CN108349974B - Substituted pyridine amide compound and application thereof - Google Patents

Abstract

A substituted pyridine amide compound and application thereof. Specifically discloses a deuterated pyridine amide compound shown in formula (I) and a pharmaceutical composition containing the compound or a crystal form, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, a hydrate or a solvate thereof. The compound can be used as a JAK inhibitor, and further can be suitable for preparing medicines for treating JAK-related diseases (such as autoimmune diseases and the like).

Description

Substituted pyridine amide compound and application thereof

Technical Field

The invention belongs to the field of medicine. The invention relates to a deuterated pyridine amide compound and application thereof, in particular to a pyridine amide compound and application thereof as a JAK inhibitor or for treating and preventing diseases related to JAK enzyme.

Background

Janus kinases (JAKs) are cytoplasmic tyrosine kinases that transduce cytokine signals from membrane receptors to STAT transcription factors. Four JAK family members have been described in the prior art: JAK1, JAK2, JAK3 and TYK 2. When cytokines bind to their receptors, JAK family members are autophosphorylated and/or transphosphorylated to each other, followed by phosphorylation of STATs, which then migrate into the nucleus to regulate transcription. JAK-STAT intracellular signaling is applicable to interferons, most interleukins, and a variety of cytokines and endocrine factors, such as EPO, TPO, GH, OSM, LIF, CNTF, GM-CSF, and PRL.

Cartilage degeneration is a hallmark of many diseases, of which rheumatoid arthritis and osteoarthritis are the most prominent. Rheumatoid Arthritis (RA) is a chronic degenerative disease of the joints characterized by inflammation and destruction of the joint structure. When the disease is not inhibited, substantial disability and pain, and even premature death, result from loss of joint functionality. Therefore, the aim of RA treatment is not only to delay the disease but also to obtain remission, thereby terminating joint destruction. In addition to the severity of the disease outcome, the high prevalence of RA (globally 0.8% of adults are afflicted) means a high socioeconomic impact.

Osteoarthritis (OA) is the most common form of arthritis, characterized by loss of articular cartilage, usually accompanied by hypertrophy of bone and pain.

Osteoarthritis is difficult to treat. Currently, there is no cure, and treatment focuses on relieving pain and preventing diseased joint deformity. Common treatments include the use of non-steroidal anti-inflammatory drugs. Although nutraceuticals such as chondroitin and glucosamine sulfate have been identified as safe and effective options for the treatment of osteoarthritis, recent clinical trials have shown that both treatments do not reduce pain associated with osteoarthritis. Filgotinib is a highly selective JAK1 inhibitor which is researched, discovered and developed by Galapagos, and has the advantages of quick response, high curative effect and good safety and tolerance for treating Rheumatoid Arthritis (RA) and Crohn's Disease (CD) in combination with acquired clinical data.

There is therefore still a need to develop new compounds for the treatment of degenerative joint diseases. The compounds of the invention are useful in the treatment of degenerative joint diseases such as osteoarthritis, rheumatoid arthritis and osteoporosis, in particular osteoarthritis. In addition, the present invention provides compounds, methods for their preparation and pharmaceutical compositions comprising the compounds of the invention and a suitable pharmaceutical carrier. The invention also provides the use of a compound of the invention in the manufacture of a medicament for the treatment of degenerative joint diseases.

Disclosure of Invention

The invention aims to provide a novel compound with JAK inhibitor effect and a preparation method thereof.

The invention provides a deuterated picolinamide compound shown as a formula (I), and physiologically acceptable salts, solvates, hydrates, prodrugs, tautomers and stereoisomers thereof, including mixtures of the compounds in all proportions.

In the formula:

each R is independently selected from the group consisting of "hydrogen (H), deuterium (D)";

and the physiologically acceptable salts, solvates, hydrates, prodrugs, tautomers and stereoisomers thereof, including the mixtures of these compounds in all ratios.

In another alternative, the deuterium isotope content of deuterium at the deuterium position is at least greater than the natural deuterium isotope content (0.015%), preferably greater than 30%, more preferably greater than 50%, more preferably greater than 75%, more preferably greater than 95%, more preferably greater than 99%.

In particular, R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²The deuterium isotope content in each deuterated position is at least 5%, preferably greater than 10%, more preferably greater than 15%, more preferably greater than 20%, more preferably greater than 25%, more preferably greater than 30%, more preferably greater than 35%, more preferably greater than 40%, more preferably greater than 45%, more preferably greater than 50%, more preferably greater than 55%, more preferably greater than 60%, more preferably greater than 65%, more preferably greater than 70%, more preferably greater than 75%, more preferably greater than 80%, more preferably greater than 85%, more preferably greater than 90%, more preferably greater than 45%, more preferably greater than 50%, more preferably greater than 55%, more preferably greater than 60%, more preferably greater than 65%, more preferably greater than 70%, more preferably greater than 75%, more preferably greaterPreferably greater than 95%, more preferably greater than 99%.

In another alternative, R of the compound of formula (I)¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹And R²²Preferably, at least one of R comprises deuterium, more preferably two of R comprises deuterium, more preferably three of R comprises deuterium, more preferably four of R comprises deuterium, more preferably five of R comprises deuterium, more preferably six of R comprises deuterium, more preferably seven of R comprises deuterium, more preferably eight of R comprises deuterium, more preferably nine of R comprises deuterium, more preferably ten of R comprises deuterium, more preferably eleven of R comprises deuterium, more preferably twelve of R comprises deuterium, more preferably thirteen of R comprises deuterium, more preferably fourteen of R comprises deuterium, more preferably fifteen of R comprises deuterium, more preferably sixteen of R comprises deuterium, more preferably seventeen of R comprises deuterium, more preferably eighteen of R comprises deuterium, more preferably nineteen of R comprises deuterium, more preferably twenty of R comprises deuterium.

In another alternative, R¹、R²、R³、R⁴、R⁵Each independently is deuterium or hydrogen.

Preferably, R¹、R²、R³、R⁴And R⁵Is deuterium.

In another alternative, R⁶、R⁷、R⁸Each independently is deuterium or hydrogen

Preferably, R⁶Is deuterium.

Preferably, R⁷Is deuterium.

Preferably, R⁸Is deuterium.

In another alternative, R⁹、R¹⁰、R¹¹、R¹²Each independently is deuterium or hydrogen.

Preferably, R⁹Is deuterium.

Preferably, R¹¹Is deuterium.

In another alternative, R¹³、R¹⁴Each independently of the otherAnd is deuterium or hydrogen.

Preferably, R¹³、R¹⁴Is deuterium.

In another alternative, R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²Each independently is deuterium or hydrogen.

Preferably, R¹⁵、R¹⁶Is deuterium.

Preferably, R¹⁷、R¹⁸Is deuterium.

Preferably, R¹⁹、R²⁰Is deuterium.

Preferably, R²¹、R²²Is deuterium.

Preferably, R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²Is deuterium.

In another alternative, the compound is selected from the group consisting of, but not limited to, the following compounds or pharmaceutically acceptable salts thereof:

the compounds of the present invention do not include non-deuterated compounds.

The compounds of the present invention are novel JAK inhibitors that exhibit significantly improved potency in vivo compared to structurally similar compounds. In a particular embodiment, the compounds of the invention are JAK1 and JAK2 inhibitors.

In another aspect, the invention provides a pharmaceutical composition comprising a compound of the invention and a pharmaceutical carrier, excipient or diluent. Moreover, the compounds of the present invention used in the pharmaceutical compositions and methods of treatment disclosed herein are pharmaceutically acceptable for preparation and use. In this aspect of the invention, the pharmaceutical composition may also contain other active ingredients suitable for use in combination with the compounds of the invention.

In another aspect of the invention, the invention provides a method of treating a mammal susceptible to or infected with those conditions listed herein, particularly conditions associated with aberrant JAK activity, such as inflammation, autoimmune diseases, proliferative diseases, transplant rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6, comprising administering a therapeutically effective amount of a pharmaceutical composition or compound of the invention as described herein. In a particular embodiment, the disorder is associated with aberrant JAK1 and JAK2 activity.

In another aspect, the present invention provides a compound of the invention for use in the treatment or prevention of a disorder selected from those listed herein, in particular those disorders that may be associated with abnormal JAK activity, such as inflammation, autoimmune diseases, proliferative diseases, transplant rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL 6.

In yet another therapeutic method aspect, the invention provides a method of treating a mammal susceptible to or infected with a condition associated with aberrant JAK activity described herein, comprising administering a pharmaceutical composition or compound of the invention described herein in an amount effective to treat the condition or prevent the condition. In a particular aspect, the disorder is etiologically associated with aberrant JAK1 and JAK2 activity.

In another aspect, the present invention provides compounds of the invention for use in the treatment or prevention of a disorder associated with aberrant JAK activity.

In another aspect, the present invention provides methods for synthesizing the compounds of the present invention using the representative synthetic schemes and routes disclosed hereinafter.

It is therefore a primary object of the present invention to provide novel compounds which can modify the activity of JAKs and thereby prevent or treat any disease which may be associated therewith. In a particular aspect, the compounds of the invention may modulate the activity of JAK1 and JAK 2.

It is another object of the present invention to provide compounds that can treat or ameliorate the symptoms of diseases or diseases such as inflammation, autoimmune diseases, proliferative diseases, transplant rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and diseases associated with hypersecretion of IL6 that may be associated with the activity of JAKs, particularly JAK1 and JAK 2.

It is another object of the present invention to provide pharmaceutical compositions useful in the treatment or prevention of various disease states including diseases associated with JAK activity such as inflammation, autoimmune diseases, proliferative diseases, transplant rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and diseases associated with hypersecretion of IL 6. In a particular embodiment, the disease is particularly associated with JAK1 and JAK2 activity.

Detailed description of the preferred embodiment

Detailed Description

Definition of

The following terms are intended to have the meanings given below and are used to understand the description herein and the scope of the invention.

When describing the present invention, it may include compounds, pharmaceutical compositions comprising such compounds, and methods of using such compounds and compositions, unless otherwise indicated, the following terms, if any, have the following meanings. It is also to be understood that any of the groups described herein may be substituted with a variety of substituents, and that substituted groups are included within their respective definitions. Unless otherwise indicated, the term "substituted" is defined as follows. It is also to be understood that the terms "group" and "radical" are used interchangeably herein.

