WO2019091441A1 - 5-amino pyrazole carboxamide compound as btk inhibitor and preparation method thereof - Google Patents

Abstract

The present invention provides a novel 5-amino pyrazole carboxamide compound as shown in formula (I), a preparation method thereof, a pharmaceutical composition comprising the compound, and a use thereof. (I)

Description

5-aminopyrazolecarboxamide compound as BTK inhibitor and preparation method thereof Technical field

The invention belongs to the technical field of medicinal chemistry, in particular to a novel high-efficiency, selective and good pharmacokinetic property, 5-aminopyrazolecarboxamide compound as a BTK inhibitor and preparation method thereof .

Background technique

Protein kinases are the largest family of biological enzymes in the human body, including over 500 proteins. In particular, for tyrosine kinases, the phenolic function on the tyrosine residue can be phosphorylated to exert important biosignaling effects. The tyrosine kinase family has members that control cell growth, migration, and differentiation. Abnormal kinase activity has been elucidated in close association with many human diseases, including cancer, autoimmune diseases, and inflammatory diseases.

Bruton's tyrosine kinase (BTK) is a cytoplasmic non-receptor tyrosine kinase belonging to the TEC kinase family (a total of five members BTK, TEC, ITK, TXK, BMX). The BTK gene is located on Xq21.33-Xq22 of the X-chromosome and shares 19 exons spanning 37.5 kb of genomic DNA.

In addition to T cells and plasma cells, BTK expression plays an essential role in almost all hematopoietic cells, especially in the development, differentiation, signaling and survival of B lymphocytes. B cells are activated by the B cell receptor (BCR), and BTK plays a decisive role in the BCR signaling pathway. Activation of BCR on B cells causes activation of BTK, which in turn leads to an increase in downstream phospholipase C (PLC) concentration and activates the IP3 and DAG signaling pathways. This signaling pathway promotes cell proliferation, adhesion and survival. Mutations in the BTK gene result in a rare hereditary B cell-specific immunodeficiency disease known as X-Iinked agammaglobulinemia (XLA). In this disease, the function of BTK is inhibited, resulting in the production or maturation of B cells. Men with XLA disease have almost no B cells in their bodies, and there are few circulating antibodies, which are prone to serious or even fatal infections. This strongly proves that BTK plays an extremely important role in the growth and differentiation of B cells.

Small molecule BTK inhibitors bind to BTK, inhibit BTK autophosphorylation, and prevent BTK activation. This can block the signal transduction of the BCR pathway, inhibit the proliferation of B lymphoma cells, destroy the adhesion of tumor cells, and promote the apoptosis of tumor cells. And induce apoptosis. This makes BTK a compelling drug target in B cell-associated cancers, especially for B-cell lymphomas and leukemias such as non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL), and Recurrent or refractory mantle cell lymphoma (MCL) and the like.

In addition to fighting against B-cell lymphoma and leukemia, BTK inhibitors also inhibit B cell autoantibodies and cytokine production. In autoimmune diseases, B cells present autoantigens, promote the activation and secretion of T cells, cause inflammatory factors, cause tissue damage, and activate B cells to produce a large number of antibodies, triggering autoimmune responses. The interaction of T and B cells forms a feedback regulatory chain, leading to uncontrolled autoimmune response and aggravation of histopathological damage. Therefore, BTK can be used as a drug target for autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus (SLE), and allergic diseases (such as diseases such as esophagitis).

In addition, BTK inhibitors have been reported to be useful in combination with chemotherapeutic agents or immunological checkpoint inhibitors, and have shown superior therapeutic effects in a variety of solid tumors in clinical trials.

Among the currently marketed drugs, Ibrutinib is an irreversible BTK inhibitor developed by Pharmacyclics and Johnson & Johnson, and was approved by the FDA in November 2013 and February 2014 for the treatment of mantle cell lymphocytes. Tumor (MCL) and chronic lymphocytic leukemia (CLL). Ibrutinib has been designated by the FDA as a "breakthrough" new drug that works by reacting with the thiol group of cysteine in BTK and forming a covalent bond that inactivates the BTK enzyme. However, ibrutinib is easily metabolized during metabolism (digested by metabolic enzymes to be dihydroxylated or inactivated by other thiol-containing enzymes, cysteine, glutathione, etc.) Affect the efficacy. The clinically administered dose reached 560 mg per day, which increased the burden on the patient. In addition, Ibrutinib also has a certain inhibitory effect on some kinases other than BTK, especially the inhibition of EGFR can lead to more serious rash, diarrhea and other adverse reactions. Therefore, there is still a need in the art to develop a new class of BTK inhibitors that are more efficient, selective, and have good pharmacokinetic properties for the treatment of related diseases.

Summary of the invention

The present inventors have developed a novel 5-aminopyrazole carboxamide derivative which is an effective, safe and highly selective inhibitor of protein kinase BTK.

It is an object of the present invention to provide a novel 5-aminopyrazolecarboxamide derivative. It is a new covalent bond inhibitor that improves its affinity with the target by altering its reactivity with cysteine to improve efficacy, selectivity and safety.

A second object of the present invention is to provide a process for the preparation of the above derivatives.

A third object of the present invention is to provide a pharmaceutical composition containing the above derivative.

A fourth object of the invention is to provide the use of the above derivatives.

Specifically, in an embodiment of the present invention, the present invention provides a novel 5-aminopyrazolecarboxamide compound or a pharmaceutically acceptable salt of the formula (I):

Here,

n, m are independently taken from 0, 1 or 2;

L is O, -C(O)NH-, -CH ₂ -, S, S(O), NH or S(O) ₂ ;

A is derived from a substituted or unsubstituted heterocyclic ring, a substituted or unsubstituted benzene ring, or a substituted or unsubstituted heteroaryl ring, and a linking site to the mother nucleus and L may be optionally selected;

B is independently taken from a substituted or unsubstituted aliphatic ring, a substituted or unsubstituted heterocyclic ring, a substituted or unsubstituted benzene ring, or a substituted or unsubstituted heteroaryl ring, and the linking site with L may be optionally selected ;

R ₁ and R ₂ are each independently selected from hydrogen, C1-C4 alkyl, halogen, cyano, or R ₁ and R ₂ together with the carbon atom to which they are attached form a ternary carbocyclic or quaternary carbocyclic ring, or R ₁ And R _{2 are} combined into an oxo group;

Y is selected from cyano group,

R ₃ , R ₄ , R ₅ and R ₆ are each independently selected from hydrogen, unsubstituted C1-C4 alkyl, hydroxy-substituted C1-C4 alkyl, C1-C4 alkoxy C1-4 alkyl, halogen, a cyano group, or -(CH ₂ ) _q N(R ^a R ^b ), where q is 1, 2, 3, or 4, and R ^a and R ^b are each independently selected from hydrogen, unsubstituted C1-C4 alkane base;

And stipulates that when one of R ₁ and R ₂ is hydrogen and the other is methyl, and Y is a cyano group, A is a benzene ring, L is O, m is 1 and n is 2, B is not substituted or unsubstituted a benzene ring; when both R ₁ and R ₂ are hydrogen, and A is a benzene ring, m is 1 and n is 2, B is not a substituted or unsubstituted benzene ring, and a substituted or unsubstituted pyridine; when R ₁ And R ₂ is hydrogen, and A is a pyridine ring, m is 1 and n is 2, B is not a substituted or unsubstituted benzene ring; when R ₁ and R ₂ are both hydrogen, and A is a benzene ring, L is When O, m is 1 and n is 1, B is not a substituted or unsubstituted benzene ring.

In one embodiment of the present invention, the present invention provides a 5-aminopyrazolecarboxamide compound of the formula (I), wherein n, m are independently taken from 0, 1 or 2; Is O, -C(O)NH-, -CH ₂ -, NH or S, more preferably O, -C(O)NH-, NH.

