CN110563733A

CN110563733A - Imidazopyrazines as selective BTK inhibitors

Info

Publication number: CN110563733A
Application number: CN201910865228.XA
Authority: CN
Inventors: 朱孝云; 蒋维平
Original assignee: An Dikang (wuxi) Biotechnology Co Ltd
Current assignee: An Dikang (wuxi) Biotechnology Co Ltd
Priority date: 2019-09-12
Filing date: 2019-09-12
Publication date: 2019-12-13

Abstract

The present invention relates to imidazopyrazines as selective Bruton's Tyrosine Kinase (BTK) inhibitors. Specifically, the invention discloses deuterated compounds of (S) -4- (8-amino-3- (1- (butyl-2-alkynoyl) pyrrolidine-2-yl) imidazo [1,5-a ] pyrazine-1-yl) -N- (pyridine-2-yl) benzamide and optical isomers thereof, or pharmaceutically acceptable salts or pharmaceutical compositions containing the compounds, and application of the deuterated compounds in treating BTK mediated diseases.

Description

Imidazopyrazines as selective BTK inhibitors

Technical Field

Bruton's Tyrosine Kinase (BTK) is a non-receptor protein kinase of the Tec family expressed in B cells and bone marrow cells. BTK consists of Pleckstrin Homology (PH), Tec Homology (TH), Src homology 3(SH3), Src homology 2(SH2) and tyrosine kinase or Src homology 1(TK or SH1) domains. The function of BTK in signaling pathways activated by the engagement of B Cell Receptors (BCRs) in mature B cells with FCER1 on mast cells is well-defined. Functional mutations in BTK in humans result in primary immunodeficiency disease (X-linked agammaglobuinaemia), characterized by: there is a defect in the hindrance between the progenitor B cell and pre-B cell (pro-and pre-B cell) stages during B cell development. This results in the almost complete absence of B lymphocytes, resulting in a significant reduction of serum immunoglobulins of all kinds. These findings support a key role for BTK in the regulation of autoantibody production in autoimmune diseases.

BTK is expressed in many B-cell lymphomas and leukemias. Other diseases where dysfunctional B cells play an important role are B cell malignancies as described in Hendriks et al, nat. rev. cancer, 2014, 14, 219-231. The reported role of BTK in regulating B-cell proliferation and apoptosis suggests the potential of BTK inhibitors in the treatment of B-cell lymphomas. Thus, BTK inhibitors have evolved as potential therapies for many of these malignancies, as described in D' Cruz et al, oncotargetsan Therapy 2013, 6, 161-176. International patent application publication WO2013/010868 discloses BTK inhibitors comprising (S) -4- (8-amino-3- (1- (but-2-ynoyl) pyrrolidin-2-yl) imidazo [1,5-a ] pyrazin-1-yl) -N- (pyridin-2-yl) benzamide (code ACP-196) for use in therapy. ACP-196 as a second generation BTK covalent inhibitor has the characteristics of strong effect and high selectivity, but is easy to metabolize in vivo, the drug exposure is not high, the blood cell distribution is low, and the headache side effect is obvious.

The compound disclosed by the invention is a deuterated compound of ACP-196, and the deuterated compound overcomes the defects of the ACP-196 and has better medicinal properties through deuteration at a specific site: high drug exposure, long half-life in vivo, improved in vivo efficacy, reduced toxic and side effects, high blood cell distribution, reduced headache side effects, etc.

Detailed Description

Briefly, the present invention is directed to compounds useful as BTK inhibitors. The compounds of the present invention have the following structural formula (I) or are stereoisomers, prodrugs, active metabolites or pharmaceutically acceptable salts, solvates or crystalline forms thereof:

I. compound (I)

Wherein,

R¹Is hydrogen or deuterium;

R²is hydrogen or deuterium;

R³Is hydrogen or deuterium;

R⁴is hydrogen or deuterium;

R⁵Is hydrogen or deuterium;

R⁶Is hydrogen or deuterium;

R⁷Is hydrogen or deuterium;

L is-CH₃or-CD₃。

in another aspect, the invention relates to compounds of formula (I) wherein,

R¹Is hydrogen or deuterium;

R²Is hydrogen or deuterium;

R³is hydrogen or deuterium;

R⁴Is hydrogen or deuterium;

R⁵Is hydrogen or deuterium;

R⁶Is hydrogen or deuterium;

R⁷is hydrogen or deuterium;

L is-CH₃or-CD₃；

And R is¹、R²、R³、R⁴、R⁵、R⁶、R⁷At least one of which is deuterium.

In another aspect, the invention relates to compounds of formula (I) wherein,

R¹Is hydrogen or deuterium;

R²Is hydrogen or deuterium;

R³Is hydrogen or deuterium;

R⁴Is hydrogen or deuterium;

R⁵is hydrogen;

R⁶is hydrogen;

R⁷Is hydrogen;

L is-CH₃or-CD₃。

In another aspect, the invention relates to compounds of formula (I) wherein,

R¹Is hydrogen or deuterium;

R²Is hydrogen or deuterium;

R³is hydrogen or deuterium;

R⁴Is hydrogen or deuterium;

R⁵Is hydrogen;

R⁶Is hydrogen;

R⁷Is hydrogen;

L is-CH₃or-CD₃；

and R is¹、R²、R³、R⁴At least one of which is deuterium.

In still another aspect, the imidazopyrazine compound of formula I, a stereoisomer, a prodrug, an active metabolite, or a pharmaceutically acceptable salt, solvate, or crystalline form thereof according to the present invention is represented by any one of the following structural formulas:

The compounds of the invention are generally used in the form of the free acid or free base. Alternatively, the compounds of the present invention may be used in the form of acid or base salts. Acid addition salts of the free amino compounds of the present invention may be prepared by methods well known in the art and may be prepared from organic and inorganic acids. Suitable organic acids include maleic acid, fumaric acid, benzoic acid, ascorbic acid, succinic acid, methanesulfonic acid, acetic acid, trifluoroacetic acid, oxalic acid, propionic acid, tartaric acid, salicylic acid, citric acid, gluconic acid, lactic acid, mandelic acid, phenylacetic acid, aspartic acid, stearic acid, palmitic acid, glycolic acid, glutamic acid, and benzenesulfonic acid. Suitable inorganic acids include hydrochloric, hydrobromic, sulfuric, phosphoric and nitric acids. Base salts include salts formed with carboxylate anions and include salts formed with organic and inorganic cations such as those selected from alkali metal ions, alkaline earth metal ions (e.g., lithium, sodium, potassium, magnesium, barium, calcium), and ammonium ions, and substituted derivatives thereof (e.g., dibenzylammonium, benzylammonium, 2-hydroxyethylammonium, and the like). Thus, the term "pharmaceutically acceptable salts" of formula (I) shall include and all acceptable salt forms.

In addition, prodrugs are also included within the scope of the present invention. A prodrug is any covalently bound carrier that releases a compound of the general formula (I) in vivo when the prodrug is administered to a patient. Prodrugs are generally prepared by modifying functional groups in a manner that enables the modification to be cleaved, either by conventional exchange or in vivo, to yield the parent compound. Prodrugs include, for example, compounds of the present invention wherein a hydroxy, amino, or sulfhydryl group is bonded to any group that, when administered to a patient, is cleaved to yield the hydroxy, amino, or sulfhydryl group.

Thus, representative examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol and amine functional groups of compounds of formula (I). Further, in the case of carboxylic acid (-COOH), esters such as methyl ester, ethyl ester, propyl ester, and the like may be included. In the case of hydroxyl groups, mixed anhydrides such as methoxy, ethoxy, propoxy, tert-butoxy, and the like may be included. The effect of long-acting is achieved by the compounds of formula (I) and their metabolites forming chain fatty ethers, long-chain fatty esters or long-chain fatty amides via hydroxyl or amino groups with long-chain fatty alcohols or acids, such as pentanol or acid, hexanol or acid, heptanol or acid, octanol or acid, nonanol or acid, decanol or acid, lauryl alcohol or acid, stearyl alcohol or acid, and the like.

for stereoisomers, the compounds of formula (I) may have chiral centers and may exist as racemates, racemic mixtures, as well as individual enantiomers or diastereomers. All isomeric forms are included within the present invention, including mixtures thereof. Furthermore, certain crystalline forms of the compounds of formula (I) may exist in the form of polymorphs, which are also included in the present invention.

furthermore, some of the compounds of formula (I) may also form solvates with water or other organic solvents. Such solvates are likewise included within the scope of the present invention. It is known to those skilled in the art that charged compounds form hydrated species when lyophilized with water, or solvated species when concentrated in a solution containing a suitable organic solvent. The compounds of the present invention include hydrates or solvates of the listed compounds.

in another embodiment, modifications of the compounds of formula (I) or metabolites thereof using PROTAC technology are also within the scope of the invention. Specifically, the PROTAC technology designs a bifunctional small molecule, one end of which is a compound of the general formula (I), and the other end of which is connected with a ligand of E3 ubiquitin ligase through a connecting chain, to form a target-induced protein degradation complex. Because this degradation has a catalytic effect, a lower dosage can achieve efficient degradation and even solve the problem of resistance to BTK inhibitors. The compound of the general formula (I) and the protein degradation connecting arm comprise long-chain ethylene glycol with the length of 2-10, long-chain propylene glycol with the length of 2-10 and long-chain fatty alkane with the length of 2-10. A ligand of E3 ubiquitin ligase represented by any of the following structural formulae:

II. Application method

the use of a compound of the invention having the structural formula (I) or being a stereoisomer, prodrug, active metabolite or pharmaceutically acceptable salt, solvate or crystalline form thereof, in the manufacture of a medicament for the treatment of a Btk-mediated disease or a Btk-mediated condition.

the use of a compound of the invention having the structural formula (I) or being a stereoisomer, prodrug, active metabolite or a pharmaceutically acceptable salt, solvate or crystalline form thereof, in the manufacture of a medicament for the treatment of chronic B cell disorders in which T cells play a major role.

