overnight. Et ₂ O (150 ml) was added and stirred for 30 minutes. The resulting suspension was filtered. The solid was washed with Et ₂ O (100 mL), collected and dried to give the title compound 206 (82 g, 61%) as an off-white solid. LCMS (m/z): 130.8 [M+1] ⁺ .

^1H NMR (400 MHz, CD3OD): δ 3.36 (s, 6H), 3.60 (s, 3H), 5.34 (s, 1H), 8.92 (s, 1H).

Step f: 2-aminopyrimidine-5-carboxylic acid methyl ester (compound 207)

To a mixture of guanidine hydrochloride (42.2 g, 0.44 mol) in dimethylformamide (DMF) (300 ml) was added compound 206 (80 g, 0.40 mol). The resulting mixture was heated at 100°C for 1 hour. The reaction mixture was filtered and then cooled. The filter cake was washed with 50 mL DMF and the combined filtrate was concentrated to leave a residue which was suspended in cold EtOH and washed with cold EtOH (50 mL) to give compound 207 (38 g; 61.5%) as a yellow solid. LCMS (m/z): 154.2 [M+1] ⁺ ; 195.1 [M+42]+.

1H NMR (400 MHz, CD3OD): δ 3.88 (s, 3H), 8.77 (s, 2H).

Step g: methyl 2-chloropyrimidine-5-carboxylate (compound R-2-2)

Compound 207 (7 g, 0.046 mol) was added to a mixture of concentrated hydrochloric acid (15.2 ml) and CH ₂ Cl ₂ (60 ml). After cooling, ZnCI ₂ (18.6 g; 0.138 mol) was added at 15-20°C. The mixture was stirred at 15-20°C for 0.5 hours and cooled to 5-10°C. NaNO ₂ (9.5 g; 0.138 mol) was added in portions while maintaining the internal temperature at 5-10°C. The reaction was carried out for approximately 2 hours. The reaction mixture was poured into ice water (50 ml). The organic layer was separated and the aqueous phase was extracted with CH ₂ Cl ₂ (30 ml x 2). The combined organic extracts were concentrated to give the crude product (4.2 g). The crude compound was suspended in hexane (20 ml), heated at 60°C for 30 minutes and filtered. The filtrate was concentrated to give the title compound R-2-2 (3.5 g, 44.4%) as an off-white solid. LCMS (m/z): 214.1 [M+42]+.

1H NMR (400 MHz, CDCl ₃ ): δ 4.00 (s, 3H), 9.15 (s, 2H).

Step h: 5-bromo-2-methoxypyridine (compound 303)

A solution of 2-methoxypyridine (100 g; 0.92 mol), N-bromosuccinimide (NBS) (180 g; 1.0 mol) in acetonitrile (1.0 L) was stirred at reflux for 21 hours. TLC (thin layer chromatography ) showed the completion of the reaction. The reaction mixture was cooled to room temperature and concentrated. Approximately 900 ml of solvent was collected. The resulting suspension was filtered and washed with n-hexane (approximately 400 ml). The filtrate was concentrated again to give the crude product. The crude product was distilled under reduced pressure (30° C./about 0.3 mm Hg (40 Pa)) to give the title compound as a clear oil (146 g; 84%). LCMS (m/z): 190.0 [M+1] ⁺ .

1H NMR (400 MHz, CDCl ₃ ): δ 3.90 (s, 3H), 6.65 (d, J = 8.8 Hz, 1H), 7.62 (dd, J = 8.8 Hz; 2.4 Hz, 1H) , 8.19 (s, 1H).

Step i: 6-methoxypyridin-3-ylboronic acid (R-3-1)

To a solution of compound 303 (20 g, 0.11 mol) in anhydrous tetrahydrofuran (THF) (180 ml), H-BuLi (59 ml; 2 M solution in THF) was added dropwise at -78°C, the resulting mixture was stirred for 1 hour Triisopropyl borate (37 ml) was added at -78°C, the reaction mixture was warmed to room temperature and continued stirring overnight. TLC (hexanes/ethyl acetate, 5:1) indicated completion of the reaction. The pH value of the mixture was adjusted to 3-4 using 4 N. HCl solution (90 ml). The precipitate was collected by filtration to give crude compound R-3-1 (21 g; 128%). Crude compound R-3-1 (21 g) was dissolved in water (200 ml) and the pH of the solution was adjusted to 8-9 using concentrated ammonia solution, the precipitate was collected by filtration to obtain pure title compound R-3-1 as white solid (11 g; 67%). LCMS (m/z): 154.1 [M+1] ⁺ .

1H NMR (400 MHz, dimethyl sulfoxide (DMSO)-d6): δ 3.86 (s, 3H), 6.76 (d, J = 8.4 Hz, 1H), 7.99 (dd, J = 8.4 Hz; 2.0 Hz, 1H), 8.05 (br, 2H), 8.52 (d, J = 2.0 Hz, 1H).

Step j: 2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (compound R-3-2)

Mixture of compound 303 (55 g; 0.29 mol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane) (90 g; 0.35 mol), potassium acetate (57 g; 0.58 mol) and bis(triphenylphosphine) palladium(II) chloride (2.2 g; 3 mmol) in anhydrous dioxane (500 ml) were heated at 108 °C in N ₂ atmosphere overnight. The reaction mixture was concentrated and purified by column chromatography, eluting with hexanes/ethyl acetate to give the title compound R-3-2 (58 g, 84%).

^1H NMR (400 MHz, DMSO- _d6 ): δ 1.30 (s, 12H), 3.88 (s, 3H), 6.81 (d, J = 8.0 Hz, 1H), 7.88 (dd, J = 8, 0 Hz, 2.0 Hz, 1H), 8.41 (d, J = 2.0 Hz, 1H).

Step k: thieno[3,2-d]pyrimidine-2,4(1H,3H)-dione (compound 102)

Urea method: mixture of methyl 3-aminothiophene-2-carboxylate (compound 101) (90.0 g; 573 mmol; 1.0 eq.) and urea (277.6 g; 4.6 mol; 8.0 eq.) was heated at 190°C for 3-4 hours and cooled to room temperature. Aq. was added to the reaction mixture. NaOH solution (10%, 800 ml). After stirring at ambient temperature for 1 hour, the solid was removed by filtration. The filtrate was acidified to pH 3-4 using HCl and the precipitated solid was collected by filtration, washed with water and dried in vacuo to give the desired product, compound 102, as an off-white solid (87 g; 89%). Melting point: 280-285°C. LCMS (m/z): 169.0 [M+1]+.

1H NMR (400 MHz, DMSO-d6): δ 6.92 (d, J = 5.2 Hz, 1H), 8.05 (d, J = 5.2 Hz, 1H), 11.0-11.5 (br, 2H).