In one embodiment of the present invention, there is provided a 5-aminopyrazolecarboxamide compound of the formula (I), wherein A is derived from a substituted or unsubstituted heterocyclic ring, substituted or unsubstituted a substituted benzene ring, or a substituted or unsubstituted heteroaryl ring, and a linking site to the parent nucleus and L may be optionally selected; B is independently derived from a substituted or unsubstituted aliphatic ring, a substituted or unsubstituted heterocyclic ring a substituted or unsubstituted benzene ring, or a substituted or unsubstituted heteroaryl ring, and a linking site with L may be optionally selected; here:

The substituted benzene ring means that any position on the phenyl group is substituted with an optional substituent selected from the group consisting of hydrogen, methyl, methoxy, fluoro, chloro, trifluoromethyl, trifluoromethyl. An oxy or cyano group; preferably, the substituted benzene ring is a fluoro substituted phenyl group, or a chloro substituted phenyl group, more preferably a 2,4-difluorophenyl group, or a 4-chlorophenyl group;

The unsubstituted heteroaryl ring means furan, pyrrole, thiophene, oxazole, isoxazole, pyrazole, imidazole, thiazole, isothiazole, oxadiazole, triazole, thiadiazole, tetrazolium, pyridine , pyrimidine, pyrazine, pyridazine, triazine; said substituted heteroaryl ring means that any position on the above group is substituted by an optional substituent selected from the group consisting of hydrogen, methyl, methoxy a radical, a fluorine, a chlorine, a trifluoromethyl group, a trifluoromethoxy group or a cyano group; more preferably, the substituted pyridine is a chloropyridine, particularly preferably a 4-chloro-pyridin-2-yl group;

The unsubstituted aliphatic ring means cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane; the substituted aliphatic ring means any position on the above group is optionally selected Substituted by a substituent selected from hydrogen, methyl, methoxy, fluoro, chloro, trifluoromethyl, trifluoromethoxy or cyano;

The unsubstituted heterocyclic ring means tetrahydrofuran, tetrahydropyran, tetrahydropyrrole, piperidine,

Wherein w is taken from 0, 1 or 2; the substituted heterocyclic ring means that any position on the above group is substituted by an optional substituent selected from the group consisting of hydrogen, methyl, methoxy, and fluorine. , chlorine, trifluoromethyl, trifluoromethoxy or cyano.

In one embodiment of the present invention, the present invention provides a 5-aminopyrazolecarboxamide compound of the formula (I), wherein, preferably, both R ₁ and R ₂ are hydrogen, or one of them Is hydrogen and the other is C1-C4 alkyl (methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl), or R ₁ and R ₂ are attached to them The carbon atoms together form a cyclopropyl group; more preferably, both R ₁ and R ₂ are hydrogen, or one of them is hydrogen and the other is methyl, or R ₁ and R ₂ together with the carbon atom to which they are attached form a cyclopropane base.

In a preferred embodiment of the present invention, the present invention provides a 5-aminopyrazolecarboxamide compound represented by formula (II) or a pharmaceutically acceptable salt thereof:

Here, L, A, B and Y are as defined above for the formula (I);

It is also stated that when A is a benzene ring, B is not a substituted or unsubstituted benzene ring, and a substituted or unsubstituted pyridine; when A is a pyridine ring, B is not a substituted or unsubstituted benzene ring.

In a more preferred embodiment of the present invention, the 5-aminopyrazolecarboxamide compound of the formula (II) provided by the present invention is one of the following compounds:

Here, the definitions of L, B and Y in the formula (II-1) are as in the above formula (I);

It is also stated that B is not a substituted or unsubstituted benzene ring, and a substituted or unsubstituted pyridine.

In a preferred embodiment of the present invention, the present invention provides a 5-aminopyrazolecarboxamide compound represented by formula (III) or a pharmaceutically acceptable salt thereof:

Here, L, A, B and Y are as defined above for the formula (I);

It is also specified that when Y is a cyano group, A is a benzene ring, and L is O, B is not a substituted or unsubstituted benzene ring.

In a more preferred embodiment of the present invention, the 5-aminopyrazolecarboxamide compound of the formula (III) provided by the present invention is one of the following compounds:

Here, the definitions of L, B and Y in the formulae (III-1) and (III-2) are as in the above formula (I);

It is also stated that when Y is a cyano group and L is O, B is not a substituted or unsubstituted benzene ring.

In a preferred embodiment of the present invention, the present invention provides a 5-aminopyrazolecarboxamide compound represented by formula (IV) or a pharmaceutically acceptable salt thereof:

Here, L, A, B, and Y are as defined in the above formula (I).

In a more preferred embodiment of the present invention, the 5-aminopyrazolecarboxamide compound of the formula (IV) provided by the present invention is the following compound:

Here, L, B and Y in the formula (IV-1) are as defined in the above formula (I).

In a more preferred embodiment of the invention, the invention provides formula (I), (II), (II-1), (III), (III-1), (III-2), (IV) Or (IV-1), wherein L is O;

B is

with

Y is -CN,

Wherein R ₃ , R ₄ , R ₅ and R ₆ are each independently selected from hydrogen, unsubstituted C1-C4 alkyl, hydroxy-substituted C1-C4 alkyl, C1-C4 alkoxy C1-4 alkyl, Halogen, cyano, or -(CH ₂ ) _q N(R ^a R ^b ), where q is 1, 2, 3, or 4, and R ^a and R ^b are each independently selected from hydrogen, unsubstituted C1- C4 alkyl.

In a particularly preferred embodiment of the invention, the 5-aminopyrazole carboxamide compound provided by the invention is selected from one of the following compounds:

In an embodiment of the invention, the "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable acid addition salt and a pharmaceutically acceptable base addition salt:

The "pharmaceutically acceptable acid addition salt" refers to a salt formed with an inorganic or organic acid capable of retaining the bioavailability of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobromides, sulfates, phosphates, and the like; organic acid salts include, but are not limited to, formate, acetate, propionate, glycolate, gluconate , lactate, oxalate, maleate, succinate, fumarate, tartrate, citrate, glutamate, aspartate, benzoate, methanesulfonate , p-toluenesulfonate and salicylate. These salts can be prepared by methods known in the art.

The "pharmaceutically acceptable base addition salt" refers to a salt capable of maintaining the bioavailability of the free acid without other side effects. These salts are prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, calcium and magnesium salts and the like. Salts derived from organic bases include, but are not limited to, ammonium salts, triethylamine salts, lysine salts, arginine salts, and the like. These salts can be prepared by methods known in the art.

In a second aspect, the present invention provides a process for the preparation of the 5-aminopyrazolecarboxamide compound represented by the above formula (I), comprising the steps of:

(1) reacting a compound of the formula (V) with a compound of the formula (VI) to give a compound of the formula (VII);

(2) subjecting a compound of the formula (VII) to a hydrolysis reaction to obtain a compound of the formula (VIII);

(3) a compound of the formula (VIII) is subjected to a deprotection group PG to give a compound of the formula (IX);

(4) reacting a compound of the formula (IX) with a compound of the formula (X) to give a compound of the formula (I);

Substituents R ₁ , R ₂ , L, A, B, Y in the above formula (V), formula (VI), formula (VII), formula (VIII), formula (IX) and formula (X) And n, m are as defined above for formula (I), PG is an amino protecting group (suitable amino protecting groups include acyl (eg acetyl), carbamate (eg 2', 2', 2'- Trichloroethoxycarbonyl, Cbzbenzyloxycarbonyl or BOC-tert-butoxycarbonyl) and arylalkyl (for example Bnbenzyl) which may be hydrolyzed, if appropriate (for example using diacids such as hydrogen chloride) Solution or trifluoroacetic acid in dichloromethane) or by reduction (eg hydrogenolysis of benzyl or benzyloxycarbonyl, or reduction of 2', 2', 2'-trichloroethoxy by zinc in acetic acid Other suitable amino protecting groups include trifluoroacetyl (-COCF ₃ ) which can be removed by base catalyzed hydrolysis to remove benzyloxycarbonyl or tert-butoxycarbonyl groups, as will be appreciated by those skilled in the art by reference to TW Greene 'Protective Groups in Organic Synthesis' (4th edition, J. Wiley and Sons, 2006)), R ₃ is a C1-C4 alkyl group (preferably ethyl), and X is chlorine, bromine or a hydroxyl group.

In the step (1), the reaction is preferably carried out under basic conditions, wherein the base is selected from the group consisting of organic bases and inorganic bases which are common in the art; the organic base may be selected, for example, from DIPEA, triethylamine, pyridine, DBN, DBU. , piperidine, 4-dimethylaminopiperidine, etc.; the inorganic base may be selected, for example, from lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, barium hydroxide, lithium carbonate, sodium carbonate, carbonic acid Potassium, cesium carbonate, sodium hydrogencarbonate, and the like.

In the step (2), the reaction is preferably carried out under acidic conditions, wherein the acid may be selected, for example, from hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, hydrofluoric acid, formic acid, acetic acid, trifluoroacetic acid, benzene. Formic acid, phenylacetic acid, methanesulfonic acid, benzenesulfonic acid, and the like. The reaction temperature may be in the range of 0-100 ° C, preferably in the range of 0-50 ° C, more preferably in the range of 20-30 ° C.

In the step (3), the reaction can be carried out under the conditions of deprotection groups which are common in the art.