The use of a compound of the invention having the structural formula (I) or being a stereoisomer, prodrug, active metabolite, or pharmaceutically acceptable salt, solvate or crystalline form thereof, in the manufacture of a medicament for the treatment of a Btk-mediated disease or a Btk-mediated condition, including, but not limited to, the treatment of B-cell lymphoma caused by chronic active B-cell receptor signaling.

The compounds of the present invention have the structural formula (I) or are stereoisomers, prodrugs, active metabolites or pharmaceutically acceptable salts, solvates or crystalline forms thereof in the manufacture of a method for the treatment of solid tumor cancer, hematological malignancies, inflammatory diseases, autoimmune disorders, immunological disorders and other diseases

The pharmaceutical compositions described herein may be used in methods of treating diseases. In preferred embodiments, they are used to treat hyperproliferative disorders. They may also be used to treat other conditions described herein and in the following paragraphs.

in some embodiments, the present invention provides methods of treating a hyperproliferative disorder in a mammal, comprising: administering to the mammal a therapeutically effective amount of a compound having the structural formula (I) or a pharmaceutical composition which is a stereoisomer, prodrug, active metabolite, or pharmaceutically acceptable salt, solvate, or crystalline form thereof. In a preferred embodiment, the mammal is a human.

In some embodiments, the increase is excessiveThe reproductive disorder is cancer. In a preferred embodiment, the cancer is selected from the group consisting of chronic lymphocytic leukemia, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, andMacroglobulinemia.

In a preferred embodiment, the cancer is selected from the group consisting of non-Hodgkin's lymphoma (e.g., diffuse large B-cell lymphoma), acute myelogenous leukemia, thymus, brain, lung, squamous cell carcinoma, skin, eye, retinoblastoma, intraocular melanoma, oral and pharyngeal cancers, bladder, stomach, membrane, breast, cervical, head and neck, kidney, liver, ovary, prostate, colorectal, bone (e.g., metastatic bone), esophagus, testis, gynecological, thyroid, CNS, PNS, AIDS-related cancers (e.g., lymphoma and Kaposi's sarcoma), virally-induced cancers, e.g., cervical (human papilloma virus), B-cell lymphoproliferative diseases, and nasopharyngeal (Epstein-Barr virus), Kaposi's sarcoma and primary effusion lymphoma (Kaposi herpes virus), Hepatocellular carcinoma (hepatitis B and c viruses) and T cell leukemia (human T cell leukemia virus-1), B cell acute lymphoblastic leukemia, Burkitt leukemia, juvenile myelomonocytic leukemia, hairy cell leukemia, hodgkin's disease, multiple myeloma, mast cell leukemia, and mastocytosis.

in preferred embodiments, the methods relate to treating a non-cancerous hyperproliferative disorder, e.g., benign hyperplasia of the skin (e.g., psoriasis), restenosis, or a prostate disorder (e.g., Benign Prostatic Hypertrophy (BPH)). In some embodiments, the hyperproliferative disorder is an inflammatory, immune, or autoimmune disorder.

In some embodiments, the hyperproliferative disorder is selected from tumor angiogenesis, chronic inflammatory diseases, rheumatoid arthritis, atherosclerosis, inflammatory bowel diseases, skin diseases, e.g., psoriasis, eczemaAnd scleroderma, diabetes, diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, hemangioma, glioma and melanoma, ulcerative colitis, atopic dermatitis, pouchitis, spondyloarthritis, uveitis, Behcet's disease, rheumatoid arthritis, giant cell arteritis, sarcoidosis, kawasaki's disease, juvenile idiopathic arthritis, hidradenitis, syphilis, and combinations thereof,Syndrome, psoriatic arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, crohn's disease, lupus, and lupus nephritis.

The compounds of the present invention have structural formula (I) or are stereoisomers, prodrugs, active metabolites or pharmaceutically acceptable salts, solvates or crystalline forms thereof, which may be used alone or in combination with at least one other therapeutic agent in therapy. In some embodiments, the compounds, stereoisomers thereof, and pharmaceutically acceptable salts thereof, may be used in combination with at least one additional therapeutic agent. The at least one additional therapeutic agent may be selected, for example, from anti-hyperproliferative agents, anti-cancer agents, and chemotherapeutic agents. The compounds and/or one pharmaceutically acceptable salt disclosed herein may be administered with at least one other therapeutic agent in a single dosage form or as separate dosage forms. When administered as a separate dosage form, the at least one additional therapeutic agent can be administered prior to, concurrently with, or subsequent to the administration of the compound and/or one of the pharmaceutically acceptable salts disclosed herein.

Therapeutic agents include compounds used in "targeted therapy" and conventional chemotherapy. Suitable therapeutic agents may be selected from, for example, agents that cause apoptosis, polynucleotides (e.g., ribozymes); polypeptides (e.g., enzymes); a drug; a biological mimetic; an alkaloid; an alkylating agent; anti-tumor antibiotics; an antimetabolite; a hormone; a platinum compound; monoclonal antibodies that bind to anti-cancer drugs, toxins and/or radionuclides; biological response modifiers (e.g., interferons, such as IFN-a, and from interleukins, such as IL-2); adoptive immunotherapies; a hematopoietic growth factor; agents that cause tumor cell differentiation (e.g., all-trans retinoic acid); a gene therapy agent; antisense therapeutic agents and nucleotides; a tumor vaccine; and angiogenesis inhibitors.

