Combined therapy using a phosphoinositide 3-kinase inhibitor having a zinc-binding moiety

Abstract

The present invention provides a method for treating cancer in a subject in need thereof, the method comprising administering to the subject: (a) a compound of formula I or a pharmaceutically acceptable salt thereof: wherein R is hydrogen or acyl; and (b) a PD-1 signal transduction inhibitor; wherein the compound of formula I or a pharmaceutically acceptable salt thereof and the PD-1 signal transduction inhibitor are administered in a therapeutically effective combined amount. The present invention also provides a pharmaceutical composition, which comprises a compound of formula I or a pharmaceutically acceptable salt thereof, a PD-1 signal transduction inhibitor, and a pharmaceutically acceptable carrier or excipient.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 729,648, filed Sep. 11, 2018. The entire teachings of the above application are incorporated herein by reference. Background of the Invention

[0003] Treatment regimens for treating cancer generally involve combination therapy using more than two agents. In particular, targeted therapies can be combined to more effectively treat various types of cancer and inhibit the development of cancer cells resistant to treatment. In the field of cancer drug development, there is a need for particularly effective drug combinations for treating specific types of cancer. Summary of the Invention

[0004] The present invention relates to combination therapy for treating cancer using a compound of formula I or a pharmaceutically acceptable salt thereof and a PD-1 signal transduction inhibitor:

[0005]

[0006] wherein R is hydrogen or acyl. The acyl is preferably R1C(O)-, where R1 is a substituted or unsubstituted C1-C 24 alkyl, preferably C1-C 10 alkyl, more preferably C1-C6 alkyl; a substituted or unsubstituted C2-C 24 alkenyl, preferably C2-C 10 alkenyl, more preferably C2-C6 alkenyl; a substituted or unsubstituted C2-C 24 alkynyl, preferably C2-C 10 alkynyl, more preferably C2-C6 alkynyl; a substituted or unsubstituted aryl, preferably a substituted or unsubstituted phenyl; or a substituted or unsubstituted heteroaryl, and A is an optionally substituted phenyl, an optionally substituted pyridyl or an optionally substituted pyrimidinyl. For example, in one embodiment, the present invention provides a method of preventing or treating cancer in a subject in need thereof. The method comprises administering to the subject a compound of formula I or a pharmaceutically acceptable salt thereof and a PD-1 signal transduction inhibitor, wherein the compound of formula I and the PD-1 signal transduction inhibitor are administered in a therapeutically effective combined amount. Preferably, the compound of formula I or its salt and the PD-1 signal transduction inhibitor are administered to the subject in a synergistic amount.

[0007] The present invention also relates to a pharmaceutical composition comprising: a compound of formula I or a pharmaceutically acceptable salt thereof, a PD-1 signal transduction inhibitor, and a pharmaceutically acceptable excipient or carrier.

[0008] In this text, the compound of formula I (especially the compound of formula I in which R is hydrogen and A is 2-methoxy-5-pyridyl) is also referred to as Compound 1, which has beneficial properties as a therapeutic agent, such as for treating cancers and other diseases and disorders related to PI3 kinase activity and / or HDAC activity. For example, Compound 1 has effective inhibitory activity against the molecular targets PI3K and HDAC in vitro and effective anti-proliferative activity against a variety of cancer cells. As observed in animal models, Compound 1 has significant oral bioavailability. After oral administration or intravenous administration in mice bearing xenograft tumors, the compound shows significant uptake by tumor tissues and pharmacodynamic activity in tumor tissues. After oral or intravenous administration, Compound 1 also shows significant anti-tumor activity in a mouse xenograft tumor model. For example, as shown by genotoxicity tests using the Ames test, the compound also has good safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] As shown in the drawings, the foregoing and other objects, features and advantages of the present invention will become apparent from the following more specific description of the preferred embodiments of the present invention.

[0010] Figure 1 is a graph showing the relationship between tumor volume and time in a mouse CT26.WT xenograft model as described in Example 11.

[0011] Figure 2 is a graph showing the relationship between tumor volume and time in a mouse A20 xenograft model as described in Example 12. DETAILED DESCRIPTION

[0012] The present invention relates to methods and compositions for the combined treatment of cancer, the compositions comprising a compound of formula I or a pharmaceutically acceptable salt thereof and a PD-1 signal transduction inhibitor. In a preferred embodiment of the compound of formula I, A is a phenyl, pyridyl or pyrimidinyl group substituted with methoxy, amino or N-methylamino. More preferably, A is one of the following groups:

[0013]

[0014] In certain embodiments of the compound of formula I, A is one of the above groups and R is hydrogen.

[0015] In a preferred embodiment, the compound of formula I is selected from the following Compound 1, Compound 2 and Compound 3 and their pharmaceutically acceptable salts:

[0016]

[0017] The present invention provides a method for preventing or treating cancer in a subject in need thereof. The method comprises the step of administering to the subject a compound of formula I or a pharmaceutically acceptable salt thereof and a PD-1 signal transduction inhibitor, wherein the compound of formula I or a pharmaceutically acceptable salt thereof and the PD-1 signal transduction inhibitor are administered in a therapeutically effective combined amount.

[0018] The PD-1 signal transduction inhibitor can be any compound that inhibits PD-1 signal transduction. For example, the PD-1 signal transduction inhibitor can inhibit PD-1 and / or an activating ligand of PD-1 (such as PD-L1 or PD-L2). The PD-1 signal transduction inhibitor can be a small molecule, polynucleotide or protein, such as an antibody. Preferably, the PD-1 signal transduction inhibitor is a monoclonal antibody, more preferably a humanized monoclonal antibody or a fully human monoclonal antibody. In one embodiment, the PD-1 signal transduction inhibitor is an anti-PD-1 monoclonal antibody. In another embodiment, the PD-1 signal transduction inhibitor is an anti-PD-L1 monoclonal antibody. In another embodiment, the PD-1 signal transduction inhibitor is an anti-PD-L2 monoclonal antibody. Suitable PD-1 signal transduction inhibitors include, but are not limited to, those described in US 8,008,449, WO 2006 / 121168, WO 2009 / 114335, US 8,354,509, US 8,609,089, US 2010 / 0028330, US 2012 / 0114649, WO 2007 / 005874, WO2010 / 077634, US7,943,743, US 2012 / 0039906 and WO / 2011 / 066342, the entire contents of which are incorporated herein by reference. Examples of suitable PD-1 signal transduction inhibitors include YW243.55.S70, MPDL3280A, MEDI-4736, MSB-0010718C, MDX-1105 and AMP-224. Preferred PD-1 signal transduction inhibitors include: pembrolizumab (KEYTRUDA TM ), nivolumab (OPDIVO TM ), atezolizumab (TECENTRIQ TM ), avelumab (BAVENCIO TM ), durvalumab (IMFINZI TM ) and pidilizumab.

[0019] In a particularly preferred embodiment of the methods and compositions of the present invention, the compound of formula I is compound 1.

[0020] In a particularly preferred embodiment of the present invention, the PD-1 signal transduction inhibitor is pembrolizumab or nivolumab.

[0021] In certain embodiments of the method of the present invention, the compound of formula I or a pharmaceutically acceptable salt thereof and the PD-1 signal transduction inhibitor are administered to a subject simultaneously as separate components. In certain embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof and the PD-1 signal transduction inhibitor are administered to the subject simultaneously by the same or different routes of administration.

[0022] In certain embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof and the PD-1 signal transduction inhibitor are administered to a subject sequentially as separate components. In certain embodiments, the compound of formula I and the PD-1 signal transduction inhibitor are administered to the subject sequentially by the same or different routes of administration. In one embodiment, the PD-1 signal transduction inhibitor is administered to the subject after the compound of formula I or a pharmaceutically acceptable salt thereof has been administered to the subject. In another embodiment, the PD-1 signal transduction inhibitor is administered to the subject before the compound of formula I or a pharmaceutically acceptable salt thereof has been administered to the subject.

[0023] In certain embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof and the PD-1 signal transduction inhibitor are administered as separate components, and each composition is independently administered transmucosally, orally, rectally, vaginally, sublingually, intravenously, intramuscularly, subcutaneously, bucally, intranasally, intracisternally, intraperitoneally or by ear. In certain embodiments, one or both compositions are administered as a suppository or a hydrogel. In a preferred embodiment, both compositions are administered orally.

[0024] In certain embodiments where the compound of formula I or a pharmaceutically acceptable salt thereof and the PD-1 signal transduction inhibitor are administered as separate components, their timing of administration is such that the pharmacological activities of the agents overlap in time to achieve a combined therapeutic effect. For example, the compound of formula I or a pharmaceutically acceptable salt thereof and the PD-1 signal transduction inhibitor may be administered sequentially at time intervals of more than about 60 minutes. For example, the time between the sequential administration of the compound of formula I or a pharmaceutically acceptable salt thereof and the PD-1 signal transduction inhibitor may be more than 60 minutes, more than 2 hours, more than 5 hours, more than 10 hours, more than 1 day, more than 2 days, more than 3 days or more than 1 week. The optimal timing of administration will depend on the rates of absorption, distribution, metabolism and / or excretion of the compound of formula I or a pharmaceutically acceptable salt thereof and the PD-1 signal transduction inhibitor.

[0025] The compound of formula I or a pharmaceutically acceptable salt thereof may be administered first, or a PD-1 signal transduction inhibitor may be administered first. For example, a PD-1 signal transduction inhibitor may be administered to a subject after the time of administration of the compound of formula I or a pharmaceutically acceptable salt thereof. In such a case, it is desirable to administer the PD-1 signal transduction inhibitor before the time at which about 50% (e.g., about 40%, about 30%, about 20%, about 10%, about 5%) of the compound of formula I has been metabolized or excreted by the subject. In another example, a first dose of the compound of formula I or a pharmaceutically acceptable salt thereof is administered to a subject, then a single dose of a PD-1 signal transduction inhibitor is administered, and then another dose of the compound of formula I or a pharmaceutically acceptable salt thereof is administered.

[0026] In certain embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof and a PD-1 signal transduction inhibitor are administered in a single dosage form, which is administered transmucosally, orally, rectally, vaginally, sublingually, intravenously, intramuscularly, subcutaneously, buccally, intranasally, intrathecally, intraperitoneally or by ear. Preferably, the single dosage form is administered orally.

[0027] The compound of formula I may be administered about once a week, about once a day or more than once a day. In one embodiment, the compound of formula I or a pharmaceutically acceptable salt thereof is administered orally. In another embodiment, the compound of formula I or a pharmaceutically acceptable salt thereof is administered parenterally (e.g., intravenously). The compound of formula I or a pharmaceutically acceptable salt thereof may be administered in a daily dose of about 1 mg to about 1,500 mg. For example, the compound of formula I or a pharmaceutically acceptable salt thereof may be administered in a daily dose of about 200 mg. In one embodiment, the compound of formula I or a pharmaceutically acceptable salt thereof is administered in a dose of about 1 mg / kg body weight to about 250 mg / kg body weight.

