CN111518080A

CN111518080A - 1,2, 4-triazole compound

Info

Publication number: CN111518080A
Application number: CN202010554939.8A
Authority: CN
Inventors: 王义汉; 李焕银
Original assignee: Shenzhen Targetrx Inc
Current assignee: Shenzhen Targetrx Inc
Priority date: 2017-10-12
Filing date: 2018-09-30
Publication date: 2020-08-11
Also published as: CN109232538A; CN109232538B; WO2019072130A1

Abstract

The invention relates to a 1,2, 4-triazole compound shown in a formula (I) and pharmaceutically acceptable salts thereof. The compounds have activity as inhibitors of apoptosis signal-regulated kinase 1 ("ASK 1"), and are therefore useful in the treatment of ASK1 mediated conditions including chronic liver disease, cardiovascular disease, metabolic disorders, respiratory disorders, gastrointestinal disorders, and neurodegenerative diseases. The invention also provides a pharmaceutical composition containing the compound and application thereof.

Description

1,2, 4-triazole compound

The application is a divisional application of an invention patent application with the application date of 2018, 09.month and 30, the application number of 201811159093.7 and the invention name of 'a 1,2, 4-triazole compound'.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a 1,2, 4-triazole compound, a composition containing the compound and application of the compound. In particular, the present invention relates to certain deuterium substituted 5- (4-cyclopropyl-1H-imidazol-1-yl) -2-fluoro-4-methyl-N- [6- (4-isopropyl-4H-1, 2,4, -triazol-3-yl) -2-pyridine ] -benzamides, which are useful in the treatment of ASK1 mediated diseases and have superior pharmacokinetic properties.

Background

Many current drugs suffer from poor absorption, distribution, metabolism and/or excretion (ADME) properties, which prevents their wider use or limits their use for certain indications. Poor ADME properties are also a significant cause of drug candidate failure in clinical trials. One such problem is rapid metabolism, which results in the removal of many drugs from the body too rapidly, which would otherwise be highly effective in the treatment of disease. A common solution for rapid drug clearance is frequent or high dose administration to achieve sufficiently high plasma drug levels. However, this poses a number of potential therapeutic problems, such as poor patient compliance with the dosing regimen, side effects becoming more acute at higher doses, and increased treatment costs. Rapidly metabolized drugs may also expose patients to undesirable toxic or reactive metabolites.

Another limitation of ADME that affects many drugs is the formation of toxic or bioreactive metabolites. Thus, some patients receiving the drug may suffer from toxicity, or the safe dose of such a drug may be limited such that the patient receives a sub-optimal amount of the active agent. In some cases, varying the dosing interval or formulation method may help to reduce clinical adverse effects, but the formation of such undesirable metabolites is often inherent in the metabolism of the compound.

One potentially attractive strategy to improve the metabolic performance of drugs is deuterium modification. In this approach, attempts have been made to slow drug metabolism or reduce the formation of undesirable metabolites by replacing one or more hydrogen atoms with deuterium atoms. Deuterium is a safe, stable, non-radioactive isotope of hydrogen. Deuterium forms a stronger bond with carbon than hydrogen. In selected cases, the increased bond strength imparted by deuterium can positively affect the ADME properties of a drug, thereby creating the potential to improve drug efficacy, safety, and/or tolerability. Also, because the size and shape of deuterium is substantially the same as the size and shape of hydrogen, replacement of hydrogen by deuterium would not be expected to affect the biochemical potency and selectivity of the drug compared to the original chemical entity containing only hydrogen.

During the past 35 years, the effect of deuterium substitution on metabolic rate was reported for a very small percentage of approved drugs (see, e.g., Foster, AB, Adv Drug Res, 1985, 14:1-40 ("Foster"); Fisher, MB et al, Curr Opindrug Discov Devel, 2006, 9: 101-09 ("Fisher)). The results are variable and unpredictable. For some compounds, deuteration causes a decrease in metabolic clearance in vivo. For other compounds, there was no metabolic change. Still other compounds exhibit increased metabolic clearance. Variability in the deuterium effect has also led the skilled person to suspect or abandon a viable drug design strategy for deuterium modification as an inhibitor of adverse metabolism (see pages 35 of Foster and 101 of Fisher).

The effect of deuterium modification on the metabolic properties of a drug is not predictable, even in the case of deuterium atom penetration into known metabolic sites. One can only determine if and how the metabolic rate differs from its non-deuterated counterpart by actually preparing and testing a deuterated drug. Many drugs have multiple sites where metabolism can occur. The sites where deuterium substitution is required and the effect on metabolism, if any, seen, the degree of deuteration necessary will vary for each drug.

The present invention relates to novel derivatives of Selonsertib and pharmaceutically acceptable salts thereof. The invention also provides compositions comprising the compounds of the invention and the use of such compositions in methods of treatment of diseases and disorders beneficially treated by administration of an inhibitor of ASK1 (apoptosis signal-regulating kinase 1).

Seloserteib, also known as GS-4997 and chemically known as 5- (4-cyclopropyl-1H-imidazol-1-yl) -2-fluoro-4-methyl-N- [6- (4-isopropyl-4H-1, 2,4, -triazol-3-yl) -2-pyridine ] -benzamide (having the structure shown below) is a small molecule inhibitor developed by Gilide pharmaceutical companies and can effectively reduce the pathological function of ASK 1. Results of a second phase clinical trial with Selonsertib in the treatment of nonalcoholic steatohepatitis (NASH) showed that anti-fibrotic effects were achieved in NASH patients only after 24 weeks. Currently, the study of Selonsertib for the treatment of NASH is in the third clinical stage, and the study for the treatment of alcoholic hepatitis is in the second clinical stage.

Phosphorylation of ASK1 protein can lead to apoptosis or other cellular responses depending on the cell type. ASK1 activation and signaling have been reported to play an important role in a wide range of diseases including: neurodegenerative disorders, cardiovascular disorders, inflammatory disorders and metabolic disorders. In addition, ASK1 is implicated in organ damage following mediation of ischemia and reperfusion of the heart, brain and kidneys (Watanabe et al (2005) BBRC 333, 562-567; Zhang et al (2003) Life Sci 74-37-43; Terada et al (2007) BBRC 364: 1043-49).

Even though Selonsertib is already present, there is still a need for effective compounds with improved pharmacokinetic and/or pharmacodynamic behaviour in the treatment of diseases related to the activation of ASK 1.

Disclosure of Invention

Aiming at the technical problems, the invention discloses a 1,2, 4-triazole compound, a composition containing the compound and application thereof, wherein the compound has stronger ASK1 inhibitor activity.

In contrast, the invention adopts the following technical scheme:

in a first aspect, the present invention relates to a 1,2, 4-triazole compound of formula (I), or a pharmaceutically acceptable salt thereof:

wherein the content of the first and second substances,

R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、Y¹、Y²、Y³、Y⁴、Y⁵and Y⁶Each independently is from hydrogen or deuterium;

X¹、X²and X³Each independently is CH₃、CH₂D、CHD₂Or CD₃。

Provided that if X is¹、X²And X³Each is CH₃Then R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、Y¹、Y²、Y³、Y⁴、Y⁵And Y⁶Is deuterium.

