CN118056832A - Pyrazolopyrimidine derivative having BTK inhibitory effect - Google Patents

Pyrazolopyrimidine derivative having BTK inhibitory effect Download PDF

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CN118056832A
CN118056832A CN202311543961.2A CN202311543961A CN118056832A CN 118056832 A CN118056832 A CN 118056832A CN 202311543961 A CN202311543961 A CN 202311543961A CN 118056832 A CN118056832 A CN 118056832A
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pharmaceutically acceptable
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鲍荣肖
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Tianjin Zhengcheng Pharmaceutical Technology Co ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
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    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems

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Abstract

The present invention relates to pyrazolopyrimidine derivative having a Bruton's Tyrosine Kinase (BTK) inhibiting effect. Pyrazolopyrimidine derivative of the invention is a deuterated compound, and a pharmaceutically acceptable salt, hydrate or solvate thereof. The invention also relates to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a pyrazolopyrimidine derivative of the invention as well as a pharmaceutically acceptable crystalline form, salt, hydrate or solvate. Pyrazolopyrimidine derivative having a BTK inhibiting effect as disclosed herein is used alone or in combination with other therapeutic agents to treat autoimmune diseases or disorders, xenogenic immune diseases or disorders, cancer, and inflammatory diseases or disorders.

Description

Pyrazolopyrimidine derivative having BTK inhibitory effect
Technical Field
The present invention relates to pyrazolopyrimidine derivative having BTK inhibitory effect.
Background
Bruton's Tyrosine Kinase (BTK) is an enzyme that mediates cell signaling and is found in plasma cells such as B cells. B cells are activated by B Cell Receptor (BCR), while BTK has an important role in BCR-mediated signaling pathways. After the BCR on the B cells is activated, BTK is activated, so that the concentration of downstream phospholipase C is increased, IP3 and DAG signal channels are activated, proliferation, adhesion and survival of the cells are promoted, and the B cell activation method plays an important role in the development process of B cell lymphomas. Aberrant BCR-mediated signaling can cause deregulated B-cell activation and/or formation of pathogenic autoantibodies that lead to a variety of autoimmune and/or inflammatory diseases. Mutations in BTK in humans result in X-linked agarobroteinemia (XLA) (Conley et al, annu. Rev. Immunol.27:199-227, 2009). This disease is associated with impaired B cell maturation, reduced immunoglobulin production, impaired T cell-independent immune responses, and a significant decrease in sustained calcium signaling upon BCR stimulation. Abnormal activation of BTK plays an important role in the pathogenesis of B cell lymphomas, meaning that inhibition of BTK is useful in the treatment of B cell lymphomas, leukemias and other hematological malignancies (Davis et al, nature 463:88-92,2010, mohamed et al, immunol. Rev.228:58-73, 2009; rokosz et al, experet. Opin. Ther. Targets 12:883-903, 2008; uckun et al, anti-CANCER AGENTS Med. Chem.7:624-632,2007; lou et al, J. Med. Chem.55 (10): 4539-4550, 2012).
Ibutenib (ibrutinib ) is a Bruton's Tyrosine Kinase (BTK) inhibitor and is useful for the treatment of chronic lymphocytic leukemia, mantle cell lymphoma, small lymphocytic lymphoma, and other diseases. The united states Food and Drug Administration (FDA) has accelerated approval of PHARMACYCLICS company and us prednisone Imbruvica (ebutinib) for marketing in month 11 2013. Ebutinib inhibits Btk irreversibly by selective covalent binding to the active site cysteine residue (Cys 481) of the target protein Btk, thereby effectively preventing tumor migration from B cells to lymphoid tissues adapted to the tumor growth environment.
The metabolic pathways of ibutinib are complex, ELLEN SCHEERS et al studied the metabolism of ibutinib in healthy male volunteers and identified a total of 40 metabolites, the relative proportions of which are not quite clear (ELLEN SCHEERS et al Drug Metab Dispos 43:289-297). And the effect of the absorption, distribution, metabolism and/or excretion of ibutenib on the efficacy and/or toxicity is still not very clear.