Examples of therapeutic agents include erlotinib (b)Genentech/OSI Pharm.); bortezomib (b)Millennium Pharm.); fulvestrant (f)AstraZeneca); sunitinib (A)pfizer); letrozole (C)Novartis); imatinib mesylate (Novartis); PTK787/ZK 222584 (Novartis); oxaliplatin (Eloxatin (r) of Sanofi); 5-FU (5-fluorouracil); calcium folinate rapamycin (Sirolimus,Wyeth); lapatinib (A)GSK572016, Glaxo Smith Kline); lonafarnib (SCH 66336); sorafenib (Bayer); dolafinil (zuozu jing); irinotecan (Pfizer) and Gefitinib (b)AstraZeneca); AG1478, AG1571(SU 5271, Sugen); tyroxate (AZD9291 AstraZeneca); alkylating agents, e.g. thiotepa andCyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimine and methyl melamine (melaminee) such as hexamethyl melamine, blue ethylene melamine, triethylenephosphoramide, triethylenethiophosphoramide and trihydroxymethyl melamine; polyacetyls (e.g., bullatacin (bullatacin) and bullatacin); camptothecin (e.g., a synthetic analog of topotecan); bryostatins; callystatin; CC-1065 and its synthetic analogs of Aldocosan, Kazelesin and Bizelesin. Cryptophycin (such as cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycins and their synthetic analogs, such as KW-2189 and CB1-TM 1; eleutherobin (eleutherobin); pancratistatin; sarcodictyin; soft sponges (spongistatin); nitrogen mustards, such as chlorambucil, chloromazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine oxide hydrochloride, melphalan, novembichin, benzene mustarch cholesterol (phenesterine), prednimustine, trimesic cyclophosphamide, uracil mustard; nitrosoureas such as carmustine, chlorourethrin, fotemustine, lomustine, nimustine and ranimustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin γ 11 and calicheamicin ω 11(Angew chem. lntl. Ed. Engl. (1994)33: 183-186)); a dynemicin, such as dynemicin a; diphosphonates, such as clodronate; an esperamicin; and neocarzinostatin chromophores, and related chromophoric groups of chromoprotein enediyne antibiotics, aclacinomycin, actinomycin (actinomycin), aurramycin, azaserine, bleomycin, actinomycin C (cactinomycin), carabicin, caminomycin, carcinomycin (carzinophilin), chromomycin, actinomycin D (dactinomycin), daunorubicin (unomycin), ditorexin (detorubicin), 6-diazo-5-oxo-L-norleucine, and related chromophoric protein enediyne type antibiotics,(doxorubicin), morpholinyl-doxorubicin, cyanomorpholine-doxorubicin, 2-pyrrolinyl-doxorubicin and deoxydoxorubicin), epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), maytansinoids (marcellmycin), mitomycins such as mitomycin C, mycophenolic acid, nogalamycin (nogalamycin), olivomycin (olivomycin), pelomycin, pofiomycin (porfiromycin), puromycin, trirubicin (quelamycin), rodobicin (rodorubicin), streptonigrin, streptogramin, tubercidin (tubicidin), ubenimex, setrestatin (zinostatin), zorubicin (zorubicin); antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thioguanine (thiamirine), thioguanine: pyrimidine analogs such as cyclocytidine, azacitidine, 6-azauridine (6-azauridine), carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as carposterone (calusterone), drostanolonepropionate (dromostanoloneprionate), epithioandrostanol, mepiquane (mepiquitane), testolactone; anti-adrenaline, such as aminoglutethimide, mitotane, trostane; folic acid supplements such as folinic acid (Frolinic acid); a glucal lactone; an aldehydic phosphoramide glycoside; aminolevulinic acid, eniluracil (eniluracil); an acrylic acid ester; bestrabuucil; bisantrene; edatraxate; defofamine; decarbonylation of colchicine (demecolcine); diziquone (diazizquone); elformithine; ammonium etitanium acetate (etiolate); an epothilone; etoglut (etoglucid); gallium nitrate; a hydroxyurea; lentinan; lonidainine; maytansinoids (maytansinoids), such as maytansine (maytansine) and ansamitocins (ansamitocins); mitoguazone (mitoguzone); mitoxantrone; mopidanmol; nitrerine; pentostatin; phenamet (phenamett); doxorubicin; losoxantrone (losoxantrone); podophylically oxalic acid (podophylically); 2-ethyl hydrazide; (ii) procarbazine;polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane (rizoxane); rhizomycin (rhizoxin); sizofuran; germanium spiroamines (spirogyranium); tenuazonic acid (tenuazonic acid); triimine quinone (triaziquone); 2,2' -trichlorotriethylamine; distamycin (e.g., T-2 toxin, veracurin A, fisetin A (roridin A), and serpentin (anguidine)); a urethane; vindesine; (ii) a dacarbazine; mannitol mustard; dibromomannitol (mitobronitol); dibromogalactitol (mi-tolactol); myogenesis (piproman); a polycytidysine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa is a taxane, for example,(paclitaxel; Bristol-Myers Squibb Oncology, Princeton, N.J.),(hydrogenated herb oil free), albumin engineered paclitaxel nanoparticle formulations (Amcran Pharmaceutical Partners, Schaumberg, lll.), and(doxetaxel；Rhone-Poulenc Rorer,Antony,France)；chloranmbucil；(gemini capecitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin, carboplatin; vinblastine etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;(vinorelbine); brewing with rice support; (ii) teniposide; edatrexate (edatrexate); daunomycin; aminopterin; capecitabineibandronic acid; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoids, such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the foregoing.

The therapeutic agent may also be selected from, for example, (i) anti-hormonal agents used to modulate or inhibit hormonal effects on tumors, such as anti-estrogens, and Selective Estrogen Receptor Modulators (SERMs), including, for example, tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifene, troxifene, keoxifene, LY117018, onapristone, and(toremifine citrate); (ii) aromatase inhibitors which inhibit aromatase and which modulate estrogen production in the adrenal gland, e.g., 4(5) -imidazole, aminoglutethimide, dihydrocarzinone, dihydrocarzin,(megestrol acetate),(exemestane; Pfizer), formastanie, fadrozole,(vorozole), letrozole, and anastrozole; (iii) antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and troxacitabine (troxaci tabine) (1, 3-dioxolane nucleoside platinum cytosine analogues); (iv) protein kinase inhibitors; (v) a lipid kinase inhibitor; (vi) antisense oligonucleotides, such as gene expression in signaling pathways that inhibit abnormal cell proliferation, such as PKC- α, Ralf, and H-Ras; (vii) ribozymes, such as VEGF expression inhibitors (e.g.,) And inhibitors of HER2 expression; (viii) vaccines, e.g. gene therapy vaccines, e.g.AndrIL-2; topoisomerase 1 inhibitors, e.g.rmRH; (ix) anti-angiogenic agents, e.g. bevacizumab (C)Genentech); and (x) pharmaceutically acceptable salts, acids and derivatives of any of the foregoing.

The therapeutic agent may also be selected from therapeutic antibodies, such as alemtuzumab (Campath), bevacizumab (bGenentech), cetuximab (Imclone); panitumumab (A)Amgen), rituximab (Genentech/Biogen Idec), pertuzumab (2C4, Genentech), trastuzumab (genentech), tositumomab (Bexxar, Corixia), and antibody drug conjugate, gemtuzumab ozogamicin (b)Wyeth)。

Humanized monoclonal antibodies having potential therapeutic effects which bind to the compounds of the present invention and stereoisomers thereof, pharmaceutically acceptable salts thereof, may be selected, for example, from alemtuzumab, aprezumab, aselizumab, atlizumab, bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumab, cedeluzumab, certuzumab, cidfutuzumab, ciduzumab, daclizumab, eculizumab (eculizumab), efuzumab, epratuzumab (epratuzumab), eculizumab (felvizumab), fontolizumab (fonticumab), gemuzumab, oxepirubicin, indouzumab, iotuzumab, flunomizumab, facillipin (ipilimumab), lapidarubizumab, rituzumab (rituximab), gemumab, gemuzumab, rituximab, rituzumab (rituximab), rituzumab (rituximab), rituzumab, rituximab, rituzumab, rituximab, inozumab, and the like, Pexizumab (pexizumab), ralizumab, ranibizumab, relivizumab, relilizumab, resyvizumab, rovelizumab, lullizumab (ruplizumab), sibutrumab (sibutrumab), hipril zumab (siplizumab), soltelizumab, tikituzumab (tacatuzumab tetani), taduzumab (taduzumab), talibizumab, tefibuzumab, tolbizumab, toralizumab, trastuzumab, tuzumab cellullein, tucusuzumab, umazumab, urotuzumab, ursuzumab (vizizumab), and viilizumab (viilizumab).

III、Preparation

Pure forms or suitable pharmaceutical compositions of the compounds of the present invention or pharmaceutically acceptable salts thereof may be administered by any acceptable mode of administration of agents that serve similar utilities. The pharmaceutical compositions of the present invention may be prepared by combining a compound of the present invention with a suitable pharmaceutically acceptable carrier, diluent or excipient, and may be formulated into solid, semi-solid, liquid or gaseous form preparations such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres and aerosols. Typical routes of administration for the pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal administration. As used herein, the term parenteral includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. The pharmaceutical compositions of the present invention are formulated to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. One or more dosage units in the composition to be administered to a subject or patient, wherein, for example, a capsule can be a single dosage unit and a container containing a compound of the invention in aerosol form can hold a plurality of dosage units. The actual methods of preparing the dosage forms are known, or will be known, to those skilled in the art. The compositions to be administered will contain, in any event, a therapeutically effective amount of a compound of the present invention, or a pharmaceutically acceptable salt thereof, in order to treat the disease or condition of interest in accordance with the teachings of the present invention.

Pharmaceutical compositions

the pharmaceutical compositions of the present invention may be in solid or liquid form. In one aspect, the carrier is a microparticle, such that the composition is in the form of, for example, a tablet or powder. The carrier can be a liquid and the composition can be, for example, an oral syrup, an injectable liquid, or an aerosol suitable for administration, for example, by inhalation. When intended for oral administration, the pharmaceutical composition is preferably selected from solid or liquid forms, wherein semi-solid, semi-liquid, suspension and gel forms are included in the forms considered herein to be solid or liquid. For oral solid compositions, the pharmaceutical compositions can be formulated in the form of powders, granules, compressed tablets, pills, capsules, chewable tablets, powder tablets, and the like. Such solid compositions typically contain one or more inert diluents or edible carriers. In addition, one or more binders such as carboxymethyl cellulose, ethyl cellulose, microcrystalline cellulose, xanthan gum or gelatin; excipients, such as starch, lactose or dextrin; disintegrating agents, such as alginic acid, sodium alginate, Primogel, corn starch, and the like; lubricants, such as magnesium stearate or hydrogenated vegetable oil (Sterotex); glidants, such as colloidal silicon dioxide; sweetening agents, such as sucrose or saccharin; a flavoring agent, such as peppermint, methyl salicylate, or orange flavoring; and a colorant.

When the pharmaceutical composition is in the form of a capsule, for example a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier, for example polyethylene glycol or an oil. The pharmaceutical composition may be in liquid form, such as a tincture, syrup, solution, emulsion, or suspension. Such liquids may be administered orally, or delivered by injection, as two examples. When intended for oral administration, the compositions preferably contain one or more of sweeteners, preservatives, dyes/colorants and flavor enhancers in addition to the compounds of the present invention. In compositions intended for administration by injection, one or more of surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizing agents and isotonicity agents may be included.