The indefinite articles "a" and "an" may be used herein to indicate one or more than one (i.e. at least one) of the grammatical object of the article, e.g. "an analogue" means one or more than one analogue.

The term "JAK" as used herein relates to the Janus kinase (JAKs) family, which is a cytoplasmic tyrosine kinase that transduces cytokine signals from membrane receptors to STAT transcription factors. The prior art describes four JAK family members: JAK1, JAK2, JAK3 and TYK2, the term JAK may denote all JAK family members or one or more JAK family members as indicated above and below.

The term "pharmaceutically acceptable" means approved or approved by a regulatory agency of the federal or a continent government or a corresponding agency in a country outside the united states, or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

The term "pharmaceutically acceptable salt" means a salt of a compound of the invention which is pharmaceutically acceptable and which possesses the desired pharmacological activity of the parent compound. In particular, such salts are non-toxic and may be inorganic or organic acid addition salts and base addition salts. In particular, such salts include: (1) acid addition salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, hydroxyacetic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3- (4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1, 2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo [2.2.2] -oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when an acidic proton is present in the parent compound, with a metal ion, such as an alkali metal ion, alkaline earth metal ion, or aluminum ion, in place of the acidic proton; or coordinated with an organic base such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, and the like. Salts further include, for example, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the compound contains a basic functional group, salts of non-toxic organic or inorganic acids such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate, and the like are formed.

The term "pharmaceutically acceptable cation" refers to an acceptable cationic counterion to an acidic functional group. Such as sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium cations, and the like.

By "pharmaceutically acceptable medium" is meant a diluent, adjuvant, excipient, or carrier with which the compound of the invention is administered.

"solvate" refers to a form of a compound that is associated with a solvent, typically by a solvolysis reaction. This physical association includes hydrogen bonding. Conventional solvents include water, ethanol, acetic acid, and the like. The compounds of the invention may be prepared, for example, in crystalline form and may be solvated or hydrated. Suitable solvates include pharmaceutically acceptable solvates, such as hydrates, and also includes stoichiometric and non-stoichiometric solvates. In some cases, the solvate can be isolated, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. "solvates" includes both solution phase and isolatable solvates. Representative solvates include hydrates, ethanolates, and methanolates.

By "therapeutically effective amount" is meant an amount of a compound that, when administered to an individual for the treatment of a disease, is sufficient to be therapeutically effective for the treatment of the disease. The "therapeutically effective amount" may vary depending on the compound, the disease and its severity, the age, weight, etc. of the individual being treated.

"preventing" or "prevention" means reducing the risk of acquiring or developing a disease or disorder, even if at least one of the clinical symptoms of the disease does not develop in an individual who may be exposed to a pathogenic agent or who is predisposed to the disease prior to its onset. The term "prevention" relates to "prevention", meaning a measure or method, which is aimed at preventing, rather than treating or curing, a disease.

The present invention also includes isotopically-labeled compounds, and examples of isotopes that can be listed for compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, respectively²H，³H，¹³C，¹⁴C，¹⁵N，¹⁷O，¹⁸O，³¹P，³²P，³⁵S，¹⁸F and³⁶and (4) Cl. The compounds of the present invention, or enantiomers, diastereomers, isomers, or pharmaceutically acceptable salts or solvates thereof, wherein isotopes or other isotopic atoms containing such compounds are within the scope of the present invention. Certain isotopically-labelled compounds of the invention, e.g.³H and¹⁴among these, the radioactive isotope of C is useful in tissue distribution experiments of drugs and substrates. Tritium, i.e.³H and carbon-14, i.e.¹⁴C, their preparation and detection are relatively easy, and are the first choice among isotopes. Isotopically labeled compounds can be prepared by conventional methods by substituting readily available isotopically labeled reagents for non-isotopically labeled reagents using the protocols set forth in the examples.

Non-limiting examples of prophylactic measures include administration of a vaccine; low molecular weight heparin is administered to hospital patients at risk for thrombosis, for example, due to immobility; and administering antimalarial drugs such as chloroquine prior to traveling to a geographic area where malaria is prevalent or at high risk of contracting malaria.

In one embodiment, "treatment" of any disease or condition means ameliorating the disease or condition (i.e., arresting the appearance, extent, or severity of the disease or alleviating at least one clinical symptom thereof). In another embodiment, "treating" or "treatment" refers to improving at least one physical metric, which may not be perceptible to the individual. In another embodiment, "treating" or "treatment" refers to modulating the disease or disorder, either physically (e.g., stabilization of a perceptible symptom), physiologically (e.g., stabilization of a physical indicator), or both.

In a further embodiment, "treating" or "treatment" means slowing the progression of the disease.

The term "inflammation" as used herein denotes a group of conditions including rheumatoid arthritis, osteoarthritis, juvenile idiopathic arthritis, psoriasis, allergic airway diseases (e.g. asthma, rhinitis), inflammatory bowel diseases (e.g. crohn's disease, colitis), endotoxin-driven disease states (e.g. complications after cardiac bypass surgery or chronic endotoxin states due to e.g. chronic heart failure) and related diseases involving cartilage, such as joint disease. The term particularly denotes rheumatoid arthritis, osteoarthritis, allergic airway diseases (e.g. asthma) and inflammatory bowel disease.

The term "autoimmune disease" as used herein denotes a group of diseases including obstructive airways disease, including conditions such as COPD, asthma (e.g. intrinsic asthma, extrinsic asthma, dust asthma, asthma infantis), in particular chronic or chronically developed asthma (e.g. late asthma and airway hyperresponsiveness), bronchitis (including bronchial asthma), Systemic Lupus Erythematosus (SLE), multiple sclerosis, type I diabetes and its associated complications, atopic eczema (atopic dermatitis), contact dermatitis, also including eczematous dermatitis, inflammatory bowel disease (e.g. crohn's disease and ulcerative colitis), atherosclerosis and amyotrophic lateral sclerosis. The term particularly denotes COPD, asthma, systemic lupus erythematosus, type I diabetes and inflammatory bowel disease.

The term "proliferative disease" as used herein denotes a condition such as cancer (e.g. uterine leiomyosarcoma or prostate cancer), myeloproliferative disorders (e.g. polycythemia vera, primary thrombocytosis and myelofibrosis), leukemia (e.g. acute myeloid leukemia and acute lymphocytic leukemia), multiple myeloma, psoriasis, restenosis, sclerosing dermatitis or fibrosis. The term particularly denotes cancer, leukemia, multiple myeloma and psoriasis.

The term "cancer" as used herein means a malignant or benign growth of cells in the skin or body organs such as, but not limited to, the breast, prostate, lung, kidney, pancreas, stomach, or intestine. Cancer tends to invade adjacent tissues and spread (metastasize) to distant organs, such as bone, liver, lung or brain. The term cancer as used herein includes metastatic tumor cell types such as, but not limited to, melanoma, lymphoma, leukemia, fibrosarcoma, rhabdomyosarcoma, and mast cell tumor, as well as tissue cancer types such as, but not limited to, colorectal cancer, prostate cancer, small cell lung cancer and non-small cell lung cancer, breast cancer, pancreatic cancer, bladder cancer, renal cancer, gastric cancer, glioblastoma, primary liver cancer, ovarian cancer, prostate cancer, and uterine leiomyosarcoma.

The term "leukemia" as used herein means neoplastic diseases of the blood and hematopoietic organs. Such diseases can lead to bone marrow and immune system dysfunction, which makes the host highly susceptible to infection and bleeding. The term leukemia particularly denotes acute myeloid leukemia and acute lymphocytic leukemia.

The term "transplant rejection" as used herein means acute or chronic rejection of an allograft or xenograft of cells, tissues or solid organs, such as pancreatic islets, stem cells, bone marrow, skin, muscle, corneal tissue, neuronal tissue, heart, lung, cardiopulmonary union, kidney, liver, intestine, pancreas, trachea or esophagus, or graft versus host disease.

The term "disease involving impaired cartilage turnover" as used herein includes conditions such as osteoarthritis, psoriatic arthritis, juvenile rheumatoid arthritis, gouty arthritis, septic or infectious arthritis, reactive arthritis, reflex sympathetic dystrophy, pain dystrophy, pedicure's syndrome or costal chondritis, fibromyalgia, osteochondritis, neurogenic or neuropathic arthritis, arthropathies, endemic forms of arthritis such as endemic osteoarthritis, mseni's disease and handigoud disease, degeneration caused by fibromyalgia, systemic lupus erythematosus, scleroderma and ankylosing spondylitis.

The term "congenital cartilage malformation" as used herein includes disorders such as hereditary chondrolysis, chondrodysplasia and pseudochondrodysplasia, in particular but not limited to, auricular malformations, anulus, metaphyseal chondrodysplasia and related disorders.

The term "diseases associated with hypersecretion of IL 6" as used herein includes disorders such as Castleman's disease, multiple myeloma, psoriasis, kaposi's sarcoma and/or mesangial proliferative glomerulonephritis.

"Compounds of the invention" and equivalent expressions are meant to include the compounds of the formulae depicted herein, including pharmaceutically acceptable salts and solvates, such as hydrates, as well as solvates of pharmaceutically acceptable salts, as the context permits. Similarly, references to intermediates (whether or not they are themselves claimed) include their salts and solvates, where the context allows.

The acid and acid-derived forms of the other derivatives of the compounds of the invention are active, but acid-sensitive forms can generally provide more favorable solubility, histocompatibility or delayed release in mammalian organisms (Bundgard, h. design of Prodrugs, p. 7-9, 21-24, Elsevier, Amsterdam 1985).

The compounds of the present invention are novel JAK inhibitors. In particular, the compounds are potent inhibitors of JAK1 and JAK 2.

Pharmaceutical composition

When used as a medicament, the compounds of the invention are generally administered in the form of a pharmaceutical composition. Such compositions may be prepared in a manner well known in the pharmaceutical art and comprise at least one active compound. Typically, the compounds of the present invention are administered in a pharmaceutically effective amount. The amount of the compound actually administered will generally be determined by a physician, in the light of the relevant circumstances, including the condition being treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.