In the step (4), the reaction is carried out under basic conditions, wherein the base is selected from the group consisting of organic bases and inorganic bases which are common in the art. The organic base may be selected, for example, from DIPEA, triethylamine, pyridine, DBN, DBU, piperidine, 4-dimethylaminopiperidine, N-methylmorpholine or the like; the inorganic base may be selected, for example, from lithium hydroxide or sodium hydroxide. , potassium hydroxide, calcium hydroxide, magnesium hydroxide, barium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, barium carbonate, sodium hydrogencarbonate, and the like.

In an embodiment of the present invention, the present invention provides a process for producing a 5-aminopyrazolecarboxamide compound represented by the above formula (I), wherein the compound of the formula (VI) can be obtained by:

Wherein the substituent R ₄ in the above compound is a C ₁ -C ₄ alkyl group (preferably a methyl group); X is chlorine or bromine, preferably chlorine; and R ₃ is a C ₁ -C ₄ alkyl group ( Preferably ethyl); L, A and B are as defined for the compound of formula (I);

More specifically, the compound (VI-1) is subjected to hydrolysis reaction under basic conditions to obtain a compound (VI-2), which is then reacted with an acid halide reagent to obtain a compound (VI-3) wherein the acid halide reagent includes a chloride Sulfone, oxalyl chloride, triphosgene, phosphorus oxychloride, phosphorus pentachloride, and the like. Compound (VI-3) is then reacted with malononitrile under basic conditions to obtain compound (VI-4) wherein the base is selected from the group consisting of n-butyl lithium, methyl lithium, hexamethyldisilazide sodium, and six Methyldisilazide potassium, sodium amide, sodium hydride, sodium methoxide, sodium ethoxide, sodium hydroxide, potassium hydroxide, magnesium hydroxide, lithium hydroxide, calcium hydroxide, barium hydroxide, sodium carbonate, potassium carbonate, carbonic acid Hydrazine, sodium hydrogencarbonate, triethylamine, trimethylamine, pyridine, DBN, DBU, piperidine, 4-dimethylaminopiperidine, N-methylmorpholine and the like. Then, the compound VI-4 is reacted with (R ₃ O) ₃ CH in the presence of acetic anhydride to obtain the compound (VI), and the reaction temperature is in the range of room temperature to 150 ° C, preferably in the range of 50 to 150 ° C, It is preferably in the range of 100 to 150 °C.

In an embodiment of the present invention, the above 5-aminopyrazolecarboxamide compound provided by the present invention is a production method represented by the formula (I), wherein the compound of the formula (V) can be produced by referring to the following method:

Wherein m, n, R ₁ and R ₂ are as defined for the compound of formula (I). PG ₁ and PG ₃ are defined as PG. PG ₂ is a hydroxy protecting group including, but not limited to, an ether protecting group (eg, benzyl, methoxymethyl, methoxyethyl), a silyl ether protecting group (eg, TBS, TES, TBDMS, etc.), an ester-forming protecting group (benzoyl, p-nitrobenzoyl, trifluoroacetyl) and the like. A common hydroxy protecting group can be referred to TW Greene 'Protective Groups in Organic Synthesis' (4th edition, J. Wiley and Sons, 2006).

More specifically, the compound of the formula (V) can be produced by referring to the following methods:

or

or

The oxidizing agent used in the oxidation reaction includes RuCl ₃ H ₂ O/NaIO ₄ , diacetoxyiodobenzene/t-butane hydroperoxide, potassium permanganate, iodine/sodium hydrogencarbonate, potassium dichromate, etc. The reagents used in the hydrogenation reaction include lithium aluminum hydride, aluminum hydride, platinum dioxide, diisobutylaluminum hydride, sodium borohydride, copper chrome oxide, Raney nickel, and the like.

More specifically, the compound of the formula (V) can be produced by referring to the following methods:

In a third aspect, the present invention provides a pharmaceutical composition comprising an effective amount of one or more of the above 5-aminopyrazolecarboxamide compounds of the present invention or a pharmaceutically acceptable salt thereof, the pharmaceutical composition further comprising A pharmaceutically acceptable carrier.

The pharmaceutical composition of the present invention can be formulated into solid, semi-solid, liquid or gaseous preparations such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres and Aerosol.

The pharmaceutical compositions of the present invention can be prepared by methods well known in the pharmaceutical art. For example, practical methods for preparing pharmaceutical compositions are known to those skilled in the art, for example, see The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000).

Routes of administration of the pharmaceutical compositions of the invention include, but are not limited to, oral, topical, transdermal, intramuscular, intravenous, inhalation, parenteral, sublingual, rectal, vaginal, and intranasal. For example, dosage forms suitable for oral administration include capsules, tablets, granules, and syrups and the like. The compound of the formula (I) of the present invention contained in these preparations may be a solid powder or granule; a solution or suspension in an aqueous or non-aqueous liquid; a water-in-oil or oil-in-water emulsion or the like. The above dosage forms can be prepared from the active compound with one or more carriers or excipients via conventional pharmaceutical methods. The above carriers need to be compatible with the active compound or other excipients. For solid formulations, commonly used non-toxic carriers include, but are not limited to, mannitol, lactose, starch, magnesium stearate, cellulose, glucose, sucrose, and the like. Carriers for liquid preparations include, but are not limited to, water, physiological saline, aqueous dextrose, ethylene glycol, polyethylene glycol, and the like. The active compound can form a solution or suspension with the above carriers. The particular mode of administration and dosage form will depend on the physicochemical properties of the compound itself, as well as the severity of the disease being applied. Those skilled in the art will be able to determine the specific route of administration based on the above factors in combination with their own knowledge. For example, see: Li Jun, "Clinical Pharmacology", People's Health Publishing House, 2008.06; Ding Yufeng, on clinical dosage forms and rational use of drugs, Medical Herald, 26 (5), 2007; Howard C. Ansel, Loyd V. Allen, Jr., Nicholas G. Popovich, Jiang Zhiqiang, “Pharmaceutical Formulation and Drug Delivery System”, China Medical Science and Technology Press, 2003.05.

The pharmaceutical compositions of the present invention may be presented in unit dosage forms containing a predetermined amount of active ingredient per unit dose. Preferred unit dosage compositions are those containing a daily or sub-dose, or an appropriate fraction thereof, of the active ingredient. Thus, such unit doses can be administered more than once a day. Preferred unit dosage compositions are those containing a daily or sub-dose (more than one administration per day) as hereinbefore described, or an appropriate fraction thereof.

The pharmaceutical compositions of this invention are formulated, quantified, and administered in a manner consistent with medical practice. A "therapeutically effective amount" of a compound of the invention is determined by the particular condition to be treated, the individual being treated, the cause of the condition, the target of the drug, and the mode of administration. Generally, the dose for parenteral administration may be 1-200 mg/kg/day, and the dose for oral administration may be 1-1000 mg/kg/day.

The range of effective dosages provided herein is not intended to limit the scope of the invention, but rather to the preferred dosage range. However, the most preferred dosages can be adjusted for individual individuals, as will be appreciated and determined by those skilled in the art (see, for example, Berkow et al., Merck Handbook, 16th Ed., Merck Corporation, Rahway, NJ, 1992). .

In a fourth aspect, the present invention provides the above 5-aminopyrazolecarboxamide compound or a stereoisomer, tautomer, solvate or pharmaceutically acceptable salt thereof for use in the prevention or treatment of BTK Use in drugs that cause disease.

The present invention provides a method for inhibiting BTK activity comprising administering to a biological system the above 5-aminopyrazolecarboxamide compound of the present invention or a stereoisomer, tautomer, solvate thereof or pharmaceutically acceptable salt thereof Or a pharmaceutical composition comprising the above 5-aminopyrazolecarboxamide compound of the present invention or a stereoisomer, tautomer, solvate thereof or a pharmaceutically acceptable salt thereof.

In some embodiments, the biological system is an enzyme, a cell, or a mammal.

The present invention also provides a method for preventing or treating a disease mediated by BTK, comprising administering a therapeutically effective amount of one or more of the above 5-aminopyrazolecarboxamide compounds of the present invention or a combination thereof to a patient in need thereof a stereoisomer, tautomer, solvate or pharmaceutically acceptable salt thereof and one or more drugs selected from the group consisting of an immunomodulator, an immunological checkpoint inhibitor, a glucocorticoid, a non-purine Anti-inflammatory drugs, Cox-2 specific inhibitors, TNF-α binding proteins, interferons, interleukins and chemotherapeutic drugs.