As a preferred embodiment of the present invention, the compound of formula (I) contains at least one deuterium atom, more preferably two deuterium atoms, more preferably three deuterium atoms, more preferably four deuterium atoms, more preferably six deuterium atoms, more preferably seven deuterium atoms, more preferably nine deuterium atoms.

As a preferred embodiment of the present invention, the deuterium isotope content of deuterium at the deuterated position is at least 0.015% greater than the natural deuterium isotope content, preferably greater than 30%, more preferably greater than 50%, more preferably greater than 75%, more preferably greater than 95%, more preferably greater than 99%.

Specifically, in the present invention R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、Y¹、Y²、Y³、Y⁴、Y⁵、Y⁶、X¹、X²And X³The 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 95%, more preferably greater than 99%.

In another embodiment, R of the compound of formula (I)¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、Y¹、Y²、Y³、Y⁴、Y⁵、Y⁶、X¹、X²And X³Preferably, at least one of the deuterium containing, more preferably two deuterium containing, more preferably three deuterium containing, more preferably four deuterium containing, more preferably five deuterium containing, more preferably six deuterium containing, more preferably seven deuterium containing, more preferably eight deuterium containing, more preferably nine deuterium containing, more preferably ten deuterium containing, more preferably eleven deuterium containing, more preferably twelve deuterium containing, more preferably thirteen deuterium containing, more preferably fourteen deuterium containing, more preferably fifteen deuterium containing, more preferably sixteen deuterium containing, more preferably seventeen deuterium containing, more preferably eighteen deuterium containing, more preferably nineteen deuterium containing, more preferably twenty one deuterium containing, more preferably twenty two deuterium containing, more preferably twenty three deuterium containingDeuterium. In particular, the compounds of formula (I) contain at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty-one, twenty-two, twenty-three deuterium atoms.

As a preferred embodiment of the present invention, R¹、R²、R³、R⁴、R⁵、R⁶、R⁷And R⁸Each independently from hydrogen or deuterium.

In another preferred embodiment, R¹、R³And R⁴Is deuterium.

In another preferred embodiment, R⁵Is deuterium.

In another preferred embodiment, R¹、R³、R⁴And R⁵Is deuterium.

As a preferred embodiment of the present invention, Y¹、Y²、Y³、Y⁴、Y⁵And Y⁶Each independently from hydrogen or deuterium.

In another preferred embodiment, Y¹Is deuterium.

In another preferred embodiment, Y²、Y³、Y⁴、Y⁵And Y⁶Is deuterium.

As a preferred embodiment of the present invention, X¹、X²And X³Each independently is CH₃、CH₂D、CHD₂Or CD₃。

In another preferred embodiment, X¹、X²Is a CD₃。

In another preferred embodiment, X³Is a CD₃。

As a preferred embodiment of the present invention, the compound is selected from the group consisting of:

in another preferred embodiment, the compound does not include non-deuterated compounds.

In a second aspect of the present invention, the present invention also discloses a pharmaceutical composition comprising a pharmaceutically acceptable excipient and the 1,2, 4-triazole compound described above, or a pharmaceutically acceptable salt thereof.

In a third aspect of the present invention, the present invention also discloses a preparation method of the pharmaceutical composition as described above, comprising the following steps: mixing a pharmaceutically acceptable excipient with a 1,2, 4-triazole-type compound, or a pharmaceutically acceptable salt thereof, as described above, to form a pharmaceutical composition.

In another preferred embodiment, the pharmaceutical composition is an injection, a sachet, a tablet, a pill, a powder or a granule.

In another preferred embodiment, the pharmaceutical composition further comprises an additional therapeutic agent, wherein the additional therapeutic agent is an agent for treating cancer, cardiovascular disease, inflammation, infection, immune disease, metabolic disease or organ transplantation.

In a fourth aspect of the invention, there is also provided a method of treating a disease mediated at least in part by ASK1 in a patient in need thereof, comprising administering to the patient in need thereof a therapeutically effective amount of a compound of the first aspect of the invention, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

In particular embodiments, the diseases treatable by the compounds of the invention are selected from diabetes, diabetic nephropathy, renal disease, renal fibrosis, pulmonary fibrosis, Idiopathic Pulmonary Fibrosis (IPF), hepatic fibrosis, pulmonary hypertension, nonalcoholic steatohepatitis, liver disease, alcoholic liver disease, inflammatory disorders, autoimmune diseases, proliferative diseases, transplant rejection, diseases involving impairment of cartilage metabolism, congenital cartilage malformations, or diseases associated with hypersecretion of IL 6.

In another preferred embodiment, the treatable disease is non-alcoholic steatohepatitis or alcoholic liver disease.

In another preferred embodiment, the treatable disease is pulmonary hypertension or pulmonary fibrosis.

In another preferred embodiment, the treatable disease is diabetic nephropathy, renal disease or renal fibrosis.

It is understood that within the scope of the present invention, each of the above-described technical features, embodiments of the present invention, and each of the technical features specifically described below (e.g., examples) may be combined with each other to constitute a new or preferred technical solution. Not to be reiterated herein, but to the extent of space.

Detailed Description

Compared with the non-deuterated compound, the compound of the deuterated ASK1 inhibitor and the pharmaceutically acceptable salt thereof have better pharmacokinetic and/or pharmacodynamic properties, so that the compound is more suitable to be used as an ASK1 inhibitor compound and is further more suitable to be used for preparing medicines for treating ASK 1-mediated related diseases. The present invention has been completed based on this finding.

Definition of

Herein, "deuterated", unless otherwise specified, means that one or more hydrogens of a compound or group are replaced with deuterium; deuterium can be mono-, di-, poly-, or fully substituted. The terms "deuterated one or more" and "deuterated one or more" are used interchangeably.

Herein, unless otherwise specified, "non-deuterated compound" means a compound containing deuterium at an atomic ratio of deuterium not higher than the natural deuterium isotope content (0.015%).

The invention also includes isotopically-labeled compounds, equivalent to those disclosed herein as the original compound. Examples of isotopes that can be listed as 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.

Compound (I)

The present invention provides compounds of formula (I), or a pharmaceutically acceptable salt, prodrug, hydrate, or solvate, crystal form, stereoisomer, or isotopic variant thereof:

wherein the content of the first and second substances,

R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、Y¹、Y²、Y³、Y⁴、Y⁵、Y⁶each independently is from hydrogen or deuterium;

X¹、X²、X³each independently is CH₃、CH₂D、CHD₂Or CD₃；

As a preferred embodiment of the present invention, the deuterium isotope content of deuterium at the deuterium position is at least 0.015% greater than the natural isotope content, 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 a particular embodiment of formula (I), "R¹、R²、R³、R⁴、R⁵、R⁶、R⁷And R⁸Each independently from hydrogen or deuterium "comprising R¹Selected from hydrogen or deuterium, R²Selected from hydrogen or deuterium, R³Selected from hydrogen or deuterium, and so on, up to R⁸Selected from hydrogen or deuterium. More specifically, includes R¹Is hydrogen or R¹Is deuterium, R²Is hydrogen or R²Is deuterium, R³Is hydrogen or R³Deuterium, and so on, until R⁸Is hydrogen or R⁸Is a technical scheme of deuterium.