The liquid pharmaceutical compositions of the present invention, whether in solution, suspension or other similar form, may include one or more adjuvants selected from sterile diluents such as water for injection, physiological saline solutions, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils (e.g., synthetic mono-or diglycerides which may be used as a solvent or suspending medium), polyethylene glycols, glycerol, propylene glycol and the like; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for adjusting tonicity such as sodium oxide or dextrose. Parenteral formulations can be packaged in ampules, disposable syringes or multi-dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. The injectable pharmaceutical composition is preferably sterile.

Liquid pharmaceutical compositions of the invention intended for parenteral or oral administration should contain an amount of a compound of the invention such that a suitable dosage is obtained. The pharmaceutical compositions of the invention may be intended for topical administration, in which case the carrier preferably comprises a solution, emulsion, ointment or gel base. For example, the base may comprise one or more of paraffin oil, lanolin, polyethylene glycol, beeswax, mineral oil, diluents (e.g., water and alcohol), and emulsifiers and stabilizers. Thickeners may be present in the pharmaceutical compositions for topical administration. If transdermal administration is intended, the composition may comprise a transdermal patch or an iontophoretic device.

The pharmaceutical compositions of the invention may be intended for rectal administration, in the form of suppositories for example, which will melt in the rectum and release the drug. Compositions for rectal administration may contain an oily base as a suitable non-irritating excipient. Such bases include, but are not limited to, lanolin, cocoa butter, and polyethylene glycols.

the pharmaceutical compositions of the present invention may include a variety of substances that alter the physical form of the solid or liquid dosage unit. For example, the composition may include a material that forms a coating shell around the active ingredient. The material forming the coating shell is generally inert and may be selected from, for example, sugars, shellac, and other enteric coating agents. Alternatively, the active ingredient may be encapsulated in a gelatin capsule.

The pharmaceutical compositions of the invention in solid or liquid form may include an agent that binds to the compound of the invention and thereby aids in the delivery of the compound. Suitable agents with this capability include monoclonal or polyclonal antibodies, proteins or liposomes.

the pharmaceutical compositions of the present invention may be comprised of dosage units that can be administered in the form of an aerosol. The term aerosol is used to denote a variety of systems ranging from the jelly-like variety to systems consisting of pressurized packs. Delivery may be by liquefied or compressed gas, or by a suitable pump system that dispenses the active ingredient. Aerosols of the compounds of the invention may be delivered as single, two, or three phase systems to deliver the active ingredient. The delivery side of the aerosol comprises the necessary containers, actuators, valves, sub-containers, etc., which together may form a kit. Preferred aerosols are determined by one of skill in the art without undue experimentation.

the pharmaceutical compositions of the present invention can be prepared by methods well known in the pharmaceutical arts. For example, pharmaceutical compositions to be administered by injection may be prepared by combining a compound of the invention with sterile distilled water to form a solution. Surfactants may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with the compounds of the present invention, thereby facilitating dissolution or uniform suspension of the compounds in an aqueous delivery system.

The compounds of the present invention, or pharmaceutically acceptable salts thereof, are administered in a therapeutically effective amount, which will vary depending on a variety of factors, including the activity of the particular compound employed; metabolic stability and length of action of the compound; the age, weight, general health, sex, and diet of the patient; mode and time of administration; the rate of excretion; a pharmaceutical composition; the severity of the particular disorder or condition; and individuals undergoing therapy.

The compounds of the present invention or pharmaceutically acceptable derivatives thereof can also be administered simultaneously with, before, or after the administration of one or more other therapeutic agents. Such combination therapy includes the administration of a single pharmaceutical dosage formulation containing a compound of the present invention and one or more other active agents, as well as the administration of separate pharmaceutical dosage formulations of a compound of the present invention with each active agent itself. For example, a compound of the invention and another active agent can be administered to a patient in a single orally administered composition (e.g., a tablet or capsule), or each agent can be administered as a separate orally administered formulation. In the case of separate administration formulations, the compound of the invention and the one or more additional active agents may be administered substantially at the same time (i.e., simultaneously) or at separately staggered times (i.e., sequentially); combination therapy is understood to include all such regimens.

The pharmaceutical combination of the present invention can be administered in a dose adjusted depending on the disease state, the administration route, the age or body weight of the patient. For oral administration to adults, the dosage is usually 0.1-100 mg/kg/day, preferably 1-20 mg/kg/day. The appropriate dose of the present invention should be set in consideration of the age, body weight, condition, administration route, etc. of the patient, and the oral administration is usually in the range of 0.05-100 mg/kg/day, preferably 0.1-10 mg/kg/day. When administered parenterally, the dosage of the pharmaceutical composition of the present invention varies widely depending on the route of administration, but is usually in the range of 0.005 to 10 mg/kg/day, preferably 0.01 to 1 mg/kg/day.

the dose of the compound of the present invention varies depending on the administration method, age, body weight, condition of a patient and the kind of disease, but in general, in the case of oral administration, the dose per 1 day for an adult is about 0.05mg to 3000mg, preferably about 0.1mg to 1000mg, and more preferably 10mg to 500 mg. Can be administered separately as required. In addition, in the case of parenteral administration, the dose is about 0.01mg to 1000mg, preferably about 0.05mg to 100mg per 1 day for an adult. It is to be understood that in the present invention, combinations of substituents and/or variables of the formulas are permissible only if they result in stable compounds.

general Synthesis

the imidazopyrazines of the invention can be prepared by methods of common general knowledge in the field of organic chemistry. If desired, the reaction product is optionally purified using conventional techniques including, but not limited to, filtration, distillation, crystallization, chromatographic separation and purification. Such materials are optionally characterized by conventional means including physical constants and spectral data.

Imidazopyrazines of formula I can be prepared by the general synthetic route shown in scheme I.

VI. examples

certain preferred embodiment aspects of the invention are shown by way of illustration in the following non-limiting examples. The reagents are commercially available or prepared according to literature procedures.

mass spectrum: electrospray mass spectra were recorded on a Waters single quadrupole mass spectrometer using flow injection in alternating positive and negative ion mode. The mass range was 120-2000Da, the capillary voltage was set at 4500V, and nitrogen was used for atomization.

LC-MS spectrometer detector, Waters, PDA (200-320nm), mass detector: ZQ

Eluent: a is acetonitrile containing 0.05 percent of trifluoroacetic acid; acetonitrile/water-1/9 (v/v) with 0.05% trifluoroacetic acid.

Example 1.

The synthetic route is as follows:

Intermediate 2: (3-chloropyrazin-2-yl) methylamine, hydrochloride

3-chloropyrazine-2-carbonitrile (32g) was dissolved in 300ml of acetic acid, 14g of Raney nickel water as a 50% slurry, slowly added to the above solution, and stirred at room temperature overnight with 4.5bar of hydrogen. Filtering with diatomite, spin-drying the filtrate, adding water into ethyl acetate to obtain a solid, dissolving the solid in ethyl acetate at 50 ℃, introducing hydrogen chloride gas into the ethyl acetate solution, separating out the solid, filtering, washing a filter cake with ethyl acetate, and drying in vacuum at 50 ℃ to obtain 23g of (3-chloropyrazin-2-yl) methylamine hydrochloride.

Intermediate 3: L-N-Cbz-2-deuteroproline

11.6g of L-2-deuterium proline (purchased from Nanjing Lei positive medicine science and technology Co., Ltd., deuterium substitution rate of 98.7%) is dissolved in 50ml of water, 25.5g of Cbz-Cl THF solution is dropwise added at room temperature, 2mol/L of sodium hydroxide solution is dropwise added at the same time, ph9-10 is maintained, reaction is carried out at room temperature for 1 hour after dropwise addition is finished, dichloromethane is added for extraction, a dichloromethane layer is dried in a spinning mode, and L-N-Cbz-2-deuterium proline is obtained, wherein the yield is 77%.