Potassium cyanate (KOCN) method: to a mixture of 3-aminothiophene-2-carboxylate (compound 101) (100.0 g; 636.9 mmol; 1.0 eq.), acetic acid (705 ml) and water (600 ml) a solution of potassium cyanate (154.8 g; 1.91 mol; 3.0 eq.) in water (326 ml) was slowly added over a period of 1 hour. The resulting mixture was stirred at room temperature for 20 hours, filtered and washed with water (500 ml). The filter cake was charged into a reactor of suitable size and 2 M aqueous sodium hydroxide solution (1.65 L) was added, the suspension was stirred for 2 hours and the formation of the desired product was confirmed by LCMS. The mixture was cooled to 10°C and a 3 M aqueous solution of hydrochloric acid (1.29 L) was added to pH 5.0-6.0. The suspension was filtered, washed with water (700 ml) and dried in a vacuum oven at 50°C for 24 hours, obtaining compound 102 (100 g; 94%) as an off-white solid. LCMS (m/z): 169.1 [M+1] ⁺ .

1H NMR (400 MHz, DMSO-d6): δ 6.92 (d, J = 5.2 Hz, 1H), 8.04 (d, J = 5.2 Hz, 1H), 11.14 (s, 1H), 11.51 (s , 1H).

Step i: 2,4-dichlorothieno[3,2-d]pyrimidine (compound 103)

Phosphorus oxychloride (152 ml; 1.67 mol; 7.0 eq.) was slowly added to a cold solution of compound 102 (40 g; 238 mmol; 1.0 eq.) and N,N-dimethylaniline (22.5 ml; 179 mmol; 0.75 eq.) in acetonitrile (250 ml), maintaining the temperature below 20°C. The mixture was then heated to 85°C and stirred for 24 hours. The reaction mixture was cooled to 15°C and then slowly poured into a mixture of ice and cold water (360 ml). The resulting suspension was filtered and washed with cold water (200 ml). The filter cake was dried in a vacuum oven at 40°C for 24 hours to give compound 103 (40.5 g, 83%) as an off-white solid. Melting point: 245-250°C. LCMS (m/z): 205.0 [M+1] ⁺ .

1H NMR (400 MHz, DMSO-d6): δ 7.75 (d, J = 5.2 Hz, 1H), 8.71 (d, J = 5.2 Hz, 1H).

Step m: 2-chloro-4-morpholothieno[3,2-d]pyrimidine (compound 104)

To a mixture of compound 103 (34.2 g; 167 mmol; 1.0 eq.) and methanol (500 ml) morpholine (31.2 ml; 367 mmol; 2.2 eq.) was slowly added. The reaction mixture was stirred at room temperature overnight. The precipitate was collected by filtration, washed with methanol and dried in vacuo to give the desired product, compound 104, as a light yellow solid (39 g; 91%). Melting point: 250-255°C. LCMS (m/z): 256.0 [M+1] ⁺ .

1H NMR (400 MHz, DMSO- _d6 ): δ 3.76 (t, J = 5.2 Hz, 4H), 3.92 (t, J = 5.2 Hz, 4H), 7.42 (d, J = 5.2 Hz, 1H), 8.32 (d, J = 5.2 Hz, 1H).

Step n: 2-chloro-4-morpholinothieno[3,2-d]pyrimidine-6-carbaldehyde (compound 105)

n-BuLi (in hexanes; 2.4 M solution; 40.8 ml; 102 mmol; 1.3 eq.) under nitrogen atmosphere. The resulting suspension was left to warm to -60°C to transform it into a clear brown solution. The reaction mixture was then cooled again to -78°C and DMF (anhydrous; 9.1 mL; 118 mmol; 1.5 eq.) was added slowly. The resulting solution was stirred at -78°C for 0.5 hours, heated to 0°C for 1 hour and slowly poured into a mixture of aq. HCl solution (0.25 M; 660 ml) and ice water (320 ml). The resulting suspension was stirred at 0-10°C for 0.5 h, filtered, washed with cold water and dried in vacuo to give compound 105 as a yellow solid (22 g; 98%). Melting point: 260-265°C. LCMS (m/z): 284.0 [M+1] ⁺ .

^1H NMR (400 MHz, DMSO- _d6 ): δ 3.77 (t, J = 5.2 Hz, 4H), 3.96 (t, J = 5.2 Hz, 4H), 8.30 (s, 1H), 10.21 (s, 1H).

Step o: (2-chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidin-6-ylmethyl)methylamine (compound 106)

To a solution of compound 105 (20.0 g; 70.4 mmol; 1.0 eq.) in methanol (125 ml) was added a solution of methylamine in methanol (27% (v/v); 75 ml; 563, 2 mmol) in a nitrogen atmosphere. The reaction mixture was stirred at room temperature overnight and the solvent was removed in vacuo to give a crude solid, which was dissolved in methanol (550 ml) and THF (220 ml) under nitrogen. Sodium borohydride (8 g; 211.2 mmol) was added in portions and the reaction mixture was stirred at room temperature.

- 17 044974 at room temperature overnight. The reaction mixture was evaporated in vacuo and water (300 ml) was added. The aqueous mixture was extracted with methylene chloride, the combined extracts were dried over Na ₂ SO ₄ and concentrated. The residue was dissolved in 6 M HCl solution (230 ml) and stirred for 30 min. The aqueous solution was washed several times with methylene chloride and the pH was adjusted to 9-10 using NaOH (4 N solution). The precipitated solid was collected by filtration and dried (60°C, 6 hours) to give a light yellow solid (18 g; 85%). Melting point: 240-245°C. LCMS (m/z): 299 [M+1]+.

^1H NMR (400 MHz, DMSO-d6): δ 2.32 (s, 3H), 3.74 (t, J = 5.2 Hz, 4H), 3.88 (t, J = 5.2 Hz, 4H), 3.96 ( s, 2H), 7.24 (s, 1H).

Step p(a): 2-[(2-chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidin-6-ylmethyl)methylamino]pyrimidine-5-carboxylic acid ethyl ester (compound 107- 1)

Diisopropylethylamine (220 mL, 1.26 mol) was added to a mixture of compounds 106 (10 g; 33.6 mmol) and R-2-1 (6.8 g; 36.4 mmol) in CH3CN (400 ml) at room temperature. The resulting mixture was stirred at room temperature overnight. The mixture was then evaporated and then methylene chloride (300 ml) was added. The organic phase was washed with water, dried over Na ₂ SO ₄ and concentrated in vacuo to give a residue. Ethyl acetate was added to the residue, and the resulting mixture was stirred at ice-water bath temperature for 50 minutes. The resulting solid was collected by filtration to give the title product 107-1 as a white solid (10.6 g; 70%). LCMS: 449 [M+1] ⁺ .

1H NMR (400 MHz, DMSO-d6): δ 1.30 (t, J = 7.2 Hz, 3H), 3.25 (s, 3H), 3.71 (t, J = 5.2 Hz, 4H), 3.83 (t , J = 4.8 Hz, 4H), 4.29 (m, 2H), 5.21 (s, 2H), 7.39 (s, 1H), 8.87 (s, 2H).

Step p(b): 2-[(2-chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidin-6-ylmethyl)methylamino]pyrimidine-5-carboxylic acid methyl ester (compound 107- 2)

A mixture of compound 106 (25 g; 84 mmol), CH3CN (500 ml) and R-2-2 (16 g; 92 mmol) was stirred at room temperature. Diisopropylethylamine (DIPEA) (500 ml; 2.9 mol) was added. The solution was stirred overnight and evaporated. After addition of methylene chloride (500 ml), the organic phase was washed with water, dried using Na ₂ SO ₄ and concentrated in vacuo. Ethyl acetate (200 ml) was added to the residue and the mixture was stirred in an ice water bath for 50 minutes. The title product was collected as a white solid (29.4 g; 81%). LCMS (m/z): 435.2

[M+1] ⁺ .