Currently, some drugs have poor drug metabolism and kinetic properties, such as: absorption, distribution, metabolism and/or excretion prevent their wider use or limit their use in specific indications. For example, due to the short elimination half-life of drugs in vivo, rapid clearance, a solution is often employed to administer drugs frequently or at high doses to achieve sufficiently high drug plasma levels. However, this introduces a number of potential treatment problems, such as patient compliance with the dosing interval, and higher dosing, side effects are more severe and increase the cost of treatment.
Attempts have been made to improve the drug's metabolism and kinetic properties by modifying (modifying) the drug with deuterium, or by substituting one or more hydrogen atoms with deuterium atoms to reduce the formation of undesired metabolites, etc. However, due to the complex metabolic processes of biological systems, the pharmacokinetic properties of drugs in organisms are affected by various factors, and also show corresponding complexity. Changes in the pharmacokinetic properties of deuterated drugs exhibit great contingency and unpredictability compared to the corresponding non-deuterated drugs. For some compounds, deuteration slows their metabolic clearance in vivo and half-life increases; for other compounds, deuteration does not cause metabolic changes; for other compounds, deuteration accelerates metabolic clearance and half-life shortens (Blake,MI et al,J Pharm Sci,1975,64:367-91;Foster,AB,Adv Drug Res 1985,14:1-40;Kushner,DJ et al,Can J Physiol Pharmacol 1999,79-88;Fisher,MB et al,Curr Opin Drug Discov De ve l,2006,9:101-09).
Even when deuterium atoms are incorporated into known metabolic sites, the effect of deuterium modification (deuterium modification) on the metabolism of the drug is not predictable. Only by actually preparing and testing deuterated drugs can it be determined whether and how the rate of metabolism will differ from the corresponding chemical entity that is not deuterated. Many drugs have multiple sites of possible metabolism. The location (site) where deuterium substitution is required and the degree of deuteration necessary to effect metabolism, if any, is found to be different for each drug (Fukuto et al.J.Med. Chem.1991,34,2871-76).
As previously mentioned, the effect of deuterium modification (deuterium modification) on the metabolism of the drug is unpredictable. Ibutenib and its in vivo metabolites have the disadvantage of being at risk of hepatotoxicity, causing clinical concerns. Liver toxicity is not only related to chemical structure but also to the clinical dosage.
Therefore, aiming at the defects of the prior art, new compounds are designed, the exposure of the new compounds in vivo is improved, the dosage and/or the frequency of the drugs are reduced, and the hepatotoxicity is reduced; and/or through structural modification, reduce the hepatotoxicity of its original form or metabolite, or reduce the generation of toxic metabolite, thus achieve the goal of attenuation and synergy.
According to the invention, various deuterated ibutinib is actually prepared and tested, and chemical entities different from the ibutinib are determined, so that a deuterated medicament with good pharmacokinetic properties, reduced use dosage and/or reduced toxic and side effects is obtained.
Disclosure of Invention
The object of the present invention is to provide pyrazolopyrimidine derivative having BTK inhibiting effect (formula i), a crystalline form thereof, a salt thereof, a hydrate thereof or a solvate thereof:
Wherein:
R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15、R16、R17 Or R 18 are each independently selected from hydrogen (H) or deuterium (D), provided that at least one of R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15、R16、R17 or R 18 is deuterium.
Pyrazolopyrimidine derivative of the invention is a deuterated compound, and a pharmaceutically acceptable crystal form, salt, hydrate or solvate thereof, and the compound of the invention has a structure selected from the group consisting of compound DI101, compound DI102, compound DI103, compound DI104, compound DI105, compound DI106 and compound DI107.