In another embodiment of formula (I), "Y¹、Y²、Y³、Y⁴、Y⁵And Y⁶Each independently from hydrogen or deuterium "comprising Y¹Selected from hydrogen or deuterium, Y²Selected from hydrogen or deuterium, Y³Selected from hydrogen or deuterium, and so on, up to Y⁶Selected from hydrogen or deuterium. More specifically, including Y¹Is hydrogen or Y¹Is deuterium, Y²Is hydrogen or Y²Is deuterium, Y³Is hydrogen or Y³Deuterium, and so on, up to Y⁶Is hydrogen or Y⁶Is a technical scheme of deuterium.

In another embodiment of formula (I), "X¹、X²、X³Each independently is CH₃、CH₂D、CHD₂Or CD₃"includes X¹Is selected from CH₃、CH₂D、CHD₂Or CD₃，X²Is selected from CH₃、CH₂D、CHD₂Or CD₃，X³Is selected from CH₃、CH₂D、CHD₂Or CD₃The technical scheme of (1). More specifically, including X¹Is CH₃、X¹Is CH₂D、X¹Is CHD₂Or X¹Is a CD₃，X²Is CH₃、X²Is CH₂D、X²Is CHD₂Or X²Is a CD₃，X³Is CH₃、X³Is CH₂D、X³Is CHD₂Or X³Is a CD₃The technical scheme of (1).

In a preferred embodiment, the present invention relates to a compound of formula (I), or a pharmaceutically acceptable salt, prodrug, hydrate or solvate, crystal form, stereoisomer or isotopic variant thereof, wherein R is⁷And R⁸、Y²-Y⁶Is hydrogen, R¹-R⁸And Y¹Each independently of the other being hydrogen or deuterium, X¹、X²And X³Each independently is CH₃、CH₂D、CHD₂Or CD₃With the proviso that said compound contains at least one deuterium atom.

In another preferred embodiment, R²And R⁶Is hydrogen.

In another preferred embodiment, R¹、R³And R⁴Each independently selected from hydrogen or deuterium. In another more preferred embodiment, R¹、R³And R⁴Is hydrogen. In another more preferred embodiment, R¹、R³And R⁴Is deuterium.

In another preferred embodiment, R⁵Is hydrogen or deuterium. In another more preferred embodiment, R⁵Is hydrogen. In another more preferred embodiment, R⁵Is deuterium.

In another preferred embodiment, X³Is CH₃。

In another preferred embodiment, X¹And X²Each independently selected from CH₃Or CD₃. In another more preferred embodiment, X¹And X²Is CH₃. In addition toIn a more preferred embodiment, X¹And X²Is a CD₃。

In another preferred embodiment, Y¹Is hydrogen or deuterium. In another more preferred embodiment, Y¹Is hydrogen. In another more preferred embodiment, Y¹Is deuterium.

The term "pharmaceutically acceptable salts" refers, inter alia, to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, the pharmaceutically acceptable salts are described in detail by Berge et al in J.pharmaceutical Sciences (1977)66: 1-19. Pharmaceutically acceptable salts of the compounds of the present invention include salts derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts with inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or with organic acids, such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid. Salts formed using methods conventional in the art, e.g., ion exchange methods, are also included. Other pharmaceutically acceptable salts include: adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cypionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, gluconate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, picrate, etc, Stearate, succinate, sulfate, tartrate, thiocyanate,P-toluenesulfonate, undecanoate, valerate, and the like. Pharmaceutically acceptable salts derived from suitable bases include alkali metals, alkaline earth metals, ammonium and N⁺(C_1-4Alkyl radical)₄And (3) salt. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium salts, and the like. Other pharmaceutically acceptable salts include, if appropriate, non-toxic ammonium, quaternary ammonium and amine cations formed with counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.

The term "solvate" refers to a complex of a compound of the present invention coordinated to solvent molecules in a specific ratio. "hydrate" refers to a complex formed by coordination of a compound of the present invention with water.

The term "prodrug" includes a class of compounds which may be biologically active or inactive in nature and which, when administered by an appropriate method, undergo a metabolic or chemical reaction in the human body to convert it to a compound of formula (I), or a salt or solution of a compound of formula (I). The prodrug includes (but is not limited to) carboxylate, carbonate, phosphate, nitrate, sulfate, sulfone ester, sulfoxide ester, amino compound, carbamate, azo compound, phosphoramide, glucoside, ether, acetal and other forms of the compound.

Pharmaceutical compositions and methods of administration

The compound has excellent activity of inhibiting ASK1 kinase, so the compound and various crystal forms, pharmaceutically acceptable salts, hydrates or solvates thereof, and a pharmaceutical composition containing the compound as a main active ingredient can be used for treating, preventing and relieving ASK1 mediated diseases. According to the prior art, the compounds of the invention are useful for the treatment of the following diseases: chronic liver diseases, cardiovascular diseases, metabolic disorders, respiratory disorders, gastrointestinal disorders, neurodegenerative diseases, and the like.

The pharmaceutical composition of the present invention comprises the compound of the present invention or a pharmacologically acceptable salt thereof in a safe and effective amount range and a pharmacologically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 0.5-2000mg of a compound of the invention per dose, more preferably, 1-500mg of a compound of the invention per dose. Preferably, said "dose" is a capsule or tablet.

"pharmaceutically acceptable excipient" refers to a non-toxic carrier, adjuvant, or vehicle that does not destroy the pharmacological activity of the compounds formulated together. Pharmaceutically acceptable carriers, adjuvants or vehicles that may be used in the compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, silica gel, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, duodenal, rectal, parenteral (intravenous, intramuscular, or subcutaneous), and topical administration.

Solid dosage forms for oral administration include capsules, tablets, pills and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or solubilizers, for example, starch, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) disintegrating agents, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, e.g., paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glycerol monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be delayed in release in a certain part of the digestive tract. Examples of embedding components which can be used are polymeric substances and wax-like substances. If desired, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compounds, the liquid dosage forms may contain inert release agents conventionally employed in the art, such as water or other solvents, solubilizing agents and emulsifiers, for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide, and oils, especially cottonseed, groundnut, corn germ, olive, castor and sesame oils, or mixtures of these materials.

In addition to these inert diluents, the compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.

Dosage forms for topical administration of the compounds of the present invention include ointments, powders, patches, sprays, and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if necessary.