1H NMR (400 MHz, DMSO-d6): 3.25 (s, 3H), 3.71 (t, J = 5.2 Hz, 4H), 3.82-3.84 (m, 7H), 5.21 (s, 2H), 7.39 ( s, 1H), 8.87 (s, 2H).

Step q(a): ethyl-2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidin- 5-carboxylate (compound 108-1)

Method A: mixture of compound 107-1 (12 g; 26.7 mmol), compound R-3-1 (4.9 g; 32 mmol), NaHCO ₃ (6.7 g; 80.1 mmol) and bis(triphenylphosphine chloride) )palladium(P) (188 mg; 0.267 mmol) in a solvent mixture of toluene (80 ml), ethanol (50 ml) and water (10 ml) was heated at 108°C for 4.5 hours under N ₂ atmosphere. TLC indicated completion of the reaction. The reaction mixture was then cooled to room temperature and water (20 ml) was added. The resulting solid was collected by filtration and then suspended in ethanol (100 ml). The suspension was stirred at room temperature for 30 minutes and filtered. The collected solid was washed with ethanol and dried in vacuo to give the title compound 108-1 as a white solid (10 g; 72%).

Method B: mixture of compound 107-1 (1.5 g; 3.34 mmol), compound R-3-2 (1.6 g; 6.68 mmol), NaHCO ₃ (0.84 g; 10.0 mmol ) and bis(triphenylphosphine)palladium(II) chloride (118 mg; 0.167 mmol) in a solvent mixture of toluene (24 ml), ethanol (15 ml) and water (3 ml) was heated at 108 ° C in an atmosphere of N ₂ overnight . The reaction mixture was partitioned between dichloromethane and water. The organic layer was separated and washed with brine, dried over Na2SO4, filtered and evaporated in vacuo to leave a residue which was purified by column chromatography, eluted with hexanes/ethyl acetate to give 108-1 as a white solid (1.7 g; 98%) .

T.pl. 198-202°C. LCMS: 522.30 [M+1] ⁺ .

1H NMR (400 MHz, DMSO-d6): δ 1.31 (t, J = 7.2 Hz, 3H), 3.28 (s, 3H), 3.76 (t, J = 4.4 Hz, 4H), 3.93 (t , J = 4.4 Hz, 4H), 3.94 (s, 3H), 4.30 (q, J = 7.2 Hz, 2H), 5.24 (s, 2H), 6.92 (d, J = 8.8 Hz, 1H), 7.47 (s, 1H), 8.57 (dd, J = 8.8 Hz, 2.0 Hz, 1H), 8.88 (s, 2H), 9.15 (d, J = 2.0 Hz, 1H).

Step q(b): methyl-2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6yl)methyl)(methyl)amino)pyrimidin-5- carboxylate (compound 108-2)

Solid NaHCO was added to a mixture of compounds 107-2 (20 g; 46.0 mmol), B-3-1 (9.2 g; 60.2 mmol; 1.3 eq.) in dioxane (540 ml) at room temperature ₃ (11.6 g; 138.1 mmol; 3 eq.), after which water (40 ml) was added. The resulting mixture was degassed by passing N2 through the surface of the solution. Bis(triphenylphosphine)palladium(II) chloride (323 mg; 0.46 mmol; 0.01 eq.) was then added and the resulting mixture was heated at 108°C for 15 hours. TLC and LCMS indicated completion of the reaction. The reaction mixture was filtered through celite while it was still hot (above 90°C) and washed with dioxane (70 ml). The filtrate was gradually cooled to room temperature, and during the cooling period

- 18 044974 small white crystals formed. The suspension was filtered and washed with dioxane (80 ml) to give the title compound 108-2 as a white solid (18 g; 78%). LCMS (m/z):

508.3 [M+1]+.

¹ H NMR (400 MHz, DMSO-d6): δ 3.28 (s, 3H), 3.76 (t, J = 4.8 Hz, 4H), 3.82 (s, 3H); 3.92 (m, 4H), 3.93 (s, 3H), 5.20 (s, 2H), 6.91 (d, J = 8.8 Hz, 1H), 7.47 (s, 1H), 8.57 (dd, J = 8, 8 Hz, 2.4 Hz, 1H), 8.88 (s, 2H), 9.15 (d, J = 2.0 Hz, 1H).

Step r: N-hydroxy-2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6yl)methyl)(methyl)amino)pyrimidin-5-carboxamide (connection 1)

Preparation of a methanol solution of hydroxylamine

A mixture of NH2OH.HCl (80 g; 1.12 mol) in MeOH (400 ml) was heated at 60-65°C for 1 hour to form a clear solution. It was then cooled in an ice water bath. A solution of KOH (96 g; 1.68 mol) in MeOH (240 ml) was added dropwise to the cold mixture, maintaining the temperature of the reaction mixture at 0-10°C. The resulting mixture was stirred at 0°C for 30 minutes and then filtered through a constant pressure funnel filled with anhydrous Na ₂ SO ₄ (700 g). The filtrate was collected under ice bath conditions and stored in a refrigerator for future use.

Obtaining Compound 1 from Compound 108-1

Compound 108-1 (10 g; 19 mmol) was suspended in the above freshly prepared methanol solution of hydroxylamine (1.79 M; 350 ml). Dichloromethane (100 ml) was added to this mixture. The reaction flask was sealed and the mixture was stirred at room temperature for 5 hours, after which it turned into a clear solution. The reaction mixture was stirred for an additional 9 hours and filtered to remove any undissolved solid. The pH of the filtrate was adjusted to 6-7 by adding acetic acid to form a solid precipitate. The solid was collected by filtration, and washed with water and a minimal amount of methanol, dried in vacuum at 60°C for 5 hours, obtaining compound 1 as a white solid (9.2 g; 96%). T.pl. 177180°C. LCMS: 509.3 [M+1] ⁺ .

1H NMR (400 MHz, DMSO-d6): δ 3.24 (s, 3H), 3.76 (t, J = 5 Hz, 4H), 3.92 (t, J = 5 Hz, 4H), 3.92 (s, 3H), 5.20 (s, 2H), 6.90 (d, J = 8.8 Hz, 1H), 7.44 (s, 1H), 8.57 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 8.75 (s, 2H ), 9.01 (s, 1H), 9.14 (d, J = 2.0 Hz, 1H), 11.08 (s, 1H).