In an embodiment of the invention, the BTK mediated diseases include autoimmune diseases, inflammatory diseases, xenogeneic immune conditions or diseases, thromboembolic diseases, and cancer. In some embodiments, the cancer comprises B-cell chronic lymphocytic leukemia, acute lymphocytic leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute myeloid leukemia, diffuse large B-cell lymphoma , multiple myeloma, mantle cell lymphoma, small lymphocytic lymphoma, Waldenstrom's macroglobulinemia, solid tumor. In some embodiments, the autoimmune disease and inflammatory disease are selected from the group consisting of rheumatoid arthritis, osteoarthritis, juvenile arthritis, chronic obstructive pulmonary disease, multiple sclerosis, systemic lupus erythematosus, psoriasis , psoriatic arthritis, Crohn's disease, ulcerative colitis, and irritable bowel syndrome. In some embodiments, the xenogeneic immune condition or disease comprises graft versus host disease, transplantation, blood transfusion, allergic reaction, allergy, type I hypersensitivity, allergic conjunctivitis, allergic rhinitis or atopy dermatitis.

Experimental data demonstrate that the present invention provides that the above 5-aminopyrazole carboxamide compound is an effective and safe inhibitor of protein kinase BTK.

DRAWINGS

Figure 1 shows that the compound of the present invention significantly inhibited the growth of the diffuse large B-cell lymphoma cell line TMD-8 in vivo and showed the same antitumor effect as the control compound Ibrutinib.

Detailed ways

The experiments, synthetic methods, and intermediates involved are illustrative of the invention and are not intended to limit the scope of the invention.

The starting materials used in the experiments of the present invention are either purchased from a reagent supplier or prepared from known starting materials by methods well known in the art. The examples herein apply the following conditions unless otherwise stated:

The unit of temperature is Celsius (°C); the definition of room temperature is 18-25 ° C;

Drying the organic solvent with anhydrous magnesium sulfate or anhydrous sodium sulfate; using a rotary evaporator to spin dry under reduced pressure (for example: 15 mmHg, 30 ° C);

For rapid column chromatography, 200-300 mesh silica gel was used as a carrier, and TLC was used for thin layer chromatography;

Normally, the progress of the reaction is monitored by TLC or LC-MS;

The identification of the final product was performed by nuclear magnetic resonance (Bruker AVANCE 300, 300 MHz) and LC-MS (Bruker esquine 6000, Agilent 1200 series).

Example 1

(R)-5-amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1-(4-cyano-4-azaspiro[2.5]oct-6 Of -1)-H-pyrazole-4-carboxamide (Compound 1)

step one

To a solution of the intermediate compound 11 (167 g, 479 mmol, 1 eq) in EtOH (1670 mL), DIPEA (185 g, 1.44 mol, 250 mL, 3 eq). Intermediate compound 17 (187 g, 575 mmol, 1.2 eq) was added to the mixture. The mixture was then stirred at 25 ° C for 12 h under N 2 atmosphere. LCMS (ET 14245-55-P1A2, product: RT = 1.723 min) showed the reaction was completed. The reaction was filtered to give the product. The product was used in the next step without purification. Intermediate compound 18 (243 g, 407 mmol, yield 85%, purity 93.1%) was obtained as white solid.

Step two

The intermediate compound 18 (121g, 218mmol, 1eq) was stirred in H ₂ SO ₄ (1200mL) at the 30 ℃ 36h. TLC (DCM: MeOH = 10:1, Rf = 0.9) indicated that compound 18 was consumed completely and only one desired point formed (DCM: MeOH = 10:1, Rf = 0.2). The multiple batches of the reaction mixture were combined, and the combined mixture was poured into MTBE (20 L), and the solid was precipitated and then filtrate was collected by suction filtration. The pH of the filtrate with aqueous ammonia was adjusted to 10 and extracted with EtOAc (2L x 10), dried over Na ₂ SO _4, filtered, and concentrated under reduced pressure to give intermediate compound 19 (crude 311G, corresponding to 238g product) as a yellow solid .

Step three

To a solution of the intermediate compound 19 (199 g, 453 mmol, 1 eq), EtOAc (EtOAc (EtOAc) Then BrCN (52.8 g, 499 mmol, 36.7 mL, 1.1 eq) was added and stirred at 15 °C for 2 h. TLC (DCM: MeOH = 10:1, _Rf = 0.2) showed that Compound 19 was completely reacted and only one desired point (DCM: MeOH = 10:1, _Rf = 0.6). The multiple batches of the reaction mixture were combined and the resulting mixture was filtered to remove cesium carbonate. The filtrate was then concentrated under reduced pressure to remove DMF. The residue was diluted with water (2 L) andEtOAcEtOAc The combined organic layers were washed with EtOAc EtOAc m. Acetonitrile (1 L) was added to the residue,yield white solid, filtered, and washed with acetonitrile (200 mL×2) to give Compound 1 (140 g, 302 mmol, yield 55%, purity 97.0%).

¹ H NMR: CDCl ₃ 400 MHz δ 8.05 (d, J = 2.4 Hz, 1H), 7.60 (dd, J = 2.4, 8.8 Hz, 1H), 7.51 (d, J = 8.8 Hz, 2H), 7.15 (d, J = 8.8 Hz, 2H), 6.86 (d, J = 8.8 Hz, 1H), 5.60 (s, 2H), 5.23 (br.s., 2H), 4.22-4.16 (m, 1H), 3.59-3.41 ( m, 2H), 2.39-2.24 (m, 2H), 2.12-2.09 (m, 1H), 1.23-1.10 (m, 2H), 0.80-0.74 (m, 2H), 0.62-0.61 (m, 1H).

Example 2

Preparation of (R)-6-mercapto-4-azaspiro[2.5]octane-4-carboxylic acid benzyl ester (intermediate compound 11)

step one

To a solution of the starting material compound 1 (21.0 kg, 104 mol, 1.00 eq) in EtOAc ( 315L), EtOAc (2, EtOAc, EtOAc, EtOAc, EtOAc, The mixture was then stirred at 25 ° C for 5 hours (220 r / min). TLC (petroleum ether: ethyl acetate = 5:1, Rf = 0.04). The mixture was added to water (100 L) and extracted. The organic phase (50.0 L) was washed with water. The organic phase was separated to give the title compound 2 (164 g, EtOAc)

Step two

(315L) was added at 10 ~ 25 ℃ ethyl acetate solution of intermediate compound 2 (32.9kg, 104mol, 1.00eq) in _{H 2 O (660L), RuCl} 3 .H 2 O (8.23kg, 36.5mol, 0.35 Eq) and NaIO ₄ (89.2 kg, 417 mol, 23.1 L, 4.00 eq), then the mixture was stirred at 25 ° C for 5 hours (220 r / min). TLC (petroleum ether: ethyl acetate = 5:1, Rf = 0.50). The mixture was added to water (100 L) and extracted, then the aqueous phase was washed with ethyl acetate (200L). The combined organic phases were washed with sodium thiosulfate (200 L) and brine (150L). The five batches of the organic phase were combined, dried over anhydrous sodium sulfate and filtered. The concentrated solution was used to remove the solvent (50 ° C, -0.1 mp) to afford EtOAc (EtOAc) or,

Using nitromethane (MeNO ₂ ) or acetonitrile as the reaction solvent, diacetoxyiodobenzene (DIB, 3.0 eq) and hydrogen peroxide tert-butanol (TBHP, 4.0 eq) were added to the reaction solvent at around 0 °C. After stirring, the intermediate compound 2 (1.0 eq) was added, and the mixture was stirred at room temperature overnight. After the reaction was completed, the mixture was quenched with water, and the mixture was extracted with ethyl acetate. The organic phase was washed twice with brine and brine Drying, chromatography on silica gel to give intermediate compound 3. or,

The intermediate compound 3 can be obtained by oxidizing the intermediate compound 2 using water/ethyl acetate as a reaction solvent and KMnO ₄ as an oxidizing agent.

Step three

To a solution of the intermediate compound 3 (85.9 kg, 260 mol, 1.00 eq) in EtOAc (430 L), EtOAc (6. The mixture was then stirred (220 r/min) at 45 ° C for 14 hours. TLC (petroleum ether: ethyl acetate = 2:1, Rf = 0.54). The solution was concentrated to remove most of the solvent (50 ° C, -0.1 MPa). DMSO (240 L) was then added to the mixture and stirred (220 r/min) for 1 hour. The solution was concentrated (50 ° C, ~ 0.1 MPa) to remove the low boiling solvent (EtOAc). A solution of Intermediate Compound 4 (60.0 kg, crude) in DMSO (240L) was obtained.