In a further aspect of the present invention there is provided a pharmaceutical composition comprising a therapeutically effective amount of any of the compounds according to claim 1 or 2 or a tautomer thereof, a mixture of forms, and pharmaceutically acceptable crystalline forms, salts, hydrates or solvates thereof, and a pharmaceutically acceptable carrier and/or excipient.
In a further aspect of the invention, a compound according to any one of claims 1 or 2 or a tautomer thereof, a mixture of forms, and pharmaceutically acceptable crystalline forms, salts, hydrates, or solvates thereof, or a pharmaceutical composition according to claim 3, is a prophylactic and/or therapeutic agent for BTK-related diseases.
In a further aspect of the invention, the BTK-related disease prevented and/or treated according to any one of the compounds as shown in claim 1 or 2 or a tautomer thereof, a mixture of forms thereof, and a pharmaceutically acceptable crystalline form, salt, hydrate or solvate thereof, or a pharmaceutical composition according to claim 3 is an allergic disorder, an autoimmune disease, an inflammatory disease, a thromboembolic disease or cancer.
In a further preferred embodiment of the present invention, the pharmaceutical composition is used for treating rheumatoid arthritis, psoriatic arthritis, infectious arthritis, progressive chronic arthritis, teratogenic arthritis, osteoarthritis, traumatic arthritis, gouty arthritis, reiter's syndrome, polychondritis, acute synovitis, spondylitis, glomerulonephritis with nephrotic syndrome, glomerulonephritis without nephrotic syndrome, autoimmune blood system disorder, hemolytic anemia, aplastic anemia, idiopathic thrombocytopenia, neutropenia, autoimmune gastritis, autoimmune inflammatory bowel disease, ulcerative colitis, crohn's disease, host versus graft disease, allograft rejection, chronic thyroiditis, graves 'disease, scleroderma, type I diabetes, type II diabetes, acute active hepatitis chronic active hepatitis, pancreatitis, primary biliary cirrhosis, myasthenia gravis, multiple sclerosis, systemic lupus erythematosus, psoriasis, atopic dermatitis, contact dermatitis, itching, skin sunburn, vasculitis, behcet's disease, chronic renal insufficiency, stevens-Johnson syndrome, inflammatory pain, idiopathic steatorrhea, cachexia, sarcoidosis, guillain-Barre syndrome, uveitis, conjunctivitis, keratoconjunctivitis, otitis media, periodontal disease, interstitial pulmonary fibrosis, asthma, bronchitis, rhinitis, sinusitis, pneumoconiosis, pulmonary insufficiency syndrome, emphysema, pulmonary fibrosis, sandy lung, chronic inflammatory lung disease, chronic obstructive pulmonary disease, proliferative diseases, B cell lymphomas caused by chronic active B cell receptor signaling, and bone diseases associated with multiple myeloma.
In yet another aspect of the invention, the BTK-related disease is a B-cell proliferative disease selected from the group consisting of chronic lymphocytic lymphoma, non-hodgkin's lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, and chronic lymphocytic leukemia.
Detailed Description
The inventor researches find that pyrazolopyrimidine derivative and pharmaceutically acceptable crystal forms, salts, hydrates or solvates thereof have better pharmacokinetics and/or safety compared with ibutinib, a compound DI901 and/or a compound DI902, and are more suitable for preparing and treating diseases related to BTK. The present invention has been completed on the basis of this finding.
As used herein, the term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention with acids or bases that are suitable for use as medicaments. Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is the salts of the compounds of the present invention with acids. Suitable salts forming acids include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid, and the like; acidic amino acids such as aspartic acid and glutamic acid.
The compounds of the invention or pharmaceutically acceptable derivatives thereof may also be administered simultaneously, prior to, or subsequent to the administration of one or more other therapeutic agents. Such combination therapies include the administration of a single pharmaceutical dosage formulation comprising a compound of the invention and one or more other active agents, as well as the administration of a separate pharmaceutical dosage formulation of the compound of the invention with each active agent itself. For example, a compound of the invention may be administered to a patient with another active agent in a single orally administered composition (e.g., a tablet or capsule), or each agent may be administered in a separate orally administered formulation. Where separate administration formulations are used, the compounds of the invention and one or more additional active agents may be administered at substantially the same time (i.e., simultaneously) or at separate staggered times (i.e., sequentially); combination therapy should be understood to include all such regimens.