[0028] However, it should be understood that the dosage frequency and total daily dose of the compound of formula I or a pharmaceutically acceptable salt thereof and a PD-1 signal transduction inhibitor for an individual patient can be determined by a person skilled in the art (e.g., the attending physician within the scope of reasonable medical judgment). The specific dose for any particular patient will depend on a variety of factors, including: the disorder being treated and the severity of the disorder; the activity of the particular compound used; the specific composition used; the age, weight, general health, sex and diet of the patient; the time of administration, route of administration and rate of excretion of the particular compound used; the duration of the treatment; drugs used in combination with or concurrently with the particular compound used; and similar factors well known in the medical arts.

[0029] The term "cancer" refers to any cancer caused by the proliferation of neoplastic cells, such as tumors, neoplasms, malignant epithelial tumors, sarcomas, leukemias, lymphomas, etc. For example, cancers include but are not limited to mesothelioma, leukemia, and lymphoma, such as cutaneous T-cell lymphoma (CTCL), non-cutaneous peripheral T-cell lymphoma, lymphoma associated with human T-cell leukemia virus (HTLV) (such as adult T-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, lymphoma, and multiple myeloma, non-Hodgkin lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), Hodgkin lymphoma, Burkitt lymphoma, adult T-cell leukemia lymphoma, acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), or hepatocellular carcinoma. Other examples include: myelodysplastic syndromes; childhood solid tumors, such as brain tumors, neuroblastoma, retinoblastoma, Wilms' tumor, bone tumors, and soft tissue sarcomas; common adult solid tumors, such as head and neck cancers (such as oral cancer, laryngeal cancer, nasopharyngeal cancer, and esophageal cancer), genitourinary cancers (such as prostate cancer, bladder cancer, kidney cancer, uterine cancer, ovarian cancer, testicular cancer), lung cancers (such as small cell cancer and non-small cell cancer), breast cancer, pancreatic cancer, melanoma, and other skin cancers, gastric cancer, brain tumors, tumors associated with Gorlin's syndrome (such as medulloblastoma, meningioma, etc.), and liver cancer. Other exemplary forms of cancer treatable with the compounds of the present invention include but are not limited to: bone cancer or smooth muscle cancer, gastric cancer, small intestine cancer, rectal malignancy, salivary gland cancer, endometrial cancer, adrenal cancer, anal cancer, rectal cancer, parathyroid cancer, and pituitary cancer.

[0030] For example, other cancers for which the compounds described herein can be used for prevention, treatment, and research are colon cancer, familial adenomatous polyposis malignancies, and hereditary non-polyposis 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 cancer, adenocarcinoma, thyroid cancer (medullary thyroid cancer and papillary thyroid cancer), kidney cancer, renal parenchymal cancer, cervical cancer, corpus cancer, endometrial cancer, choriocarcinoma, testicular cancer, urogenital organ cancer, melanoma, brain tumors (such as glioblastoma), astrocytoma, meningioma, medulloblastoma, and peripheral neuroectodermal tumors, gallbladder cancer, bronchial cancer, multiple myeloma, basal cell carcinoma (basalioma), teratoma, retinoblastoma, choroidal melanoma, seminoma, rhabdomyosarcoma, craniopharyngioma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, Ewing sarcoma, and plasmacytoma. In one aspect of the present invention, the present invention provides the use of one or more compounds of the present invention in the preparation of a medicament for treating cancer.

[0031] In one embodiment, the cancer to be treated is a hematological cancer. Hematological cancers include leukemia, lymphoma, and multiple myeloma. Examples include lymphocytic leukemia, such as acute lymphocytic leukemia, including precursor B acute lymphoblastic leukemia, precursor T acute lymphoblastic leukemia, Burkitt leukemia, and acute biphenotypic leukemia; chronic lymphocytic leukemia, including B-cell prolymphocytic leukemia; and myeloid leukemia, such as acute myeloid leukemia, including acute promyelocytic leukemia, acute granulocytic leukemia, and acute megakaryocytic leukemia; chronic myeloid leukemia, including chronic monocytic leukemia; acute monocytic leukemia. Other leukemias include: hairy cell leukemia; T-cell prolymphocytic leukemia; large granular lymphocytic leukemia; and adult T-cell leukemia.

[0032] Lymphomas include Hodgkin lymphoma and non-Hodgkin lymphoma, including B-cell lymphoma, T-cell lymphoma, NK-cell lymphoma, and precursor lymphoid neoplasms. B-cell lymphomas include Burkitt lymphoma / leukemia, diffuse large B-cell lymphoma, B-cell chronic lymphocytic leukemia / small cell lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma (such as Waldenström macroglobulinemia), splenic marginal zone lymphoma, hairy cell leukemia, plasmacytic neoplasms, plasma cell myeloma (also known as multiple myeloma), plasmacytoma, monoclonal immunoglobulin deposition disease, heavy chain disease, extranodal marginal zone B-cell lymphoma (also known as MALT lymphoma), nodal marginal zone B-cell lymphoma, follicular lymphoma, primary cutaneous follicle center lymphoma, mantle cell lymphoma, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK+ large B-cell lymphoma, fibroblastic lymphoma, primary effusion lymphoma, large B-cell lymphoma arising in HHV8-related multicentric Castleman disease, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK+ large B-cell lymphoma, fibroblastic lymphoma, primary effusion lymphoma, and large B-cell lymphoma arising in HHV8-related multicentric Castleman disease.

[0033] T-cell and NK-cell lymphomas include cutaneous T-cell, T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, aggressive NK-cell leukemia, adult T-cell leukemia / lymphoma, nasal-type extranodal NK / T-cell lymphoma, enteropathy-associated T-cell lymphoma, hepatosplenic T-cell lymphoma, blastic NK-cell lymphoma, mycosis fungoides / Sezary syndrome, primary cutaneous CD30-positive T-cell lymphoproliferative diseases such as primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, peripheral T-cell lymphoma, and unless otherwise specified, angioimmunoblastic T-cell lymphoma and anaplastic large cell lymphoma.

[0034] In a preferred embodiment, the cancer to be treated is non-Hodgkin lymphoma, more preferably B-cell lymphoma. In a particularly preferred embodiment, the cancer to be treated is diffuse large B-cell lymphoma (DLBCL), such as ABC subtype DLBCL, GCB subtype DLBCL, double hit DLBCL, or double expresser DLBCL (Quintanilla-Martinez, L., Hematol. Oncol. 2015, 33: 50-55). In certain embodiments, the cancer is relapsed or refractory DLBCL.

[0035] In one embodiment, the present invention provides the use of a compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cancer in combination with a PD-1 signal transduction inhibitor. In another embodiment, the present invention provides the use of a compound of formula I or a pharmaceutically acceptable salt thereof and a PD-1 signal transduction inhibitor in the preparation of a medicament for treating cancer. In a preferred embodiment, the compound of formula I is compound 1 or a pharmaceutically acceptable salt thereof, and the PD-1 signal transduction inhibitor is pembrolizumab or nivolumab.

[0036] The present invention also relates to a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof and a PD-1 signal transduction inhibitor. In one embodiment, the compound of formula I is compound 1, compound 2 or compound 3, and the PD-1 signal transduction inhibitor is pembrolizumab or nivolumab.

[0037] The compound of formula I or a pharmaceutically acceptable salt thereof and the PD-1 signal transduction inhibitor can be administered by any suitable means, including but not limited to parenteral, intravenous, intramuscular, subcutaneous, implant, oral, sublingual, buccal, nasal, pulmonary, transdermal, topical, vaginal, rectal and transmucosal administration, etc. Topical administration may also involve the use of transdermal administration, such as transdermal patches or iontophoresis devices. Pharmaceutical formulations include solid, semi-solid or liquid preparations (tablets, pills, lozenges, capsules, suppositories, creams, ointments, aerosols, powders, liquids, emulsions, suspensions, syrups, injections, etc.), which contain a compound of formula I or a pharmaceutically acceptable salt thereof, a PD-1 signal transduction inhibitor or both, and are suitable for the selected mode of administration. In one embodiment, the pharmaceutical composition is administered orally and is thus formulated in a form suitable for oral administration, i.e., as a solid or liquid preparation. Suitable solid oral preparations include tablets, capsules, pills, granules, pellets, sachets and effervescent agents, powders, etc. Suitable liquid oral preparations include solutions, suspensions, dispersions, emulsions, oils, etc. In one embodiment of the present invention, the composition is formulated in a capsule. According to this embodiment, in addition to the active compound and an inert carrier or diluent, the composition of the present invention further comprises a hard gelatin capsule.

[0038] Any inert excipient commonly used as a carrier or diluent can be used in the formulations of the present invention, such as gums, starches, sugars, cellulosic materials, acrylates, or mixtures thereof. A preferred diluent is microcrystalline cellulose. The composition may also contain a disintegrating agent (e.g., sodium croscarmellose) and a lubricant (e.g., magnesium stearate), and may also contain one or more additives selected from the group consisting of binders, buffers, protease inhibitors, surfactants, solubilizers, plasticizers, emulsifiers, stabilizers, thickeners, sweeteners, film-forming agents, or any combination thereof. Additionally, the compositions of the present invention can be in the form of controlled-release or immediate-release formulations.

[0039] For liquid formulations, pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions, emulsions, or oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol / water solutions, emulsions or suspensions, including salt solutions and buffer media. Examples of oils are oils of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower seed oil, and cod liver oil. The solution or suspension may also contain the following ingredients: sterile injectable diluents (e.g., water), salt solutions, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate, or phosphate, and agents for adjusting tonicity such as sodium chloride or glucose. The pH can be adjusted with an acid or a base (e.g., hydrochloric acid or sodium hydroxide).

[0040] In addition, the composition may further comprise binders (such as gum arabic, corn starch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone), disintegrants (such as corn starch, potato starch, alginic acid, silica, croscarmellose sodium, crospovidone, guar gum, sodium starch glycolate, Primogel), buffers of various pH and ionic strengths (such as tris-HCl, acetate, phosphate), additives to prevent adsorption to surfaces (such as albumin or gelatin), detergents (such as Tween 20, Tween 80, Pluronic F68, bile salts), protease inhibitors, surfactants (such as sodium lauryl sulfate), penetration enhancers, solubilizers (such as glycerol, polyethylene glycol, polyethylene glycol), glidants (such as colloidal silica), antioxidants (such as ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (such as hydroxypropyl cellulose, hydroxypropyl methyl cellulose), thickeners (such as carbomer, colloidal silica, ethyl cellulose, guar gum), sweeteners (such as sucrose, aspartame, citric acid), flavoring agents (such as mint, methyl salicylate or orange flavoring), preservatives (such as thimerosal, benzyl alcohol, parabens), lubricants (such as stearic acid, magnesium stearate, polyethylene glycol, sodium dodecyl sulfate), glidants (such as colloidal silica), plasticizers (such as diethyl phthalate, triethyl citrate), emulsifiers (such as carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymer coatings (such as poloxamer or poloxamine), coatings and film formers (such as ethyl cellulose, acrylate, polymethacrylate) and / or adjuvants.