The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

When the pharmaceutical composition is used, a safe and effective amount of the compound of the present invention is suitable for mammals (such as human beings) to be treated, wherein the administration dose is a pharmaceutically-considered effective administration dose, and for a human body with a weight of 60kg, the daily administration dose is usually 0.5-2000mg, preferably 1-500 mg. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

Methods of treating diseases

Therapeutic agents useful as inhibitors of ASK1 signaling have the following potential: treating or improving the life of neurodegenerative, cardiovascular, inflammatory, autoimmune and metabolic disorders in a patient in need of treatment of the disease or disorder. In particular, ASK1 inhibitors have the following potential: treating cardiorenal diseases, including kidney diseases, diabetic nephropathy, chronic kidney diseases, fibrotic diseases (including lung and kidney fibrosis), dilated cardiomyopathy, respiratory diseases (including Chronic Obstructive Pulmonary Disease (COPD) and acute lung injury), and acute and chronic liver diseases (such as nonalcoholic steatohepatitis and alcoholic hepatitis).

Various assays for identifying the ability of compounds to inhibit ASK1 kinase activity and their effectiveness as ASK1 inhibitors are known in the art and are described, for example, in U.S. patent No. 8,742,126.

Some embodiments described herein relate to the use of a form of a compound described herein or a pharmaceutical composition described herein to treat a disease in a patient in need of treatment with an ASK1 inhibitor.

Some embodiments described herein are methods of treating diabetic nephropathy or diabetic complications comprising administering a therapeutically effective amount of a compound of formula (I) or a pharmaceutical composition described herein. In some embodiments, diabetes includes type 1 and type 2 diabetes, gestational diabetes, prediabetes, insulin resistance, metabolic syndrome, impaired fasting glucose, and impaired glucose tolerance. Type 1 diabetes is also known as Insulin Dependent Diabetes Mellitus (IDDM). Type 2 is also known as non-insulin-dependent diabetes mellitus (NIDDM).

Another embodiment relates to a method of treating kidney disease or diabetic nephropathy comprising administering a therapeutically effective amount of a compound of formula (I) as described herein or a pharmaceutical composition as described herein.

Another embodiment relates to a method of treating renal fibrosis, pulmonary fibrosis or Idiopathic Pulmonary Fibrosis (IPF) comprising administering a therapeutically effective amount of a form of compound I described herein or a pharmaceutical composition described herein.

Another embodiment relates to a method of treating diabetic nephropathy, renal fibrosis, liver fibrosis or lung fibrosis comprising administering a therapeutically effective amount of a crystalline form of compound I described herein or a pharmaceutical composition described herein.

Disclosed herein are methods of treating and/or preventing a liver disease in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a form of compound I described herein or a composition thereof, optionally in combination with a therapeutically effective amount of a LOXL2 inhibitor. The presence of active liver disease can be detected by the presence of elevated enzyme levels in the blood. In particular, blood levels of alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) above the clinically accepted normal range are known to indicate ongoing liver damage. Conventional monitoring of blood levels of ALT and AST in patients with liver disease is used clinically to measure the progression of liver disease at the time of medical treatment. The reduction of elevated ALT and AST to within the accepted normal range is taken as clinical evidence, reflecting the reduction in severity of ongoing liver damage in the patient.

In certain embodiments, the liver disease is chronic liver disease. Chronic liver disease involves progressive destruction and regeneration of the liver parenchyma, leading to fibrosis and cirrhosis of the liver. In general, chronic liver disease can be caused by viruses (e.g., hepatitis b, hepatitis c, Cytomegalovirus (CMV) or Epstein Barr Virus (EBV)), toxic agents or drugs (e.g., alcohol, methotrexate, or nitrofurantoin), metabolic diseases (e.g., non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hemochromatosis, or wilson's disease), autoimmune diseases (e.g., autoimmune chronic hepatitis, primary biliary cirrhosis, or primary sclerosing cholangitis), or other causes (e.g., right heart failure).

In a particular embodiment, the liver disease is a metabolic liver disease. In one embodiment, the liver disease is non-alcoholic fatty liver disease (NAFLD). NAFLD is associated with insulin resistance and metabolic syndrome (obesity, combined hyperlipidemia, diabetes (type II) and hypertension). NAFLD is thought to cover a range of disease activities and begins as fat accumulation in the liver (hepatic steatosis).

The compounds of the present invention have a number of advantages over the non-deuterated compounds known in the prior art. The advantages of the invention include: firstly, the compound adopting the technical scheme of the invention has excellent inhibitory property on ASK1 protein kinase. Second, the metabolism of the compound in the organism is improved, giving the compound better pharmacokinetic parameters. In this case, the dosage can be varied and a long acting formulation formed, improving the applicability. Thirdly, the medicine concentration of the compound in the animal body is improved, and the medicine curative effect is improved. Fourth, certain metabolites are inhibited, increasing the safety of the compounds.

Examples

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.

In general, in the preparative schemes, each reaction is usually carried out in an inert solvent at a temperature ranging from room temperature to reflux temperature (e.g., from 0 ℃ to 100 ℃, preferably from 0 ℃ to 80 ℃). The reaction time is usually 0.1 to 60 hours, preferably 0.5 to 24 hours.

Example 15- (4-cyclopropyl-1H-imidazolyl-1-yl) -N- (6- (4-isopropyl-4H-1, 2, 4-triazole-3- ₂Group-5-d) preparation of pyridin-2-yl-3, 4-d) -2-fluoro-4-methylbenzamide (Compound T-1).

The specific synthesis steps are as follows:

step 1 synthesis of compound 2.

Compound 1(5.0g, 32.86mmol) and methanol (60mL) were added sequentially to a 100mL single neck flask equipped with magnetic stirring, stirred to dissolve, hydrazine hydrate (3.29g, 65.72mmol) was slowly added dropwise, after dropping, the reaction mixture was heated under reflux for 3 hours, then cooled to room temperature to precipitate a large amount of white solid, filtered, the filter cake was washed with cold methanol, dried to obtain 3.5g of the white solid with a yield of 70%. LC-MS (APCI): M/z ═ 153.2(M +1)⁺.¹H NMR(DMSO-d₆,300MHz)(/ppm):9.14(s,1H),7.51(t,J＝5.7Hz,1H),7.11(d,J＝5.7Hz,1H),6.61(d,J＝6.0Hz,1H),6.08(s,2H),4.48(s,2H).

Step 2 synthesis of compound 3.

To a 100mL single neck flask equipped with magnetic stirring were added compound 2(3.5g, 23mmol), toluene (Tol, 50mL), isopropylamine (9.8g, 166mmol) and N, N-dimethylformamide-dipropylacetal (DMF-DPA, 10.89g, 62mmol) in that order, and acetic acid (2.1g, 35mmol) was slowly added dropwise with stirring₂The reaction mixture was heated under reflux for 24h under an atmosphere. Cooling to room temperature, removing the solvent by evaporation under reduced pressure, adding water (40mL), precipitating a large amount of solid under stirring, filtering, washing the filter cake with isopropanol to obtain 3.1g of white solid with a yield of 66.3%. LC-MS (APCI): M/z 204.2(M +1)⁺.¹H NMR(CDCl₃,300MHz)(/ppm):8.31(s,1H),7.58-7.54(m,2H),6.56(dd,J₁＝5.4Hz,J₂＝1.5Hz,1H),5.64-5.57(m,1H),4.45(s,2H),1.52(d,J＝4.8Hz,6H).