The invention has the positive progress effects that:
(1) The compound has good BTK inhibition effect and can be effectively used for diseases related to BTK.
(2) The compound has good effect of inhibiting B cell activation and is effectively used as a B cell activation inhibitor.
(3) The deuterated pyrazolopyrimidine derivative has the advantages of low hepatotoxicity, good pharmacokinetic property, reduced dosage and/or reduced toxic and side effects, and better patentability.
Preparation method
The following more specifically describes the preparation method of the compound of the present invention, but these specific methods do not limit the present invention in any way. Methods for preparing non-deuterated pyrazolopyrimidine derivatives and physiologically compatible salts thereof for use in the present invention are known. Corresponding deuterated pyrazolopyrimidine derivatives can be synthesized using the same route starting from the corresponding deuterated starting compounds.
Taking compound DI104 as an example, a preferred preparation scheme is as follows:
the specific synthetic method is illustrated in example 1.
The invention will be further illustrated with reference to specific examples. These examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.
Example 1: synthesis of Compound DI104
Synthetic route
The first step: synthesis of Compound DI602
1H-pyrazolo [3,4-b ] pyridine (compound DI 601) (91 mg) and N-iodosuccinimide (345 mg) were mixed in CH 2Cl2 (4 ml), heated at 45℃for 16 hours, and then cooled to room temperature. The reaction mixture was purified by silica gel column eluting with hexane and ethyl acetate (1:1) to give compound DI602.
And a second step of: synthesis of Compound DI704
In a 20ml microwave vial was placed compound DI602 (441.1 mg), compound DI304 (542.6 mg), palladium acetate (19.1 mg), 2-dicyclohexylphosphine-2 ',6' -dimethoxydiphenyl (69.4 mg), potassium carbonate (701.5 mg) suspended in dioxane (8.00 ml) and water (0.80 ml). The reaction mixture was subjected to microwaves at 160 ℃ for 15 minutes. The reaction mixture was allowed to cool to room temperature. Sodium sulfate was added to the mixture, the mixture was then filtered through a silica gel plug, and the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to give compound DI704.
And a third step of: synthesis of Compound DI706
To tetrahydrofuran (5 mL) was added compound DI704 (102.3 mg), polymer-supported triphenylphosphine (332.6 mg), followed by tert-butyl 3 s-hydroxy-1-piperidinecarboxylate (compound DI 605) (202.7 mg) and diisopropyl azodicarboxylate (0.1 mL). The reaction mixture was stirred at room temperature overnight, filtered and concentrated, then purified with a silica gel column to give compound DI706.
Fourth step: synthesis of Compound DI104
Compound DI706 (483.3 mg) was added to a 4 mol/l solution of hydrochloric acid in dioxane (10 mL), stirred at room temperature for 1 hour, then concentrated to dryness, the residue was dissolved in dichloromethane, triethylamine (0.45 mL) was added, and then tridentate acryloyl chloride (compound DI 608) (0.1 mL) was added, and stirred at room temperature for 2 hours. The reaction mixture was washed with 5% aqueous citric acid solution and then brine. The organic layer was dried over magnesium sulfate and concentrated. Purifying with silica gel column to obtain compound DI104. The nuclear magnetic resonance hydrogen spectrum of the compound DI104 is ：1H-NMR(DMSO-d6)δ8.3(1H),7.7(2H),7.4(2H),7.0-7.3(4H),4.7(1H),4.5(0.5H),4.2(1H),4.1(0.5H),3.7(0.5H),3.2(1H),3.0(0.5H),2.3(1H),2.1(1H),1.9(1H),1.6(1H).