[0041] In one embodiment, the active compound is prepared with a carrier that will protect the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulation delivery systems. Biodegradable biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid. The methods for preparing such formulations are obvious to those skilled in the art. Such materials are also available from ALZA Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art (for example, as described in U.S. Patent No. 4,522,811).

[0042] For ease of administration and consistent dosage, it is particularly advantageous to formulate oral compositions in unit dosage forms. As used herein, "unit dosage form" refers to physically discrete units suitable as unit doses for the subject to be treated; each unit contains a predetermined quantity of the active compound which is calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications of the unit dosage forms of the present invention depend on and are directly dependent on: the unique properties of the active compound and the particular therapeutic effect to be achieved, as well as the limitations inherent in the art of compounding such active compounds for individual therapy.

[0043] The formulations of the present invention for oral administration may contain more than one permeation enhancer, including long-chain fatty acids or their salts, such as capric acid and sodium caprate.

[0044] In a preferred embodiment, the compound can be formulated as an aqueous solution for intravenous injection. In one embodiment, solubilizing agents can be appropriately used. Particularly preferred solubilizing agents include cyclodextrins and modified cyclodextrins, such as sulfonic acid-substituted β-cyclodextrin derivatives or their salts, including sulfobutyl-derivatized β-cyclodextrin, such as sulfobutylether-7-β-cyclodextrin sold under the trade name

[0045] The pharmaceutical composition can be contained in a container, package, or dispenser together with instructions for use.

[0046] Daily administration can be repeated continuously for periods of several days to several years. Oral treatment may last from one week to the lifetime of the patient. Preferably, administration can be carried out continuously for 5 days, after which the patient can be evaluated to determine whether further administration is required. Administration can be continuous or intermittent, such as treatment for several consecutive days followed by a rest period. The compounds of the present invention can be administered intravenously on the first day of treatment and orally on the second day and all subsequent consecutive days.

[0047] Methods for preparing pharmaceutical compositions containing the active ingredient are well known in the art, for example, by mixing, granulation, or tablet-forming methods. The active therapeutic ingredient is usually mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. For oral administration, the active agent is mixed with additives conventionally added for this purpose (such as excipients, stabilizers, or inert diluents) and converted into a form suitable for administration by conventional methods, such as tablets, coated tablets, hard or soft gelatin capsules, aqueous solutions, alcoholic solutions, or oily solutions as described above.

[0048] The amount of the compound administered to the patient is less than the amount that would cause toxicity in the patient. In certain embodiments, the amount of the compound administered to the patient is less than the amount that would result in a concentration of the compound in the patient's plasma equal to or exceeding the toxic 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 plasma is maintained at about 25 nM. In one embodiment, the concentration of the compound in the patient's plasma is maintained at about 50 nM. In one embodiment, the concentration of the compound in the patient's plasma is maintained at about 100 nM. In one embodiment, the concentration of the compound in the patient's plasma is maintained at about 500 nM. In one embodiment, the concentration of the compound in the patient's plasma is maintained at about 1000 nM. In one embodiment, the concentration of the compound in the patient's plasma is maintained at about 2500 nM. In one embodiment, the concentration of the compound in the patient's plasma is maintained at about 5000 nM. In practicing the present invention, the optimal amount of the compound to be administered to the patient will depend on the particular compound used and the type of cancer being treated.

[0049] Definitions

[0050] The following lists the definitions of various terms used to describe the present invention. Unless otherwise restricted in a particular instance, whether used alone or as part of a larger group, these definitions apply to the terms used throughout the specification and claims.

[0051] The term "acyl" refers to hydrogen, alkyl, partially or fully saturated cycloalkyl, partially or fully saturated heterocycle, aryl, and heteroaryl-substituted carbonyl. For example, acyl includes groups such as (C1-C6) alkanoyl (e.g., formyl, acetyl, propionyl, butyryl, valeryl, hexanoyl, tert-butylacetyl, etc.), (C3-C6) cycloalkylcarbonyl (e.g., cyclopropylcarbonyl, cyclobutylcarbonyl, cyclopentylcarbonyl, cyclohexylcarbonyl, etc.), heterocyclocarbonyl (e.g., pyrrolidinylcarbonyl, pyrrolid-2-one-5-carbonyl, piperidinylcarbonyl, piperazinylcarbonyl, tetrahydrofurylcarbonyl, etc.), aroyl (e.g., benzoyl), and heteroaroyl (e.g., benzothiol-2-carbonyl, benzothiol-3-carbonyl, furyl-2-carbonyl, furyl-3-carbonyl, 1H-pyrrol-2-carbonyl, 1H-pyrrol-3-carbonyl, benzo[b]benzothiol-2-carbonyl, etc.). In addition, the alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl moieties of acyl can be any of the groups described in the respective definitions. When expressed as "optionally substituted", acyl can be unsubstituted or optionally substituted with more than one substituent (usually 1 to 3 substituents), the substituents being independently selected from the substituents listed below in the definition of "substituted", or the cycloalkyl, heterocycle, aryl, and heteroaryl moieties of acyl can be substituted with the preferred and more preferred lists of substituents as described above, respectively.

[0052] The term "alkyl" includes straight-chain or branched-chain groups having from 1 to about 20 carbon atoms, or preferably from 1 to about 12 carbon atoms. More preferably, the alkyl is a "lower alkyl" having from 1 to about 10 carbon atoms. Most preferably, the alkyl is a lower alkyl having from 1 to about 8 carbon atoms. Examples of such groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, and the like.

[0053] The term "alkenyl" includes straight-chain or branched-chain groups having at least one carbon-carbon double bond, having from 2 to about 20 carbon atoms, or preferably from 2 to about 12 carbon atoms. More preferably, the alkenyl is a "lower alkenyl" group having from 2 to about 10 carbon atoms, more preferably from about 2 to about 8 carbon atoms. Examples of alkenyl groups include: vinyl, allyl, propenyl, butenyl, and 4-methylbutenyl. The terms "alkenyl" and "lower alkenyl" include groups having "cis" and "trans" orientations or "E" and "Z" orientations.

[0054] The term "alkynyl" includes straight-chain or branched-chain groups having at least one carbon-carbon triple bond, having from 2 to about 20 carbon atoms, or preferably from 2 to about 12 carbon atoms. More preferably, the alkynyl is a "lower alkynyl" group having from 2 to about 10 carbon atoms, more preferably from about 2 to about 8 carbon atoms. Examples of alkynyl include: propargyl, 1-propynyl, 2-propynyl, 1-butyne, 2-butynyl, and 1-pentynyl.

[0055] The term "aryl", used alone or in combination, refers to a carbocyclic aromatic system containing 1, 2, or 3 rings, where such rings may be connected together in a pendant fashion or may be fused. The term "aryl" includes aromatic groups such as phenyl, naphthyl, tetrahydronaphthyl, indane, and biphenyl.

[0056] The term "heteroaryl" includes unsaturated heterocyclic groups. Examples of heteroaryl include: unsaturated 3- to 6-membered, preferably 5- or 6-membered, heterocyclic monocyclic groups containing 1 to 4 nitrogen atoms, such as pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl (e.g., 4H-1,2,4-triazolyl, 1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, etc.), tetrazolyl (e.g., 1H-tetrazolyl, 2H-tetrazolyl, etc.); unsaturated fused heterocyclic groups containing 1 to 5 nitrogen atoms, such as indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolinyl, isoquinolinyl, indazolyl, benzotriazolyl, tetrazolopyridazinyl (e.g., tetrazolo[1,5-b]pyridazinyl, etc.), etc.; unsaturated 3- to 6-membered, preferably 5- or 6-membered, heterocyclic monocyclic groups containing an oxygen atom, such as pyranyl, furyl, etc.; unsaturated 3- to 6-membered heterocyclic monocyclic groups containing a sulfur atom, such as thienyl, etc.; unsaturated 3- to 6-membered, preferably 5- or 6-membered, heterocyclic groups containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, such as oxazolyl, isoxazolyl, oxadiazolyl (e.g., 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl, etc.), etc.; unsaturated fused heterocyclic groups containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms (e.g., benzoxazolyl, benzoxadiazolyl, etc.); unsaturated 3- to 6-membered, preferably 5- or 6-membered, heterocyclic monocyclic groups containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms, such as thiazolyl, thiadiazolyl (e.g., 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, etc.), etc.; unsaturated fused heterocyclic groups containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms (e.g., benzothiazolyl, benzothiadiazolyl, etc.), etc.

[0057] The term "substituted" means replacing one or more hydrogen groups in a given structure with a group of specified substituents, and the substituents include, but are not limited to: halogen, alkyl, alkenyl, alkynyl, aryl, heterocyclic group, mercapto, alkylthio, arylthio, alkylthioalkyl, arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkyloxy, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkyloxycarbonyl, carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl, heteroaryl, heterocycle and aliphatic group. It should be understood that the substituents can be further substituted.

[0058] In the case of neoplasms, tumor growth, or tumor cell growth, the term "inhibition" can be evaluated by the delay in the appearance of primary or secondary tumors, the slowing of the development of primary or secondary tumors, the reduction in the incidence of primary or secondary tumors, the slowing or reduction in the severity of secondary effects of the disease, tumor growth arrest, and tumor regression. In the present context, in extreme cases, complete inhibition is referred to as prevention or chemoprevention.

[0059] As used herein, the term "metastasis" refers to the migration of cancer cells from the site of the original tumor through the blood and lymphatic vessels to give rise to cancer in other tissues. Metastasis is also the term used for secondary cancers that grow at distant sites.

[0060] As used herein, the term "neoplasm" refers to an abnormal mass of tissue caused by excessive cell division. A neoplasm can be benign (non-cancerous) or malignant (cancerous) and can also be referred to as a tumor. The term "neoplasm" is the pathological process that leads to tumor formation.

[0061] As used herein, the term "pre-cancerous" refers to a non-malignant condition that may become malignant if not treated promptly.

[0062] The term "proliferation" refers to cells that undergo mitosis.

[0063] The term "treatment" refers to any process, action, application, therapy, etc., in which a mammal (including a human) receives medical assistance in order to directly or indirectly improve the condition of the mammal.

[0064] As used herein, the term "pharmaceutically acceptable salt" refers to those salts that are suitable, within the scope of sound medical judgment, for contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., and that are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S.M. Berge et al. described pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences 1977, 66: 1-19. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention or separately by reacting the free base functional group with a suitable organic or inorganic acid. Examples of pharmaceutically acceptable non-toxic acid addition salts include, but are not limited to, amino salts formed with inorganic or organic acids or by using other methods used in the art (such as ion exchange), inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, and organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid, lactobionic acid, or malonic acid. Other pharmaceutically acceptable salts include, but are not limited to: adipates, alginates, ascorbates, aspartates, benzenesulfonates, benzoates, bisulfates, borates, butyrates, camphorates, camphorsulfonates, citrates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, formates, fumarates, glucoheptanoates, glycerophosphates, glucuronates, hemi-sulfates, heptanoates, hexanoates, hydroiodides, 2-hydroxyethanesulfonates, lactobionates, lactates, laurates, laurylsulfates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, stearates, succinates, sulfates, tartrates, thiocyanates, p-toluenesulfonates, undecanoates, valerates, etc. Representative alkali metal or alkaline earth metal salts include: sodium, lithium, potassium, calcium, magnesium, etc. Other pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkylsulfonates having 1-6 carbon atoms, sulfonates, and arylsulfonates. It has been found that certain salts, such as sodium salts, potassium salts, and choline salts, as well as acid salts (such as sulfates and methanesulfonates), can improve the solubility of the 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 salts and potassium salts. Other preferred salts include sulfates and methanesulfonates. Particularly preferred salts of compound 1 are methanesulfonates and benzenesulfonates. A particularly preferred salt of compound 2 is a hydrochloride salt.