Step 3 synthesis of compound 4.

Compound 3(200mg, 984umol), heavy water (10mL) and Pd/C (50mg, 10%) were added to a 50mL sealed tube equipped with magnetic stirring in this order, and hydrogen was bubbled for 5 minutes under stirring, then the reaction mixture was sealed, warmed to 110 ℃ and stirred under heat overnight. Cooled to room temperature, dichloromethane (20mL) was added, filtered, the layers separated, the aqueous phase extracted with dichloromethane (20mL x2), the organic phases combined, dried over anhydrous sodium sulphate, filtered, concentrated and separated by a silica gel column to give 120mg of a white solid in 59.1% yield. LC-MS (APCI): M/z 207.3(M +1)⁺.¹H NMR(CDCl₃,300MHz)(/ppm):7.56(s,1H),5.62-5.59(m,1H),4.50(s,1H),1.51(d,J＝5.4Hz,6H).

Step 4 synthesis of compound 7.

Compound 6(5.0g, 24.5mmol) and toluene (50mL) were added sequentially to a 100mL single-neck flask equipped with magnetic stirring, the mixture was stirred until a solution was cleared, Compound 5(4.4g, 27.0mmol) and N, N-diisopropylethylamine (DIPEA,8.5mL, 51.5mmol) were added dropwise, and the reaction solution was heated under reflux for 2 hours. Cooling to room temperature, adding water (50mL), separating the layers, and successively adding saturated NH to the organic phase₄Cl water solution (20mL), saturated NaHCO₃Washing with water solution (20mL) and saturated brine (20mL), drying with anhydrous sodium sulfate, filtering, concentrating, recrystallizing the residue in n-hexane, filtering, and oven drying to obtain off-white solid 3.1g, with yield 44.1%. LC-MS (APCI) m/z 286.2&288.2(M+1)⁺.¹H NMR(DMSO-d₆,300MHz)(/ppm):7.07(d,J＝6.9Hz,1H),6.52(d,J＝4.5Hz,1H),5.29(t,J＝4.2Hz,1H),4.18(d,J＝4.2Hz,2H),4.52-4.51(m,1H),2.11(s,3H),0.98-0.88(m,4H).

Step 5 synthesis of compound 8.

To a 50mL single neck flask equipped with magnetic stirring was added compound 7(3.1g, 10.8mmol) and dichloromethane (DCM, 15mL) in sequence, stirred to dissolve and cool to 0 deg.C, formic acid (15mL) and acetic anhydride (Ac) were added slowly dropwise₂O, 4.1mL, 43.3mmol), reaction solution N₂The reaction was stirred at 0 ℃ for 3 hours under an atmosphere. Adding water (20mL), adjusted to pH 9 with 40% aqueous NaOH, the organic phase was separated, the aqueous phase was extracted with dichloromethane (30mLx2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered and the concentrate was used directly in the next reaction. LC-MS (APCI) 314.1. m/z&316.1(M+1)⁺.¹H NMR(DMSO-d₆,300MHz)(/ppm):8.15(d,J＝9.6Hz,1H),7.61(d,J＝5.4Hz,1H),7.41(d,J＝7.2Hz,1H),4.68(d,2H),2.50(s,3H),2.12-1.98(m,1H),0.96-0.85(m,4H).

Step 6 synthesis of compound 9.

A50 mL single neck flask equipped with magnetic stirring was charged with Compound 8(3.39g, 10.8mmol) and glacial acetic acid (30mL) in sequence, the solution was stirred, ammonium acetate (2.57g, 42.1mmol), N₂The reaction mixture was heated to reflux overnight under an atmosphere. Cooled to room temperature, the acetic acid was evaporated under reduced pressure, water (20mL) was added, the pH was adjusted to 9 with 40% aqueous NaOH, dichloromethane was extracted (30mLx3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was separated by silica gel column to give 1.7g of a white solid with a yield of 53.4%. LC-MS (APCI) M/z 295.1 and 297.1(M +1)⁺.¹H NMR(CDCl₃,300MHz)(/ppm):7.41(d,J＝4.8Hz,1H),7.37(s,1H),7.07(d,J＝6.6Hz,1H),6.72(s,1H),2.14(s,3H),1.90-1.85(m,1H),0.90-0.78(m,4H).

Step 7 synthesis of compound 10.

To a 50mL three-necked flask equipped with magnetic stirring was added compound 9(1.7g, 5.8mmol), vacuum was applied and N was added₂Substitution 3 times, N₂Anhydrous THF (30mL) was added dropwise under an atmosphere, the solution stirred, cooled to 0 deg.C, isopropyl magnesium chloride (4.3mL, 8.6mmol, 2M) was added dropwise slowly, the reaction was stirred for 1 hour at constant temperature, and then the reaction mixture was purged with CO₂Slowly introducing CO into the reaction liquid by a balloon of gas₂After reaction for 1 hour, water (20mL) was added to quench the reaction, the pH was adjusted to 5 with 6M aqueous HCl, ethyl acetate was extracted (30mLx3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was recrystallized from ether to give 600mg of a white solid with a yield of 40.0%. LC-MS (APCI) M/z 261.1(M +1)⁺.¹H NMR(DMSO-d₆,300MHz)(/ppm):9.17(s,1H),7.95(d,J＝4.8Hz,1H),7.70(s,1H),7.52(d,J＝8.4Hz,1H),2.24(s,3H),2.05-1.91(m,1H),1.01-0.85(m,4H).

Step 8 Synthesis of Compound T-1.

To a 50mL three-necked flask equipped with magnetic stirring was added compound 10(100mg, 0.384mmol), vacuum was applied and N was added₂Substitution of N₂Under the atmosphere, dry dichloromethane (2mL) and DMF (2mL) were added dropwise via syringe, the solution was stirred and dissolved, the reaction solution was cooled to 0 deg.C, oxalyl chloride (0.33mL, 0.653mmol, 2M dichloromethane solution) was added dropwise slowly, the ice bath was removed, and the reaction was stirred at room temperature for 1 hour. The solvent was evaporated under reduced pressure and dry dichloromethane (10mL), N, was added₂The solution was stirred under an atmosphere, cooled to 0 deg.C, and a solution of Compound 4(94mg, 0.461mmol) in dichloromethane (2mL) was added dropwise slowly, followed by DIPEA (0.19mL, 1.15mmol), the ice bath was removed, and the reaction was stirred at room temperature for 2 hours. The reaction was quenched by the addition of water (20mL), the organic layer was separated, the aqueous phase was extracted with dichloromethane (20mLx2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was separated on a silica gel column to give an off-white solid 60mg, yield 35.1%. LC-MS (APCI): M/z 446.4(M +1)⁺.¹H NMR(CDCl₃,300MHz)(/ppm):9.06(d,J＝6.0Hz,1H),8.40-8.36(m,2H),8.07-8.05(m,2H),7.91(t,J＝6.0Hz,1H),7.45(s,1H),7.18(d,J＝9.0Hz,1H),6.78(s,1H),5.51-5.44(m,1H),2.28(s,3H),1.91-1.88(m,1H),1.59(d,J＝5.1Hz,6H),0.90-0.81(m,4H).