The pharmaceutical compositions of the present invention may be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intra-articular, intravenous, intramuscular, and intranasal. Depending on the intended route of delivery, the compounds of the invention are preferably formulated as injectable or oral compositions or all ointments, lotions or patches for transdermal administration.

Compositions for oral administration may take the form of bulk liquid solutions or suspensions or bulk powders. More commonly, however, the compositions are presented in unit dosage form for accurate administration. The term "unit dosage form" refers to physically discrete units suitable for administration as unitary units in human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient, vehicle or carrier. Typical unit dosage forms include pre-filled, pre-measured ampoules or syringes of liquid compositions or, in the case of solid compositions, pills, tablets, capsules and the like. In such compositions, the compounds of the present invention are generally a minor component (from about 0.1% to about 50%, preferably from about 1% to about 40% by weight), with the remainder being various vehicles or carriers and processing aids to assist in forming the desired administration form.

Liquid forms suitable for oral administration may include suitable aqueous or non-aqueous media containing buffers, suspending and dispersing agents, coloring agents, flavoring agents, and the like. Solid forms may include, for example, any of the following or compounds of similar nature: a binder, such as microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch or lactose; disintegrants, for example alginic acid, Primogel or corn starch; lubricants, such as magnesium stearate; glidants, such as colloidal silicon dioxide; sweetening agents, such as sucrose or saccharin; or a flavoring agent, such as peppermint, methyl salicylate, or citrus flavoring.

Injectable compositions are typically based on injectable sterile saline or phosphate buffered saline or other injectable carriers known in the art. As noted above, the active compound in the composition is typically a minor component, typically about 0.05% to 10% by weight, with the remainder being an injectable carrier or the like.

Transdermal compositions are typically formulated as topical ointments or creams containing the active ingredient in an amount generally ranging from about 0.01% to about 20% by weight, preferably from about 0.1% to about 10% by weight and more preferably from about 0.5% to about 15% by weight. When formulated as an ointment, the active ingredient is typically mixed with a paraffinic or water-miscible ointment base. Alternatively, the active ingredient may be formulated in a cream with an oil-in-water cream base, for example. Such transdermal formulations are well known in the art and typically contain other ingredients to enhance the epidermal penetration stability of the active ingredient or formulation. All such known transdermal formulations and ingredients are included within the scope of the present invention.

The compounds of the present invention may also be administered by transdermal means. Thus, transdermal administration may be accomplished using a patch of reservoir or porous membrane type or solid matrix variant.

The components of the above orally administrable, injectable or topically administrable compositions are merely representative. Other materials and processing techniques are described in Remington's Pharmaceutical Sciences, 17 th edition, 1985, Mack Publishing Company, Iston, part 8 of Pa, which is incorporated herein by reference.

The compounds of the present invention may also be administered in a sustained release form or from a slow release drug delivery system. Representative sustained release materials are described in Remington's Pharmaceutical Sciences (Remington pharmacology).

The following formulation examples illustrate representative pharmaceutical compositions that can be prepared according to the present invention. However, the present invention is not limited to the following pharmaceutical compositions.

Formulation 1 tablet

The compounds of the invention may be mixed as a dry powder with a dry gelatin binder in a ratio of about 1: 2 weight ratio. A small amount of magnesium stearate was added as a lubricant. The mixture was formulated into 240-270mg tablets (each containing 80-90mg of the active amide compound) in a tablet press.

Preparation 2 Capsule

The compounds of the invention may be mixed as a dry powder with a starch diluent in a ratio of about 1:1 weight ratio. The mixture was filled into 250mg capsules (each containing 125mg of active amide compound).

Formulation 3 liquid

The compound of the present invention (125mg) may be mixed with sucrose (1.75g) and xanthan gum (4mg), and the resulting mixture may be mixed, passed through a 10 mesh u.s. sieve, and then mixed with an aqueous solution of microcrystalline cellulose and sodium carboxymethylcellulose (11: 89, 50mg) previously prepared. Sodium benzoate (10mg), flavours and colours were diluted with water and added with stirring. Sufficient water may then be added with stirring. Sufficient water was then added to make a total volume of 5 mL.

Formulation 4 tablets

The compounds of the invention may be mixed as a dry powder with a dry gelatin binder in a ratio of about 1: 2 weight ratio. A small amount of magnesium stearate was added as a lubricant. The mixture was formulated into 450-900mg tablets (150-300mg of active amide compound) in a tablet press.

Preparation 5 injection

The compounds of the present invention may be dissolved or suspended in a buffered sterile saline injectable aqueous medium to a concentration of about 5 mg/mL.

Formulation 6 topical application

Stearyl alcohol (250g) and white petrolatum (250g) were melted at about 75 ℃, then a mixture of the compound of the present invention (50g), methyl paraben (0.25g), propyl paraben (0.25g), sodium lauryl sulfate (10g) and propylene glycol (120g) dissolved in water (about 370g) was added and the resulting mixture was stirred until coagulated.

Method of treatment

The compounds of the present invention may be useful as therapeutic agents for the treatment of conditions associated with or caused by abnormal activity of JAK in mammals. In particular, such disorders are associated with abnormal activity of JAK1 and/or JAK 2. Accordingly, the compounds of the present invention and the pharmaceutical compositions of the present invention find use as therapeutic agents for the prevention and/or treatment of inflammation, autoimmune diseases, proliferative diseases, transplant rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and diseases associated with hypersecretion of IL6 in mammals, including humans.

In a further method of treatment aspect, the invention provides a method of treating a mammal susceptible to or infected with inflammation. The method comprises administering one or more of the pharmaceutical compositions or compounds of the invention described herein in an amount effective to treat the disorder or prevent the disorder. In particular embodiments, the inflammation is selected from rheumatoid arthritis, osteoarthritis, allergic airway diseases (e.g., asthma), and inflammatory bowel disease.

In another aspect, the invention provides a compound of the invention for use in the treatment, prevention or prophylaxis of inflammation. In particular embodiments, the inflammation is selected from rheumatoid arthritis, osteoarthritis, allergic airway diseases (e.g., asthma), and inflammatory bowel disease.

In another method of treatment, the invention provides a method of treating a mammal susceptible to or infected with an autoimmune disease. The method comprises administering one or more of the pharmaceutical compositions or compounds of the invention described herein in an amount effective to treat the disorder or prevent the disorder. In particular embodiments, the autoimmune disease is selected from COPD, asthma, systemic lupus erythematosus, type I diabetes, and inflammatory bowel disease.

In another aspect, the invention provides a compound of the invention for use in the treatment, prevention or prophylaxis of an autoimmune disease. In particular embodiments, the autoimmune disease is selected from COPD, asthma, systemic lupus erythematosus, type I diabetes, and inflammatory bowel disease.

In a further method of treatment, the invention provides a method of treating a mammal susceptible to or infected with a proliferative disease, particularly cancer (e.g. a solid tumor such as uterine leiomyosarcoma or prostate cancer), leukemia (e.g. AML or ALL), multiple myeloma and/or psoriasis.

In another aspect, the invention provides a compound of the invention for use in the treatment, prevention or prophylaxis of a proliferative disease, in particular cancer (e.g. a solid tumor such as uterine leiomyosarcoma or prostate cancer), leukemia (e.g. AML or ALL), multiple myeloma and/or psoriasis.

In another method of treatment, the invention provides a method of treating a mammal susceptible to or infected with transplant rejection. In particular embodiments, the present invention provides methods of treating organ transplant rejection.

In another aspect, the invention provides a compound of the invention for use in the treatment, prevention or prophylaxis of transplant rejection. In particular embodiments, the present invention provides methods of treating organ transplant rejection.

In a method of treatment, the invention provides a method of treatment, prevention or prophylaxis in a mammal susceptible to or infected with a disease involving impaired cartilage turnover, the method comprising administering a therapeutically effective amount of a compound of the invention described herein or one or more pharmaceutical compositions of the invention.

In another aspect, the invention provides a compound of the invention for use in the treatment, prevention or prophylaxis of a disease in which impaired cartilage turnover is involved.

The present invention also provides a method of treating congenital cartilage malformations, comprising administering an effective amount of one or more of the pharmaceutical compositions or compounds of the present invention described herein.

In another aspect, the present invention provides a compound of the invention for use in the treatment, prevention or prophylaxis of congenital cartilage malformations.

As a further aspect of the invention, the present invention provides compounds of the invention for use as medicaments, in particular for the treatment or prevention of the above-mentioned conditions and diseases. Also provided herein is the use of a compound of the invention in the manufacture of a medicament for the treatment or prevention of one of the conditions and diseases described above.

A particular embodiment of the method comprises administering to an individual suffering from inflammation an effective amount of a compound of the invention for a time sufficient to reduce the level of inflammation in the patient and preferably to stop the progression of said inflammation. A particular embodiment of the method comprises administering to an individual patient suffering from or susceptible to the development of rheumatoid arthritis, an effective amount of a compound of the invention for a time sufficient to reduce or prevent, respectively, inflammation of the joints of said patient, and preferably to stop the progression of said inflammation.

Another particular embodiment of the method comprises administering to an individual suffering from a disease condition characterized by degeneration of cartilage or joints, such as rheumatoid arthritis and/or osteoarthritis, an effective amount of a compound of the invention for a time sufficient to reduce and preferably stop the progression of said degeneration itself. A particular embodiment of the method comprises administering to an individual patient suffering from or susceptible to developing osteoarthritis an effective amount of a compound of the invention for a time sufficient to reduce or prevent, respectively, cartilage degradation in the joint of said patient, and preferably to stop the progression of said degradation itself. In particular embodiments, the compounds will exhibit cartilage anabolic and/or anti-catabolic properties.

The injection dosage level ranges from about 0.1 mg/kg/hour to at least 10 mg/kg/hour, over a period of about 1 to about 120 hours, especially 24 to 96 hours. Pre-filled injectables of about 0.1mg/kg to about 10mg/kg or more can also be administered to achieve adequate steady-state levels. The maximum total dose is not expected to exceed about 2 g/day for 40 to 80kg human patients.