[0065] As used herein, "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with the administration of a drug, such as sterile pyrogen-free water. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences (a standard reference in the art), the contents of which are incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline solution, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and anhydrous carriers, such as fixed oils, may also be used. The use of such media and agents for active pharmaceutical substances is well known in the art. Unless any conventional media or agent is incompatible with the active compound, it is contemplated for use in the compositions. Supplementary active compounds may also be incorporated into the compositions.

[0066] As used herein, the term "precancerous" refers to a non-malignant condition that may become malignant if not treated in a timely manner.

[0067] As used herein, the term "subject" refers to an animal. Preferably, the animal is a mammal. More preferably, the mammal is a human. Subjects also refer to, for example, dogs, cats, horses, cows, pigs, guinea pigs, fish, birds, and the like.

[0068] The compounds of the present invention can be modified by the addition of appropriate functional groups to enhance selective biological properties. Such modifications are known in the art and can include those that increase bioper-meability to a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral bioavailability, increase solubility to permit administration by injection, alter metabolism, and alter the rate of excretion.

[0069] Drug composition

[0070] The pharmaceutical composition of the present invention comprises a therapeutically effective amount of a compound of Formula I (e.g., Compound 1) or a pharmaceutically acceptable salt thereof and a PD-1 signal transduction inhibitor formulated together with one or more pharmaceutically acceptable carriers or excipients.

[0071] Preferably, a pharmaceutically acceptable carrier or excipient is any type of non-toxic inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid. Some examples of materials that can be used as pharmaceutically acceptable carriers are: sugars such as lactose, glucose and sucrose; cyclodextrins such as α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose 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; diols 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; Ringer's solution; ethanol and phosphate buffer solutions, and other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, and coloring agents, release agents, coating agents, sweetening agents, flavoring agents and fragrances, preservatives and antioxidants may also be present in the composition, according to the judgment of the formulator.

[0072] The pharmaceutical compositions of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir, preferably by oral administration or by injection. The pharmaceutical compositions of the present invention can contain any conventional non-toxic pharmaceutically acceptable carrier, adjuvant or vehicle. In some cases, the pH of the formulation can be adjusted with a pharmaceutically acceptable acid, base or buffer to enhance the stability of the formulated compound or its delivery form. As used herein, the term parenteral includes: subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intra-synovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

[0073] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compound, the liquid dosage forms may also contain inert diluents commonly used in the art (such as water or other solvents), solubilizing agents and emulsifying agents such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils (especially cottonseed oil, arachis oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan and mixtures thereof. In addition to the inert diluent, the oral compositions may also contain adjuvants such as wetting agents, emulsifying agents and suspending agents, sweetening agents, flavoring agents and fragrances.

[0074] Injectable preparations can be formulated using suitable dispersing or wetting agents and suspending agents in accordance with known techniques, such as sterile injectable aqueous or oleaginous suspensions. Sterile injectable preparations can also be sterile injectable solutions, suspensions or emulsions in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Acceptable carriers and solvents that can be used are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. In addition, sterile fixed oils are commonly used as a solvent or suspending medium. For this purpose, any mild fixed oil can be used, including synthetic mono- or di-glycerides of fatty acids. In addition, fatty acids (such as oleic acid) are used in the preparation of injectables.

[0075] Injectable preparations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium before use.

[0076] To prolong the action of a drug, it is often necessary to slow the absorption of a drug injected subcutaneously or intramuscularly. This can be achieved by using a liquid suspension of a poorly water-soluble crystalline or amorphous material. The rate of absorption of the drug then depends on its rate of dissolution, which in turn depends on crystal size and crystal form. Alternatively, delayed absorption of a parenterally administered drug form is achieved by dissolving or suspending the drug in an oil vehicle. Injectable depot forms of drugs are prepared by forming a microcapsule matrix of the drug in a biodegradable polymer (such as poly(lactic-co-glycolic acid)). The rate of release of the drug can be controlled according to the ratio of the drug to the polymer and the nature of the particular polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Injectable depot preparations of drugs can also be prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0077] Compositions for rectal or vaginal administration are preferably suppositories, which can be prepared by mixing the compounds of the present invention with a suitable non-irritating excipient or carrier (such as cocoa butter, polyethylene glycol or suppository wax), which are solid at ambient temperature but liquid at body temperature. Thus, they melt in the rectal or vaginal cavity and release the active compound.

[0078] Oral solid dosage forms include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert pharmaceutically acceptable excipient or carrier (such as sodium citrate or dibasic calcium phosphate) and / or the following components: a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; b) binders, such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and acacia; c) humectants, such as glycerol; d) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; e) solution retarding agents, such as paraffin wax; f) absorption promoters, such as quaternary ammonium compounds; g) wetting agents, such as cetyl alcohol and glyceryl monostearate; h) absorbents, such as kaolin and bentonite; i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also contain buffering agents.

[0079] Similar types of solid compositions can also be used as fillers in soft and hard gelatin capsules, using excipients such as lactose or milk sugar and high molecular weight polyethylene glycol, etc.

[0080] Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells (such as enteric coatings and other coatings known in the pharmaceutical formulation art). They may optionally contain emulsifying agents and may also have compositions that release one or more active ingredients optionally in a delayed manner, only or preferably in certain parts of the intestine. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0081] Dosage forms for topical or transdermal administration of the compounds of the present invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The active ingredient is mixed under sterile conditions with a pharmaceutically acceptable carrier and any desired preservatives or buffering agents that may be required. Ophthalmic preparations, ear drops, eye ointments, powders, and solutions are also contemplated within the scope of the present invention.

[0082] In addition to the active compounds of the present invention, ointments, pastes, creams, and gels may also contain excipients such as animal and vegetable fats, oils, waxes, paraffin wax, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0083] In addition to the compounds of the present invention, powders and sprays may also contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, and polyamide powder, or mixtures of these substances. Sprays may also contain conventional propellants, such as chlorofluorocarbons.

[0084] Transdermal patches have the additional advantage of providing controlled delivery of a compound to the body. Such dosage forms can be prepared by dissolving or dispensing the compound in a suitable medium. Penetration enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by providing a rate-controlling membrane or by dispersing the compound in a polymeric matrix or gel.

[0085] For pulmonary delivery, the therapeutic compositions of the present invention are formulated and administered to a patient in the form of solid or liquid particles by direct administration (e.g., inhalation into the respiratory system). Solid or liquid particle forms of the active compounds prepared for practicing the present invention include particles of inhalable size: i.e., particles small enough to pass through the mouth and larynx and enter the bronchioles and alveoli of the lungs upon inhalation. Delivery of nebulized therapeutic agents (especially nebulized antibiotics) is known in the art (e.g., see U.S. Patent No. 5,767,068 to Van Devanter et al., U.S. Patent No. 5,508,269 to Smith et al., and WO 98 / 43650 to Montgomery, the entire contents of which are incorporated herein by reference). There is also a discussion of pulmonary delivery of antibiotics in U.S. Patent No. 6,014,969 (the contents of which are incorporated herein by reference).

[0086] A "therapeutically effective amount" of a combination of a compound of Formula I and a PD-1 signal transduction inhibitor refers to an amount of each compound that, when combined, confers a therapeutic effect on a subject being treated at a reasonable benefit / risk ratio applicable to any medical treatment. The therapeutic effect can be objective (i.e., measurable by some test or marker) or subjective (i.e., the subject gives signs of or feels an effect). The effective dose will also vary depending on the route of administration and the likelihood of co-use with other drugs. However, it is to be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of reasonable medical judgment. The specific therapeutically effective dose level for any particular patient will depend on a variety of factors, including the disease being treated and the severity of the disease; the activity of the particular compound used; the specific composition employed; the age, weight, general health, sex, and diet of the patient; the time of administration, route of administration, and rate of excretion of the particular compound used; the duration of the treatment; drugs used in combination with or concurrently with the particular compound employed; and factors well known in the medical arts. In a preferred embodiment, a therapeutically effective amount of a combination of a compound of Formula I or a pharmaceutically acceptable salt thereof and a PD-1 signal transduction inhibitor exhibits a synergistic effect in the type of cancer to be treated.

[0087] In the combination therapy of the present invention, the total daily dose of each compound administered to a human or other animal in a single dose or divided doses can be, for example, an amount from 0.01 mg / kg body weight to 50 mg / kg body weight or more typically from 0.1 mg / kg body weight to 25 mg / kg body weight. A single dose composition can contain such an amount or a submultiple thereof to make up the daily dose. Generally, the treatment regimen of the present invention comprises administering from about 10 mg to about 1000 mg of the compound of the present invention to a patient in need of such treatment in a single dose or multiple doses per day.

[0088] For example, each compound in the combination therapy of the present invention can be administered by injection, intravenously, intraarterially, subcutaneously, intraperitoneally, intramuscularly or sublingually; or, orally, buccally, nasally, transmucosally, topically, in an ophthalmic formulation or by inhalation; administered every 4 to 120 hours or as required by the particular drug, in a dose range from about 0.1 mg / kg body weight to about 500 mg / kg body weight or from 1 mg / dose to 1000 mg / dose. The methods herein contemplate administering an effective amount of the compound or compound composition to achieve a desired or specified effect. Generally, the pharmaceutical compositions of the present invention will be administered from about 1 to about 6 times per day, or as a continuous infusion. Such administration can be used for chronic or acute treatment. The amount of the active ingredient which can be combined with a pharmaceutical excipient or carrier to produce a single dosage form will vary depending on the host to be treated and the particular mode of administration. A typical formulation will contain from about 5% to about 95% active compound (w / w). Alternatively, such formulations can contain from about 20% to about 80% active compound.

[0089] Doses lower or higher than those described above may be required. The specific dose and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the particular compound employed, age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition or symptom, the patient's predisposition to the disease, condition or symptom and the judgment of the treating physician.

[0090] After improvement of the patient's condition, a maintenance dose of the compound, composition or combination of the present invention can be administered if necessary. Subsequently, the dose administered or the frequency of administration or both can be reduced, depending on the symptoms, to a level that maintains the improved condition when the symptoms have been reduced to the desired level. However, once the symptoms of the disease recur, the patient may require long-term intermittent treatment.