Example 25- (4-cyclopropyl-1H-imidazolyl-1-yl) -N- (6- (4-isopropyl-4H-1, 2, 4-triazole-3- Yl) pyridin-2-yl) -2-fluoro-4-methylbenzamide-6-d (Compound T-2).

The specific synthesis steps are as follows:

step 1 synthesis of compound 12.

To a 20mL microwave tube equipped with magnetic stirring was added compound 6(2.0g, 9.8mmol) and heavy water (10mL), and slowly added with stirringDCl (0.817mL, 9.8mmol, 12M), the reaction mixture was heated to 160 ℃ under microwave for 1.5 hours. Cooling to room temperature, saturated NaHCO₃The pH was adjusted to 10, extracted with dichloromethane (20mLx3), the organic phases combined, dried over anhydrous sodium sulfate, filtered and concentrated to give a brown solid 1.59g with 79.1% yield. LC-MS (APCI): M/z ═ 205.1(M +1)⁺.¹H NMR(DMSO-d₆,300MHz)(/ppm):6.93(d,J＝7.2Hz,1H),4.94(s,2H),1.99(s,3H).

Step 2 synthesis of compound 13.

Compound 12(1.59g, 7.75mmol) and toluene (30mL) were added sequentially to a 100mL single-neck flask equipped with magnetic stirring, the mixture was dissolved by stirring, compound 5(1.39g, 8.53mmol) and DIPEA (2.7mL, 16.28mmol) were added dropwise, and the reaction mixture was heated under reflux for 2 hours. Cooled to room temperature, water (30mL) was added, the layers were separated and the organic phase was successively saturated with NH₄Cl water solution (10mL), saturated NaHCO₃Washing with water solution (10mL) and saturated brine (10mL), drying with anhydrous sodium sulfate, filtering, concentrating, recrystallizing the residue in n-hexane, filtering, and oven drying to obtain off-white solid 1.5g, with yield 67.4%. LC-MS (APCI) m/z 287.2&289.2(M+1)⁺.

Step 3 synthesis of compound 14.

To a 50mL single neck flask equipped with magnetic stirring was added compound 13(1.5g, 5.22mmol) and dichloromethane (10mL) in sequence, the mixture was stirred to dissolve and cooled to 0 deg.C, and formic acid (15mL) and acetic anhydride (2.1mL, 20.9mmol) and reaction solution N were added dropwise slowly₂The reaction was stirred at 0 ℃ for 3 hours under an atmosphere. The reaction was quenched by addition of water (10mL), adjusted to pH 9 with 40% aqueous NaOH, the organic phase separated, extracted with aqueous dichloromethane (20mLx2), the organic phases combined, dried over anhydrous sodium sulfate, filtered and the concentrate used directly in the next reaction. LC-MS (APCI) m/z 315.1&317.1(M+1)⁺.

Step 4 synthesis of compound 15.

To a 50mL single neck flask equipped with magnetic stirring was added compound 14(1.65g, 5.24mmol) and glacial acetic acid (15mL) in sequence, the mixture was stirred to a clear solution, ammonium acetate (1.25g, 20.5mmol), N was added₂The reaction was heated to reflux overnight under an atmosphere. Cooling to room temperature, and removing by evaporation under reduced pressureAcetic acid, water (10mL), 40% aqueous NaOH to adjust pH to 9, dichloromethane extraction (20mLx3), combined organic phases, dried over anhydrous sodium sulfate, filtered, and the residue was chromatographed on silica gel to give 1.7g of a white solid in 53.4% yield. LC-MS (APCI) M/z 296.1 and 298.1(M +1)⁺.¹H NMR(CDCl₃,300MHz)(/ppm):7.38(s,1H),7.09(d,J＝6.9Hz,1H),6.73(s,1H),2.15(s,3H),1.91-1.87(m,1H),0.91-0.78(m,4H).

Step 5 synthesis of compound 16.

To a 50mL three-necked flask equipped with magnetic stirring was added compound 15(820mg, 2.77mmol), evacuated and N₂Substitution 3 times, N₂Anhydrous THF (15mL) was added dropwise under an atmosphere, the solution stirred, cooled to 0 deg.C, isopropyl magnesium chloride (2.77mL, 5.54mmol, 2M) was added slowly dropwise, the reaction was stirred for 1 hour at constant temperature, then the reaction was quenched by filling with CO₂Slowly introducing CO into the reaction liquid by a balloon of gas₂After 1 hour of reaction, the reaction was quenched by addition of water (20mL), adjusted to pH 5 with 6M aqueous HCl, extracted with ethyl acetate (30mLx3), the combined organic phases were dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was recrystallized from ether to give 200mg of a white solid with a yield of 27.6%. LC-MS (APCI) M/z 262.1(M +1)⁺.¹H NMR(DMSO-d₆,300MHz)(/ppm):8.69(s,1H),7.53(s,1H),7.50(d,J＝8.7Hz,1H),2.24(s,3H),1.96-1.93(m,1H),0.96-0.93(m,2H),0.80-0.77(m,2H).

Step 6 Synthesis of Compound T-2.

To a 50mL three-necked flask equipped with magnetic stirring was added compound 16(130mg, 0.497mmol), evacuated and N₂Substitution three times, N₂Under the atmosphere, dry dichloromethane (2mL) and DMF (2mL) were respectively added dropwise via a syringe, the solution was stirred and dissolved, the reaction solution was cooled to 0 ℃, oxalyl chloride (0.42mL, 0.846mmol, 2M dichloromethane solution) was slowly added dropwise, the ice bath was removed after the dropwise addition, and the reaction was stirred at room temperature for 1 hour. The solvent was evaporated under reduced pressure and dry dichloromethane (10mL), N, was added₂The solution was stirred under an atmosphere, cooled to 0 deg.C, a solution of Compound 3(122mg, 0.597mmol) in dichloromethane (2mL) was added slowly dropwise, followed by DIPEA (0.25mL, 1.49mmol), the ice bath was removed and the reaction stirred at room temperature for 2 hours. Water (20mL) was added to quench the reaction, which was separatedThe organic layer was extracted with aqueous dichloromethane (20ml x2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated and the residue was separated on a silica gel column to give an off-white solid 60mg with a yield of 35.1%. LC-MS (APCI) M/z 447.4(M +1)⁺.¹H NMR(CDCl₃,300MHz)(/ppm):9.06(d,J＝12.3Hz,1H),8.41-8.38(m,2H),8.07(d,J＝5.7Hz,1H),7.93(t,J＝6.0Hz,1H),7.49(s,1H),7.20(d,J＝9.6Hz,1H),6.78(s,1H),5.52-5.46(m,1H),2.29(s,3H),2.00-1.88(m,1H),1.59(d,J＝5.1Hz,6H),0.91-0.81(m,4H).