Step four

KOH (103 kg, 1.56 kmol, purity 85.0%, 6.00 eq) was stirred (220 r/min) in DMSO (300 L) for 2 hours, and intermediate compound 4 (30.0 kg, 261 mol, 1.00 eq) of DMSO was added to the mixture. (120 L) solution was stirred (220 r/min) for 2 hours, then benzyl bromide (134 kg, 782 mol, 92.8 L, 3.00 eq) was added and stirred (220 r/min) for 5 hours at 15 to 25 °C. TLC (dichloromethane:methanol = 5:1, Rf = 0.10). The mixture was added to dichloromethane (500 L) and filtered, and the filter cake was washed with dichloromethane (250L*3). The filtrate was extracted with brine (400 L*2). The two organic phases were combined, dried over anhydrous sodium sulfate and filtered. Concentrate under reduced pressure (50 ° C, -0.1 MPa). The crude product was purified by column chromatography (SiO _2, petroleum ether: ethyl acetate = 20 to 1:1) purification. Activated carbon (5.00 kg) was added to the product and stirred (180 r/min) at 40 ° C for 8 hours. Then, the mixture was filtered, and the filtrate was evaporated. mjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjj

1H NMR: CDCl ₃ 400MHz δ7.11-7.31 (m, 10H), 4.27-4.66 (m, 4H), 3.62-3.79 (m, 1H), 3.16-3.31 (m, 2H), 2.57-2.73 (m, 1H) ), 2.29-2.44 (m, 1H), 1.82-2.07 (m, 2H)

Step five

To a solution of the intermediate compound 5 (35.0 kg, 118 mol, 1.00 eq.) in THF (280 L) was added EtMgBr (3M, 158L, 4.00 eq) at -10 to 0 ° C, then at -10 to 0 ° C A solution of Ti(Oi-Pr) ₄ (40.4 kg, 142 mol, 42.0 L, 1.20 eq) in THF (70.0 L) was added to the solution, and the mixture was stirred at 180 ° C (180 r / min) for 10 hours. TLC (petroleum ether: ethyl acetate = 0:1, Rf = 0.45) indicated that the material was consumed completely. To the mixture was added EtOAc (500 L) and a saturated solution of NH ₄ Cl (350 L), and the mixture was stirred (180 r / min) for 4 hr. The mixture was then filtered and the filter cake was washed with EtOAc (EtOAc). The aqueous phase was then extracted with EtOAC (200 L). The combined organic phases were extracted with brine (200 L), dried over anhydrous sodium sulfate Concentrate under reduced pressure (50 ° C, -0.1 MPa). The crude product was purified by column chromatography (SiO _2, petroleum ether: ethyl acetate = 500:1 to 50:1) to give intermediate compound 6 (16.2kg, 52.7mol, yield 44.5%) as a yellow solid.

1H NMR: CDCl ₃ 400MHz δ 7.27-7.47 (m, 10H), 4.53-4.66 (m, 2H), 3.85-4.05 (m, 2H), 3.79 (tt, J = 8.80, 4.14 Hz, 1H), 2.98 ( Dt, J = 13.6, 1.84 Hz, 1H), 2.76 (dd, J = 13.6, 8.78 Hz, 1H), 2.12 - 2.25 (m, 1H), 1.75 - 2.01 (m, 2H), 1.37 (br, d, J = 7.60 Hz, 1H), 0.67-0.82 (m, 2H), 0.43-0.58 (m, 2H).

Step six

Pd/C (20.0 g, 13.0 mmol, purity 30.0%) was added to MeOH (1. <RTI ID=0.0></RTI></RTI><RTIID=0.0></RTI></RTI><RTIgt; Eq), stirred (180 r/min) at 45 ° C for 10 hours under H ₂ (50 psi). TLC (petroleum ether: ethyl acetate = 1:1, Rf = 0.99) indicated that starting material was consumed. The mixture was filtered, and EtOAc EtOAc (EtOAc m.

Step seven

To a solution of the intermediate compound 7 (2.60 kg, 15.9 mol, 1.00 eq, HCl) in THF (26.0 L) and H ₂ O (26.0 L) was added NaHCO ₃ (5.34 kg, 63.6 mol, 2.47). L, 4.00 eq) and CbzCl (3.52 kg, 20.7 mol, 2.94 L, 1.30 eq). The mixture was then stirred (180 r/min) at 25 ° C for 10 hours. TLC (dichloromethane:methanol = 10:1, Rf = 0.08) The four batches of the reaction mixture were combined. The combined mixture was taken up in water (3 EtOAc)EtOAc. Concentration under reduced pressure (50 ° C, -0.1 MPa) gave Intermediate Compound 8 (18.5 g, crude) as a yellow oil.

Step eight

To a solution of the intermediate compound 8 (1.50 kg, 5.74 mol, 1.00 eq) and compound a (1.66 kg, 6.31 mol, 1.10 eq) in THF (15 L) was added PPh ₃ (2.11 kg, 8.04) at 0 to 10 °C. Mol, 1.40 eq) and DIAD (1.62 kg, 8.04 mol, 1.56 L, 1.40 eq). The mixture was then stirred (180 r/min) for 5 hours at 25 °C. TLC (petroleum ether: ethyl acetate = 1:1, Rf = 0.46) showed that material was consumed completely. 11 batches of the reaction mixture were combined. The combined mixture was concentrated under reduced pressure (50 ° C, -0.1 MPa). The crude product was purified by column chromatography (SiO _2, petroleum ether: ethyl acetate = 100 to 1:1) to give a yellow oil of intermediate compound 9 (17.2kg, 34.0mol, yield 53.9%).

Compound a_1 (15.0 kg, 101 mol, 1.00 eq) was added to a solution of compound a 2 (13.5 kg, 102 mol, 1.01 eq) in toluene (300 L), and the mixture was stirred at 220 ° C (220 r / min) for 6 hours. TLC (petroleum ether: ethyl acetate = 3:1, Rf = 0.45). The mixture was cooled to 10 ° C, and the mixture was filtered. EtOAc mjjjjjjjjjj

1H NMR: DMSO 400MHz δ 9.88 (s, 1H), 7.87-8.02 (m, 4H), 1.21-1.53 (m, 9H).

Step nine

Was added NH ₂ NH ₂ · H solution (50.0 L) in MeOH in a solution of intermediate compound 9 (5.00kg, 9.89mol, 1.00eq) 2 O (657g, 12.9mol, 638mL, purity 98.0%, 1.30eq), The mixture was stirred (180 r/min) at 70 ° C for 6 hours. TLC (petroleum ether: ethyl acetate = 2:1, Rf = 0.72). The mixture was filtered, and the filtrate was concentrated under reduced pressure (50 ° C, -0.1 MPa). Five batches of the reaction mixture were combined. The combined mixture was added to EtOAc (20.0 mL) and EtOAc (EtOAc) 41.8 mol, yield 84.6%, purity 100%), which was a white solid.

1H NMR: MeOD 400MHz δ7.22-7.42 (m, 5H), 5.10 (s, 2H), 3.79-3.95 (m, 2H), 3.18-3.37 (m, 2H), 2.04-2.18 (m, 1H), 1.64 -1.78(m,1H), 1.43(br,s,9H),1.20(s,1H),1.01-1.12(m,1H),0.88(dt,J=9.56,6.37Hz,1H),0.46-0.64 (m, 2H)

Step ten

To a solution of the intermediate compound 10 (5.00 kg, 13.3 mol, 1.00 eq) in EtOAc (20.0 <RTI ID=0.0></RTI> ) 10 hours. LCMS (product: RT = 1.105 min) showed complete material consumption. The three batches of the reaction mixture were combined. The combined mixture was filtered, and the filter cake was concentrated under reduced pressure (50 ° C, -0.1 MPa) to give a crude product. The crude product was purified along with another batch of 700 g of crude material. The mixture was added to EtOAc (24.0 L) and EtOAc (EtOAc) (EtOAc). The mixture was then cooled to 25 ° C, the mixture was filtered, and the filtered cake was evaporated. mjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjj

1H NMR: DMSO 400MHz δ 7.29-7.42 (m, 4H), 5.09 (s, 2H), 3.94-4.08 (m, 1H), 2.95-3.14 (m, 2H), 1.96-2.09 (m, 1H), 1.53 -1.70 (m, 2H), 1.21-1.37 (m, 1H), 0.99-1.12 (m, 1H), 0.80 (dt, J = 9.6, 6.04 Hz, 1H), 0.49-0.70 (m, 2H).