[0091] Examples

[0092] The compounds and methods of the present invention will be better understood in conjunction with the following examples, which are for illustrative purposes only and do not 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 those changes and modifications, including but not limited to those related to the chemical structure, substituents, derivatives, formulations, and / or methods of the present invention, can be made without departing from the spirit of the present invention and the scope of the appended claims.

[0093] The synthesis of Compound 1 and its mesylate, sodium salt, potassium salt, and choline salt is shown in the following figure.

[0094]

[0095]

[0096] Intermediate 107-1 or 107-2 can be prepared by reacting 106 with R-2-1 or R-2-2 respectively. The synthetic schemes of R-2-1 and R-2-2 are shown as follows:

[0097]

[0098] Or by another method:

[0099]

[0100] Intermediate 108-1 and 108-2 can be prepared by the coupling reaction of 107-1 or 107-2 with R-3-1 or R-3-2, wherein R-3-1 and R-3-2 can be prepared according to the following scheme:

[0101]

[0102] Example 1: Preparation of N-Hydroxy-2-(((2-(6-Methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidine-5-carboxamide (Compound 1)

[0103] Step a: (Z)-Ethyl 2-(Ethoxymethyl)-3-methoxyacrylate (Compound 202)

[0104] Sodium (40.9 g, 1.78 mol) was carefully added in portions to ethanol (750 mL). After all the sodium metal had disappeared, the solution was concentrated to give NaOEt powder. While stirring, hexane (1.0 L) was added and the mixture was cooled with an ice-water bath. A mixture of 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 with cooling in an ice-water bath. The resulting mixture was heated at 50 °C for 2 h. Triethylammonium chloride (122 g) and sodium hydroxide (20 g) were added to the mixture. Then the mixture was stirred at room temperature for 4 h and filtered. The filtrate was washed with water and dried over Na2SO4. 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.

[0105] Step b: Ethyl 2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (Compound 203)

[0106] 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 under reflux overnight. After evaporating about 50% of the reaction 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: 171 [M+1] + . 1 H NMR (400 MHz, CDCl3): 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).

[0107] Step c: Ethyl 2-oxo-1,2-dihydropyrimidine-5-carboxylate (Compound 204)

[0108] 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 under reflux for 2 h, cooled to room temperature, and further cooled to 0 - 5 °C, then filtered to give the title compound 204 (38 g, 54%) as a yellow solid. LCMS: 169 [M+1] + . 1 H 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).

[0109] Step d: Ethyl 2-chloropyrimidine-5-carboxylate (Compound R-2-1)

[0110] A mixture of Compound 204 (38.0 g, 153 mmol), phosphorus trichloride (300 mL), and N,N-dimethylaniline (3 mL) was heated under 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 with sodium carbonate, and it was extracted with EtOAc. The combined organic matter was washed with ice water and brine, dried over Na2SO4, evaporated, and purified by column chromatography (eluted with EtOAc / hexane, 10%), to obtain Compound R-2-1 (15 g, 52%) as a white solid. LCMS: 187 [M+1] + 。 1 1H NMR (400 MHz, CDCl3): δ 1.36 (t, J = 7.5 Hz, 3H), 4.39 (q, J = 7.5 Hz, 2H), 9.08 (s, 2H).

[0111] Step e: Sodium (Z)-2-(dimethoxymethyl)-3-methoxy-3-oxoprop-1-enoate (Compound 206)

[0112] 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 then methyl 3,3-dimethoxypropionate (205) (100 g, 0.675 mol) was added dropwise. The resulting mixture was stirred for 0.5 hour, and 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, then cooled to 0 °C. The reaction mixture was slowly warmed to 25 °C and stirred overnight. Et2O (150 mL) was added and stirred for 30 minutes. The resulting suspension was filtered. The solid was washed with Et2O (100 mL), collected, and dried to obtain the title compound 206 (82 g, 61%) as an off-white solid. LCMS (m / z): 130.8 [M+1] + 。 1 1H NMR (400 MHz, CD3OD): δ 3.36 (s, 6H), 3.60 (s, 3H), 5.34 (s, 1H), 8.92 (s, 1H).

[0113] Step f: Methyl 2-aminopyrimidine-5-carboxylate (Compound 207)

[0114] To a mixture of guanidine hydrochloride (42.2 g, 0.44 mol) in DMF (300 mL) was added compound 206 (80 g, 0.40 mol). The resulting mixture was heated at 100 °C for 1 h. The reaction mixture was filtered and then cooled. The filter cake was washed with 50 mL of DMF, and the combined filtrates were concentrated to give 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] + . 1 1H NMR (400 MHz, CD3OD): δ 3.88 (s, 3H), 8.77 (s, 2H).

[0115] Step g: Methyl 2-chloropyrimidine-5-carboxylate (Compound R-2-2)

[0116] Compound 207 (7 g, 0.046 mol) was added to a mixture of concentrated hydrochloric acid (15.2 mL) and CH2Cl2 (60 mL). After cooling, ZnCl2 (18.6 g, 0.138 mol) was added at 15 - 20 °C. The mixture was stirred at 15 - 20 °C for 0.5 h and then cooled to 5 - 10 °C. NaNO2 (9.5 g, 0.138 mol) was added portionwise while maintaining the internal temperature at 5 - 10 °C. The reaction continued for about 2 h. The reaction mixture was poured into ice water (50 mL). The organic layer was separated, and the aqueous phase was extracted with CH2Cl2 (30 mL * 2). The combined organic extracts were concentrated to give a crude product (4.2 g). The crude compound was suspended in hexane (20 mL), heated at 60 °C for 30 min, and then 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] + . 1 1H NMR (400 MHz, CDCl3): δ 4.00 (s, 3H), 9.15 (s, 2H).

[0117] Step h: 5-Bromo-2-methoxypyridine (Compound 303)

[0118] A solution of 2-methoxypyridine (100 g, 0.92 mol) and NBS (180 g, 1.0 mol) in acetonitrile (1.0 L) was refluxed with stirring for 21 h. TLC showed that the reaction was complete. The reaction mixture was cooled to room temperature and concentrated. Approximately 900 mL of the solvent was collected. The resulting suspension was filtered and washed with n-hexane (ca. 400 mL). The filtrate was concentrated again to give the crude product. The crude product was distilled under reduced pressure (30 °C / ca. 0.3 mmHg) to give the title compound (146 g, 84%) as a clear oil. LCMS (m / z): 190.0 [M+1] + 。 1 1H NMR (400 MHz, CDCl3): 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).

[0119] Step i: 6-Methoxypyridin-3-ylboronic acid (R-3-1):

[0120] At -78 °C, n-butyllithium (59 mL, 2 M in THF) was added dropwise to a solution of compound 303 (20 g, 0.11 mol) in anhydrous THF (180 mL), and the resulting mixture was stirred for 1 h. Triisopropyl borate (37 mL) was added at -78 °C, and the reaction mixture was warmed to room temperature and stirred overnight. TLC (hexane / ethyl acetate = 5:1) showed that the reaction was complete. The pH of the mixture was adjusted to 3 - 4 with 4N HCl (90 mL). The precipitate was collected by filtration to give the crude compound R-3-1 (21 g, 128%). The crude compound R-3-1 (21 g) was dissolved in water (200 mL), and the pH of the solution was adjusted to 8 - 9 with concentrated ammonia. The precipitate was collected by filtration to give the pure title compound R-3-1 (11 g, 67%) as a white solid. LCMS (m / z): 154.1 [M+1] + 。 1 1H NMR (400 MHz, 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).

[0121] Step j: 2-Methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (Compound R-3-2)

[0122] Under a N2 atmosphere, a mixture of compound 303 (55 g, 0.29 mol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(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) was heated at 108 °C overnight. The reaction mixture was concentrated and purified by column chromatography, eluting with hexane / ethyl acetate to afford the title compound R-3-2 (58 g, 84%). 1 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).

[0123] Step k: Thieno[3,2-d]pyrimidine-2,4(1H,3H)-dione (Compound 102)

[0124] Urea method: A mixture of methyl 3-aminothiophene-2-carboxylate (101) (90.0 g, 573 mmol, 1.0 equiv) and urea (277.6 g, 4.6 mol, 8.0 equiv) was heated at 190 °C for 3 - 4 h and cooled to room temperature. Aqueous NaOH solution (10%, 800 mL) was added to the reaction mixture. After stirring at ambient temperature for 1 h, the solid was removed by filtration. The filtrate was acidified to pH 3 - 4 with 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] + . 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).

[0125] KOCN method: Within 1 hour, an aqueous solution (326 mL) of potassium cyanate (154.8 g, 1.91 mol, 3.0 equivalents) was slowly added to a mixture of 3-aminothiophene-2-carboxylate (101) (100.0 g, 636.9 mmol, 1.0 equivalent), acetic acid (705 mL) and water (600 mL). The resulting mixture was stirred at room temperature for 20 hours, filtered, and rinsed with water (500 mL). The filter cake was charged into a reactor of appropriate size, 2M aqueous sodium hydroxide solution (1.65 L) was added, and the slurry was stirred for 2 hours. LCMS confirmed the formation of the desired product. The mixture was cooled to 10 °C, and 3M aqueous hydrochloric acid solution (1.29 L) was added until pH = 5.0 to 6.0. The slurry was filtered, rinsed with water (700 mL), and dried in a vacuum oven at 50 °C for 24 hours to obtain compound 102 (100 g, 94%), as an off-white solid. LCMS (m / z): 169.1 [M+1] + 。 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).

[0126] Step l: 2,4-Dichlorothieno[3,2-d]pyrimidine (Compound 103)

[0127] Phosphorus oxychloride (152 mL, 1.67 mol, 7.0 equivalents) was slowly added to a cold acetonitrile solution (250 mL) of compound 102 (40 g, 238 mmol, 1.0 equivalent) and N,N-dimethylaniline (22.5 mL, 179 mmol, 0.75 equivalent), while 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 onto an ice-water mixture (360 mL). The resulting slurry was filtered and rinsed with cold water (200 mL). The filter cake was dried in a vacuum oven at 40 °C for 24 hours to obtain compound 103 (40.5 g, 83%), as an off-white solid. Melting point: 245 - 250 °C. LCMS (m / z): 205.0 [M+1] + 。 1 1H NMR (400 MHz, DMSO-d6): δ 7.75 (d, J = 5.2 Hz, 1H), 8.71 (d, J = 5.2 Hz, 1H).

[0128] Step m: 2-Chloro-4-morpholinothieno[3,2-d]pyrimidine (Compound 104)

[0129] Morpholine (31.2 mL, 367 mmol, 2.2 equiv) was slowly added to a mixture of Compound 103 (34.2 g, 167 mmol, 1.0 equiv) and methanol (500 mL). The reaction mixture was stirred overnight at room temperature. 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] + . 1 H 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).