For the prevention and/or treatment of long-term conditions, such as degenerative conditions, the treatment regimen typically extends over months or years, and thus oral administration is preferred for patient convenience and tolerability. For oral administration, a typical regimen is 1 to 5, particularly 2 to 4, usually 3 oral doses per day. With these modes of administration, each dose provides about 0.01 to about 20mg/kg of a compound of the invention, for a particular dose, each provides about 0.1 to about 10mg/kg, specifically about 1 to about 5 mg/kg.

Transdermal administration is generally selected to provide similar or lower blood levels than achieved with injectable administration.

When used to prevent the onset of inflammatory conditions, the compounds of the present invention are administered to a patient at risk of developing the condition, usually under the direction and supervision of a physician, at dosage levels as described above. Patients at risk of developing a particular disorder typically include those with a family history of the disorder, or those identified as particularly susceptible to developing the disorder by genetic testing or screening.

The compounds of the invention may be administered as the sole active agent, or they may be administered in combination with other active agents, including other compounds that have the same or similar therapeutic activity and are determined to be safe and effective for such combined administration. In particular embodiments, co-administration of two (or more) active agents allows for a significant reduction in the dosage of each active agent used, thereby reducing the side effects seen.

In one embodiment, the compounds of the invention are co-administered with additional therapeutic agents for the treatment and/or prevention of inflammatory diseases, particular active agents including, but not limited to, immunomodulators, such as azathioprine, corticosteroids (e.g., prednisolone or dexamethasone), cyclophosphamide, cyclosporine a, tacrolimus, mycophenolate mofetil, moclomab-CD 3(OKT3, for example), ATG, aspirin, acetaminophen, ibuprofen, naproxen, and piroxicam.

In one embodiment, the compounds of the present invention are co-administered with other therapeutic agents for the treatment and/or prevention of arthritis (e.g., rheumatoid arthritis), particular active agents including, but not limited to, analgesics, non-steroidal anti-inflammatory drugs (NSAIDS), steroids, synthetic DMARDS (such as, but not limited to, methotrexate, leflunomide, sulfasalazine, auranofin, disodium aurothionine, penicillamine, chloroquine, hydroxychloroquine, azathioprine, and cyclosporine), and biologicals such as, but not limited to, infliximab, etanercept, adalimumab, rituximab, and abacavir.

In one embodiment, the compounds of the invention are co-administered with other therapeutic agents for the treatment and/or prevention of proliferative disorders, specific active agents including, but not limited to: methotrexate, folinic acid, doxorubicin, prednisone, bleomycin, cyclophosphamide, 5-fluorouracil, paclitaxel, docetaxel, vincristine, vinblastine, vinorelbine, doxorubicin, tamoxifen, toremifene, megestrol acetate, anastrozole, goserelin, anti-HER 2 monoclonal antibodies (e.g., Herceptin (TM)), capecitabine, raloxifene hydrochloride, EGFR inhibitors (e.g., Tarceva)^TM、Erbitux^TM) VEGF inhibitors (e.g. Avastin)^TM) Proteasome inhibitors (e.g. Velcade)^TM) Or hsp90 inhibitors (e.g., 17-AAG). In addition, the compounds of the present invention may be administered in combination with other treatments including, but not limited to, radiation therapy or surgery. In particular embodiments, the proliferative disorder is selected from cancer, a myeloproliferative disease, or leukemia.

In one embodiment, the compounds of the invention are co-administered with other therapeutic agents for the treatment and/or prevention of autoimmune diseases, particular active agents include, but are not limited to: glucocorticoids, cell proliferation inhibitors (e.g., purine analogs), alkylating agents (e.g., nitrogen mustards (cyclophosphamide), nitrosoureas, platinum compounds, and the like), antimetabolites (e.g., methotrexate, azathioprine, and mercaptopurine), cytotoxic antibiotics (e.g., dactinomycin anthracycline, mitomycin C, bleomycin, and plicamycin), antibodies (e.g., anti-CD 20, anti-CD 25, or anti-CD 3(OTK3) monoclonal antibodies, cyclosporine, tacrolimus, rapamycin, interferons, TNF binding proteins, etanercept or adalimumab, mycophenolate mofetil, fingolimod, and multispheromone.

In one embodiment, the compounds of the present invention are co-administered with other therapeutic agents for the treatment and/or prevention of transplant rejection, particular active agents include, but are not limited to: calcineurin inhibitors (e.g. cyclosporine or tacrolimus, mTOR inhibitors (e.g. sirolimus, everolimus), antiproliferatives (e.g. azathioprine, mycophenolic acid), corticosteroids (e.g. prednisolone, hydrocortisone), antibodies (e.g. monoclonal anti-IL-2 ra receptor antibodies, basiliximab, daclizumab), polyclonal anti-T-cell antibodies (e.g. anti-thymocyte globulin (ATG), anti-lymphocyte globulin.

In one embodiment, the compounds of the present invention are co-administered with other therapeutic agents for the treatment and/or prevention of asthma and/or rhinitis and/or COPD, particular active agents include, but are not limited to: beta is a₂Adrenoceptor agonists (e.g. salbutamol, levalbuterol, terbutaline and bitolterol), epinephrine (e.g. inhalation or tablet), anticholinergic (e.g. ipratropium bromide), glucocorticoids (e.g. oral or inhalation), long-acting beta₂Combinations of agonists (e.g. salmeterol, formoterol, bambuterol and delayed release oral salbutamol), inhaled steroids and long-acting bronchodilators (e.g. fluticasone/salmeterol, budesonide/formoterol), leukotriene antagonists and synthesis inhibitors (e.g. montelukast, zafirlukast and zileuton), mediator release inhibitors (e.g. cromoglycate and ketotifen), biological modulators of the IgE response (e.g. omalizumab), antihistamines (e.g. cetirizine, cinnarizine, fexofenadine and vasoconstrictors (e.g. oxymetazoline, xylonazole)Quinoline, naphazoline, and tramazoline).

Additionally, the compounds of the invention may be administered in combination with emergency therapy for asthma and/or COPD, such therapy including oxygen or heliox administration, nebulized salbutamol or terbutaline (optionally in combination with an anticholinergic), synthetic steroids (oral or intravenous, e.g., prednisone, prednisolone, methylprednisolone, dexamethasone or hydrocortisone), intravenous salbutamol, non-specific beta-agonists, injected or inhaled (e.g., epinephrine, isotetralin, isoproterenol, metaproterenol), anticholinergics (e.g., glycopyrrolate, atropine, ipratropium), methylxanthines (theophylline, aminophylline, benzylamine), inhalation anesthetics (isoflurane, halothane, enflurane), ketamine and intravenous magnesium sulfate.

In one embodiment, the compounds of the present invention are co-administered with other therapeutic agents for the treatment and/or prevention of Inflammatory Bowel Disease (IBD), and particular active agents include, but are not limited to: glucocorticoids (e.g., prednisone, budesonide), synthetic disease modifying immunomodulators (e.g., methotrexate, leflunomide, sulfasalazine, mesalazine, azathioprine, 6-mercaptopurine, and cyclosporine), and biologics disease modifying immunomodulators (e.g., infliximab, adalimumab, rituximab, and abacavir).

In one embodiment, the compounds of the invention are co-administered with other therapeutic agents for the treatment and/or prevention of systemic lupus erythematosus, particular active agents include, but are not limited to: anti-rheumatic drugs that alleviate diseases such as antimalarial drugs (e.g., hydroxychloroquine), immunosuppressants (e.g., methotrexate and azathioprine), cyclophosphamide and mycophenolic acid; immunosuppressive drugs and analgesics such as non-steroidal anti-inflammatory drugs, anesthetics (e.g., dextropropoxyphene and codeine co-acetaminophen), opioids (e.g., hydrocodone, oxycodone, mexican or methadone), and fentanyl transdermal patches.

In one embodiment, the compounds of the present invention are co-administered with other therapeutic agents for the treatment and/or prevention of psoriasis, particular active agents include, but are not limited to: topical therapies such as bath solutions, moisturizers, medicated creams and coal tar containing ointments, dithranol, corticosteroids such as desoximetasone, fluocinonide, vitamin D analogues (e.g. calcipotriol), Arganoiland retinoids (acitrexate, avilam, tazarotene), systemic therapies such as methotrexate, cyclosporine, retinoids, thioguanine, hydroxyurea, sulfasalazine, mycophenolate mofetil, azathioprine, tacrolimus, fumarates or biologicals such as alfacast, etanercept, adalimumab, infliximab, truer and ustrocumab (IL-12 and IL-23 blockers). In addition, the compounds of the present invention may be administered in combination with other therapies, including, but not limited to, phototherapy or photochemotherapy.

It will be apparent to the skilled person that co-administration includes any manner of delivering two or more therapeutic agents to a patient as part of the same treatment regimen. While two or more active agents may be administered simultaneously in a single formulation, this is not required. The active agents may be administered in different formulations and at different times.

Synthesis method

General rule

The compounds of the present invention are prepared from readily available starting materials using the following general procedures and procedures. It is to be understood that where typical or preferred process conditions (i.e., reaction temperatures, times, molar ratios of reactants, solvents, pressures, etc.) are given, other process conditions may also be employed unless otherwise indicated. Optimum reaction conditions may vary depending on the particular reactants or solvents used, but such conditions can be determined by one skilled in the art using routine optimization procedures.

In addition, it will be apparent to those skilled in the art that conventional protecting groups may be required to prevent certain functional groups from undergoing undesirable reactions. The selection of suitable protecting groups for particular functional groups and suitable conditions for protection and deprotection are well known in the art. For example, T.W.Greene and P.G.M.Wuts, Protecting Groups in Organic Synthesis, second edition, Wiley, New York, 1991 and the references cited therein describe a number of Protecting Groups and their introduction and removal.

The following methods describe in detail the preparation of the compounds of the invention and the compounds of the comparative examples listed above. The compounds of the invention and the compounds of the comparative examples can be prepared by those skilled in the art of organic synthesis using known or commercially available starting materials and reagents.