Example 3

Preparation of 2-((4-((5-chloropyridin-2-yl)oxy)phenyl)(ethoxy)methylene)malononitrile (Compound 7)

step one

Methyl 4-hydroxybenzoate (1A) (2.64 kg, 17.3 mol, 0.950 eq) and 5-chloro-2-fluoropyridine (12) (2.40 kg, 18.3 mol) of N,N-dimethylformamide (2.50 L) To the solution was added cesium carbonate (6.50 kg, 65 mmol). The reaction solution was stirred at 110 ° C for 12 hours, and subjected to thin layer chromatography (petroleum ether: ethyl acetate = 5:1, product R _f = 0.5). Seven batches of the reaction mixture were combined. The combined mixture was taken up in water (70 L), filtered and dried filtered. A white solid (30.0 kg, yield 88.7%, purity 93%) of methyl 4-((5-chloropyridin-2-yl)oxy)benzoate (13) was obtained.

MS: m/z 264.2 [M+1]

Step two

To a solution of the intermediate compound 13 (2.00 kg, 7.59 mol) in THF (4.00 L), LiOH.H ₂ O (4M, 3.98 L). The mixture was stirred at 45 ° C for 12 hours. TLC (petroleum ether: ethyl acetate = 5:1, material Rf = 0.5) showed that the reaction was completed. 15 batches of the reaction were processed together. The reaction mixture was concentrated under reduced pressure to remove THF. The residue was diluted with H ₂ O 50.0L, 4M HCl was added to the mixture until pH 2-3, filtered and the filter cake was dried under vacuum. The solid was dried to give EtOAc (EtOAc m.

MS: m/z 250.1 [M+1]

Step three

Intermediate compound 14 (3.50 kg, 14.0 mol) was added to a solution of 8OCl ₂ (8.61 kg, 72.4 mol). The mixture was stirred at 80 ° C for 12 hours. TLC (petroleum ether: ethyl acetate = 5:1, product Rf = 0.5) showed that the reaction was completed. Three batches of reactions were processed together. The reaction was concentrated to give a crude material. Intermediate compound 15 (10.0 kg, purity 98.9%) was obtained as a white solid.

Step four

To a solution of the intermediate compound 15 (3.33 kg, 12.4 mol) and malononitrile (903 g, 13.7 mol, 860 mL) in THF (10.0 L) was added NaH (745 g, 18.6 mol, purity 60%). The mixture was stirred at 25 ° C for 2 hours. TLC (petroleum ether: ethyl acetate = 0:1, product Rf = 0.2) showed that the reaction was completed. 4 M HCl was added to the mixture at pH 5-6 at 0 ° C, then the reaction mixture was concentrated under reduced pressure to remove THF. It was then extracted with EtOAc (5.00 L*1, 250 L*2). The combined organic layers were washed (2.50 L) with brine, dried over Na ₂ SO _4, filtered and concentrated under reduced pressure to give the crude product. The crude product was triturated with MTBE (2.50 L). The resulting precipitate was collected by filtration and dried in vacuo. Intermediate compound 16 (3.20 kg, 10.6 mol, yield 85.4%, purity 98.7%) was obtained as a yellow solid.

Step five

Ac solution of intermediate compound 16 (5.00kg, 16.8mol) of ₂ O _(20.0L) was added triethyl orthoformate (22.3kg, 150mol, 25.0L). The mixture was stirred at 120 ° C for 12 hours. TLC (petroleum ether: ethyl acetate = 0:1, product Rf = 0.7) showed that the reaction was completed. The two batches of reactions were processed together. Concentration under reduced pressure gave a residue. The residue was purified by column chromatography (SiO _2, petroleum ether: ethyl acetate = 4 to 0:1) purification. Multiple batches of the reaction crude product were combined and further purified. The combined crude product was recrystallized from i-PrOH (10.0 L) at 90 °C. Intermediate compound 17 (10.0 kg, theoretical 19.9 kg, yield 50.8%) was obtained as a pale yellow solid.

¹ H NMR: DMSO 400 MHz 8.28 (m, 1 H), 8.02 - 8.28 (m, 1 H), 7.74 - 7.77 (m, 2H), 7.37-7.40 (m, 2H), 7.21 - 7.24 (m, 1H), 4.20 -4.26 (m, 2H), 1.27-1.32 (m, 3H).

Example 4

5-amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1-((3R,6S)-1-cyano-6-methylpiperidin-3- Preparation of (1)-H-pyrazole-4-carboxamide (Compound 2)

Compound 2 was prepared in a similar manner to Compound 1 starting from the corresponding starting material. MS: m/z 452.2 [M+1]

Example 5

5-amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1-((3R,6R)-1-cyano-6-methylpiperidin-3- Preparation of -1H-pyrazole-4-carboxamide (Compound 3)

Compound 3 was prepared in a similar manner to Compound 1 starting from the corresponding starting material. MS: m/z 452.2 [M+1]

Example 6

(R)-5-Amino-1-(4-cyano-4-azaspiro[2.5]oct-6-yl)-3-(4-(2,4-difluorophenoxy)phenyl) Preparation of -1H-pyrazole-4-carboxamide (Compound 4)

Compound 4 was prepared in a similar manner to Compound 1 starting from the corresponding starting material.

¹ H-NMR (400 MHz, CDCl ₃ ): δ ppm 7.48 (d, J = 8.4 Hz, 2H), 7.15-7.09 (m, 1H), 7.02-6.95 (m, 3H), 6.92-6.88 (m, 1H) , 5.75 (s, 2H), 5.29 (s, 2H), 4.32-4.26 (m, 1H), 3.65-3.52 (m, 2H), 2.42-2.30 (m, 2H), 2.18-2.15 (m, 1H) , 1.19-1.14 (m, 1H), 0.87-0.67 (m, 4H).

Example 7

(R)-5-amino-1-(4-(2-butynyl)-4-azaspiro[2.5]oct-6-yl)-3-(4-((5-chloropyridine-2-) Preparation of oxy)phenyl)-1H-pyrazole-4-carboxamide (Compound 5)

Compound 5 was prepared in a similar manner to Compound 1 starting from the corresponding starting material.

¹ H-NMR (400 MHz, MeOD): δ ppm 8.08 (s, 1H), 7.85 (d, J = 8.8 Hz, 1H), 7.61-7.57 (m, 2H), 7.23-7.20 (m, 2H), 7.05 ( d, J=8.8 Hz, 1H), 4.40-4.37 (m, 1H), 4.22-3.90 (m, 2H), 3.60-3.31 (m, 1H), 2.34-2.31 (m, 1H), 2.16-2.09 ( m, 2H), 2.06 (s, 3H), 1.14 - 0.75 (m, 4H).

MS: m/z 505.1 [M+H].

Example 8

(R)-5-amino-1-(4-(2-butynyl)-4-azaspiro[2.5]oct-6-yl)-3-(4-(2,4-difluorophenoxy) Of phenyl)-1)-pyrazole-4-carboxamide (Compound 6)

Compound 6 was prepared in a similar manner to Compound 1 starting from the corresponding starting material.

^{1 H-NMR (400MHz, CDCl} 3): δppm 7.52 (d, J = 8.0Hz, 1H), 7.13-7.10 (m, 1H), 7.03-6.95 (m, 3H), 6.91-6.88 (m, 1H) , 5.65 (s, 2H), 5.18 (s, 2H), 4.56-3.92 (m, 3H), 3.25-3.20 (m, 1H), 2.52-2.51 (m, 1H), 2.21-2.14 (m, 2H) , 2.09 (s, 3H), 1.08-0.88 (m, 2H), 0.72-0.65 (m, 2H).

MS: m/z 506.4 [M+H].

Example 9

Kinase Activity Inhibition Experiment (BTK)

Inhibition of BTK kinase activity by the compounds disclosed herein was tested in a time-resolved fluorescence resonance energy transfer method. Recombinant Btk and the compounds disclosed herein at room temperature in assay buffer containing 50 mM Tris pH 7.4, 10 mM MgCl ₂ , 2 mM MnCl ₂ , 0.1 mM EDTA, 1 mM DTT, 20 nM SEB, 0.1% BSA, 0.005% tween-20 Incubate for 1 hour in advance. The reaction was initiated by the addition of ATP (at ATP Km concentration) and the peptide substrate (Biotin-AVLESEEELYSSARQ-NH2). After 1 hour incubation at room temperature, an equal volume of p-Tyr66 antibody containing 50 mM HEPES pH 7.0, 800 mM KF, 20 mM EDTA, 0.1% BSA, attached Eu cryptate, and streptavidin-labeled XL665 were added. Stop the solution to stop the reaction. The plates were incubated for an additional hour at room temperature and then the TR-FRET signal (ex337 nm, em 620 nm / 665 nm) was read on a BMG PHERAstar FS instrument. Based on the ratio of the fluorescence at 615 nm to the fluorescence at 665 nm, the residual enzyme activity in the case of an increase in compound concentration was calculated. The IC50 of each compound was obtained by fitting the data to the four-parameter logistic equation of Graphpad Prism software.