[0130] Step n: 2-Chloro-4-morpholinothieno[3,2-d]pyrimidine-6-carbaldehyde (Compound 105)

[0131] At -78 °C and under nitrogen, n-butyllithium (2.4 M in hexanes, 40.8 mL, 102 mmol, 1.3 equiv) was slowly added to a suspension of Compound 104 (20 g, 78.4 mmol, 1.0 equiv) in anhydrous THF (320 mL). The resulting slurry was warmed to -60 °C to become a clear brown solution. The reaction mixture was then cooled again to -78 °C and DMF (anhydrous, 9.1 mL, 118 mmol, 1.5 equiv) was slowly added. The resulting solution was stirred at -78 °C for 0.5 h, warmed to 0 °C over 1 h, and slowly poured into a mixture of aqueous HCl (0.25 M, 660 mL) and ice water (320 mL). The resulting slurry was stirred at 0 - 10 °C for 0.5 h, filtered, washed with cold water, and dried in vacuo to give Compound 105 (22 g, 98%) as a yellow solid. Melting point: 260 - 265 °C. LCMS (m / z): 284.0 [M+1] + . 1 H 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).

[0132] Step o: (2-Chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidin-6-ylmethyl)-methylamine (Compound 106)

[0133] Under a nitrogen atmosphere, a methanol solution (27% v / v, 75 mL, 563.2 mmol) of methylamine was added to a methanol (125 mL) solution of Compound 105 (20.0 g, 70.4 mmol, 1.0 equiv.). The reaction mixture was stirred overnight at room temperature, and the solvent was removed in vacuo to give a crude solid product, which was dissolved in methanol (550 mL) and THF (220 mL) under nitrogen. Sodium borohydride (8 g, 211.2 mmol) was added portionwise, and the reaction mixture was stirred overnight at room temperature. The reaction mixture was evaporated in vacuo, and water (300 mL) was added. The aqueous mixture was extracted with dichloromethane, and the combined extracts were dried over Na2SO4 and concentrated. The residue was dissolved in 6 M HCl (230 mL) and stirred for 30 minutes. The aqueous solution was washed several times with dichloromethane, and the pH was adjusted to 9 - 10 with NaOH (4 N). The precipitated solid was collected by filtration and dried (60 °C, 6 h) to give a pale yellow solid (18 g, 85%). Melting point: 240 - 245 °C. LCMS (m / z): 299 [M+1] + 。 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).

[0134] Step p(a): Ethyl 2-[(2-chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidin-6-ylmethyl)-methyl-amino]-pyrimidine-5-carboxylate (Compound 107-1)

[0135] Diisopropylethylamine (220 mL, 1.26 mol) was added to a mixture of 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 overnight at room temperature. The mixture was then evaporated, and dichloromethane (300 mL) was added. The organic phase was washed with water, dried over Na2SO4, 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] + 。 11H 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).

[0136] Step p(b): Methyl 2-[(2-chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidin-6-ylmethyl)-methyl-amino]-pyrimidine-5-carboxylate (Compound 107-2)

[0137] 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 adding dichloromethane (500 mL), the organic phase was washed with water, dried over Na2SO4 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 (29.4 g, 81%) was collected as a white solid. LCMS (m / z): 435.2 [M+1] + . 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).

[0138] Step q(a): Ethyl 2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidine-5-carboxylate (Compound 108-1)

[0139] Method A: Under N2 atmosphere, a mixture of Compound 107-1 (12 g, 26.7 mmol), R-3-1 (4.9 g, 32 mmol), NaHCO3 (6.7 g, 80.1 mmol) and bis(triphenylphosphine)palladium(II) chloride (188 mg, 0.267 mmol) in a mixed solvent of toluene (80 mL), ethanol (50 mL) and water (10 mL) was heated at 108 °C for 4.5 h. TLC showed the completion of the reaction. Then the reaction mixture was 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 min 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%).

[0140] Method B: Under N2 atmosphere, a mixture of Compound 107-1 (1.5 g, 3.34 mmol), R-3-2 (1.6 g, 6.68 mmol), NaHCO3 (0.84 g, 10.0 mmol) and bis(triphenylphosphine)palladium(II) chloride (118 mg, 0.167 mmol) in a mixed solvent of toluene (24 mL), ethanol (15 mL) and water (3 mL) was heated overnight at 108 °C. The reaction mixture was partitioned between dichloromethane and water. The organic layer was separated, washed with brine, dried over Na2SO4, filtered and evaporated in vacuo to give a residue which was purified by column chromatography eluting with hexane / ethyl acetate to give Compound 108-1 (1.7 g, 98%) as a white solid.

[0141] Melting point: 198 - 202 °C. LCMS: 522.30 [M+1] + 。 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).

[0142] Step q(b): Methyl 2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidine-5-carboxylate (Compound 108-2)

[0143] At room temperature, solid NaHCO3 (11.6 g, 138.1 mmol, 3 equiv) was added to a mixture of Compound 107-2 (20 g, 46.0 mmol) and B-3-1 (9.2 g, 60.2 mmol, 1.3 equiv) in dioxane (540 mL), and then water (40 mL) was added. The resulting mixture was degassed by passing N2 over the surface of the solution. Then bis(triphenylphosphine)palladium(II) chloride (323 mg, 0.46 mmol, 0.01 equiv) was added, and the resulting mixture was heated at 108 °C for 15 h. TLC and LCMS indicated completion of the reaction. The reaction mixture was filtered through Celite while still hot (>90 °C) and washed with dioxane (70 mL). The filtrate was gradually cooled to room temperature, and white microcrystals formed during cooling. The suspension was filtered and washed with dioxane (80 mL) to give the title compound 108-2 (18 g, 78%) as a white solid. LCMS (m / z): 508.3 [M+1] + 。 1 1H 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).

[0144] Step r: N-Hydroxy-2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidine-5-carboxamide (Compound 1)

[0145] Preparation of hydroxylamine methanol solution

[0146] A mixture of NH2OH·HCl (80 g, 1.12 mol) in MeOH (400 mL) was heated at 60 - 65 °C for 1 h to form a clear solution. Then it was cooled in an ice-water bath. A solution of KOH (96 g, 1.68 mol) in MeOH (240 mL) was added dropwise to this cold mixture while maintaining the reaction temperature at 0 - 10 °C. The resulting mixture was stirred at 0 °C for 30 min and then filtered through a constant-pressure funnel filled with anhydrous Na2SO4 (700 g). The filtrate was collected under an ice bath and stored in the refrigerator for future use.

[0147] Preparation of Compound 1 from Compound 108-1

[0148] Compound 108-1 (10 g, 19 mmol) was suspended in the freshly prepared hydroxylamine methanol solution (1.79 M, 350 mL) described above. Dichloromethane (100 mL) was added to the mixture. The reaction flask was sealed and the mixture was stirred at room temperature for 5 h, then it became a clear solution. The reaction was stirred for an additional 9 h. Any insoluble solids were removed by filtration. The pH of the filtrate was adjusted to 6 - 7 by adding acetic acid, forming a solid precipitate. The solid was collected by filtration, washed with water and a minimal amount of methanol, and dried in vacuo at 60 °C for 5 h to give Compound 1 (9.2 g, 96%) as a white solid. Melting point: 177 - 180 °C. LCMS: 509.3 [M+1] + . 1 H 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).

[0149] Preparation of Compound 1 from Compound 108-2

[0150] At room temperature, the freshly prepared hydroxylamine methanol solution (1.79 M, 744 mL) was added to a suspension of Compound 108-2 (31 g, 61.1 mmol) in dichloromethane (310 mL). The reaction flask was sealed and the reaction mixture was stirred at room temperature for 5 h. The reaction mixture became a clear solution. The reaction solution was filtered to remove any insoluble solids. Then water (310 mL) was added to the filtrate, and no solid was formed during the addition. Acetic acid (18.5 mL) was added with stirring to adjust the pH to 10.20 (monitored continuously by a pH meter). During the addition of acetic acid, the internal temperature did not change. The resulting reaction mixture was stirred for an additional 4 h. A white solid gradually formed. The suspension was filtered and washed with a minimal amount of methanol (100 mL × 3). The collected white solid was resuspended in methanol (620 mL) and water (124 mL) to form a suspension. Additional acetic acid (11 g) was added to the above suspension to adjust the pH to 5 - 6. A change in the form of the solid was observed. The suspension was stirred for an additional 2 h, filtered through filter paper, and washed with a minimal amount of methanol (100 mL × 3). The collected white solid was dried in an oven (50 °C) for 12 h to give the title Compound 1 (23.6 g, 76.0%) as a white solid. Melting point: 255 - 259 °C. LCMS (m / z): 509.3 [M+1]+ . 1 1H NMR (400 MHz, DMSO-d6): 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).

[0151] Example 2: Preparation of N-Hydroxy-2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidine-5-carboxamide mesylate (mesylate of Compound 1)

[0152] Method A: A solution of methanesulfonic acid (114 mg, 1.18 mmol) in MeOH (3 mL) was added to a mixture of Compound 1 (300 mg, 0.59 mmol) and MeOH / Et2O (3 / 1, 40 mL) at 0 °C. The resulting mixture was stirred at 0 °C for 3 hours. The precipitate was collected by filtration and washed with Et2O to give Compound 2 (260 mg, 73%) as a white solid.

[0153] Method B: A suspension of methanesulfonic acid (341 mg, 3.55 mmol) in 2 mL of MeOH was added to a suspension of Compound 1 (1.5 g, 2.95 mmol) in dichloromethane / MeOH (40 mL / 10 mL) at room temperature (15 °C) to form 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 was continued at room temperature for 3 hours. The resulting precipitate was collected by filtration to give Compound 2 (1.45 g, 83%) as a white solid.

[0154] Melting point: 179 - 185 °C. LCMS: 509.3 [M+1] + . 11H 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).

[0155] Example 3: Preparation of Sodium N-Hydroxy-2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidine-5-carboxamide (Sodium Salt of Compound 1)

[0156] To a suspension of Compound 1 (300 mg, 0.59 mmol) in methanol (30 mL) at 0 °C was slowly added t-BuONa (85 mg, 0.88 mmol). The resulting mixture was warmed to room temperature and stirred for an additional 2 h. The reaction was concentrated, the residue was triturated and washed with ethanol, and then filtered to give Compound 3 (230 mg, 73%) as a white solid. Melting point: 178 - 183 °C. LCMS: 509.3 [M+1] + . 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).

[0157] Example 4: Preparation of Potassium N-Hydroxy-2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidine-5-carboxamide (Potassium Salt of Compound 1)

[0158] At 0 °C, t-BuOK (132 mg, 1.17 mmol) was added to a mixture of Compound 1 (400 mg, 0.78 mmol) in methanol (50 mL) under N2. The mixture was stirred at 0 °C for 1 h and then continued to stir at room temperature for 1.5 h. The insoluble solid was removed by filtration and the filtrate was cooled to -20 °C. Et2O (100 mL) was added to the filtrate. The resulting mixture was stirred at -20 °C for 1 h. Hexane (70 mL) was added and the mixture was continued to stir at -20 °C for 2 h. The solid was collected by filtration and dried in vacuo to give Compound 4 (150 mg, 35%) as a white solid. Melting point: 174 - 179 °C. LCMS: 509.3 [M+1] + 。 1 H 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).