Unless otherwise indicated, all reagents were of commercial grade and were used as received without further purification. Commercially available anhydrous solvents are used for the reaction under an inert atmosphere. Reagent grade solvents were used in all other cases unless otherwise stated.

The following abbreviations are used in the experimental section:

DCM dichloromethane

DiPEA N, N-diisopropylethylamine

MeCN acetonitrile

BOC tert-butoxycarbonyl

DMF N, N-dimethylformamide

TFA trifluoroacetic acid

THF tetrahydrofuran

NMR nuclear magnetic resonance

DMSO dimethyl sulfoxide

DPPA diphenylphosphoryl azide

LC-MS liquid chromatography-mass spectrometry

Ppm parts per million of Ppm

EtOAc ethyl acetate

PdCl₂dppf [1, 1' -bis (diphenylphosphino) ferrocene]Palladium dichloride (II)

TEA Triethylamine

The following more specifically describes the preparation of the compounds of formula (I) according to the invention, but these particular methods do not limit the invention in any way. The compounds of the present invention may also be conveniently prepared by optionally combining various synthetic methods described in the present specification or known in the art, and such combinations may be readily carried out by those skilled in the art to which the present invention pertains.

The compounds of the present invention have a number of advantages over the non-deuterated compounds known in the prior art. The main advantages of the invention include: (1) the compounds of the present invention have excellent inhibitory activity against JAK kinases; (2) the metabolism of the compound in organisms is changed by the deuteration technology, so that the compound has better pharmacokinetic parameter characteristics, and in the case, the dosage can be changed, a long-acting preparation is formed, and the applicability is improved; (3) deuterium is used for replacing hydrogen atoms in the compound, and due to the deuterium isotope effect, the medicine concentration of the compound in an animal body can be improved, so that the medicine curative effect is improved; replacement of hydrogen atoms in compounds with deuterium may increase the safety of the compounds as certain metabolites are inhibited.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Parts and percentages are parts and percentages by weight unless otherwise indicated.

Example 1N- (5- (4- ((1, 1-dioxo-4-thiomorpholine-2, 2,6,6-d4) methyl) phenyl) - [1,2,4] Triazolo [1,5-a]Pyridin-2-yl) cyclopropanecarboxamides (Compound 13)

Step 1: synthesis of 1- (6-bromopyridin-2-yl) -3-ethoxycarbonyl-thiourea (Compound 3).

Ethoxycarbonyl isothiocyanate (6.80mL, 57.8mmol) was slowly added dropwise to a solution of 2-amino-6-bromopyridine (10.0g, 57.8mmol) in dichloromethane (100mL) at 5 ℃ over 15 minutes, and after completion of the addition, the reaction mixture was warmed to room temperature and stirred for reaction overnight. The solvent was removed under reduced pressure, the solid was filtered, washed with petroleum ether and dried in vacuo to give 16.9g of a yellow solid in yield: 96.1 percent.

Step 2: synthesis of 5-bromo- [1,2,4] triazolo [1,5-a ] pyridin-2-amine (Compound 4).

DIPEA (27.0mL, 165.6mmol) was added dropwise to a solution of hydroxylamine hydrochloride (19.2g, 276.0mmol) in ethanol/methanol (v: v ═ 1:1, 170mL) at room temperature, and the reaction was stirred at room temperature for 1 h. 1- (6-bromopyridin-2-yl) -3-ethoxyformyl-thiourea (16.8g, 55.2mmol) was added slowly in portions and the reaction was refluxed for 3 hours. The reaction was cooled to room temperature and the solid was filtered. The filtrate was concentrated under reduced pressure, and 30mL of water was added. The resulting precipitate was filtered. The solids were combined, washed with water (30mL), ethanol/methanol (v/v ═ 1:1,60mL) and dried in vacuo to give 9.8g of a white solid, yield: 83.3 percent.¹H NMR(300MHz,DMSO-d₆):δ7.40–7.30(m,2H),7.21(dd,J＝6.5,2.2Hz,1H),6.29(s,2H)。

And step 3: synthesis of N- (5-bromo- [1,2,4] triazolo [1,5-a ] pyridin-2-yl) cyclopropanecarboxamide (Compound 6).

Triethylamine (5.80mL, 41.0mmol) and cyclopropanecarbonyl chloride (3.80mL, 41mmol) were slowly added dropwise to 5-bromo- [1,2,4] respectively at 5 deg.C]Triazolo [1,5-a]Pyridin-2-amine (3.5g, 16.4mmol) in dry acetonitrile solvent (75 mL). The reaction solution was warmed to room temperature and stirred until the reaction of the starting materials was complete (12 h). The solvent was removed under reduced pressure and the residue was taken up in ammonia in methanol (7N, 30mL) and the diacylate was hydrolyzed with stirring at room temperature (6 h). The solvent was removed under reduced pressure, ether (20mL) and acetone (20mL) were added, the solid was filtered, washed with water (20mL), acetone (20mL), ether (20mL), and dried in vacuo to give 2.7g of a brown solid, yield: 58.7 percent. LC-MS (APCI) M/z 281.1(M + H)⁺。

And 4, step 4: synthesis of 4- (4-bromobenzyl) 1, 1-dioxo-4-thiomorpholine (Compound 9).

Triethylamine (2.18mL, 15.7mmol) was added dropwise to a mixture of p-bromobenzyl bromide (1.28g, 5.10mmol) and thiomorpholine 1, 1-dioxide hydrochloride (944mg, 5.50mmol) in DMF (15mL), and the reaction was stirred at room temperature overnight. The reaction was diluted with ethyl acetate (50mL), washed with water (30 mL. times.3) and brine (30mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressureCondensation gave 1.0g of a white solid, yield: and (4) 64.5%. Directly used for the next reaction.¹H NMR(300MHz,MeOD-d₄):δ7.53–7.47(m,2H),7.34–7.27(m,2H),4.90(s,2H),3.16–3.06(m,4H),3.00–2.93(m,4H)。

And 5: synthesis of 4- (4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxolan-2-yl) benzyl) thiomorpholine-1, 1-dioxide (Compound No. 10).

DMSO (15mL) was added to 4- (4-bromobenzyl) -1, 1-dioxo-4-thiomorpholine (1.00g, 3.30mmol) and pinacol diboron (1.00g, 3.90mmol), potassium acetate (970mg, 9.86mmol), Pd (dppf) Cl under nitrogen₂(170mg) the reaction was stirred overnight at 100 ℃ under nitrogen. Cool to room temperature, dilute with water (25mL), extract with ethyl acetate (30mL × 2), wash the organic layer with saturated brine (20mL), anhydrous sulfuric acid as a white brown solid, yield: 47.4 percent. LC-MS (APCI) M/z 352.2(M + H)⁺。

Step 6: synthesis of N- [5- [4- [ (1, 1-dioxo-4-thiomorpholinyl) methyl ] phenyl ] - [1,2,4] triazolo [1,5-a ] pyridin-2-yl ] cyclopropanecarboxamide (Compound 11).

In N₂Under protection, 1, 4-dioxane (6mL) and water (1.5mL) were injected into N- (5-bromo- [1,2,4]]Triazolo [1,5-a]Pyridin-2-yl) cyclopropanecarboxamide (140mg, 0.50mmol), 4- (4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxolan-2-yl) benzyl) thiomorpholine-1, 1-dioxide (210mg, 0.60mmol), Pd (dppf) Cl₂(15mg) and potassium carbonate (207mg, 1.50mmol), and the reaction was carried out overnight at 100 ℃. Cooling to the greenhouse, filtering through celite, washing the filter cake with dichloromethane, drying the filtrate with anhydrous sodium sulfate, removing the solvent, and column separating the concentrate (eluent: dichloromethane/methanol (v/v) ═ 25:1) to give 140mg of a brown solid, yield: 65.8 percent. LC-MS (APCI) M/z 426.5(M + H)⁺；¹H NMR(300MHz,DMSO-d₆):δ11.05(s,1H),7.99(d,J＝8.3Hz,2H),7.75–7.66(m,2H),7.52(d,J＝8.2Hz,2H),7.29(dd,J＝6.1,2.4Hz,1H),3.77(s,2H),3.21–3.08(m,4H),2.99–2.85(m,4H),2.14–1.88(m,1H),0.81(d,J＝6.2Hz,4H)。

And 7: synthesis of 4- (4- (2-amino- [1,2,4] triazolo [1,5-a ] pyridin-5-yl) phenyl) thiomorpholine-1, 1-dioxo-2, 2,6,6-d4 (Compound 12).

Sodium methoxide (30mg, 0.50mmol) was added to N- [5- [4- [ (1, 1-dioxo-4-thiomorpholinyl) methyl group]Phenyl radical]-[1,2,4]Triazolo [1,5-a]Pyridin-2-yl]Cyclopropanecarboxamide (35mg, 0.08mmol) in deuterated methanol (CD)₃OD-d₄5mL) under nitrogen, and the reaction was refluxed overnight. The reaction was quenched with heavy water (10mL), extracted with dichloromethane (10mL × 4), the organic phase was washed with saturated brine (15mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the concentrate was subjected to column separation (eluent: dichloromethane/methanol (v/v) ═ 10:1) to give 28mg of a beige solid, yield: 96.8 percent. LC-MS (APCI) M/z 362.2(M + H)⁺。

And 8: n- (5- (4- ((1, 1-dioxo-4-thiomorpholine-2, 2,6, 6-d)₄) Methyl) phenyl) - [1,2,4]Triazolo [1,5-a]Pyridin-2-yl) cyclopropanecarbonyl (Compound 13).