Preparation of compound 1 from compound 108-2

To a suspension of compound 108-2 (31 g; 61.1 mmol) in dichloromethane (310 ml) was added the above freshly prepared methanol solution of hydroxylamine (1.79 M; 744 ml) at room temperature. The reaction flask was sealed and the reaction mixture was stirred at room temperature for 5 hours. The reaction mixture turned into a clear solution. The reaction solution was filtered to remove any undissolved solid. Water (310 ml) was then added to the filtrate and no solid was formed during the addition. Acetic acid (18.5 ml) was added to adjust the pH to 10.20 (with continuous monitoring on a pH meter) while stirring. No change in internal temperature was observed during the addition of acetic acid. The resulting reaction mixture was continued to stir for another 4 hours. A white solid gradually formed. The suspension was filtered and washed with a minimum amount of methanol (100 ml x 3). The collected white solid was resuspended in methanol (620 ml) and water (124 ml) to form a suspension. An additional amount of acetic acid (11 g) was added to the above suspension to adjust the pH to 5-6. A change in solid form was observed. The suspension was continued to stir for another 2 hours, filtered through filter paper and washed with a minimum amount of methanol (100 ml x 3). The collected white solid was dried in an oven (50°C) for 12 hours to obtain the title compound 1 as a white solid (23.6 g; 76.0%). Melting point: 255-259°C. LCMS (m/z): 509.3 [M+1] ⁺ .

1H NMR (400 MHz, DMSO-d ₆ ): δ 3.24 (s, 3H), 3.76 (t, J = 5.2 Hz, 4H), 3.92 (t, J = 5.2 Hz, 4H), 3.92 ( s, 3H), 5.20 (s, 2H), 6.91 (d, J = 8.4 Hz, 1H), 7.45 (s, 1H), 8.57 (dd, J = 8.4 Hz; 2.4 Hz, 1H ), 8.75 (s, 2H), 9.07 (s, 1H), 9.14 (d, J = 2.4 Hz, 1H), 11.14 (s, 1H).

Example 2. Preparation of N-hydroxy-2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidin- 5-carboxamide methanesulfonate (methanesulfonate salt of compound 1)

Method A: to a mixture of compound 1 (300 mg; 0.59 mmol) and MeOH/Et ₂ O (3/1, 40 ml) was added a solution of methanesulfonic acid (114 mg; 1.18 mmol) in MeOH (3 ml) at 0°C. The resulting mixture was stirred at 0°C for 3 hours. The precipitate was collected by filtration and washed with Et ₂ O, obtaining compound 2 as a white solid (260 mg; 73%).

Method B: To a suspension of compound 1 (1.5 g; 2.95 mmol) in dichloromethane/MeOH (40 ml/10 ml) was added methanesulfonic acid (341 mg; 3.55 mmol) in 2 ml MeOH at room temperature ( 15°C), obtaining a clear solution. The reaction mixture was stirred at room temperature overnight. The reaction mixture remained clear. Ethyl acetate (40 ml) was added to the mixture and stirring continued for 3 hours at room temperature. The resulting precipitate was collected by filtration to give compound 2 as a white solid (1.45 g; 83%).

Melting point: 179-185°C. LCMS: 509.3 [M+1]+.

^1H NMR (400 MHz, DMSO- _d6 ): δ 2.35 (s, 3H), 3.26 (s, 3H), 3.78 (t, J = 9.6 Hz, 4H), 3.95 (s, 3H), 4.03 (t, J = 9.2 Hz, 4H), 5.24 (s, 2H), 6.99 (d, J = 8.8 Hz, 1H), 7.50 (s, 1H), 8.54 (dd, J = 8.8 Hz; 2.4 Hz, 1H), 8.76 (s, 2H),

9.12 (d, J = 2.4 Hz, 1H), 11.11 (br, 1H).

Example 3. Preparation of N-hydroxy-2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidin-5- carboxamide sodium salt (sodium salt of compound 1)

To a suspension of compound 1 (300 mg; 0.59 mmol) in methanol (30 ml) at 0° C. tert-BuONa (85 mg; 0.88 mmol) was slowly added. The resulting mixture was warmed to room temperature and continued stirring for 2 hours. The reaction mixture was concentrated, the residue was triturated and washed with ethanol, then filtered to give compound 3 as a white solid (230 mg; 73%). Melting point: 178-183°C. LCMS: 509.3 [M+1] ⁺ .

1H NMR (400 MHz, DMSO-d6): δ 3.17 (s, 3H), 3.75 (s, 4H), 3.92 (s, 7H), 5.16 (s, 2H), 6.90 (d, J = 8.4 Hz , 1H), 7.42 (s, 1H), 8.57 (d, J = 8.0 Hz, 1H), 8.65 (s, 2H), 9.14 (s, 1H).

Example 4. Preparation of N-hydroxy-2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidin-5- carboxamide potassium salt (potassium salt of compound 1)

To a mixture of compound 1 (400 mg; 0.78 mmol) in methanol (50 ml) was added tert-BuOK (132 mg; 1.17 mmol) at 0° C. under N2 atmosphere. The mixture was stirred at 0°C for 1 hour and continued to stir at room temperature for 1.5 hours. Undissolved solid was removed by filtration and the filtrate was cooled to -20°C. Et ₂ O (100 ml) was added to the filtrate. The resulting mixture was stirred at -20°C for 1 hour. Hexanes (70 ml) were added and the mixture continued to stir at -20°C for 2 hours. The solid was collected by filtration and dried in vacuo to give compound 4 as a white solid (150 mg; 35%). Melting point: 174-179°C. LCMS: 509.3 [M+1] ⁺ .

1H NMR (400 MHz, DMSO- _d6 ): δ 3.16 (s, 3H), 3.74-3.76 (m, 4H), 3.90-3.93 (m, 7H), 5.15 (s, 2H), 6.90 (d, J = 8.4 Hz, 1H), 7.43 (s, 1H), 8.39 (br, 1H), 8.58 (d, J = 8.8 Hz, 1H), 8.62 (s, 2H), 9.15 (s, 1H) .

Example 5. Preparation of N-hydroxy-2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidin-5- carboxamide as choline salt (choline salt of compound 1)

To a solution of compound 1 (200 mg; 0.39 mmol) in DCM (dichloromethane)/MeOH (60 ml/12 ml) was added choline hydroxide (106 mg; 0.39 mmol; 45% solution in MeOH). The mixture was stirred at room temperature for 2 hours and then concentrated to remove approximately 30 ml of solvent. Ethyl acetate (60 ml) was added and the mixture was stirred at room temperature for 2 hours. After a small amount of precipitate had formed, the mixture was concentrated to remove approximately 40 ml of the solvent and additional ethyl acetate (60 ml) was added. The mixture was stirred at room temperature for 2 hours and filtered to give compound 5 as a white solid (180 mg; 76%). Melting point: 181-185°C. LCMS: 509.3 [M+1] ⁺ .

1H NMR (400 MHz, DMSO-d ₆ ): δ 3.11 (s, 9H), 3.17 (s, 3H), 3.40 (t, J = 4.8 Hz, 2H), 3.75 (t, J = 4.8 Hz, 4H), 3.84 (br, 2H), 3.90-3.93 (m, 7H), 5.15 (s, 2H), 6.89 (d, J = 8.8 Hz, 1H), 7.41 (s, 1H), 8.57 (dd, J = 8.8 Hz; 2.4 Hz, 1H), 8.64 (s, 2H), 9.14 (d, J = 2.0 Hz, 1H).