Intermediate 4: (S) -2- ((3-chloropyrazin-2-yl) methylcarbamoyl) -2-deuterium pyrrolidine-1-carboxylic acid benzyl ester 10g (3-chloropyrazin-2-yl) methylamine, hydrochloride and 10.5g L-N-Cbz-2-deuterium proline are suspended in 400ml dichloromethane, the reaction liquid is cooled to 0-5 ℃, 24ml triethylamine is added dropwise, stirring is carried out for 15min while maintaining the temperature at 0-5 ℃, and 17g 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethylurea hexafluorophosphate is added. The mixture was stirred at 0-5 ℃ for 1 hour. The reaction solution was washed with 0.1M hydrochloric acid, sodium bicarbonate solution, brine, dried, concentrated and subjected to flash column chromatography to give 11.9g of (S) -2- ((3-chloropyrazin-2-yl) methylcarbamoyl) -2' -deuteropyrrolidine-1-carboxylic acid benzyl ester.

intermediate 5: (S) -2- (8-Chloroimidazo [1,5-a ] pyrazin-3-yl) -2-deuteropyrrolidine-1-carboxylic acid benzyl ester

11.8g of (S) -2- ((3-chloropyrazin-2-yl) methylcarbamoyl) -2-deuteropyrrolidine-1-carboxylic acid benzyl ester was dissolved in 100ml of acetonitrile, 11ml of 1, 3-dimethyl-2-imidazolidinone was added, the temperature was reduced to 0 to 5 ℃, phosphorus oxychloride was added dropwise, the mixture was stirred for 30min while being used for 15min, the temperature was maintained at 5 to 10 ℃, and then the mixture was refluxed for 8 hours. After TLC detection reaction is completed, the reaction solution is poured into ammonia water-ice water and stirred, and the system ph8-9 is obtained. Extraction was carried out with ethyl acetate, washed with saturated brine, and concentrated by spin-drying to obtain 10g of crude product. Flash column chromatography over silica gel with petroleum ether/ethyl acetate as eluent was carried out to obtain 8.6g of (S) -2- (8-chloroimidazo [1,5-a ] pyrazin-3-yl) -2-deuteropyrrolidine-1-carboxylic acid benzyl ester.

intermediate 6: (S) -2- (1-bromo-8-chloroimidazo [1,5-a ] pyrazin-3-yl) -2-deuteropyrrolidine-1-carboxylic acid benzyl ester

44.5g of intermediate 5 are dissolved in 500ml of DMF, 22g of NBS are added in portions, the reaction is stirred at room temperature for 4.5h, and the completion of the reaction is checked by TLC. Adding the reaction solution into water at 10-15 deg.C, extracting with ethyl acetate, washing with saturated salt solution twice, and concentrating to dry. Flash column chromatography over silica gel with petroleum ether/ethyl acetate as eluent was carried out to obtain 42.6g of (S) -2- (1-bromo-8-chloroimidazo [1,5-a ] pyrazin-3-yl) -2-deuterium pyrrolidine-1-carboxylic acid benzyl ester.

Intermediate 7: (S) -2- (1-bromo-8-aminoimidazo [1,5-a ] pyrazin-3-yl) -2-deuteropyrrolidine-1-carboxylic acid benzyl ester

Adding 6.3g of intermediate 6 into a 200ml pressure reactor, suspending the mixture in 80ml of isopropanol, introducing ammonia gas for 20min until the mixture is saturated, closing the pressure reactor, heating the mixture to 110 ℃, reacting for 4hr, cooling the mixture to room temperature, introducing ammonia gas for 20min again, closing the pressure reactor, heating the mixture to 110 ℃, reacting for 4hr, and repeating for 3 times in total. After the reaction, dichloromethane extraction, water washing 2 times, saturated salt water washing 2 times. Concentration gave 4.9g of benzyl (S) -2- (1-bromo-8-aminoimidazo [1,5-a ] pyrazin-3-yl) -2-deuteropyrrolidine-1-carboxylate.

Intermediate 8: n- (pyridin-2-yl) -4- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzamide

dissolving 20g of 4- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzoic acid in 350ml of dichloromethane, cooling to 0-5 ℃, adding 1ml of DMF, dropwise adding 16ml of oxalyl chloride, and stirring at 0-5 ℃ for 20 min; the mixture was warmed to room temperature and stirred for 2 hr. The reaction was concentrated, with 2 Xdichloromethane. 21.5g of the acid chloride are obtained and used directly in the next reaction.

dissolving the above reaction solution in 20ml dichloromethane, cooling to 0-5 deg.C, adding 4.5g 2-aminopyridine into the above solution in batches, heating the reaction solution to room temperature, and stirring for 2 hr. Adding 3% citric acid aqueous solution for extraction, washing a dichloromethane layer with 3% citric acid aqueous solution, drying and concentrating to obtain the off-white solid N- (pyridine-2-yl) -4- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzamide, wherein the yield is 88%.

Intermediate 9: (S) -benzyl 2- (8-amino-1- (4- (pyridin-2-ylcarbamoyl) phenyl) imidazo [1,5-a ] pyrazin-3-yl) -2-deuteropyrrolidine-1-carboxylate

To the reaction flask were added 8g of intermediate 7, 6.8g of intermediate 8, 90ml of 2N potassium carbonate solution, 230ml of 1, 4-dioxane, and stirred at room temperature until clear. 3.5g of Pd (dppf) Cl was added₂Heating to 87 deg.C, refluxing, stirring for 1hr, detecting by TLC to complete reaction, cooling to 25 deg.C, adding 300ml water and 300ml ethyl acetate. The mixture was filtered through celite, extracted, and the ethyl acetate layer was washed with saturated brine. The organic layer is dried, concentrated and purified by flash column chromatography on silica gel using methanol/dichloromethane as eluent to give 8.3g of (S) -2- (8-amino-1- (4- (pyridin-2-ylcarbamoyl) phenyl) imidazo [1,5-a]Pyrazin-3-yl) -2-deuteropyrrolidine-1-carboxylic acid benzyl ester, LC-Ms ESI +: 535.3[ M + H]⁺.

¹H-NMR(DMSO-d6,300MHz):2.03-2.32(4H,m),3.54-3.85(2H,m),4.87(2H,s),6.14-6.23(2H,br),6.90-6.99(1H,m),7.14-7.24(3H,m),7.38(2H,s),7.71-7.91(4H,m),8.12(2H,m),8.39-8.48(2H,m),8.76(1H,s),10.85(1H,br).

Intermediate 10: (S) -4- (8-amino-3- (2-deuteropyrrolidin-2-yl) imidazo [1,5-a ] pyrazin-1-yl) -N- (pyridin-2-yl) benzamide

9g of intermediate 9, adding 230ml of 33 percent hydrobromic acid/acetic acid, stirring for clarification, reacting at room temperature for 1 hour, detecting complete reaction by TLC, dropwise adding sodium bicarbonate solution to adjust the pH to 9, adding water and dichloromethane, extracting, washing with sodium bicarbonate for 2 times, washing with water for 2 times, and concentrating a dichloromethane layer to obtain 5.81g of (S) -4- (8-amino-3- (2-deuteropyrrolidine-2-yl) imidazo [1,5-a ] in an amount of 5.81g]pyrazin-1-yl) -N- (pyridin-2-yl) benzamide, LC-Ms ESI +: 401.2[ M + H]⁺.

example 1: (S) -4- (8-amino-3- [ (1-but-2-ynoyl) -2-deuteropyrrolidin-2-yl ] imidazo [1,5-a ] pyrazin-1-yl) -N- (pyridin-2-yl) benzamide

4g of 2-alkynylbutyric acid and 6.2g N-hydroxybenzotriazole are mixed in 30ml of ethyl acetate, the temperature is reduced to-5 ℃, and 20ml of DCC (9.5g) ethyl acetate solution is added dropwise. Heating to 0-5 deg.C, and reacting for 4 hr. Filtering, washing a filter cake with ethyl acetate, and concentrating under reduced pressure to obtain a benzotriazole 2-alkyne butyl ester intermediate.

2g of intermediate 10 was suspended in 40ml of dichloromethane and cooled to 0-5 ℃. 1g of benzotriazole 2-propargyl butyl ester intermediate is dissolved in 20ml of dichloromethane and is dropwise added to the suspension of the intermediate 10. Reacting at 0-5 deg.C for 1hr, and detecting by TLC to complete reaction. The solution was warmed to room temperature, quenched with water, separated, and washed with brine. The organic layer was concentrated under reduced pressure to give a yellow solid. Purification by flash column chromatography on silica gel with ethyl acetate-methanol as eluent gave example 1as a yellow solid, 1.8 g. Deuteration rate 98.6%, LC-Ms ESI +: 467.1[ M + H]⁺

¹H-NMR(DMSO-d6,300MHz):1.65-2.02(3H,s),2.09-2.16(1H,m),2.19-2.45(2H,m),3.40(1H,m),3.50-3.85(2H,m)，6.14-6.23(2H,br),7.14-7.18(2H,m),7.75-7.91(4H,m),8.14(2H,m),8.23-8.45(2H,m),10.85(1H,br).

Example 2.

The synthetic route is as follows:

Intermediate 12: L-N-Cbz-2, 3, 3, 4, 4, 5, 5-heptadeuteroproline

The process for the preparation of intermediate 12 is as described for the preparation of intermediate 3, except that L-2-deuteroproline is replaced by L-2, 3, 3, 4, 4, 5, 5-heptadeuteroproline (available from south beijing plus pharmaceutical science co., ltd., deuteration rate 96.8%).

intermediate 13: (S) -2- ((3-Chloropyrazin-2-yl) methylcarbamoyl) -2, 3, 3, 4, 4, 5, 5-heptadeuteropyrrolidine-1-carboxylic acid benzyl ester

intermediate 13 was prepared as described for intermediate 4 except that L-N-Cbz-2-deuteroproline was substituted for intermediate 12.