According to the above experimental methods, the compounds of the present invention exhibit strong kinase inhibitory activity (IC50 < 1000 nM), and some of the preferred compounds have strong kinase inhibitory activity (IC50 < 100 nM). The specific results are shown in the table below.

The kinase inhibitory activity levels are classified as A, B, C, specifically A (IC ₅₀ <100 nM), B (100 nM < IC ₅₀ <1000 nM).

Example 10

In vitro kinase selectivity assay

The detection platform of EGFR and ITK kinase activity was established by time-resolved fluorescence resonance energy transfer method. The detection platform of LCK, SRC and LYN kinase activity was established by Z'-Lyte method. The detection platform of TEC and JAK3 kinase activity was established by Lance Ultra method. The inhibitory effects of the compounds disclosed herein on different kinase activities were tested separately. Each compound activity data were determined at 11 concentrations of the compound IC ₅₀ value calculated using Graphpad Prism software.

According to the above experimental method, the compound of the present invention exhibited a strong kinase selectivity, which was significantly superior to the control compound ibrutinib. See the table below for the results.

The kinase inhibitory activity levels are classified into A, B, C, specifically A (IC ₅₀ <100 nM), B (100 nM < IC ₅₀ <1000 nM), C (IC ₅₀ >1000 nM).

Example 11

B cell inhibition assay

Short exposure to BTK inhibitors in vitro is sufficient to inhibit B cell activation in normal human B cells. This protocol mimics the predicted exposure of cells to inhibitors in vivo and shows that inhibition of B cells is maintained despite the washing inhibitor.

B cells were purified from healthy donor blood by negative selection using the RosetteSep Human B Cell Enrichment Mix. Cells were plated in growth medium (10% RPMI + 10% fetal bovine serum) and inhibitors of the indicated concentrations were added. After incubating for 1 hour at 37 ° C, the cells were washed three times, and each wash was used for 8-fold dilution in growth medium. The cells were then stimulated with 10 μg/mL IgM F(ab') 2 for 18 hours at 37 °C. Cells were subsequently stained with anti-CD69-PE antibody and analyzed by flow cytometry using standard conditions.

According to the above method, the compounds of the examples of the present invention have strong inhibitory activity against B cells, and their IC50 values are less than 10 nM.

Example 12

T cell inhibition assay

T cells were purified from healthy donor blood by negative selection using the RosetteSep Human T Cell Enrichment Mix. Cells were plated in growth medium (10% RPMI + 10% fetal bovine serum) and inhibitors of the indicated concentrations were added. After incubating for 1 hour at 37 ° C, the cells were washed three times, and each wash was used for 10-fold dilution in growth medium. The cells were then challenged with anti-CD3/CD28 coated beads (bead/cell ratio of 1:1) for 18 hours at 37 °C. Cells were subsequently stained with anti-CD69-PE antibody and analyzed by flow cytometry using standard conditions. The compounds of the examples of the present invention have a weak inhibitory activity or no inhibition on T cells, and have an IC50 value of more than 4000 nM, as determined by the above method.

Example 13

Human whole blood B cell inhibition experiment

Human whole blood (hWB) was obtained from healthy volunteers and blood was collected by venipuncture into a Vacutainer tube that was anticoagulated with sodium heparin. Test compounds were diluted to 10 times the required initial drug concentration in PBS), followed by three-fold serial dilutions in 10% DMSO in PBS to give a 9 point dose response curve. 5.5 μL of each compound dilution was added to the aiil 96-well V-bottom plate in duplicate; 5.5 μL of 10% DMSO in PBS was added to the control and non-stimulated wells. Human whole blood (100 μL) was added to each well, and after mixing, the plates were incubated for 30 minutes at 37 C, 5% CO ₂ , 100% humidity. Add F (ab') 2 anti-human IgM (Southern Biotech) (10 μL of 500 μg/mL solution, 50 μg/mL final concentration) to each well (without stimulation holes) with stirring, and incubate the plate for an additional 20 hours. . At the end of 20 hours incubation, the samples were labeled with fluorescent probes 20μL APC mouse anti-human CD69 (BD Pharmingen) at _{37C, 5% CO 2, 100} % humidity for 30 minutes. Induction controls, unstained, and single stains are included for compensation adjustment and initial voltage settings. The sample was then lysed with 1 ml of IX Pharmingen Lyse Buffer (BD Pharmingen) and the plate was centrifuged at 1500 rpm for 5 minutes. The supernatant was removed by aspiration, and the remaining pellet was again lysed with an additional 1 ml of IX Pharmingen Lyse Buffer, and the plate was centrifuged as before. The supernatant was aspirated and the remaining pellet was washed in FACs buffer (PBS + 1% FBQ. After centrifugation and the supernatant was removed, the pellet was resuspended in 150 μL of FACs buffer. Transfer the sample to a suitable one. 96-well plates run on the HTS 96-well system of the BD LSR II flow cytometer. Data were acquired using excitation and emission wavelengths appropriate for the fluorophore used and percent positive cells were obtained using Cell Quest Software. Results were initially analyzed using FACS analysis (Flow Jo) Analysis. IC50 values were calculated using XLfit v3, Equation 201.

According to the above method, the compound of the present invention has strong inhibitory activity against B cells in human whole blood, and its IC50 value is less than 200 nM.

Example 14

Study on the stability of compounds in liver microsomes

1. A compound of the present invention was dissolved in acetonitrile to prepare a stock solution having a concentration of 0.5 mM.

2. Add 2 μL stock solution to a 1.5 ml centrifuge tube, then add 148 μL phosphate buffer (100 mM, pH 7.4) and 10 μL liver microsome (protein concentration 20 mg/ml) suspension [BD Gentest], liver microsomes. The genus was human, dog, rat, and mouse; the control group was added with 158 μL of phosphate buffer (100 mM, pH 7.4).

3. Prepare the mixed system in step 2, pre-incubated for 3 minutes in a 37 ° C water bath, then add 40 μL of NADPH production system (containing NADP +: 6.5 mM, glucose 6-phosphate: 16.5 mM, MgCl ₂ : 16.5 mM, glucose 6 - Phosphate dehydrogenase: 2 U/ml) The reaction was initiated and incubated for 1 hour in a 37 ° C water bath.

4. After the reaction was carried out for 1 hour, the centrifuge tube was taken out from the water bath, and the reaction was terminated by adding 400 μL of acetonitrile, then vortexed for 3 minutes, finally centrifuged (13,000 rpm, 4 ° C) for 5 minutes, and the supernatant was taken for detection of the remaining drug by HPLC. Concentration Cr.

5. Parallel preparation 0 minute reaction sample preparation method: The prepared mixed system in step 2 was pre-incubated for 3 minutes in a 37 ° C water bath, and then 400 μL of acetonitrile was added, followed by 40 μL of NADPH generation system. After vortexing for 3 minutes, centrifugation (13,000 rpm, 4 ° C) for 5 minutes, and the supernatant was taken to detect the drug concentration C0 by HPLC.

6. After 60 minutes of incubation, the remaining percentage of drug in the incubation system is calculated as follows:

Drug surplus (%) = Cr / C0 × 100%

In accordance with the experimental methods described above, the compounds of the examples of the present invention exhibited better microsomal stability with a residual percentage >30% in liver microsomes of various genera.

Example 15

Evaluation of compound inhibition of CYP enzyme

CYP enzyme metabolism is the main pathway for drug biotransformation, and its quantity and activity directly affect the activation and metabolism of drugs in the body. As a major metabolic enzyme of exogenous compounds, cytochrome CYP is an important drug phase I metabolizing enzyme that catalyzes the oxidation and reductive metabolism of various exogenous compounds. The CYP enzyme plays a very important role in the elimination of the drug, and is also the main factor in the drug interaction caused by the combination.

METHODS: This experiment used the cocktail probe drug method to simultaneously determine the inhibitory effect of compounds on five CYP450 enzymes in human liver microsomes. The human microsomes were from BD Gentest.