Example 6. Preparation of N-hydroxy-2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidin-5- carboxamide sulfate (sulfate salt of compound 1)

To a suspension of compound 1 (200 mg; 0.39 mmol) in DCM/MeOH (30 ml/7.5 ml) was added sulfuric acid (77 mg; 0.79 mmol; in 1 ml MeOH) to obtain a clear solution. The reaction mixture was stirred at room temperature overnight. A precipitate formed and tert-butyl methyl ether (60 ml) was then added. The resulting mixture was continued to stir for 1 hour at room temperature. The solid was collected by filtration to give compound 6 as a white solid (180 mg; 76%). Melting point: 243-246°C. LCMS: 509.3 [M+1] ⁺ .

1H NMR (400 MHz, DMSO-d6): δ 3.26 (s, 3H), 3.78 (t, J = 4.8 Hz, 4H), 3.96 (s, 3H), 4.03 (t, J = 4.4 Hz , 4H), 5.24 (s, 3H), 6.98 (d, J = 8.4 Hz, 1H), 7.50 (s, 1H), 8.54 (dd, J = 8.8 Hz; 2.4 Hz, 1H) , 8.76 (s, 2H), 9.12 (d, J = 2.0 Hz, 1H), 11.06 (br, 1H).

Example 7. N-hydroxy-2-(methyl((2-(6-(methylamino)pyridin-3-yl)-4-morpholinothieno[3,2d]pyrimidin-6-yl)methyl)amino)pyrimidin-5- carboxamide (compound 2)

Step 7a: (2-chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidin-6-ylmethyl)methylamine (compound 0503)

To a solution of compound 0112 (20.0 g; 70.4 mmol) in methanol (125 ml) was added a solution of methylamine in methanol (27% (v/v); 75 ml; 563.2 mmol) under nitrogen atmosphere . The reaction mixture was stirred at room temperature overnight and the solvent was removed in vacuo to give a crude solid, which was dissolved in methanol (550 ml) and THF (220 ml) under nitrogen. Portions

- 20 044974 sodium borohydride (8 g; 211.2 mmol) was added and the reaction mixture was stirred at room temperature overnight. The reaction mixture was evaporated in vacuo and water (300 ml) was added. The aqueous mixture was extracted with methylene chloride, the combined extracts were dried over Na ₂ SO ₄ and concentrated. The residue was dissolved in 6 M HCl solution (230 ml) and stirred for 30 min. The aqueous solution was washed several times with methylene chloride and the pH was adjusted to 9-10 using NaOH (4 N solution). The precipitated solid was collected by filtration and dried (60°C, 6 hours) to give a light yellow solid (18 g; 85%).

LCMS: 299 [M+1]+.

^1H NMR (400 MHz, DMSO-d6): δ 2.32 (s, 3H), 3.74 (t, J = 5.2 Hz, 4H), 3.88 (t, J = 5.2 Hz, 4H), 3.96 ( s, 2H), 7.24 (s, 1H).

Step 7b: 2-[(2-chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidin-6-ylmethyl)methylamino]pyrimidine 5-carboxylic acid ethyl ester (compound 0504)

A mixture of compound 0503 (10 g; 33.6 mmol), CH3CN (400 ml) and 0305 (6.8 g; 36.4 mmol) was stirred at room temperature. Diisopropylethylamine (DIPEA) (220 ml; 1.26 mol) was then added and the solution was stirred overnight and evaporated. After adding methylene chloride (300 ml), the organic phase was washed with water, dried over Na ₂ SO ₄ and concentrated in vacuo to leave a residue. Ethyl acetate was added to the residue and the mixture was stirred in an ice water bath for 50 minutes. The title product 0504 was collected as a white solid (10.6 g; 70%). LCMS: 449 [M+1] ⁺ ;

1H NMR (400 MHz, DMSO-d6): δ 1.30 (t, J = 7.2 Hz, 3H), 3.25 (s, 3H), 3.71 (t, J = 5.2 Hz, 4H), 3.83 (t , J = 4.8 Hz, 4H), 4.29 (m, 2H), 5.21 (s, 2H), 7.39 (s, 1H), 8.87 (s, 2H).

Step 7c: Ethyl 2-(methyl((2-(6-(methylamino)pyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6yl)methyl)amino)pyrimidine-5-carboxylate ( connection 0603-111)

N-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine mixture (0602-227) (351 mg; 1.5 mmol), compounds 0504 (314 mg, 0.7 mmol), NaHCO ₃ (176 mg, 2.1 mmol) and bis(triphenylphosphine) palladium dichloride (Pd(PPh ₃ ) ₂ Cl ₂ ; 24.6 mg; 0.035 mmol) were dissolved in mixture of toluene/EtOH/H ₂ O (2.5 ml/1.6 ml/0.7 ml). The reaction mixture was then stirred at 120°C in a microwave reactor for 2 hours. Water (8 ml) was added to this mixture and extracted with ethyl acetate (15 ml x 3). The organic layer was dried, concentrated, purified by column chromatography (methanol in dichloromethane, 5% (v/v)) to give the title compound 0603-111 (150 mg; 41%) as a white solid. LCMS: 521 [M+1] ⁺ .

1H NMR (400 MHz, DMSO-d ₆ ): δ 1.28 (t, J = 7.2 Hz, 3H), 2.81 (d, J = 4.4 Hz, 3H), 3.24 (s, 3H), 3.73 ( d, J = 4.4 Hz, 4H), 3.86 (d, J = 4.4 Hz, 4H), 4.27 (q, J = 7.2 Hz, 2H), 5.20 (s, 2H), 6.48 (d , J = 8.4 Hz, 1H), 6.91 (d, J = 4.4 Hz, 1H), 7.39 (s, 1H), 8.25 (d, J = 8.4 Hz, 1H), 8.86 (s, 2H), 8.90 (s, 1H).

Step 7d: N-hydroxy-2-(methyl((2-(6-(methylamino)pyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)amino)pyrimidin- 5-carboxamide (compound 2)

Compound 2 was prepared as a brown solid (21 mg; 14%) from compound 0603-236 (150 mg; 0.29 mmol) and freshly prepared methanolic hydroxylamine solution (6 ml) using a similar procedure to that described in Example 1: i.e. pl. 193-195°C. LCMS: 508 [M+1] ⁺ .

1H NMR (400 MHz, DMSO-d6): δ 2.83 (d, J = 4.8 Hz, 3H), 3.23 (s, 3H), 3.74 (m, 4H), 3.89 (m, 4H), 5.20 (s , 2H), 6.50 (d, J = 8.8 Hz, 1H), 6.92 (d, J = 5.2 Hz, 1H), 7.39 (s, 1H), 8.27 (dd, J = 8.8 Hz; 2.0 Hz, 1H), 8.75 (s, 2H), 9.01 (d, J = 2.0 Hz, 1H), 9.07 (br, 1H).