The procedure for the preparation of intermediate 14 was as for the preparation of intermediate 5 except that benzyl (S) -2- ((3-chloropyrazin-2-yl) methylcarbamoyl) -2-deuteropyrrolidine-1-carboxylate was replaced with intermediate 13.

Intermediate 15 was prepared as described for intermediate 6 except intermediate 5 was replaced with intermediate 14.

intermediate 16 was prepared as described for intermediate 7 except intermediate 6 was replaced with intermediate 15.

Intermediate 17 was prepared as described for intermediate 9 except intermediate 7 was replaced with intermediate 16.

Intermediate 18 was prepared as described for intermediate 10 except intermediate 9 was replaced with intermediate 17.

Example 2: (S) -4- (8-amino-3- [ (1-but-2-ynoyl) -2, 3, 3, 4, 4, 5, 5-heptadeuteropyrrolidin-2-yl ] imidazo [1,5-a ] pyrazin-1-yl) -N- (pyridin-2-yl) benzamide

2g of intermediate 18 are suspended in 40ml of dichloromethane and cooled to 0-5 ℃. 1g of benzotriazole 2-propargyl butyl ester intermediate is dissolved in 20ml of dichloromethane and is dropwise added to the suspension of the intermediate 18. Reacting at 0-5 deg.C for 1hr, and detecting by TLC to complete reaction. The solution was warmed to room temperature, quenched with water, separated, and washed with brine. The organic layer was concentrated under reduced pressure to give a yellow solid. Purification by flash column chromatography on silica gel with ethyl acetate-methanol as eluent gave example 2 as a yellow solid, 1.7 g. Deuteration rate is 96.7%, LC-Ms ESI +: 473.1[ M + H]⁺

¹H-NMR(DMSO-d6,300MHz):1.66-2.04(3H,s)，6.15-6.24(2H,br),7.15-7.19(2H,m),7.75-7.91(4H,m),8.15(2H,m),8.24-8.45(2H,m),10.85(1H,br).

Example 3.

The synthetic route is as follows:

Intermediate 19: L-N-Cbz-2, 5, 5-Trideuteroproline

The preparation of intermediate 19 was as described for intermediate 3 except that L-2-deuterium proline was replaced with L-2, 5, 5-trideutero proline (available from Nanjing Renzhen medical science and technology, Inc., with a deuteration rate of 81.8%).

The procedure for the preparation of intermediate 20 was as for the preparation of intermediate 4, except that L-N-Cbz-2-deuterium proline was replaced by intermediate 19.

The procedure for the preparation of intermediate 21 was as for the preparation of intermediate 5, except that benzyl (S) -2- ((3-chloropyrazin-2-yl) methylcarbamoyl) -2-deuteropyrrolidine-1-carboxylate was replaced with intermediate 20.

The preparation of intermediate 22 was as described for intermediate 6 except that intermediate 5 was replaced with intermediate 21.

Intermediate 23 was prepared as described for intermediate 7 except intermediate 6 was replaced with intermediate 22.

Intermediate 24 was prepared as described for intermediate 9 except intermediate 7 was replaced with intermediate 23.

Intermediate 25 was prepared as described for intermediate 10 except intermediate 9 was replaced with intermediate 24.

Example 3: (S) -4- (8-amino-3- [ (1-but-2-ynoyl) -2, 5, 5-trideuteropyrrolidin-2-yl ] imidazo [1,5-a ] pyrazin-1-yl) -N- (pyridin-2-yl) benzamide

2g of intermediate 18 are suspended in 40ml of dichloromethane and cooled to 0-5 ℃. 1g of benzotriazole 2-propargyl butyl ester intermediate is dissolved in 20ml of dichloromethane and is dropwise added to the suspension of the intermediate 25. Reacting at 0-5 deg.C for 1hr, and detecting by TLC to complete reaction. The solution was warmed to room temperature, quenched with water, separated, and washed with brine. The organic layer was concentrated under reduced pressure to give a yellow solid. Flash column chromatography on silica gel eluting with ethyl acetate-methanol afforded example 3 as a yellow solid1.5 g. Deuteration rate 80.4%, LC-Ms ESI +: 469.1[ M + H]⁺

¹H-NMR(DMSO-d6,300MHz):1.65-2.04(3H,s)，2.10-2.15(1H,m),2.18-2.43(2H,m),3.39(1H,m),6.14-6.25(2H,br),7.16-7.18(2H,m),7.74-7.91(4H,m),8.14(2H,m),8.23-8.45(2H,m),10.84(1H,br).

example 4.

The synthetic route is as follows:

Example 4: (S) -4- (8-amino-3- [ (1-but-4, 4, 4-trideuterio-2-alkynoyl) -2-deuteropyrrolidin-2-yl ] imidazo [1,5-a ] pyrazin-1-yl) -N- (pyridin-2-yl) benzamide

4g of 4, 4, 4-trideuter-2-alkyne butyric acid (purchased from Nanjing Lei Zheng medicine science and technology Co., Ltd., deuteration rate of 96.6%) and 6.2g N-hydroxybenzotriazole were mixed in 30ml of ethyl acetate, the temperature was reduced to-5 ℃, and 20ml of DCC (9.5g) ethyl acetate solution was added dropwise. Heating to 0-5 deg.C, and reacting for 4 hr. Filtering, washing filter cakes with ethyl acetate, and concentrating under reduced pressure to obtain a benzotriazole 4, 4, 4-tridedeuterium-2-alkyne butyl ester intermediate.

2g of intermediate 18 are suspended in 40ml of dichloromethane and cooled to 0-5 ℃. 1g of benzotriazole 4, 4, 4-trideuterio-2-alkyne butyl ester intermediate is dissolved in 20ml of dichloromethane and is dripped into the suspension of the intermediate 10. Reacting at 0-5 deg.C for 1hr, and detecting by TLC to complete reaction. The solution was warmed to room temperature, quenched with water, separated, and washed with brine. The organic layer was concentrated under reduced pressure to give a yellow solid. Purification by flash column chromatography on silica gel with ethyl acetate-methanol as eluent gave example 4 as a yellow solid, 1.6 g. Deuterium substitution rate of 94.2%, LC-Ms ESI +: 470.1[ M + H]⁺

¹H-NMR(DMSO-d6,300MHz):2.10-2.16(1H,m),2.20-2.45(2H,m),3.42(1H,m),3.57-3.85(2H,m)，6.15-6.26(2H,br),7.15-7.18(2H,m),7.78-7.92(4H,m),8.15(2H,m),8.25-8.48(2H,m),10.88(1H,br).

example 5.

the synthetic route is as follows:

example 5: (S) -4- (8-amino-3- [ (1-but-4, 4, 4-trideuterio-2-ynoyl) -2, 3, 3, 4, 4, 5, 5-heptadeuteropyrrolidin-2-yl ] imidazo [1,5-a ] pyrazin-1-yl) -N- (pyridin-2-yl) benzamide

4g of 4, 4, 4-trideuter-2-alkyne butyric acid and 6.2g N-hydroxybenzotriazole are mixed in 30ml of ethyl acetate, the temperature is reduced to-5 ℃, and 20ml of DCC (9.5g) ethyl acetate solution is dripped. Heating to 0-5 deg.C, and reacting for 4 hr. Filtering, washing filter cakes with ethyl acetate, and concentrating under reduced pressure to obtain a benzotriazole 4, 4, 4-tridedeuterium-2-alkyne butyl ester intermediate.

2g of intermediate 18 are suspended in 40ml of dichloromethane and cooled to 0-5 ℃. 1g of benzotriazole 4, 4, 4-trideuterio-2-alkyne butyl ester intermediate is dissolved in 20ml of dichloromethane and is dripped into the suspension of the intermediate 18. Reacting at 0-5 deg.C for 1hr, and detecting by TLC to complete reaction. The solution was warmed to room temperature, quenched with water, separated, and washed with brine. The organic layer was concentrated under reduced pressure to give a yellow solid. Purification by flash column chromatography on silica gel with ethyl acetate-methanol as eluent gave example 5 as a yellow solid, 1.4 g. Deuterium substitution rate of 93.3%, LC-Ms ESI +: 476.1[ M + H]⁺

¹H-NMR(DMSO-d6,300MHz):6.16-6.28(2H,br),7.17-7.20(2H,m),7.79-7.925(4H,m),8.16(2H,m),8.27-8.49(2H,m),10.89(1H,br).

Example 6.

The synthetic route is as follows:

Example 6: (S) -4- (8-amino-3- [ (1-but-4, 4, 4-trideuterio-2-ynoyl) -2, 5, 5-trideuterio-pyrrolidin-2-yl ] imidazo [1,5-a ] pyrazin-1-yl) -N- (pyridin-2-yl) benzamide

Example 6 the procedure was as for example 5 except that intermediate 18 was replaced with intermediate 25 to give the yellow compound example 6 with a deuteration of 77.5%, LC-Ms ESI +: 472.1[ M + H]⁺

¹H-NMR(DMSO-d6,300MHz):2.08-2.15(1H,m),2.17-2.44(2H,m),3.37(1H,m),6.13-6.26(2H,br),7.14-7.17(2H,m),7.73-7.92(4H,m),8.13(2H,m),8.22-8.46(2H,m),10.83(1H,br).

example 7.