The experimental steps are as follows:

The reaction was carried out in 100 mM phosphate buffer in a total volume of 200 μL. The concentration of the microsomes in the reaction system was 0.25 mg/mL, and the concentration of the test compound was 20 μM, 6.67 μM, 2.22 μM, 0.74 μM, 0.25 μM. The specific probe substrate and concentration were phenacetin (CYP1A2) 40 μM, respectively. Dextromethorphan (CYP2D6) 5 μM, diclofenac (CYP2C9) 10 μM, S-mefenidine (CYP2C19) 40 μM, testosterone (CYP3A4) 80 μM. The incubation system was pre-incubated for 5 minutes in a 37-degree constant temperature shaker, and the reaction was started by adding a NADPH-producing system (containing 1.3 mM NADP+, 3.3 mM glucose 6-phosphate, 0.4 U/L glucose 6-phosphate dehydrogenase, 3.3 mM MgCL2). After incubation for 45 minutes, the reaction was stopped by adding an equal volume of acetonitrile, vortexed, centrifuged at 13,000 rpm, and the supernatant was subjected to LC-MS-MS injection to determine the amount of metabolite production. The specific metabolites were acetaminophen (CYP1A2), dextrorphan (CYP2D6), 4-hydroxydiclofenac (CYP2C9), 4-hydroxyfenfenin (CYP2C19), and 6β-hydroxytestosterone (CYP3A4). The specific inhibitors were furaphylline (CYP1A2), quinidine (CYP2D6), sulfaphenazole (CYP2C9), tranylcypromine (CYP2C19), ketoconazole (CYP3A4). The final result of this experiment is to calculate the IC50 value of the half inhibitory concentration. IC50 = ((50% - low inhibition rate %) / (high inhibition rate % - low inhibition rate %)) x (high concentration - low concentration) + low concentration.

According to the above experimental method, the compounds of the examples of the present invention have only strong inhibition or no inhibition on various CYP enzymes, indicating that they have less influence on the metabolism of other drugs.

Example 16

Method for pharmacokinetic study of compounds in rats

1. Male SD rats [Hua Kangkang] were purchased and adapted for 7 days in this laboratory.

2. Nine SD rats were randomly divided into 3 groups, 3 in each group, one group was administered by intragastric administration, and the other group was administered by tail vein injection. Rats in the gavage-administered group were fasted overnight before administration.

3. After administration of the rats, blood samples were taken at the following time points by blood collection from the orbital venous plexus: IV: (before administration), 0.08 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours ,24 hours. P.O.: 0.08 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours. The blood collection at each blood collection time point was about 300 μl.

4. The collected blood samples were centrifuged at 12000 rpm for 5 minutes at 4 ° C, then the upper plasma samples were collected and stored in a refrigerator at -20 ° C for testing.

5. The experimental operation is summarized in Table 4:

Table 4. Design of pharmacokinetics of compounds in rats

6. Using LC-MS/MS (UPLC-MS/MS: liquid phase Waters Acquity UPLC (USA) and mass spectrometry 5500Q Trap (Applied Biosystem/MDS SCIEX) or HPLC-MS\MS: liquid phase Agilent 1200 series (USA) and mass spectrometry API 4000 (Applied Biosystem/MDS SCIEX)) detects the concentration of compounds in plasma. Typical test conditions are as follows:

Use the pharmacokinetic professional software WinNonlin [Model: Phoenix ^TM

6.1 Manufacturer: Pharsight Corporation] Calculate pharmacokinetic parameters. [Phoenix 1.1 User's Guide: p251-p300]

In accordance with the experimental methods described above, the compounds of the examples of the present invention exhibited better bioavailability (&gt;40%).

Example 17

hERG binding assay (Dofetillide method)

The IC50 value of the compound for hERG inhibition can be determined according to the method described in the patent US20050214870 A1. The compounds of the present invention have only a weak or no inhibitory effect on hERG and have an IC50 value greater than 1000 nM.

Example 18

Pharmacodynamic experiment

The immunodeficiency serious defect NOD.SCID mouse was purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. and was raised in the SPF animal room. After the TMD-8 cells were cultured to a sufficient amount, the cells were collected by centrifugation and washed twice with PBS. Finally, the cells were resuspended in serum-free RPMI 1640 medium and Matrigel (1:1 v/v). Using a 1 ml syringe and a 25G syringe needle, 0.2 ml of the cell suspension was injected into the right flank area of each mouse. The tumor size was measured with a vernier caliper for about one week, and the tumor volume was calculated by the following formula: tumor volume = (length x width ² )/2. When the tumor volume reached about 100-200 mm ³ , the mice were intragastrically administered for 21 days.

The compound of the present invention can significantly inhibit the growth of the diffuse large B-cell lymphoma cell line TMD-8 in vivo and exhibit the same antitumor effect as the control compound ibrutinib (see Fig. 1 for experimental results).

Claims (10)

1. A method for preparing a 5-aminopyrazolecarboxamide compound represented by formula (I),

   

   among them,

   n, m are independently taken from 0, 1 or 2;

   L is O, -C(O)NH-, -CH ₂ -, S, S(O), NH or S(O) ₂ ;

   A is derived from a substituted or unsubstituted heterocyclic ring, a substituted or unsubstituted benzene ring, or a substituted or unsubstituted heteroaryl ring, and a linking site to the parent nucleus and L is optional;

   B is independently taken from a substituted or unsubstituted aliphatic ring, a substituted or unsubstituted heterocyclic ring, a substituted or unsubstituted benzene ring, or a substituted or unsubstituted heteroaryl ring, and the attachment site to L is optional of;

   R ₁ and R ₂ are each independently selected from hydrogen, C1-C4 alkyl, halogen, cyano, or R ₁ and R ₂ together with the carbon atom to which they are attached form a ternary carbocyclic or quaternary carbocyclic ring, or R ₁ And R _{2 are} combined into an oxo group;

   Y is selected from cyano group,

   

   R ₃ , R ₄ , R ₅ and R ₆ are each independently selected from hydrogen, unsubstituted C1-C4 alkyl, hydroxy-substituted C1-C4 alkyl, C1-C4 alkoxy C1-4 alkyl, halogen, a cyano group, or -(CH ₂ ) _q N(R ^a R ^b ), where q is 1, 2, 3, or 4, and R ^a and R ^b are each independently selected from hydrogen, unsubstituted C1-C4 alkane base;

   And stipulates that when one of R ₁ and R ₂ is hydrogen and the other is methyl, and Y is a cyano group, A is a benzene ring, L is O, m is 1 and n is 2, B is not substituted or unsubstituted a benzene ring; when both R ₁ and R ₂ are hydrogen, and A is a benzene ring, m is 1 and n is 2, B is not a substituted or unsubstituted benzene ring, and a substituted or unsubstituted pyridine; when R ₁ And R ₂ is hydrogen, and A is a pyridine ring, m is 1 and n is 2, B is not a substituted or unsubstituted benzene ring; when R ₁ and R ₂ are both hydrogen, and A is a benzene ring, L is When O, m is 1 and n is 1, B is not a substituted or unsubstituted benzene ring;

   The preparation method comprises the following steps:

   (1) reacting a compound of the formula (V) with a compound of the formula (VI) to give a compound of the formula (VII);

   

   (2) subjecting a compound of the formula (VII) to a hydrolysis reaction to obtain a compound of the formula (VIII);

   

   (3) a compound of the formula (VIII) is subjected to a deprotection group PG to give a compound of the formula (IX);

   

   (4) reacting a compound of the formula (IX) with a compound of the formula (X) to give a compound of the formula (I);

   

   Substituents R ₁ , R ₂ , L, A, B, Y in the above formula (V), formula (VI), formula (VII), formula (VIII), formula (IX) and formula (X) And n, m are as defined above for formula (I), PG is a protecting group, R ₃ is a C1-C4 alkyl group, and X is chlorine, bromine or a hydroxyl group.
2. The method of claim 1 wherein said 5-aminopyrazolecarboxamide compound is as shown in formula (II):

   
3. The method according to claim 1, wherein said 5-aminopyrazolecarboxamide compound is as shown in formula (III):

   
4. The method according to claim 1, wherein said 5-aminopyrazolecarboxamide compound is as shown in formula (IV):

   
5. The method of claim 1 further comprising the preparation of a compound of formula (VI):

   

   Wherein the substituent R ₄ in the above compound is a C ₁ -C ₄ alkyl group; X is chlorine or bromine; R ₃ is a C ₁ -C ₄ alkyl group; and L, A and B are as defined in the formula (I) Compound.
6. The method according to claim 5, wherein the compound (VI-1) is subjected to hydrolysis reaction under basic conditions to obtain a compound (VI-2); and then the compound is reacted with an acid halide reagent to obtain a compound (VI-3). Then, the compound (VI-3) is reacted with malononitrile under basic conditions to obtain a compound (VI-4); then, the compound VI-4 is reacted with (R ₃ O) ₃ CH in the presence of acetic anhydride. Compound (VI).
7. The method of claim 1 further comprising the preparation of formula (V):

   

   Wherein PG, PG1, PG2, PG3, PG4 are protecting groups, and R ₁ , R ₂ , n, and m are as defined in the above formula (I).
8. The method of claim 7 comprising:

   
9. The method of claim 7 comprising:

   
10. The method of claim 7 comprising:

    

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