Example 8. 2-(((2-(4-Aminophenyl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)N-hydroxypyrimidine-5-carboxamide (compound 3)

Step 8a: N-(4-bromophenyl)acetamide (compound 0601-150)

To a solution of 4-bromoaniline (6.3 g; 63.7 mmol) in CH ₂ Cl ₂ (50 ml) was added acetyl chloride (3.75 g; 47.7 mmol) and triethylamine (TEA; 7.4 g; 73.4 mmol) at 0°C, stirred for 2 hours. The reaction mixture was washed with water, brine, dried over Na2SO4, filtered and concentrated under reduced pressure, obtaining the title compound 0601-150 (3.6 g, 46%) as brown solid. LCMS: 214 [M+1] ⁺ ;

1H NMR (400 MHz, DMSO-d ₆ ). δ 2.05 (s, 3H), 7.46 (d, J = 8.8 Hz, 2H), 7.57 (d, J = 8.8 Hz, 2H), 10.12 (s, 1H).

Step 8b: N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)acetamide (compound 0602150)

The title compound 0602-150 was obtained (2.3 g; 94%) as a white solid from compound 0601-150 (2.0 g; 9.3 mmol), bis(pinacolato)diboron (4.4 g; 17.5 mmol), potassium acetate (3.5 g; 14 mmol) and PdCl ₂ (dppf) ₂ (76 mg; 0.088 mmol), using a procedure similar to that described for compound 0602-107 (example 34). LCMS: 262 [M+1] ⁺ .

1H NMR (400 MHz, DMSO-d6) δ 1.27 (d, J = 6.8 Hz, 12H), 2.04 (s, 3H), 7.58 (s, 4H), 10.03 (s, 1H).

Step 8c: Ethyl 2-(((2-(4-aminophenyl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidine-5-carboxylate (compound 0603- 150)

A mixture of compound 0504-54 (210 mg; 0.46 mmol), compound 0602-150 (159 mg; 0.60 mmol), hydro

- 21 044974 sodium carbonate (118 mg; 1.4 mmol) and bis(triphenylphosphine) palladium(11) chloride (17 mg; 0.02 mmol) in toluene (4 ml), ethanol (2 ml) and water (1 ml ) was washed with a strong stream of nitrogen and heated under microwave irradiation at 120°C for 2 hours. The reaction mixture was partitioned between ethyl acetate and water, the organic layer was washed with brine, dried over magnesium sulfate, filtered and evaporated in vacuum. The residue was washed with dichloromethane to give ethyl 2-(((2-(4-acetamidophenyl)-4morpholothieno-[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidine-5-carboxylate (136 mg ; 53%) as a white solid. LCMS: 548 [M+1]+.

1H NMR (400 MHz, DMSO-d6): δ 1.29 (t, J = 7.2 Hz, 3H), 2.06 (s, 6H), 3.26 (s, 3H), 3.75 (m, 4H), 3.91 (m , 4H), 4.28 (q, J = 7.2 Hz, 2H), 5.22 (s, 2H), 7.45 (s, 1H), 7.67 (d, J = 8.8 Hz, 1H), 8.31 (d, J = 8.8 Hz, 1H), 8.87 (s, 1H), 10.10 (s, 1H).

To a solution of the above ethyl ester (280 mg, 0.51 mmol) in THF (10 ml) was added an aqueous solution of HCl (6 M, 15 ml) at 40°C, stirred for 2 hours, the reaction mixture was neutralized using NaHCO ₃ . and extracted with CH ₂ Cl ₂ , the organic layer was washed with water, brine, dried over Na ₂ SO ₄ , filtered and concentrated, purified by column chromatography (methanol in dichloromethane, 2% (v/v)) to give the title compound 0603-150 (180 mg; 48%) as a white solid. LCMS: 506 [M+1] ⁺ .

1H NMR (400 MHz, DMSO-d ₆ ) δ 1.29 (t, J = 7.6 Hz, 3H), 3.24 (s, 3H), 3.73 (m, 4H), 3.86 (m, 4H), 4.27 (q , J = 6.8 Hz, 2H), 5.20 (s, 2H), 6.59 (d, J = 8.8 Hz, 2H), 7.36 (s, 1H), 8.07 (d, J = 8.0 Hz, 2H), 8.86 (s, 1H).

Step 8d: 2-(((2-(4-aminophenyl)-4-morpholothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)N-hydroxypyrimidine-5-carboxamide (compound 3)

Compound 3 (43 mg; 26%) was prepared as a yellow solid from compound 0603-150 (170 mg; 0.3 mmol) and freshly prepared methanolic hydroxylamine solution (4 ml) using a procedure similar to that described in Example 1. T.pl. 183-186°C. LCMS: 493 [M+1] ⁺ ;

1H NMR (400 MHz, DMSO-d6) δ 3.22 (s, 3H), 3.74 (m, 4H), 3.87 (m, 4H), 4.27 (q, J = 6.8 Hz, 2H), 5.20 (s, 2H), 5.50 (s, 2H), 6.59 (d, J = 8.8 Hz, 2H), 7.36 (s, 1H), 8.07 (d, J = 8.0 Hz, 2H), 8.86 (s, 2H ).

Example 9 PI3-kinase activity assay

The following assays were used to determine the ability of Compound 1 to inhibit various isoforms and mutant forms of PI3K.

PI3Ka

PI3Ka activity was measured using the ADP-Glo luminescent kinase assay. PI3Ka, a complex of recombinant full-length human p110a subunit N-terminally tagged with glutathione S-transferase (GST) and untagged recombinant full-length human p85a subunit, was coexpressed in baculovirus-infected Sf9 cells using the baculovirus expression system (GenBank accession no. for p110a , U79143; for р85а, ХМ_043865). Proteins were purified by affinity chromatography in one step using glutathione agarose. A competition assay was performed by measuring the amount of adenosine diphosphate (ADP) generated from adenosine triphosphate (ATP) in the presence of purified recombinant PI3Ka (p110a/p85a) and phosphatidylinositol biphosphate (PIP2). PI3Ka was incubated with 20 μM PIP2 substrate in reaction buffer (50 mM HEPES (2-hydroxyethyl-piperazine-2-ethanesulfonic acid), pH 7.4; 150 mM NaCl, 5 mM MgCl ₂ , 3 μM Na orthovanadate, 1 mM dithiothreitol ( DTT), 10 µM ultrapure ATP and 0.5% DMSO) for 30 min at 30°C. The ADP produced in this reaction was then measured using the ADP-Glo assay. This analysis was carried out in two stages; an equal volume of ADP-GLO™ reagent (Promega) was first added to stop the kinase reaction and consume the remaining ATP. In the second step, a kinase detection reagent was added, which simultaneously promotes the conversion of ADP to ATP. Newly synthesized ATP was measured using a coupled luciferin/luciferase reaction. The IC ₅₀ value (concentration causing 50% inhibition) determined for compound 1 in this assay was less than 100 nM.

In addition, the ability of compound 1 to inhibit mutant forms of PI3Ka with the H1047R and E545K mutations was determined using the general procedure described above. The IC ₅₀ value determined for both mutant forms was less than 100 nM.