Triethylamine (32mg, 0.31mmol) and cyclopropanecarbonyl chloride (35mg, 0.31mmol) were slowly added dropwise to 4- (4- (2-amino- [1,2, 4) at 5 ℃ respectively]Triazolo [1,5-a]Pyridin-5-yl) phenyl) thiomorpholine-1, 1-dioxo-2, 2,6,6-d₄(28mg, 0.08mmol) of dry dichloromethane (5mL) and the reaction was warmed to room temperature until the reaction was complete (16 h). The reaction solvent was removed under reduced pressure, and the residue was added to a methanol solution of ammonia (7M, 5mL), and the bisacylate was hydrolyzed with stirring at room temperature (6h) to give the monoacylated target. The reaction was quenched with water (20mL), extracted with dichloromethane (20mL × 3), the organic phase was washed with saturated brine (15mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the concentrate was subjected to preparative thin layer chromatography (developing solvent: dichloromethane/methanol (v/v) ═ 12:1/15:1) to give 22mg of a beige solid, yield: 64.2 percent. LC-MS (APCI) M/z 430.10(M + H)⁺。¹H NMR(300MHz,DMSO-d₄):δ11.06(s,1H),8.00(d,J＝8.2Hz,2H),7.76–7.65(m,2H),7.53(d,J＝8.2Hz,2H),7.30(dd,J＝6.2,2.3Hz,1H),3.77(s,2H),2.92(s,4H),2.04–1.96(m,1H),0.82(d,J＝6.2Hz,4H)。

Example 2N- (5- (4- ((1, 1-dioxo-4-thiomorpholinyl) methyl-d) phenyl- [1,2,4]Triazolo compounds [1,5-a]Pyridin-2-yl) cyclopropanecarboxamides (Compound 18)

Step 1: synthesis of N- (5- (4-formylphenyl) - [1,2,4] triazolo [1,5-a ] pyridin-2-yl) cyclopropanecarboxamide (Compound 15).

N₂Under protection, 1, 4-dioxane (12mL) and water (4mL) were injected into N- (5-bromo- [1,2, 4)]Triazolo [1,5-a]Pyridin-2-yl) Cyclopropanecarboxamide (560mg, 2.00mmol), (4-formylphenyl) boronic acid (360mg, 2.40mmol), Pd (dppf) Cl₂(70mg, 0.10mmol) and potassium carbonate (850mg, 6.00mmol) were reacted at 90 ℃ overnight (16 h). Cooling to the greenhouse, filtering through celite, washing the filter cake with dichloromethane, drying the filtrate with anhydrous sodium sulfate, removing the solvent, and column separating the concentrate (eluent: dichloromethane/methanol (v/v) ═ 25:1) to give 450mg beige solid, yield: 73.5 percent. LC-MS (APCI) M/z 307.1(M + H)⁺。

Step 2: synthesis of N- (5- (4- (hydroxymethyl-d) phenyl) - [1,2,4] triazolo [1,5-a ] pyridin-2-yl) cyclopropanecarboxamide (Compound 16).

Sodium borodeuteride (74mg, 1.76mmol) was added portionwise to N- (5- (4-formylphenyl) - [1,2,4] under ice-bath]Triazolo [1,5-a]Pyridin-2-yl) cyclopropanecarboxamide (450mg, 1.47mmol) in dry methanol (5mL) was allowed to warm to room temperature for 1.5 h. The reaction was quenched by the addition of heavy water (10mL), extracted with dichloromethane (30mL × 3), the organic layer was washed with saturated brine (30mL), dried over anhydrous sodium sulfate, the solvent was removed, and the concentrate was subjected to column separation (eluent: dichloromethane/methanol (v/v) ═ 25:1) to give 400mg of beige solid, yield: 88.1 percent. LC-MS (APCI): M/z 310.2(M + H)⁺；¹H NMR(300MHz,DMSO-d₆):δ11.05(s,1H),7.97(d,J＝8.2Hz,2H),7.76–7.63(m,2H),7.50(d,J＝8.1Hz,2H),7.28(dd,J＝6.3,1.8Hz,1H),5.33(d,J＝5.7Hz,1H),4.58(d,J＝5.7Hz,1H),2.12–1.92(m,1H),0.81(d,J＝6.1Hz,4H)。

And step 3: synthesis of (4- (2- (cyclopropylcarbamoylamino) - [1,2,4] triazolo [1,5-a ] pyridin-5-yl) phenyl) methyl-d-methanesulfonate (Compound 17).

Triethylamine (0.15mL, 1.00mmol) and MsCl (methylsulfonyl chloride, 0.05mL, 0.58mmol) were added slowly, in that order, dropwise to N- (5- (4- (hydroxymethyl-d) phenyl) - [1,2,4] in an ice bath]Triazolo [1,5-a]Pyridin-2-yl) cyclopropanecarboxamide (150mg, 0.49mmol) in anhydrous dichloromethane (10mL) and the reaction was warmed to room temperature for 2 h. The reaction was quenched with water (25mL), extracted with dichloromethane (30mL x 3), and the organic layer was washed with saturated brine (30mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give 190mg of oil, which was used directly in the next reaction, yield: 100 percent. LC-MS (APCI) M/z 388.1(M + H)⁺。

And 4, step 4: synthesis of N- (5- (4- ((1, 1-dioxo-4-thiomorpholinyl) methyl-d) phenyl- [1,2,4] triazolo [1,5-a ] pyridin-2-yl) cyclopropanecarboxamide (Compound 18).

Triethylamine (0.25mL, 1.50mmol) and thiomorpholine 1, 1-dioxide hydrochloride (125mg, 0.75mmol) were added to (4- (2- (cyclopropylcarboxamide) - [1,2,4] successively under ice-bath]Triazolo [1,5-a]Pyridin-5-yl) phenyl) methyl-d-methanesulfonate (190mg, 0.49mmol) in anhydrous tetrahydrofuran (5mL) and the reaction mixture was allowed to warm to room temperature for overnight reaction. The reaction was quenched with water (25mL), extracted with dichloromethane (30mL × 3), the organic layer was washed with saturated brine (30mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the concentrated solution was subjected to column separation (eluent: dichloromethane/methanol (v/v) ═ 25:1) to give a beige solid, which was further purified by preparative thin layer chromatography (developing solvent: dichloromethane/methanol (v/v) ═ 12:1/15:1) to give 70mg of a white solid, yield: 33.5 percent. LC-MS (APCI) M/z 427.0(M + H)⁺；¹H NMR(300MHz,DMSO-d₆):δ11.06(s,1H),7.99(d,J＝8.2Hz,2H),7.78–7.64(m,2H),7.52(d,J＝8.2Hz,2H),7.29(dd,J＝6.1,2.4Hz,1H),3.74(s,1H),3.19–3.08(m,J＝4.8Hz,4H),2.99–2.85(m,J＝2.5Hz,4H),2.06–1.90(m,J＝30.2Hz,1H),0.82(d,J＝6.2Hz,4H)。

Example 3N- (5- (4- ((1, 1-dioxo-4-thiomorpholine) methyl-d 2) phenyl) - [1,2,4]Triazolo compounds [1,5-a]Pyridin-2-yl) cyclopropanecarboxamides (Compound 24)

Step 1: synthesis of 4-Carboxylic acid methyl ester phenylboronic acid (Compound 20).

Concentrated sulfuric acid (0.5mL) was added dropwise to 4-carboxyphenylboronic acid (3.50g, 21.09mmol) in dry methanol (50mL) and the reaction was refluxed overnight under nitrogen. The reaction mixture was concentrated under reduced pressure, 50mL of water was added, extraction was performed with ethyl acetate (50 × 3), and the organic layer was washed with water (50mL) and saturated brine (50mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 4.1g of a white solid, yield: 100 percent. LC-MS (APCI) M/z 181.1(M + H)⁺；¹H NMR(300MHz,DMSO-d₆):δ7.89(s,4H),7.52(s,4H),3.83(s,3H)。

Step 2: synthesis of methyl 4- (2- (cyclopropylcarbamoylamino) - [1,2,4] triazolo [1,5-a ] pyridin-5-yl) benzoate (Compound 21).

N₂Under protection, 1, 4-dioxane (9mL) and water (3mL) were injected into N- (5-bromo- [1,2, 4)]Triazolo [1,5-a]Pyridin-2-yl) Cyclopropanealkylcarboxamide (282mg, 1.00mmol), (4-Carboxylic acid methyl ester phenylboronic acid (220mg, 1.20mmol), Pd (dppf) Cl₂(40mg, 0.05mmol) and potassium carbonate (420mg, 3.00mmol) were reacted at 90 ℃ overnight under nitrogen (16 h). Cooling to the greenhouse, filtering through celite, washing the filter cake with dichloromethane, drying the filtrate with anhydrous sodium sulfate, removing the solvent, and column separating the concentrate (eluent: dichloromethane/methanol (v/v) ═ 25:1) to give 230mg beige solid, yield: 68.4 percent.

LC-MS(APCI):m/z＝337.2(M+H)⁺。

And step 3: n- (5- (4- (hydroxymethyl) -d)₂₎Phenyl) - [1,2,4]Triazolo [1,5-a]Synthesis of pyridin-2-yl) cyclopropanecarboxamide (Compound 22).

Lithium aluminum tetradeuteride (27mg, 0.63mmol) was added portionwise to 4- (2- (cyclopropylalkylcarboxamide) - [1,2,4] under ice-bath]Triazolo [1,5-a]Pyridin-5-yl) benzoic acid methyl ester (200mg, 0.60mmol) in dry tetrahydrofuran (10mL) was allowed to warm to room temperature for 1.5 h. The reaction was quenched by the addition of heavy water (10mL), extracted with dichloromethane (30mL × 3), the organic layer was washed with saturated brine (30mL), dried over anhydrous sodium sulfate, the solvent was removed, and the concentrated solution was subjected to column separation (eluent: dichloromethane/methanol (v/v) ═ 15:1) to give 143mg of a white solid, yield: 77.2 percent. LC-MS (APCI): M/z 311.2(M + H)⁺；¹H NMR(300MHz,DMSO-d₆):δ11.03(s,1H),7.95(d,J＝8.2Hz,2H),7.69(m,2H),7.48(d,J＝8.2Hz,2H),7.27(dd,J＝6.4,2.0Hz,1H),5.29(s,1H),2.02(m,1H),0.80(d,J＝6.2Hz,4H).

And 4, step 4: (4- (2- (cyclopropylcarboxamide) - [1,2,4]]Triazolo [1,5-a]Pyridin-5-yl) phenyl) methyl-d₂Synthesis of mesylate (Compound 23).