[0159] Example 5: Preparation of N-Hydroxy-2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidine-5-carboxamide choline salt (choline salt of Compound 1)

[0160] Choline hydroxide (106 mg, 0.39 mmol, 45%, in MeOH) was added to a solution of Compound 1 (200 mg, 0.39 mmol) in DCM / MeOH (60 mL / 12 mL). The mixture was stirred at room temperature for 2 h and then concentrated to remove about 30 mL of the solvent. Ethyl acetate (60 mL) was added and the mixture was stirred at room temperature for 2 h. After a small amount of precipitation occurred, the mixture was concentrated to remove about 40 mL of the solvent and additional ethyl acetate (60 mL) was added. The mixture was stirred at room temperature for 2 h, filtered to give Compound 5 (180 mg, 76%) as a white solid. Melting point: 181 - 185 °C. LCMS: 509.3 [M+1] + 。 11H NMR (400 MHz, DMSO-d6): 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).

[0161] Example 6: Preparation of N-Hydroxy-2-(((2-(6-Methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidine-5-carboxamide Sulfate (Sulfate of Compound 1)

[0162] 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 form a clear solution. The reaction mixture was stirred overnight at room temperature. Precipitation occurred, and then tert-butyl methyl ether (60 mL) was added. The resulting mixture was stirred at room temperature for an additional 1 hour. The solid was collected by filtration to give Compound 6 (180 mg, 76%) as a white solid. Melting point: 243 - 246 °C. LCMS: 509.3 [M+1] + . 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).

[0163] Example 7: N-Hydroxy-2-(methyl((2-(6-(methylamino)pyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)amino)pyrimidine-5-carboxamide (Compound 2)

[0164] Step 7a: (2-Chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidin-6-ylmethyl)-methylamine (Compound 0503)

[0165] Under a nitrogen atmosphere, a methanol solution (27% v / v, 75 mL, 563.2 mmol) of methylamine was added to a methanol (125 mL) solution of 0112 (20.0 g, 70.4 mmol). The reaction mixture was stirred overnight at room temperature, and the solvent was removed under vacuum to obtain a crude solid product, 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 overnight at room temperature. The reaction mixture was evaporated under vacuum, and water (300 mL) was added. The aqueous mixture was extracted with dichloromethane, and the combined extracts were dried over Na2SO4 and concentrated. The residue was dissolved in 6M HCl (230 mL) and stirred for 30 minutes. The aqueous solution was washed several times with dichloromethane and adjusted to pH = 9 - 10 with NaOH (4N). The precipitated solid was collected by filtration and dried (60 °C, 6 h) to obtain a pale yellow solid (18 g, 85%).

[0166] LCMS: 299 [M + 1] + 。 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).

[0167] Step 7b: Ethyl 2-[(2-chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidin-6-ylmethyl)-methyl-amino]-pyrimidine-5-carboxylate (Compound 0504)

[0168] A mixture of 0503 (10 g, 33.6 mmol), CH3CN (400 mL) and 0305 (6.8 g, 36.4 mmol) was stirred at room temperature. Then diisopropylethylamine (DIPEA) (220 mL, 1.26 mol) was added, and the solution was stirred overnight and evaporated. After adding dichloromethane (300 mL), the organic phase was washed with water, dried over Na2SO4 and concentrated under vacuum to obtain 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 (10.6 g, 70%) was collected as a white solid. LCMS: 449 [M + 1] + ; 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).

[0169] Step 7c: Ethyl 2-(methyl((2-(6-(methylamino)pyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)amino)pyrimidine-5-carboxylate (Compound 0603-111)

[0170] A mixture of N-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine (0602-227) (351 mg, 1.5 mmol), 0504 (314 mg, 0.7 mmol), NaHCO3 (176 mg, 2.1 mmol) and Pd(PPh3)2Cl2 (24.6 mg, 0.035 mmol) was dissolved in toluene / EtOH / H2O (2.5 mL / 1.6 mL / 0.7 mL). The reaction mixture was then stirred in a microwave at 120 °C for 2 h. Water (8 mL) was added to the mixture, and the mixture was extracted with ethyl acetate (15 mL × 3). The organic layer was dried, concentrated and purified by column chromatography (dichloromethane solution of methanol, 5% v / v) to give the title compound 0603-111 (150 mg, 41%) as a white solid. LCMS: 521 [M+1] + . 1 1H NMR (400 MHz, DMSO-d6): 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).

[0171] Step 7d: N-Hydroxy-2-(methyl((2-(6-(methylamino)pyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)amino)pyrimidine-5-carboxamide (Compound 2)

[0172] Compound 2 (21 mg, 14%) as a brown solid was prepared from 0603-236 (150 mg, 0.29 mmol) and freshly prepared hydroxylamine methanol solution (6 mL) using a method similar to that described in Example 1. Melting point: 193-195 °C. LCMS: 508 [M+1] + . 11H 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, 2.0 Hz, 1H), 8.75 (s, 2H), 9.01 (d, J = 2.0 Hz, 1H), 9.07 (br, 1H).

[0173] Example 8: 2-(((2-(4-Aminophenyl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl](methyl)amino)-N-hydroxypyrimidine-5-carboxamide (Compound 3)

[0174] Step 8a: N-(4-Bromophenyl)acetamide (Compound 0601-150)

[0175] To a solution of 4-bromoaniline (6.3 g, 63.7 mmol) in CH2Cl2 (50 mL) at 0 °C was added acetyl chloride (3.75 g, 47.7 mmol) and TEA (7.4 g, 73.4 mmol), and the mixture was stirred for 2 h. The reaction mixture was washed with water and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give the title compound 0601-150 (3.6 g, 46%) as a brown solid. LCMS: 214 [M+1] + ; 1 1H NMR (400 MHz, DMSO-d6): 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).

[0176] Step 8b: N-(4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)acetamide (Compound 0602-150)

[0177] Using a method similar to that described for Compound 0602-107 (Example 34), the title compound 0602-150 (2.3 g, 94%) was prepared from 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 PdCl2(dppf)2 (76 mg, 0.088 mmol) as a white solid. LCMS: 262 [M+1] + . 11H 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).

[0178] Step 8c: Ethyl 2-(((2-(4-aminophenyl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidine-5-carboxylate (Compound 0603-150)

[0179] A mixture of Compound 0504-54 (210 mg, 0.46 mmol), 0602-150 (159 mg, 0.60 mmol), sodium bicarbonate (118 mg, 1.4 mmol), and bis(triphenylphosphine)palladium(II) chloride (17 mg, 0.02 mmol) in toluene (4 mL), ethanol (2 mL), and water (1 mL) was flushed with nitrogen and heated at 120 °C for 2 hours under microwave irradiation. The reaction mixture was partitioned between ethyl acetate and water, and the organic layer was washed with brine, dried over magnesium sulfate, filtered, and evaporated in vacuo. The residue was washed with dichloromethane to afford ethyl 2-(((2-(4-acetamidophenyl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidine-5-carboxylate (136 mg, 53%) as a white solid. LCMS: 548 [M+1] + , 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).

[0180] At 40 °C, aqueous HCl solution (6 M, 15 mL) was added to a solution of the above ethyl ester (280 mg, 0.51 mmol) in THF (10 mL), and the mixture was stirred for 2 hours. The reaction mixture was neutralized with NaHCO3, extracted with CH2Cl2, and the organic layer was washed with water and brine, dried over Na2SO4, filtered, concentrated, and purified by column chromatography (dichloromethane solution of methanol, 2% v / v) to give the title compound 0603-150 (180 mg, 48%) as a white solid. LCMS: 506 [M+1] + 。 11H NMR (400 MHz, DMSO-d6): 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).

[0181] Step 8d: 2-(((2-(4-Aminophenyl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl](methyl)amino)-N-hydroxypyrimidine-5-carboxamide (Compound 3)

[0182] Using a method similar to that described in Example 1, Compound 3 (43 mg, 26%) was prepared from 0603-150 (170 mg, 0.3 mmol) and freshly prepared hydroxylamine methanol solution (4 mL) as a yellow solid. Melting point: 183 - 186 °C. LCMS: 493 [M+1] + ; 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).

[0183] Example 9: PI3 Kinase Activity Assay

[0184] The following assay was used to determine the ability of Compound 1 to inhibit various isoforms and mutants of PI3K.

[0185] PI3Kα

[0186] The PI3Kα activity was measured using the ADP-Glo luminescent kinase assay. In a baculovirus-infected Sf9 cell expression system, a complex of P13Kα, a recombinant full-length human p110α with an N-terminal GST tag, and an untagged recombinant full-length human p85α (GenBank accession number of p110α is U79143; GenBank accession number of p85α is XM_043865) was co-expressed. The protein was purified by one-step affinity chromatography using glutathione-agarose. In the presence of purified recombinant PI3Kα (p110α / p85α) and PIP2, a competition assay was performed to measure the amount of ADP produced from ATP. PI3Kα was incubated with 20 μM PIP2 substrate at 30 °C for 30 minutes in reaction buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 3 μM sodium orthovanadate, 1 mM DTT, 10 μM ultrapure ATP, and 0.5% DMSO). Then the ADP produced in the reaction was measured by the ADP-Glo assay. This assay was carried out in two steps: first, an equal volume of ADP-GLO TM reagent (Promega) was added to terminate the kinase reaction and deplete the remaining ATP. In the second step, a kinase detection reagent was added, which simultaneously converted ADP to ATP. The newly synthesized ATP was measured using the coupled luciferase / luciferin reaction. In this assay, the IC 50 of compound 1 was measured to be less than 100 nM.

[0187] The ability of compound 1 to inhibit the PI3Kα mutants H1047R and E545K was also determined using the above general method. The IC 50 of both mutants was measured to be less than 100 nM.

[0188] PI3Kβ

[0189] Measure the activity of PI3Kβ by a time-resolved fluorescence resonance energy transfer (TR-FRET) assay using homogeneous time resolved fluorescence (HTRF) technology. In a baculovirus-infected Sf21 cell expression system, co-express a complex of P13Kβ, recombinant full-length human p110β with a histidine tag at the N-terminus, and untagged recombinant full-length human p85α (GenBank accession number of p110β is NM_006219; GenBank accession number of p85α is XM_043865). Purify the protein by one-step affinity chromatography using glutathione-agarose. In the presence of purified recombinant PI3Kβ (p110β / p85α), perform a competition assay to measure the amount of PIP3 produced from PIP2. Incubate PI3Kβ with 10 μM PIP2 substrate in a reaction buffer (20 mM HEPES, pH 7.5, 10 mM NaCl, 4 mM MgCl2, 2 mM DTT, 10 μM ATP, and 1% DMSO) at 30 °C for 30 minutes. Then, mix the reaction product with a PIP3 detector protein, europium-labeled antibody, biotin-labeled PIP3 probe, and allophycocyanin-labeled streptavidin. A sensor complex is formed to generate a stable TR-FRET signal in the reaction mixture. The signal intensity decreases as the biotin-labeled probe bound to the PIP3 detector is replaced by PIP3 produced by enzymatic activity, and the amount of unbound biotin-labeled PIP3 probe in the mixture increases. Measure the TR-FRET signal using a microplate reader with background subtraction function.