₆Example 35- (4-cyclopropyl-1H-imidazolyl-1-yl) -N- (6- (4- (prop-2-yl-1, 1,1,3,3,3-d) Preparation of 4H-1,2, 4-triazol-3-yl) pyridin-2-yl) -2-fluoro-4-methylbenzamide (Compound T-3).

The specific synthesis steps are as follows:

step 1 synthesis of compound 18.

To a 50mL single neck flask equipped with magnetic stirring was added ammonium acetate (6.32g, 78mmol) and MeOD (50mL), N₂The temperature is increased and the reflux is carried out for 3 hours under the atmosphere. Concentrating under reduced pressure to dryness, adding MeOD (10mL), adding acetone-d under stirring₆(1.0g, 15.6mmol) and NaBH₃CN(980mg，15.6mmol)，N₂The reaction was stirred at room temperature under an atmosphere overnight. The reaction was quenched by addition of water (10mL), pH adjusted to 2 with 6M hydrochloric acid, ethyl acetate extraction (20mLx2), pH adjusted to 12 with aqueous 6M NaOH, dichloromethane extraction (20mLx3), organic phases combined, dried over anhydrous sodium sulfate, filtered, pH adjusted to 2 with 2M methanolic hydrochloric acid, and concentrated to dryness to give compound 18 hydrochloride, which was used directly in the next step.

Step 2 synthesis of compound 19.

A50 mL single-neck flask equipped with magnetic stirring was charged with Compound 2(300mg, 1.97mmol), toluene (10mL), Compound 18 hydrochloride (642mg, 9.86mmol), and N, N-bisMethylcarboxamide-dipropylacetal (933mg, 5.32mmol), acetic acid (296mg, 4.93mmol) was slowly added dropwise with stirring, and after addition, N was added₂The reaction mixture was heated under reflux for 3 hours under an atmosphere. Cooled to room temperature, the solvent was distilled off under reduced pressure, and the residue was passed through a silica gel column to give 170mg of a white solid in a yield of 41.2%. LC-MS (APCI): M/z 210.2(M +1)⁺.¹H NMR(CDCl₃,300MHz)(/ppm):8.32(s,1H),7.62-7.56(m,2H),6.58(dd,J₁＝5.1Hz,J₂＝1.2Hz,1H),5.59(s,1H),4.51(s,2H).

Step 3 Synthesis of Compound T-3.

To a 50mL three-necked flask equipped with magnetic stirring was added compound 10(130mg, 0.497mmol), evacuated and N₂Substitution of N₂Under the atmosphere, dry dichloromethane (2mL) and DMF (2mL) were respectively added dropwise via a syringe, the mixture was stirred and dissolved, the reaction solution was cooled to 0 ℃, oxalyl chloride (0.42mL, 0.846mmol, 2M dichloromethane solution) was slowly added dropwise, the ice bath was removed after the dropwise addition, and the reaction was stirred at room temperature for 1 hour. The solvent was evaporated under reduced pressure and dry dichloromethane (10mL), N, was added₂The solution was stirred under an atmosphere, cooled to 0 deg.C, and a solution of compound 19(122mg, 0.597mmol) in dichloromethane (2mL) was added slowly dropwise, followed by the addition of DIPEA (0.25mL, 1.49mmol) dropwise, the ice bath was removed, and the reaction was stirred at room temperature for 2 hours. The reaction was quenched by the addition of water (20mL), the organic layer was separated, the aqueous phase was extracted with dichloromethane (20mLx2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was separated on a silica gel column to give an off-white solid 60mg, yield 35.1%. LC-MS (APCI): M/z ═ 452.4(M +1)⁺.¹H NMR(CDCl₃,300MHz)(/ppm):9.05(d,J＝11.7Hz,1H),8.39-8.35(m,2H),8.07-8.04(m,2H),7.91(t,J＝6.6Hz,1H),7.45(s,1H),7.18(d,J＝9.0Hz,1H),6.77(s,1H),5.43(s,1H),2.27(s,3H),1.94-1.90(m,1H),0.90-0.80(m,4H).

₇Example 45- (4-cyclopropyl-1H-imidazolyl-1-yl) -N- (6- (4- (prop-2-yl-d) -4H-1,2, 4-tris Preparation of oxazol-3-yl) pyridin-2-yl) -2-fluoro-4-methylbenzamide (compound T-4).

The specific synthesis steps are as follows:

step 1 synthesis of compound 20.

To a 50mL single neck flask equipped with magnetic stirring was added ammonium acetate (6.32g, 78mmol) and MeOD (50mL), N₂The temperature is increased and the reflux is carried out for 3 hours under the atmosphere. Concentrating under reduced pressure to dryness, adding MeOD (10mL), adding acetone-d under stirring₆(1.0g, 15.6mmol) and NaBD₃CN(980mg，15.6mmol)，N₂The reaction was stirred at room temperature under an atmosphere overnight. The reaction was quenched by addition of water (10mL), pH adjusted to 2 with 6M hydrochloric acid, ethyl acetate extraction (20mLx2), pH adjusted to 12 with aqueous 6M NaOH, dichloromethane extraction (20mLx3), organic phases combined, dried over anhydrous sodium sulfate, filtered, pH adjusted to 2 with 2M methanolic hydrochloric acid, concentrated to dryness to give compound 20 hydrochloride, which was used directly in the next step.

Step 2 synthesis of compound 21.

A50 mL single-neck flask equipped with magnetic stirring was charged with Compound 2(300mg, 1.97mmol), toluene (10mL), Compound 20 hydrochloride (642mg, 9.86mmol), and N, N-dimethylformamide-dipropylacetal (933mg, 5.32mmol) in this order, and acetic acid (296mg, 4.93mmol) was slowly added dropwise with stirring, after completion of addition, N₂The reaction mixture was heated under reflux for 3 hours under an atmosphere. Cooled to room temperature, the solvent was distilled off under reduced pressure, and the residue was passed through a silica gel column to give 170mg of a white solid in a yield of 41.2%. LC-MS (APCI): M/z ═ 211.2(M +1)⁺.

Step 3 Synthesis of Compound T-4.