ΡΙ3Κβ

PI3KP activity was measured using time-resolved fluorescence resonance energy transfer (TR-FRET) assay using homogeneous time-resolved fluorescence (HTRF) technology. PI3KP, a complex of the N-terminally histidine-tagged recombinant full-length human p110b subunit and the unlabeled recombinant full-length human p85a subunit, was coexpressed in baculovirus-infected Sf21 cells using the baculovirus expression system (GenBank accession no. for p110b, NM_006219; for p8 5a, ХМ_043865). Proteins are purified by affinity chromatography in a single step using glutathione agarose. A competition assay was performed by measuring the amount of phosphatidylinositol triphosphate (PIP3) produced from PIP2 in the presence of purified recombinant PI3K-beta

- 22 044974 (ρ110β/ρ85α). ΡΙ3Κβ was incubated with 10 μM PIP2 substrate in reaction buffer (20 mM HEPES, pH 7.5; 10 mM NaCl, 4 mM MgCl ₂ , 2 mM DTT, 10 μM ATP, and 1% DMSO) for 30 min at 30°C. The reaction product was then mixed with a PIP3 detection protein, namely a europium-labeled antibody, a biotin-labeled PIP3 probe, and allophycocyanin-labeled streptavidin. A sensor complex is formed that generates a stable TR-FRET signal in the reaction mixture. The intensity of this signal decreases as binding of the biotin-labeled probe to the PIP3 detector is replaced by the formation of PIP3 by enzymatic activity, and the amount of unbound biotin-labeled PIP3 probe in the mixture increases. The TR-FRET signal was determined using a background subtraction microplate reader.

The IC ₅₀ value determined for compound 1 in this assay ranged from 100 to 1000 nM.

ΡΙ3Κδ

PI3K5 activity was measured using a fluorescence polarization assay. PI3K5, a complex of the N-terminally histidine-tagged recombinant full-length human p1105 subunit and the untagged recombinant full-length human p85a subunit was coexpressed in baculovirus-infected Sf9 cells using the baculovirus expression system (GenBank accession no. for p1105, NM_005026). Proteins are purified by affinity chromatography in a single step using glutathione agarose. A competition assay was performed by measuring the amount of PIP3 produced from PIP2 in the presence of purified recombinant PI3K5 (p1105/p85a). PI3K5 was incubated with 10 μM PIP2 substrate in reaction buffer (20 mM HEPES (pH 7.5), 10 mM NaCl, 4 mM MgCl2, 2 mM DTT, 10 μM ATP, and 1% DMSO) for 1 h at 30°C. The reaction product was then mixed with PIP3 detection protein and a fluorescent probe for PIP3. Polarization values (in mP) decrease as fluorescent probe binding to the PIP3 detector is replaced by PIP3 production by enzymatic activity, and the amount of unbound fluorescent probe in the mixture increases. The degree of polarization values (in mP) were determined using a microplate reader with background subtraction.

The IC ₅₀ value determined for compound 1 in this assay was less than 100 nM.

ΡΙ3Κγ

PI3Ky activity was measured using time-resolved fluorescence resonance energy transfer (TR-FRET) assay using homogeneous time-resolved fluorescence (HTRF) technology. N-terminally histidine-tagged human PI3Ky was expressed in baculovirus-infected Sf9 cells using the baculovirus expression system (GenBank accession no. AF327656). Proteins are purified by affinity chromatography in a single step using glutathione agarose. A competition assay was performed by measuring the amount of PIP3 produced from PIP2 in the presence of purified recombinant PI3Ky φ120γ). PI3Ky (2 nM) was incubated with 10 μM PIP2 substrate in reaction buffer (20 mM HEPES, pH 7.5; 10 mM NaCl, 4 mM MgCl ₂ , 2 mM DTT, 10 μM ATP and 1% DMSO) for 30 min at 30°C. The reaction product was then mixed with a PIP3 detection protein, namely a europium-labeled antibody, a biotin-labeled PIP3 probe, and allophycocyanin-labeled streptavidin. A sensor complex is formed that generates a stable TR-FRET signal in the reaction mixture. The intensity of this signal decreases as binding of the biotin-labeled probe to the PIP3 detector is replaced by the formation of PIP3 by enzymatic activity, and the amount of unbound biotin-labeled PIP3 probe in the mixture increases. The TR-FRET signal was determined using a background subtraction microplate reader.

The IC ₅₀ value determined for compound 1 in this assay ranged from 100 to 1000 nM.

Example 10 HDAC Activity Assay

Inhibition of HDAC activity was assessed using the Biomol Color de Lys system (AK-500, Biomol, Plymouth Meeting, PA). Briefly, HeLa cell nuclear extracts were used as a source of HDACs. Serial dilutions of test compounds were prepared in dimethyl sulfoxide (DMSO) to obtain different concentrations and added to HeLa cell nuclear extracts in the presence of an artificial substrate for colorimetric detection. The final assay conditions were 50 mM Tris/Cl, pH 8.0; 137 mM NaCl, 2.7 mM KCl and 1 mM MgCl ₂ . The reactions were carried out at room temperature (25°C) for 1 hour, after which a developing agent was added to stop the reaction.

Relative enzyme activity was measured in a WALLAC Victor II 1420 microplate reader as fluorescence intensity (excitation: 350–380 nm; emission: 440–460 nm). Data were analyzed using GraphPad Prism software (version 4.0a) with sigmoidal dose response curve fitting to calculate _IC50 . The IC ₅₀ value determined for compound 1 in this assay was less than 100 nM.

The activities of compound 1 against HDAC isotypes were also determined. HDAC specificity assays were performed at BPS Bioscience (San Diego, CA) following their standard operating procedures. Briefly, purified enzymes tagged with flag (in the case of human HDAC-1), NCOR2 (nuclear receptor corepressor 2) (in the case of human HDAC-3), GST (in the case of HDAC-4, -6, -7, -10 and -11 human) or His (in the case of human HDAC-2, -5, -8 and -9), were expressed in Sf9 insect cells and purified before use. As a substrate for HDAC-1,

2, 3, 6, 7, 8, 9 and 11 used HDAC substrate 3 developed at BPS Bioscience. For other HDAC enzymes, a class 2a HDAC substrate was used. All enzymatic reactions were performed in duplicate at 37°C for 30 minutes with the exception of the HDAC-11 enzyme assay, which was performed at room temperature for 3 hours.

The table below shows the results for each of HDACs 1-11, with 1C50 values shown as follows: I greater than 1000 nM; 100 nM less than 11 less than 1000 nM; 10 nM less than 111 less than 100 nM; IV is less than 10 nM.

N DAC 1 2 3 8 4 5 6 7 9 10 eleven IC50 IV IV IV II II II 111 II II IV IV

Example 11: Study of Compound 1 and Anti-PD-1 Antibody in a CTW26.WT Mouse Colon Carcinoma Cell Xenograft Mouse Model

Animals: Female Balb/c mice, 8 weeks old, fed a high-fat diet.

Connections

Compound 1: administered orally (PO) at a dose of 50 mg/kg according to the scheme: 5 days after administration followed by 2 days of rest (5+2-).

Isotype control: purified 1gG2a LEAFTM, obtained from BioLegend™, San Diego, CA; administered intraperitoneally (i.p.) at a dose of 100 mcg/mouse, once a week (2x/week).

Anti-PD-1 antibody: rat anti-mouse CD279 (PD-1) LEAFTM, clone 29F.1AI2, obtained from BioLegendTM, San Diego, CA; administered i.p. at a dose of 100 mcg/mouse, 2x/week.