The synthetic route is as follows:

Example 8: (S) -4- (8-amino-3- [ (1-but-4, 4, 4-trideuterio-2-ynoyl) -pyrrolidin-2-yl ] imidazo [1,5-a ] pyrazin-1-yl) -N- (pyridin-2-yl) benzamide

EXAMPLE 8 preparation Process referring to the preparation of example 5, except that intermediate 18 was replaced with (S) -4- (8-amino-3- (pyrrolidin-2-yl) imidazo [1, 5-a)]Pyrazin-1-yl) -N- (pyridin-2-yl) benzamide example 8, a yellow compound was obtained with a deuteration of 96.3%, LC-Ms ESI +: 469.1[ M + H]⁺

ACP-196：

ACP-196 was prepared according to the method for preparing Compound 1 of the example.

Ms:ESI 466.1[M+H]⁺.

Table compounds:

Using a similar protocol to that above, the following table compounds and their single enantiomers were prepared.

Table 1: structure of table compound

Biological activity evaluation:

test 1BTK kinase Activity and BTK-WT (wild-type) kinase Selectivity assay

(1) BTK kinase enzyme assay

The compounds of the examples were tested for inhibition of BTK kinase (aa2-659, Carna Biosciences) in an assay based on the time-resolved fluorescence resonance energy transfer (TR-FRET) method. Assay in a 384-well Low volume blackboard in a reaction mixture containing BTK kinase, 5. mu.M ATP, 2. mu.M peptide substrate and 0-10. mu.M compound in 50mM Tris pH7.4, 10mM MgCl₂、2mM MnCl₂0.1mM EDTA, 1mM DTT, 0.005% Tween-20, 20nM SEB and 0.01% BSA. The kinase was incubated with the compound for 60 minutes at room temperature and the reaction was initiated by addition of ATP and peptide substrate. After 60 minutes reaction at room temperature, an equal volume of stop/detection solution was added according to the manufacturer's instructions. The stop/detection solution contained Eu in a buffer solution containing 50mM HEPES pH7.0, 800mM KF, 20mM EDTA and 0.1% BSA³⁺Cryptate-conjugated monoclonal antibody (PT66) was directed against phosphotyrosine and XL 665-conjugated streptavidin. The plates were sealed and incubated at room temperature for 1 hour, and the TR-FRET signal (ratio of fluorescence emitted at 665nm to that emitted at 620nm, excitation at 337nm wavelength) was recorded on a PHERAStar FS plate reader (BMG Labtech). Phosphorylation of the peptide substrate results in binding of the anti-phosphotyrosine antibody to the biotinylated peptide substrate, which will give rise to a fluorescence donor (Eu)³⁺cryptate) is placed very close to the acceptor (streptavidin-XL 665), thereby resulting in a high degree of fluorescence resonance energy transfer from the donor fluorophore (at 620nm) to the acceptor fluorophore (at 665 nm). Inhibition of BTK kinase activity results in a decrease in TR-FRET signal. IC of the Compound₅₀Data were fit to a four parameter logistic equation (logistic equation) by Graphpad Prism software.

(2) BTK-WT (wild-type) kinase enzyme assay

BTK-WT (wild type) assay at 5, 25, 100 μ M university in south of Jiang for the example compounds, referenced to the BTK kinase assay method, by using the TR-FRET assay and the corresponding peptide as substrate in the south of JiangConducted by southern university. Measurement and preincubation for 1 hour to determine Compound K of example_i。

the compounds disclosed in the present invention all exhibit BTK inhibitory activity IC₅₀below 1 μ M, representative examples of which have biological activities as follows.

table 2: enzyme inhibitory activity of the compounds of the examples.

As a result: the compounds of the examples showed significant BTK inhibitory activity and showed varying degrees of selectivity for wild-type BTK. The selectivity index, represented by example compounds 1 and 2, was 3.4 times that of ACP-196. It is shown that the compounds of the examples have superior therapeutic effects and lower toxic side effects than ACP-196.

Test 2 BTKpY223 cell activity assay method:

The BTKpY223 cellular assay is an HTRF-based assay, intended to quantitatively determine endogenous levels of phosphorylation at BTK Tyr 223. Phosphorylated Tyr223 is essential for complete activation of BTK. The assay was performed in the lymphoma cell line Ramos (Ramos) cells (CRL-1596, ATCC) in the BTKpY223 assay kit (Cisbio).

Ramus cells were serum starved for 2 hours in RPMI 1640 with 0.5% FBS. After starvation, cells were incubated with example compounds to CO at different concentrations₂Detection was performed in the incubator for 1 hour. After inoculation, the cells were treated with 1mM Perovanadate (PV) or Na₃VO₄(OV) stimulation for 20 min. Then, cells were spun down and lysed with 1x lysis buffer for 10 min at room temperature (4 x lysis buffer was provided in the kit). During incubation, a 1 Xantibody mixture was prepared by diluting anti-BTK-d 2 and anti-pBTK-K in detection buffer. 2 ul/well of 1 Xantibody mixture was dispensed into optPlate-384 assay plates (Perkin Elmer). Thereafter, 18. mu.L of cell lysate was transferred to the antibody pre-loaded solution for assayand (5) fixing the plate. After gentle mixing and simple rotation, the plates were sealed and kept in the dark at room temperature for 18 hours. Fluorescence emission was measured at two different wavelengths (665nm and 620nm) on a compatible HTRF reader. The potency of compound 1 was calculated based on the inhibition of the ratio between the signal intensities at 665nm and 620 nm. IC (integrated circuit)₅₀Values were calculated with GraphPadPrism software using a sigmoidal dose response function.

Table 3: BTKpY223 cell activity of the example compounds.

As a result: the compounds of the examples have better effect on inhibiting proliferation of lymphoma cell line Ramos cells than ACP-196, especially the compounds 1, 2 and 4 have the cell activity more than 12 times that of ACP-196. Test 3 pharmacokinetic Studies of the Compounds of the examples

In the experimental process, rats are randomly grouped, 6 SD rats with the age of 7-8 weeks in each group are fasted for 8-12 h, and then 100mg/kg of sample CMC-Na solution is sequentially gavaged (ig.) respectively, wherein a small amount of drinking water is gavaged before/after administration of each rat so as to balance the water-soluble environment of the gastrointestinal tract among individuals. After administration to rats, about 300. mu.l of venous blood from rats before and 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12, 24, 36, 48, 72, 96, 144, 168h before and after administration was collected from orbital venous plexus into a blood collection tube (the blood collection tube was anticoagulated with 0.5% heparin in advance), and after centrifugation at 8000rpm for 5min, supernatant plasma was transferred, and analyzed by injection after treatment, and the concentration of plasma samples with time was determined. High performance liquid chromatography-quadrupole tandem mass spectrometry (including Shimadzu high performance liquid chromatography (LC-20A), American AB mass spectrometry (API4000), electrospray ion source, and Analyst 1.5.1 workstation).

data analysis parameter C_max(peak plasma drug concentration), T_max(time to peak) can be determined from plasma drug concentration-timeDirectly obtaining in the curve; AUC_0-tn(area under the plasma concentration-time curve from t0 to tn) was calculated by the trapezoidal method, t0 representing the instant after administration, tn representing the time of sampling of the last quantifiable concentration; AUC_0→∞(area under the plasma concentration-time curve from t0 to infinity time) calculated as eq.1, AUC_0→∞＝AUC_0→tn+ Ctn/λ z (eq.1), where Ctn is the last quantifiable plasma concentration and λ z (terminal elimination rate constant) is determined from the slope of the linear portion at the end of the log plasma concentration-time curve; the elimination half-life (t1/2) is calculated according to eq.2, t1/2 is 0.693/λ z (eq.2); the Mean Residence Time (MRT) is calculated by eq.3, MRT ═ AUMC/AUC; apparent clearance (CL/F) is calculated as eq.4, CL/F ═ Dosage/AUC0 → ∞ (eq.4), wherein Dosage denotes the dose administered; the apparent volume of distribution (V/F) is calculated by eq.5, where V/F is CL/λ z (eq.5).

Table 4: pharmacokinetic parameters of the compounds of the examples.

Example Compound #	1	2	ACP-196
				C_max(ng/ml)	933	882	763
T_max(h)	0.75	0.69	0.66
				AUC_0→12h(ng*h/ml)	2625.8	2833.1	691
t_1/2(h)	5.66	6.85	1.76
				F(％)	40.6	38.3	29.8

as a result: the half-life of the example compounds 1 and 2 is significantly higher than that of ACP-196, and the oral absorption availability is significantly improved, especially the AUC of the example compounds 1 and 2_0→12hIs 3.8 times and 4.1 times of ACP-196, greatly improving the exposure of the medicine in vivo. The compounds of the examples can be used in clinical application in a significantly reduced dosage or with a longer dosage interval and corresponding fewer toxic side effects.