To a 50mL three-necked flask equipped with magnetic stirring was added compound 10(130mg, 0.497mmol), evacuated and N₂Substitution of N₂Under the atmosphere, dry dichloromethane (2mL) and DMF (2mL) were respectively added dropwise via a syringe, the mixture was stirred and dissolved, the reaction solution was cooled to 0 ℃, oxalyl chloride (0.42mL, 0.846mmol, 2M dichloromethane solution) was slowly added dropwise, the ice bath was removed after the dropwise addition, and the reaction was stirred at room temperature for 1 hour. The solvent was evaporated under reduced pressure and dry dichloromethane (10mL), N, was added₂Stirring under atmosphere to dissolve, and coolingA solution of compound 21(122mg, 0.597mmol) in dichloromethane (2mL) was added slowly dropwise to 0 deg.C, followed by the addition of DIPEA (0.25mL, 1.49mmol) dropwise, the ice bath removed, and the reaction stirred at room temperature for 2 hours. The reaction was quenched by the addition of water (20mL), the organic layer was separated, the aqueous phase was extracted with dichloromethane (20mLx2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was separated on a silica gel column to give an off-white solid 60mg, yield 35.1%. LC-MS (APCI): M/z 453.4(M +1)⁺.¹H NMR(CDCl₃,300MHz)(/ppm):9.06(d,J＝12.0Hz,1H),8.41(d,J＝6.9Hz,1H),8.38(s,1H),8.09(d,J＝5.7Hz,2H),7.94(t,J＝6.0Hz,1H),7.47(s,1H),7.21(d,J＝9.3Hz,1H),6.80(s,1H),2.31(s,3H),1.98-1.88(m,1H),0.92-0.84(m,4H).

And (4) testing the biological activity.

(1) Kinase inhibition

Reagents and consumables:

ASK1(Invitrogen catalog number PV3809), ATP (Sigma, catalog number A7699), DMSO (Sigma, catalog number D8418-1L), 384 well plates (Greiner, catalog number 784076), HTRF KinEASE-STK Kit (Cisbio, catalog number PV3809), 5 Xkinase buffer A (Life Technologies, catalog number PV3186), kinase tracer 199(Life Technologies, catalog number PV5830),

Eu-anti-GST antibody (Life Technologies, Cat. No. PV 5594).

The specific experimental method comprises the following steps:

compound preparation: test compounds were dissolved in DMSO to make 20mM stock. Then, the cells were diluted ten times in DMSO with a gradient of 3-fold. When adding medicine, the medicine is diluted by 10 times by using buffer solution.

ASK1 kinase assay: ASK1 kinase was mixed with pre-diluted formulations of compounds at different concentrations for 10 minutes in 5x kinase buffer a, each concentration being duplicate wells. Adding corresponding substrate and ATP, reacting for 20 minutes at room temperature (wherein negative and positive controls: negative is blank control, and positive is erlotinib). Adding detection reagent (reagent in HTRF KinEASE-STK Kit) after reaction, incubating for 30 min at room temperature, detecting by Evnvision microplate reader, and determiningThe enzyme activity of the compound of the invention at each concentration is calculated, the inhibition activity of the compound with different concentrations on the enzyme activity is calculated, then the inhibition activity of the enzyme activity of the compound with different concentrations is fitted according to a four-parameter equation and Graphpad 5.0 software, and IC is calculated₅₀The value is obtained.

The compounds of the invention and the non-deuterated compound, Selonsertib, were tested in the kinase inhibition assay described above and found to have more potent or comparable activity against ASK1 kinase. The results of the inhibition of kinases by representative example compounds are summarized in table 1 below.

TABLE 1

(2) Metabolic stability evaluation

Microsome experiment: human liver microsomes: 0.5mg/mL, Xenotech; rat liver microsomes: 0.5mg/mL, Xenotech; mouse 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 example was weighed out finely and dissolved in DMSO to 5mM each.

Preparation of phosphate buffer (100mM, pH 7.4): 150mL of 0.5M potassium dihydrogenphosphate and 700mL of 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, diluted 5-fold with ultrapure water before use, and magnesium chloride was added to obtain a phosphate buffer solution (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 (pH7.4) is taken to a 50mL centrifuge tube, 812.5 mu L of human, rat and mouse liver microsomes are respectively added and mixed evenly, and liver microsome dilution liquid with the protein concentration of 0.625mg/mL is obtained. 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 each dilution of human, rat or mouse liver microsomes was added to a 96-well plate (N2), 2. mu.L of each dilution was added to 0.25mM working solution, and the mixture was mixed.

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, 30 min 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.

t_1/2(min)；CL_int(μL/min/mg)。

The compounds of the present invention and the non-deuterated compound Selonsertib were simultaneously tested and compared to evaluate their metabolic stability in human, rat and mouse liver microsomes. The half-life and intrinsic hepatic clearance as indicators of metabolic stability are shown in table 2. As shown in table 2, the compounds of the present invention can significantly improve metabolic stability by comparison with the non-deuterated compound Selonsertib in human, rat and mouse liver microsome experiments.

TABLE 2

(3) Pharmacokinetic experiment of rat

6 male Sprague-Dawley rats, 7-8 weeks old, weighing about 210g, were divided into 2 groups of 3 per group and compared for pharmacokinetic differences by intravenous or oral administration of a single dose of compound (10 mg/kg oral).

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. In the test tube there was 30. mu.L of 1% heparin salt solution. Before use, the tubes were dried overnight at 60 ℃. After completion of blood collection at the last 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 100 μ L of plasma into a clean plastic centrifuge tube, designating 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.

Experiments show that the compound has better pharmacokinetic property in animals, thereby having better pharmacodynamics and treatment effect.

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

1. A 1,2, 4-triazole compound of formula (I), or a pharmaceutically acceptable salt thereof:

wherein the content of the first and second substances,

X¹、X²、X³each independently is CH₃、CH₂D、CHD₂Or CD₃；

2. The 1,2, 4-triazole compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R is¹、R³And R⁴Is deuterium.

3. The 1,2, 4-triazole compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein R⁵Is deuterium.

4. The 1,2, 4-triazole compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein X is¹And X²Is a CD₃。

5. The 1,2, 4-triazole compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein Y is¹Is deuterium.

6. The 1,2, 4-triazole-type compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

7. a pharmaceutical composition comprising a pharmaceutically acceptable excipient and the 1,2, 4-triazole-type compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6.

8. A method of preparing the pharmaceutical composition of claim 7, comprising: mixing a pharmaceutically acceptable excipient with the 1,2, 4-triazole compound of any one of claims 1-6, or a pharmaceutically acceptable salt thereof, to form a pharmaceutical composition.

9. A method of preparing a 1,2, 4-triazole compound of any one of claims 1-6, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 7 or 8 for use in treating a disease mediated at least in part by ASK 1.

10. The method of claim 9, further comprising administering another therapeutic agent; preferably, wherein said another therapeutic agent is a LOX2 inhibitor.

11. The method of claim 9 or 10, wherein the disease is diabetes, diabetic nephropathy, kidney disease, kidney fibrosis, lung fibrosis, idiopathic lung fibrosis (IPF), liver fibrosis, lung hypertension, nonalcoholic steatohepatitis, liver disease, alcoholic liver disease, an inflammatory disorder, an autoimmune disease, a proliferative disease, transplant rejection, a disease involving impairment of cartilage metabolism, congenital cartilage malformations, or a disease associated with hypersecretion of IL 6.

12. The method of claim 11, wherein the disease is non-alcoholic steatohepatitis or alcoholic liver disease.

13. The method of claim 11, wherein the disease is pulmonary hypertension or pulmonary fibrosis.

14. The method of claim 11, wherein the disease is diabetic nephropathy, or renal fibrosis.