Cell injection

CT26.WT cells were collected from flasks and washed once in Roswell Park Memorial Institute (RPMI) 1640 medium, which does not contain any additives. Solutions were prepared at final concentrations using unsupplemented culture medium (RPMI-1640) and stored on ice until administered to mice. On day 7 or day 8, as soon as tumors were palpable, mice were assigned to groups and randomized according to body weight. Treatment was started with the antibody and Compound 1 was immediately administered.

Group distribution

Group Qty Detailed introduction 1 8 Thinner (thinner for compound 1 - 30% CAPTISOL™) 2 8 Compound 1, 50 mg/kg (5+2-) 3 8 Negative isotype control, 100 μg/mouse (one once a day (1x/day), 2x a week) 4 8 Antibody to PD-1 (1A12) 100 mcg/mouse (1x/day, 2x/week) 5 8 Compound 1, 50 mg/kg (5+2-) Negative isotype control, 100 µg/mouse (1x/day, 2x/week) 6 8 Compound 1, 50 mg/kg (5+2-) Antibody to PD-1 (1A12) 100 mcg/mouse (1x/day, 2x/week)

Study plan

Animals were monitored prior to dosing and then measurements were taken twice weekly during dosing until tumor volume reached the protocol limit or until ulceration became too severe. At the end of the study, tumor tissue and blood/plasma were collected.

results

The results of this study are presented in the table below and Fig. 1. There were 13 groups of 7 mice that were subject to evaluation. In groups 4-6, all 8 mice were assessed. When compound 1 was used alone at a dose of 50 mg/kg, very little inhibition of tumor growth was demonstrated. Anti-PD-1 antibody showed stronger tumor growth inhibition, but still below 50%. However, when using a combination of compound 1 and an anti-PD-1 antibody, almost complete inhibition of tumor growth was shown.

- 24 044974

Medicine Dosage TGI (tumor growth inhibition), %* (day 15) p-value* (Student's t-test) Number of mice (15th day) Thinner (for connection 1) - pa (data not shown) pa 7/8 Isotype control mAb (monoclonal antibody) 100 mcg pa pa 7/8 Conn. 1 50 mg/kg 3 ns (not significant) 7/8 Conn. 1 + isotype control mAb 50 mg/kg + 100 mcg 16 ns 8/8 mAb to PD-1 100 mcg 44 less than 0.05 8/8 Conn. 1 + tab k PD-1 50 mg/kg + 100 mcg 97 less 0.0001 8/8 * Relative to the group receiving the diluent (for compound 1)

Example 12 Study of Compound 1 and Anti-PD-1 Antibody in the A20 Xenograft Mouse Model

Animals: Female Balb/c mice, 4-5 weeks old, regularly fed.

Connections

Compound 1: injected p.o. at a dose of 50 mg/kg or 100 mg/kg according to the scheme: after 5 days of administration, followed by 2 days of rest (5 doses/2 rest).

Isotype control: LEAF™ purified rat iG2a obtained from BioLegend™, San Diego, CA; administered i.p. at a dose of 100 mcg/mouse, 2x/week, 3 weeks.

Anti-PD-1 antibody: rat anti-mouse CD279 (PD-1) LEAF™, clone 29F.1A12, obtained from BioLegend™, San Diego, CA; administered at a dose of 100 mcg/mouse, 2x/week, 3 weeks.

Thinner: Compound 1 Thinner - 30% Captisol™; administered according to the scheme 5 intake/2 rest, p.o., 3 weeks.

Cell injection

Syngeneic A20 cells were collected from flasks and washed once in RPMI 1640 medium containing no additives. Solutions were prepared at final concentrations using unsupplemented culture medium (RPMI-1640) and stored on ice until administered to mice. Animals were implanted with ^2x105 cells in the right flank. Administration began when tumors exceeded 100 mm ³ in size (approximately day 13) and continued for 21 days or until tumors reached a size limit of 2000 mm ³ .

Group distribution

Group Qty Detailed introduction 1 8 Diluent Isotype control 2 8 Antibody to PD-1 3 8 Compound 1, 100 mg/kg 4 8 Compound 1, 100 mg/kg Antibody to PD-1

Study plan

Dosing of animals began around day 13, when tumors were fully palpable and measurable. Dosing was continued until the tumors became ulcerated requiring killing. Tumors were selected before extensive necrosis was achieved for enzyme-linked immunosorbent assay (ELISA) and flow analysis.

results

The results of this study are presented in the table below and Fig. 2. Significant tumor growth inhibition was observed when both compound 1 at a dose of 100 mg/kg and anti-PD-1 antibody alone were used. When using the combination of compound 1 and an anti-PD-1 antibody, greater inhibition of tumor growth was observed than with any anti-PD-1 agent.

-

Claims (7)

separately. Medicine Dosage TGI, %* (13th day) p-value* (Student's t-test) Number of mice (13th day) Diluent (for connection 1) + isotype control pa pa 8/8 mAb to PD-1 100 mcg 49 ns 8/8 Conn. 1 100 mg/kg 60 <0.05 8/8 Conn. 1 + tab k PD-1 100 mg/kg + 100 mcg 75 <0.005 8/8 * Relative to the group receiving the diluent (for compound 1) + isotype control The patent and scientific literature referred to herein constitute the basis of the knowledge available to those skilled in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, documents, monographs and scientific literature cited herein are hereby incorporated by reference. While the present invention has been specifically shown and described with reference to its preferred embodiments, those skilled in the art will appreciate that, without departing from the scope of the present invention, various changes in form and detail may be made as contemplated by the appended claims. inventions. CLAIM 1. A method of treating colon carcinoma in a subject in need thereof, comprising administering to the subject: (a) compounds of formula or a pharmaceutically acceptable salt thereof and (b) an inhibitor of programmed cell death protein 1 (PD1) signaling, wherein the compound or a pharmaceutically acceptable salt thereof and an inhibitor of PD-1-mediated signaling are administered in amounts that in combination are therapeutically effective 2. The method of claim 1, wherein the inhibitor of PD-1-mediated signaling is an inhibitor of one or more of PD-1, PD-L1 and PD-L2. 3. The method of claim 2, wherein the inhibitor of PD-1-mediated signaling inhibits PD-1 or PD-L1. 4. The method of claim 2, wherein the PD-1-mediated signaling inhibitor is pembrolizumab, nivolumab, atelolizumab, avelumab, durvalumab or pidilizumab. 5. The method of claim 4, wherein the inhibitor of PD-1-mediated signaling is pembrolizumab or nivolumab. 6. Method according to any one of claims 1-5, where: (a) a compound of Formula I, or a pharmaceutically acceptable salt thereof, and a PD-1-mediated signaling inhibitor are administered to a subject simultaneously as separate compositions; or (b) a compound of Formula I, or a pharmaceutically acceptable salt thereof, and a PD-1-mediated signaling inhibitor are administered to a subject sequentially as separate compositions. compositions. 7. The method according to any one of claims 1 to 5, wherein the compound of formula I or a pharmaceutically acceptable salt thereof and an inhibitor of PD-1-mediated signaling are administered in a single composition. -