TEST 4 efficacy Studies of the example Compounds in the TMD-8 xenograft model

The tumor transplantation method comprises the following steps:

animals were pre-treated with cyclophosphamide (prepared in saline, i.p.) i.p. at 150 mpk) and disulfiram (prepared in 0.8% Tween 80/saline, 100mpk oral gavage (p.0.), 1 hour after each dose of cyclophosphamide) for 2 days, once daily. The animals were then inoculated with TMD-8 cells 24 hours after the second dose of cyclophosphamide. On the day of transplantation, the cell culture medium was replaced with fresh medium. After 4 hours, the medium was removed and the cells were harvested as described above. The cells were resuspended in cold (4 ℃) PBS and the same volume of matrigel was added to give a final concentration of 2.5X10⁷individual cells/ml. Inoculation ofPreviously, resuspended cells were placed on ice. Before cell inoculation, the right underarm area of each mouse was cleaned with 75% ethanol. Each animal was injected subcutaneously 5X10 in 200. mu.l of cell suspension via a 26-gauge needle in the right front flank⁶And (4) cells.

For in vivo efficacy studies, the volume of the transplanted tumor grows to 100-300 mm³On the left and right, animals were randomly divided into a desired number of groups of 10 mice each. Mice were treated twice a day (BID) with vehicle (0.5% carboxymethylcellulose (CMC) + 0.2% Tween 80) and different dose levels of compound for 14 days. Treatment was by oral gavage (p.o.) at a volume of 10ml/kg body weight. Body weight was assessed immediately prior to dosing and the dose volume adjusted accordingly. Tumor volume was measured twice weekly in two dimensions using calipers (in this study from day 11 post-inoculation). Tumor volume was calculated using the formula V ═ 0.5 (aXb)²) Wherein a and b are the major and minor diameters of the tumor, respectively. Statistical analysis was performed using the student T-test (student T-test). P<0.05 was considered statistically significant. The relative tumor proliferation rate T/C% (guideline reference value less than or equal to 40%) was calculated. One person was responsible for tumor measurements throughout the duration of the study. Body weight was also recorded twice weekly. After the last administration, the red ears, frequent scratching of forelimbs, crouching and reduced activity of the mice are counted. Clinical indications of mouse toxicity were also monitored daily for the duration of the study.

As a result: examples compounds 1 and 2 have a relative tumor proliferation rate T/C% significantly less for TMD-8 xenograft tumors than ACP-196, again demonstrating the advantage of the efficacy of the compounds at the animal level. The body weight of mice in the example compound group is increased, and the body weight of ACP-196 group is reduced, which shows that the example compound is lower in toxicity, and animals have better tolerance and compliance.

After 14 days of treatment, the red ears and frequent head scratching of forelimbs of the mice are shown as grade 3 in the ACP-196; the phenomenon of crouching and reduced activity is shown as level 2; both of these phenomena are associated with headache side effects of drugs. The red ears and frequent pronation of forelimbs of mice in the example group are shown as grade 1, and no crouching and reduced activity are found, which indicates that the compound of the example has small side effect on headache, higher tolerance in clinical application and better compliance of patients.

It will be appreciated by those skilled in the art that the present disclosure is not limited to the foregoing illustrative embodiments, but may be embodied in other specific forms without departing from the essential attributes thereof. It is therefore intended that the embodiments described herein are to be considered in all respects as illustrative and not restrictive, the embodiments referenced by the appended claims being other than the foregoing embodiments, the references being made to the appended claims rather than to the foregoing examples, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

All patents, patent applications, and literature references cited in this specification are hereby incorporated by reference in their entirety. In the event of inconsistencies, the present disclosure, including definitions, will be convincing.

Claims

1. The present invention provides a compound of formula I, or a stereoisomer, prodrug, active metabolite, or pharmaceutically acceptable salt, solvate, or crystalline form thereof:

Wherein:

R¹Is hydrogen or deuterium;

R²Is hydrogen or deuterium;

R³Is hydrogen or deuterium;

R⁴Is hydrogen or deuterium;

R⁵Is hydrogen or deuterium;

R⁶Is hydrogen or deuterium;

R⁷is hydrogen or deuterium;

L is-CH₃or-CD₃。

2. The compound of claim 1, wherein:

R¹Is hydrogen or deuterium;

R²is hydrogen or deuterium;

R³is hydrogen or deuterium;

R⁴Is hydrogen or deuterium;

R⁵is hydrogen or deuterium;

R⁶Is hydrogen or deuterium;

R⁷Is hydrogen or deuterium;

L is-CH₃or-CD₃；

3. The compound of claim 1, wherein:

R¹Is hydrogen or deuterium;

R²Is hydrogen or deuterium;

R³is hydrogen or deuterium;

R⁴Is hydrogen or deuterium;

R⁵Is hydrogen;

R⁶Is hydrogen;

R⁷Is hydrogen;

L is-CH₃or-CD₃；

and R is¹、R²、R³、R⁴at least one of which is deuterium.

4. The compound as provided in claim 1, or a stereoisomer, prodrug, active metabolite, or pharmaceutically acceptable salt, solvate, or crystalline form thereof, wherein the compound is represented by any one of the following structural formulae:

5. A compound according to any one of claims 1 to 4 which has activity to inhibit Btk, corresponding to an IC of 1 μ M or less in a Btk kinase assay₅₀。

6. a pharmaceutical composition comprising a therapeutically effective amount of a compound of any one of claims 1 to 4 in unit dosage form and one or more pharmaceutically acceptable carriers.

7. A combination comprising a therapeutically effective amount of a compound according to any one of claims 1 to 4 and another agent having therapeutic activity against an autoimmune and/or inflammatory disease or cancer.

8. Use of a compound of any one of claims 1 to 4, an N-oxide thereof, or a prodrug thereof, for the manufacture of a medicament for treating a bruton's tyrosine kinase (Btk) -mediated disorder, wherein the Btk-mediated disorder is an allergic disease, an autoimmune disease, an inflammatory disease, or cancer.

9. Use of a compound of any one of claims 1 to 4, an N-oxide thereof, or a prodrug thereof, for the manufacture of a medicament for treating a bruton's tyrosine kinase (Btk) -mediated condition, wherein the Btk-mediated condition is selected from: rheumatoid arthritis, psoriatic arthritis, infectious arthritis, progressive chronic arthritis, teratogenic arthritis, osteoarthritis, traumatic arthritis, gouty arthritis, Reiter's syndrome, polychondritis, acute synovitis, spondylitis, glomerulonephritis with nephrotic syndrome, glomerulonephritis without nephrotic syndrome, autoimmune hematologic disorders, hemolytic anemia, aplastic anemia, idiopathic thrombocytopenia, neutropenia, autoimmune gastritis, autoimmune inflammatory bowel disease, ulcerative colitis, Crohn's disease, host versus graft disease, allograft rejection, chronic thyroiditis, graves ' disease, scleroderma, type I diabetes, type II diabetes, acute active hepatitis, chronic active hepatitis, pancreatitis, primary biliary cirrhosis, progressive chronic arthritis, rheumatoid arthritis, osteoarthritis, traumatic arthritis, rheumatoid arthritis, leitis, leishmaniasis, leiomyelitis, rheumatoid arthritis, leishmaniasis, rheumatoid arthritis, leishmaniasis, rheumatoid arthritis, myasthenia gravis, multiple sclerosis, systemic lupus erythematosus, psoriasis, atopic dermatitis, contact dermatitis, itching due to dampness, sunburn of the skin, vasculitis, Behcet's disease, chronic renal insufficiency, Stevens-Johnson syndrome, inflammatory pain, idiopathic steatorrhea, cachexia, sarcoidosis, Guillain-Barr syndrome, uveitis, conjunctivitis, keratoconjunctivitis, otitis media, periodontal disease, pulmonary interstitial fibrosis, asthma, bronchitis, rhinitis, sinusitis, pneumoconiosis, pulmonary insufficiency syndrome, emphysema, pulmonary fibrosis, trachoma, chronic inflammatory lung disease, chronic obstructive pulmonary disease, proliferative diseases, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), Mantle Cell Lymphoma (MCL), B-cell chronic lymphocytic leukemia, acute lymphoblastic leukemia with mature B cells, chronic lymphocytic leukemia, chronic inflammatory pulmonary disease, chronic inflammatory disease, chronic obstructive pulmonary disease, chronic inflammatory disease, chronic, B cell lymphomas caused by chronic active B cell receptor signaling and bone disorders associated with multiple myeloma.

10. The use of claims 8-9, wherein the Btk-mediated condition is a B-cell proliferative disease selected from chronic lymphocytic lymphoma, non-hodgkin's lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, and chronic lymphocytic leukemia.