Triethylamine (0.12mL, 0.86mmol) and MsCl (methylsulfonyl chloride, 0.04mL, 0.52mmol) were added slowly, in that order, dropwise to N- (5- (4- (hydroxymethyl-d) under ice-bath₂₎Phenyl) - [1,2,4]Triazolo [1,5-a]Pyridin-2-yl) cyclopropanecarboxamide (135mg, 0.43mmol) in dry dichloromethane (10mL) and the reaction was warmed to room temperature for 2 h. The reaction was quenched with water (25mL), extracted with dichloromethane (30mL x 3), and the organic layer was washed with saturated brine (30mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give 170mg of oil, which was used directly in the next reaction, yield: 100 percent. LC-MS (APCI) M/z 388.1(M + H)⁺。

And 5: n- (5- (4- ((1, 1-dioxo-4-thiomorpholine) methyl-d₂) Phenyl) - [1,2,4]Triazolo [1,5-a]Synthesis of pyridin-2-yl) cyclopropanecarboxamide (Compound 24).

Triethylamine (0.25mL, 1.74mmol) and thiomorpholine 1, 1-dioxide hydrochloride (120mg, 0.70mmol) were added to (4- (2- (cyclopropylcarboxamide) - [1,2,4] successively under ice-bath]Triazolo [1,5-a]Pyridin-5-yl) phenyl) methyl-d₂To a solution of methanesulfonate (225mg, 0.58mmol) in anhydrous tetrahydrofuran (5mL), the reaction mixture was heated to room temperature and reacted overnight, and then heated to 85 ℃ and reacted at reflux overnight. The reaction was quenched with water (25mL), extracted with dichloromethane (30mL x 3), and the organic layer was washed with saturated brine (3mL x 3)0mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the concentrated solution was subjected to column separation (eluent: dichloromethane/methanol (v/v) ═ 25:1) to give a white solid, 160mg of white solid was obtained, yield: and (4) 64.6%. LC-MS (APCI) M/z 427.0(M + H)⁺；¹H NMR(300MHz,DMSO-d₆):δ11.05(s,1H),7.99(d,J＝8.1Hz,2H),7.70(m,2H),7.52(d,J＝8.0Hz,2H),7.29(dd,J＝6.0,2.1Hz,1H),3.15(m,4H),2.94(m,4H),1.99(m,1H),0.80(d,J＝6.1Hz,4H)。

EXAMPLE 4N- (5- (4- ((1, 1-dioxo-4-thiomorpholine-2, 2,6,6-d4) methyl-d 2) phenyl)- [1,2,4]Triazolo [1,5-a]Pyridin-2-yl) cyclopropanecarboxamides (Compound 26)

Step 1: 4- ((4- (2-amino- [1,2, 4)]Triazolo [1,5-a]Pyridin-5-yl) phenyl) methyl-d₂) Thiomorpholine-1, 1-dioxide-2, 2,6,6-d₄(Compound 25) Synthesis.

Sodium methoxide (120mg, 2.10mmol) was added to N- (5- (4- ((1, 1-dioxo-4-thiomorpholine) methyl-d₂) Phenyl) - [1,2,4]Triazolo [1,5-a]Pyridin-2-yl) Cyclopropanealkylcarboxamide (89mg, 0.21mmol) in deuterated methanol (CD)₃OD-d₄5mL) and the tube is sealed and reacted under the protection of nitrogen overnight. The reaction was quenched with heavy water (10mL), extracted with dichloromethane (10mL × 4), the organic phase was washed with saturated brine (15mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the concentrate was subjected to column separation (eluent: dichloromethane/methanol (v/v) ═ 10:1) to give 70mg of a beige solid, yield: 92.0 percent. ESI-MS M/z:364.2(M + H)⁺。

Step 2: n- (5- (4- ((1, 1-dioxo-4-thiomorpholine-2, 2,6, 6-d)₄) Methyl-d₂) Phenyl) - [1,2,4]Triazolo [1,5-a]Synthesis of pyridin-2-yl) cyclopropanecarboxamide (Compound 26).

Triethylamine (80mg, 0.76mmol) and cyclopropanecarbonyl chloride (80mg, 0.76mmol) were slowly added dropwise to 4- ((4- (2-amino- [1,2, 4) in that order at 5 deg.C]Triazolo [1,5-a]Pyridin-5-yl) Phenyl) methyl-d₂) Thiomorpholine-1, 1-dioxide-2, 2,6,6-d₄(70mg, 0.19mmol) of dry dichloromethane (5mL) and the reaction was warmed to room temperature until the reaction was complete (16 h). The reaction solvent was removed under reduced pressure, and the residue was added to a methanol solution of ammonia (7M, 5mL), and the bisacylate was hydrolyzed with stirring at room temperature (6h) to give the monoacylated target. The reaction was quenched with water (20mL), extracted with dichloromethane (20mL x 3), the organic phase washed with saturated brine (15mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the concentrate was subjected to preparative thin layer chromatography to give a beige solid 50mg, yield: 61.0 percent. LC-MS (APCI) M/z 432.1(M + H)⁺；¹H NMR(300MHz,DMSO-d6):δ11.05(s,1H),7.99(d,J＝8.1Hz,2H),7.69(m,2H),7.53(d,J＝8.0Hz,2H),7.29(dd,J＝6.0,2.3Hz,1H),2.95(m,4H),2.00(m,1H),0.80(d,J＝6.2Hz,4H)。

EXAMPLE 5N- (5- (4- ((1, 1-dioxo-4-thiomorpholine) methyl) phenyl-2, 6-d2) - [1,2,4]]III Azolo [1,5-a ]]Pyridin-2-yl) cyclopropanecarboxamides (Compound 33)

Step 1: 4-methylaniline-2, 6-d₂(Compound 28) Synthesis.

Concentrated deuterated hydrochloric acid (1.70mL, 20.00mmol) was added dropwise to a turbid solution of p-toluidine (2.14g, 20.00mmol) in heavy water (10mL) at room temperature to dissolve completely, and then the reaction mixture was stirred at 180 ℃ for 45min under microwave irradiation. Cooled to room temperature, adjusted to neutral ph with saturated sodium bicarbonate solution, extracted with dichloromethane (30mL x 3), the organic layer washed with saturated brine (20mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a dark brown solid 1.80g, yield: 84.1 percent. LC-MS (APCI) M/z 110.1(M + H)⁺；¹H NMR(300MHz,CDCl₃):δ7.01(s,2H),2.28(s,3H)。

Step 2: 2,6-d₂Synthesis of-4-bromotoluene (Compound 29).

HBr (40 wt% aq, 22mL) was added slowly dropwise to 4-methylaniline-2,6-d₂(3.60g, 33.0mmol) in water (16 mL). After stirring in the greenhouse for 15min, the mixture was cooled to-5 ℃ in an ice salt bath. Then, sodium nitrite (2.67g, 38.6mmol) in water (10mL) was slowly added dropwise while the reaction temperature was kept at 0 ℃ and stirred for 30 min. The reaction mixture was slowly added dropwise to a suspension of cuprous bromide (6.86g, 47.8mmol) in HBr (40 wt% aq, 30mL) and the reaction was carried out at 50 ℃ for 2 h. Cooled to the greenhouse, extracted with petroleum ether (100mL x 3), the organic layers were combined, washed with saturated sodium bicarbonate solution, water (100mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the concentrate was subjected to column separation (eluent: petroleum ether/ethyl acetate (v/v) ═ 1:0) to give 840mg of a colorless liquid, yield: 12.0 percent.

And step 3: 1-bromo-4- (bromomethyl) benzene-2, 6-d₂(Compound 30) Synthesis.

Azobisisobutyronitrile (AIBN, 45mg, 0.24mmol) was added to 2,6-d at room temperature₂4-bromotoluene (840mg, 4.85mmol) and N-bromosuccinimide (NBS, 900mg, 5.09mmol) in carbon tetrachloride (15mL) solvent under nitrogen protection at reflux overnight. The reaction mixture was concentrated under reduced pressure, 20mL of water was added, the organic layer was washed with dichloromethane (20mL x 3), saturated sodium bicarbonate (30mL) and saturated brine (30mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the concentrated solution was subjected to column separation (eluent: petroleum ether/ethyl acetate (v/v) ═ 2:1) to give 700mg of a brown-yellow oil, yield: 57.9 percent. LC-MS (APCI) M/z 251.0(M + H)⁺。

And 4, step 4: 4- (4-bromobenzyl-3, 5-d)₂) Synthesis of 1, 1-dioxo-4-thiomorpholine (Compound 31).

Triethylamine (850mg, 8.34mmol) was added dropwise to 1-bromo-4- (bromomethyl) benzene-2, 6-d₂(700mg, 2.78mmol) and thiomorpholine 1, 1-dioxide hydrochloride (477mg, 2.78mmol) in N, N-dimethylformamide (DMF,15mL), the reaction mixture was stirred at room temperature for reaction overnight. The reaction was diluted with ethyl acetate (50mL), washed with water (20mL × 3) and saturated brine (20mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the concentrated solution was subjected to column separation (eluent: petroleum ether/ethyl acetate (v/v) ═ 2:1) to give 270mg of a white solid, yield: 31.7％。ESI-MS m/z:306.1(M+H)⁺。

and 5: compound 4- (3, 5-d)₂Synthesis of (E) -4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzyl) thiomorpholine-1, 1-dioxide (Compound 32)

DMSO (5mL) was added to 4- (4-bromobenzyl-3, 5-d under nitrogen₂)1, 1-dioxo-4-thiomorpholine (270mg, 0.88mmol) and pinacol diboron (270mg, 1.06mmol), potassium acetate (260mg, 2.64mmol), Pd (dppf) Cl₂(36mg, 0.04mmol) and the reaction was stirred at 100 ℃ overnight under nitrogen. After cooling to room temperature, water (25mL) was added for dilution, extraction was performed with ethyl acetate (30mL × 2), the organic layer was washed with saturated brine (20mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the concentrated solution was subjected to column separation (eluent: petroleum ether/ethyl acetate (v/v) ═ 2:1) to obtain 240mg of a white solid, yield: 77.0 percent. LC-MS (APCI) M/z 354.2(M + H)⁺。

Step 6: n- (5- (4- ((1, 1-dioxo-4-thiomorpholinyl) methyl)]Phenyl-2, 6-d₂)-[1,2,4]Triazolo [1,5-a]Pyridin-2-yl]Synthesis of cyclopropylcarboxamide (Compound 33).