[0190] In this assay, the IC 50 of compound 1 was measured to be from 100 nM to 1000 nM.

[0191] PI3Kδ

[0192] The activity of PI3Kδ was measured using fluorescence polarization assay. In a baculovirus-infected Sf9 cell expression system, a complex of P13Kδ, a recombinant full-length human p110δ with a histidine tag at the N-terminus, and an untagged recombinant full-length human p85α (GenBank accession number of p110δ is NM_005026) was co-expressed. The protein was purified by one-step affinity chromatography using glutathione-agarose. In the presence of the purified recombinant PI3Kδ (p110δ / p85α), a competition assay was performed to measure the amount of PIP3 generated from PIP2. In the reaction buffer (20 mM HEPES (pH 7.5), 10 mM NaCl, 4 mM MgCl2, 2 mM DTT, 10 μM ATP and 1% DMSO), PI3Kδ was incubated with 10 μM PIP2 substrate at 30 °C for 1 hour. Then the reaction product was mixed with a PIP3 detector protein and a fluorescent PIP3 probe. As the binding of the fluorescent probe to the PIP3 detector was replaced by PIP3 generated by the enzymatic activity, the polarization (mP) value decreased and the amount of unbound fluorescent probe in the mixture increased. The polarization degree (mP) value was measured using a microplate reader with background subtraction function.

[0193] In this assay, the IC 50 of compound 1 was measured to be less than 100 nM.

[0194] PI3Kγ

[0195] The activity of PI3Kγ was measured by a time-resolved fluorescence resonance energy transfer (TR-FRET) assay using homogeneous time-resolved fluorescence (HTRF) technology. Human P13Kδ with a histidine tag at the N-terminus (GenBank accession number AF327656) was expressed in a baculovirus-infected Sf9 cell expression system. The protein was purified by one-step affinity chromatography using glutathione-agarose. In the presence of purified recombinant PI3Kγ (p120γ), a competition assay was performed to measure the amount of PIP3 generated from PIP2. PI3Kγ (2 nM) 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) at 30 °C for 30 minutes. The reaction product was then mixed with a PIP3 detector protein, an europium-labeled antibody, a biotin-labeled PIP3 probe, and allophycocyanin-labeled streptavidin. A sensor complex was formed to generate a stable TR-FRET signal in the reaction mixture. The signal intensity decreased as the biotin-labeled probe bound to the PIP3 detector was replaced by PIP3 generated by enzymatic activity, and the amount of unbound biotin-labeled PIP3 probe in the mixture increased. The TR-FRET signal was measured using a microplate reader with background subtraction function.

[0196] In this assay, the IC 50 of compound 1 was measured to be 100 to 1000 nM.

[0197] Example 10: HDAC Activity Assay

[0198] HDAC inhibitory activity was evaluated using the Biomol Color de Lys system (AK-500, Biomol, Plymouth Meeting, PA). Briefly, HeLa nuclear extract was used as the source of HDAC. Different concentrations of the test compound were serially diluted in dimethyl sulfoxide (DMSO) and added to the HeLa nuclear extract in the presence of a colorimetric artificial substrate. The final assay conditions included: 50 mM Tris / Cl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, and 1 mM MgCl2. The reaction was carried out at room temperature (25 °C) for 1 hour before termination by adding a developer. Relative enzyme activity was measured as fluorescence intensity (excitation: 350 - 380 nm; emission: 440 - 460 nm) in a WALLAC Victor II 1420 microplate reader. Data were analyzed using GraphPad Prism (v4.0a) with a sigmoidal dose-response curve for IC 50 calculation. In this assay, the IC 50Less than 100 nM.

[0199] The activity of Compound 1 against HDAC isotypes was also determined. The HDAC specificity assays were performed at BPS Bioscience (San Diego, CA) according to their standard operating procedures. Briefly, purified flag-(human HDAC-1), NCOR2-(human HDAC3), GST-(human HDAC4, HDAC6, HDAC7, HDAC10, and HDAC11), or His-(human HDAC2, HDAC5, HDAC8, and HDAC9)-tagged enzymes were expressed in Sf9 insect cells and purified before use. The substrate for HDAC1, HDAC2, HDAC3, HDAC6, HDAC7, HDAC8, HDAC9, and HDAC11 was HDAC Substrate 3 developed by BPS Bioscience. For other HDAC enzymes, a class 2a HDAC substrate was used. All enzymatic reactions were carried out at 37 °C for 30 minutes, in duplicate, except for the HDAC11 enzyme assay which was carried out at room temperature for 3 hours.

[0200] The following table lists the individual results for HDAC1-11, and the IC 50 values are provided as follows: I > 1000 nM; 100 nM < II < 1000 nM; 10 nM < III < 100 nM; IV < 10 nM.

[0201] HDAC 1 2 3 8 4 5 6 7 9 10 11 <![CDATA[IC 50 > IV IV IV II II II III II II IV IV

[0202] Example 11: Study of Compound 1 and anti-PD-1 in a mouse xenograft model - CTW26.WT mouse colon cancer cells

[0203] Animals: Female Balb / c mice, 8 weeks old, fed a high-fat diet.

[0204] Compound :

[0205] Compound 1: Administered orally (PO), 50 mg / kg, scheduled for 5 days of dosing followed by 2 days of rest (5 + 2-).

[0206] Isotype control: LEAF TM Purified rat IgG2a, obtained from BioLegend TM (San Diego, CA); Administered intraperitoneally (IP), 100 μg / mouse, twice a week (BIW).

[0207] PD-1 antibody: LEAF TM Rat anti-mouse CD279 (PD-1), clone 29F.1A12, obtained from BioLegend TM, (San Diego, California); IP administration, 100 μg / mouse, BIW.

[0208] Cell administration:

[0209] Collect CT26.WT cells from the flask and wash them once with RPMI 1640 without additives. Achieve the final concentration in plain medium (RPMI-1640), maintain on ice until administered to mice. On day 7 or 8, once the tumor is palpable, randomly group the mice by body weight. Start treatment with the antibody and immediately start Compound 1.

[0210] Grouping:

[0211]

[0212] Study schedule:

[0213] Monitor the animals until dosing begins, then take measurements twice a week during dosing until the tumor volume reaches the protocol limit or the ulceration is too severe. Collect tumor tissue and blood / plasma at the end of the study.

[0214] Results

[0215] The results of this study are shown in the table below and Figure 1 in. There were 7 evaluable mice in Groups 1 - 3. In Groups 4 - 6, all 8 mice were evaluable. Compound 1 alone at 50 mg / kg showed only a small tumor growth inhibition. Anti-PD-1 showed a greater tumor growth inhibitory effect, but still less than 50%. However, the combination of Compound 1 and anti-PD-1 showed almost complete inhibition of tumor growth.

[0216]

[0217] Example 12: Study of Compound 1 and anti-PD-1 in an A20 mouse xenograft model

[0218] Animals: Female Balb / c mice, 4 to 5 weeks old, fed regularly

[0219] Compound:

[0220] Compound 1: PO administration, 50 mg / kg or 100 mg / kg, scheduled for 5 days of dosing followed by 2 days off (5 + 2-).

[0221] Isotype control: LEAF TM Purified rat IgG2a, obtained from BioLegend TM (San Diego, California); IP administration, 100 μg / mouse, BIW, for 3 weeks.

[0222] Anti-PD-1 antibody: LEAF TM Rat anti-mouse CD279 (PD-1), clone 29F.1A12, obtained from BioLegend TM (San Diego, California); administered at 100 μg / mouse, BIW, for 3 weeks.

[0223] Vehicle: Compound 1 vehicle - 30% Captisol TM ; administered 5 + 2 -, PO, for 3 weeks.

[0224] Cell administration:

[0225] Collect A20 syngeneic cells from the flask and wash once with RPMI 1640 without additives. Achieve the final concentration in normal medium (RPMI-1640), maintain on ice until administered to mice. Implant 2 × 10 5 cells into the right flank of the animal. Start administration when the tumor exceeds 100 mm 3 (around day 13) and continue administration for 21 days or until the tumor reaches 2000 mm 3 limit.

[0226] Grouping:

[0227]

[0228] Research schedule :

[0229] When the tumor is fully palpable and measurable, start administering the animals around day 13. Continue administration until the tumor begins to show ulcers that require euthanasia. Collect the tumors before extensive necrosis for ELISA and flow analysis.

[0230] Results

[0231] The results of this study are presented in the table below and Figure 2 herein. Both 100 mg / kg of Compound 1 alone and anti-PD-1 alone showed significant tumor growth inhibitory effects. The combination of Compound 1 and anti-PD-1 showed a greater tumor growth inhibitory effect than either drug alone.

[0232]

[0233] The patents and scientific literature referred to herein establish the knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated herein by reference. All published foreign patents and patent applications cited herein are incorporated herein by reference. All other published references, documents, manuscripts, and scientific literature cited herein are incorporated herein by reference.

[0234] Although the present invention has been specifically shown and described with reference to preferred embodiments thereof, those skilled in the art will understand that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

Claims

Use of compound 1 represented by the following formula or a pharmaceutically acceptable salt thereof and a PD-1 signal transduction inhibitor in the preparation of a medicament for treating colon cancer 2. The use according to claim 1, wherein The PD-1 signal transduction inhibitor is one or more inhibitors among PD-1, PD-L1 and PD-L2.

3. Use according to claim 2, wherein, The PD-1 signal transduction inhibitor inhibits PD-1 or PD-L1.

4. The use according to claim 1, wherein The PD-1 signal transduction inhibitor is an anti-PD-1 monoclonal antibody, an anti-PD-L1 monoclonal antibody or an anti-PD-L2 monoclonal antibody.

5. The use according to claim 4, wherein, The PD-1 signal transduction inhibitor is pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab or pidilizumab.

6. The use according to claim 5, wherein, The PD-1 signal transduction inhibitor is pembrolizumab or nivolumab.

7. A pharmaceutical composition for treating colon cancer, wherein, The pharmaceutical composition comprises: (a) compound 1 represented by the following formula or a pharmaceutically acceptable salt thereof (b) a PD-1 signal transduction inhibitor; and (c) a pharmaceutically acceptable carrier or excipient.

8. The pharmaceutical composition according to claim 7, wherein, The PD-1 signal transduction inhibitor is one or more inhibitors among PD-1, PD-L1 and PD-L2.

9. The pharmaceutical composition according to claim 8, wherein, The PD-1 signal transduction inhibitor inhibits PD-1 or PD-L1.

10. The pharmaceutical composition according to claim 7, wherein The PD-1 signal transduction inhibitor is an anti-PD-1 monoclonal antibody, an anti-PD-L1 monoclonal antibody or an anti-PD-L2 monoclonal antibody.

11. The pharmaceutical composition according to claim 7, wherein, The PD-1 signal transduction inhibitor is pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab or pidilizumab.

12. The pharmaceutical composition according to claim 10, wherein, The PD-1 signal transduction inhibitor is pembrolizumab or nivolumab.

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