In N₂Under protection, 1, 4-dioxane (6mL) and water (1.5mL) were injected into N- (5-bromo- [1,2,4]]Triazolo [1,5-a]Pyridin-2-yl) Cyclopropanealkylcarboxamide (80mg, 0.30mmol), 4- (3,5-d₂-4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzyl) thiomorpholine-1, 1-dioxide (115mg, 0.33mmol), Pd (dppf) Cl₂(15mg, 0.02mmol) and potassium carbonate (130mg, 0.90mmol), and the reaction was carried out at 90 ℃ overnight. Cooling to the greenhouse, filtering through celite, washing the filter cake with dichloromethane, drying the filtrate with anhydrous sodium sulfate, removing the solvent, and column separating the concentrate (eluent: dichloromethane/methanol (v/v) ═ 25:1) to give 50mg beige solid, yield: 68.4 percent. LC-MS (APCI) 428.2(M + H) M/z⁺；¹H NMR(300MHz,DMSO-d₆):δ11.04(s,1H),7.69(m,2H),7.51(s,2H),7.29(m,1H),3.76(s,2H),3.14(m,4H),2.92(m,4H),2.00(m,1H),0.80(d,J＝6.0Hz,4H)。

Example 6 JAK kinase inhibition

Reagents and consumables:

recombinant human JAK1 catalytic domain (Carna, Cat.No. 08-144), recombinant human JAK2 catalytic domain (Carna, Cat.No. 08-045), recombinant human JAK3 catalytic domain (Carna, Cat.No. 08-046) ATP (Sigma, Cat.No. A7699-1G), DMSO (Sigma, Cat.No. D2650), 96-well plate (Corning, Cat.No.3365), 384-well plate (Greiner, Cat.No.784076), HTRF kit TK kit (Cisbio, Cat.No. 62TK0PEB). The experimental method comprises the following steps:

compound preparation: test compounds were dissolved in DMSO to make 20mM stock. Compounds were diluted to 0.1mM (100-fold final dilution) in DMSO and diluted in 3-fold gradients, 11 concentrations, prior to use. When adding medicine, the medicine is diluted by buffer solution into 4 times of the dilution solution with final concentration.

And (3) kinase detection: after buffer preparation, the enzyme was mixed with the compounds of different concentrations prepared by dilution in advance, and left at room temperature for 30 minutes, each concentration being double-well. The corresponding substrate and ATP were added and the reaction was carried out at room temperature for 60 minutes (negative and positive controls were set). And (3) after the reaction is finished, adding an antibody for detection, incubating at room temperature for 60 minutes, then carrying out Evnvision detection, and collecting data. Data analysis and mapping were performed according to XLfit5 software. Wherein A represents IC₅₀Less than or equal to 0.04 mu M; b represents 0.04. mu.M<IC₅₀Less than or equal to 0.1 mu M; c represents 0.1. mu.M<IC₅₀Less than or equal to 1 mu M; d represents IC₅₀>1μM；

Kinase inhibition IC in the examples₅₀(. mu.M) is summarized in Table 1 below

TABLE 1 kinase inhibition by the compounds of the examples

As shown in table 1, the compound of the present invention has excellent selective inhibitory effect on JAK enzyme, and the inhibitory effect on JAK1 (examples 1,2 and 3 are equivalent to Filgotinib activity, and examples 4 and 5 are even superior to Filgotinib activity) is superior to the inhibitory effect on JAK2 and more superior to the inhibitory effect on JAK 3. Therefore, the compounds of the present invention, while effective in reducing their toxic side effects, are useful in the treatment of conditions associated with aberrant JAK activity.

Example 7 evaluation of metabolic stability

Microsome experiment: human liver microsomes: 0.5mg/mL, Xenotech; rat liver microsomes: 0.5mg/mL, Xenotech; coenzyme (NADPH/NADH): 1mM, Sigma Life Science; magnesium chloride: 5mM, 100mM phosphate buffer (pH 7.4).

Preparing a stock solution: an amount of the compound of the example was weighed out precisely and dissolved in DMSO to 5mM each.

Preparation of phosphate buffer (100mM, pH 7.4): 150mL of 0.5M potassium dihydrogenphosphate and 700mL of a 0.5M dipotassium hydrogenphosphate solution prepared in advance were mixed, the pH of the mixture was adjusted to 7.4 with the 0.5M dipotassium hydrogenphosphate solution, the mixture was diluted 5-fold with ultrapure water before use, and magnesium chloride was added to obtain a phosphate buffer (100mM) containing 100mM potassium phosphate and 3.3mM magnesium chloride at a pH of 7.4.

NADPH regenerating system solution (containing 6.5mM NADP, 16.5mM G-6-P, 3U/mL G-6-P D, 3.3mM magnesium chloride) was prepared and placed on wet ice before use.

Preparing a stop solution: acetonitrile solution containing 50ng/mL propranolol hydrochloride and 200ng/mL tolbutamide (internal standard). 25057.5 mu L of phosphate buffer solution (pH7.4) is taken to a 50mL centrifuge tube, 812.5 mu L of human liver microsome is respectively added and mixed evenly, and liver microsome dilution liquid with the protein concentration of 0.625mg/mL is obtained. 25057.5 mu L of phosphate buffer (pH7.4) is taken to a 50mL centrifuge tube, 812.5 mu L of SD rat liver microsome is respectively added, and the mixture is mixed evenly to obtain liver microsome dilution with the protein concentration of 0.625 mg/mL.

Incubation of the samples: the stock solutions of the corresponding compounds were diluted to 0.25mM each with an aqueous solution containing 70% acetonitrile, and used as working solutions. 398. mu.L of human liver microsome or rat liver microsome dilutions were added to a 96-well plate (N2), 2. mu.L of 0.25mM working solution was added, and mixed well.

Determination of metabolic stability: 300. mu.L of pre-cooled stop solution was added to each well of a 96-well deep-well plate and placed on ice as a stop plate. The 96-well incubation plate and the NADPH regeneration system are placed in a 37 ℃ water bath box, shaken at 100 rpm and pre-incubated for 5 min. 80. mu.L of the incubation solution was taken out of each well of the incubation plate, added to the stop plate, mixed well, and supplemented with 20. mu.L of NADPH regenerating system solution as a 0min sample. Then 80. mu.L of NADPH regenerating system solution was added to each well of the incubation plate, the reaction was started, and the timer was started. The reaction concentration of the corresponding compound was 1. mu.M, and the protein concentration was 0.5 mg/mL. When the reaction was carried out for 10min, 30min and 90min, 100. mu.L of each reaction solution was added to the stop plate and vortexed for 3min to terminate the reaction. The stop plates were centrifuged at 5000 Xg for 10min at 4 ℃. And (3) taking 100 mu L of supernatant to a 96-well plate in which 100 mu L of distilled water is added in advance, mixing uniformly, and performing sample analysis by adopting LC-MS/MS.

And (3) data analysis: and detecting peak areas of the corresponding compound and the internal standard through an LC-MS/MS system, and calculating the peak area ratio of the compound to the internal standard. The slope is determined by plotting the natural logarithm of the percentage of compound remaining against time and calculating t according to the following formula_1/2And CL_intWhere V/M is equal to 1/protein concentration.

Experimental results as shown in table 2 below, the compounds of the present invention showed excellent metabolic stability in both human liver microsome and rat liver microsome experiments.

Table 2 evaluation of liver microparticle metabolism of the compounds of the examples

Example 8 pharmacokinetic evaluation in rats

8 male Sprague-Dawley rats, 7-8 weeks old, weighing about 210g, divided into 2 groups of 4 rats each, given a single oral administration of 5mg/kg dose of (a) control group: n- (5- (4- ((1, 1-dioxo-4-thiomorpholinyl) methyl) phenyl- [1,2,4] triazolo [1,5-a ] pyridin-2-yl) cyclopropylcarboxamide (b) test group, examples 1-5, the pharmacokinetic differences were compared.

Rats were fed with standard feed and given water. Fasting began 16 hours prior to the experiment. The drug was dissolved with PEG400 and dimethyl sulfoxide. Blood was collected from the orbit at 0.083 hr, 0.25 hr, 0.5 hr, 1 hr, 2 hr, 4 hr, 6 hr, 8 hr, 12 hr and 24 hr post-dose.

The rats were briefly anesthetized after ether inhalation and 300 μ L of blood was collected from the orbit into a test tube. There was 30 μ L of 1% heparin salt solution in the tube. Before use, the tubes were dried overnight at 60 ℃. After completion of blood sample collection at a subsequent time point, rats were sacrificed after ether anesthesia.

Immediately after blood collection, the tubes were gently inverted at least 5 times to ensure mixing and then placed on ice. The blood samples were centrifuged at 5000rpm for 5 minutes at 4 ℃ to separate the plasma from the erythrocytes. Pipette out 100 μ L of plasma into a clean plastic centrifuge tube, indicating the name of the compound and the time point. Plasma was stored at-80 ℃ before analysis. The concentration of the compounds of the invention in plasma was determined by LC-MS/MS. Pharmacokinetic parameters were calculated based on the plasma concentration of each animal at different time points.

The results of the experiments show that the compounds of the invention have better pharmacokinetics and thus better pharmacodynamics and therapeutic effects in animals compared to the control compounds.

It is to be understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention, and that experimental procedures not specifically identified in the examples will generally be performed under conventional conditions, or under conditions recommended by the manufacturer. Parts and percentages are parts and percentages by weight unless otherwise indicated.

The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

Claims (4)

1. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

2. a pharmaceutical composition comprising a compound of claim 1 or a pharmaceutically acceptable salt thereof and a conventional pharmaceutical carrier.

3. Use of the pharmaceutical composition of claim 2 in the manufacture of a medicament for treating, preventing or eliminating JAK kinase-associated rheumatoid arthritis.

4. Use of a compound of claim 1 in the manufacture of a medicament for the treatment of JAK kinase-associated rheumatoid arthritis.