Example 2: synthesis of Compound DI602
Fifth step: synthesis of Compound DI602
To a solution of 1H-pyrazolo [3,4-b ] pyridine (compound DI 601) (500.0 mg) in DMF (10 mL) was added iodine (2.13 g) and potassium hydroxide (943 mg) at 0deg.C. Back heating to room temperature and stirring for l hours. Then saturated sodium thiosulfate solution (l 0 mL) was added, and extracted with ethyl acetate (2X 200 mL). The combined organic layers were washed with water (3X 50 ml), brine (50 ml), dried over sodium sulfate and concentrated in vacuo to give compound DI602.
Example 3: synthesis of Compound DI102
According to "example 1: the synthesis of compound DI104 "proceeds, and compound DI302 is used instead of compound DI304 only in the second step to obtain compound DI102. The nuclear magnetic resonance hydrogen spectrum of the compound DI102 is ：1H-NMR(DMSO-d6)δ8.3(1H),7.7(2H),7.4(2H),7.0-7.3(2H),4.7(1H),4.5(0.5H),4.2(1H),4.1(0.5H),3.7(0.5H),3.2(1H),3.0(0.5H),2.3(1H),2.1(1H),1.9(1H),1.6(1H).
Example 4: synthesis of Compound DI103
According to "example 1: the synthesis of compound DI104 "proceeds, and compound DI103 is obtained only in the second step by replacing compound DI304 with compound DI 303. The nuclear magnetic resonance hydrogen spectrum of the compound DI103 is ：1H-NMR(DMSO-d6)δ8.3(1H),7.7(2H),7.0-7.3(4H),4.7(1H),4.5(0.5H),4.2(1H),4.1(0.5H),3.7(0.5H),3.2(1H),3.0(0.5H),2.3(1H),2.1(1H),1.9(1H),1.6(1H).
Example 5: synthesis of Compound DI101
According to "example 1: the synthesis of compound DI104 "proceeds, and compound DI603 is used instead of compound DI304 only in the second step to obtain compound DI101. The nuclear magnetic resonance hydrogen spectrum of the compound DI101 is ：1H-NMR(DMSO-d6)δ8.3(1H),7.7(2H),7.0-7.3(2H),4.7(1H),4.5(0.5H),4.2(1H),4.1(0.5H),3.7(0.5H),3.2(1H),3.0(0.5H),2.3(1H),2.1(1H),1.9(1H),1.6(1H).
Example 6: synthesis of Compound DI608
Sixth step: synthesis of Compound DI608
To tetradeuterated acrylic acid (compound DI 305) (0.15 ml) was added DMF (0.003 ml) followed by oxalyl chloride (0.18 ml). The reaction mixture was stirred for about 40 minutes to give tridentate acryloyl chloride (Compound DI 608)
Example 7: synthesis of Compound DI603
Seventh step, synthesis of Compound DI203
Compound DI201 (7.8 g) was taken and dissolved in anhydrous tetrahydrofuran (80 ml), sodium hydride (8.0 g) was slowly added in portions with stirring, 1-bromo-4-iodobenzene (compound DI 202) (25.8 g) was added in portions and reacted at room temperature for 15hr, the reaction was filtered, the filtrate was dried by spinning, and methylene chloride was added for dissolution. Passing through silica gel column, and using petroleum ether: ethyl acetate (1:5) to give compound DI203.
Eighth step, synthesis of Compound DI603
Compound DI203 (6.2 g) is dissolved in dry THF (120 ml), reacted for 30min at-78 ℃ under the protection of N2, N-butyllithium (2.3 g) is slowly added dropwise, the reaction is carried out for 3h at-78 ℃ after the addition is finished, triisopropyl borate (5.1 g) is slowly added dropwise, the reaction is carried out for 2h at-78 ℃ after the addition is finished, the temperature is slowly raised to about room temperature, the reaction is carried out for 15h, after the TLC monitoring reaction is finished, the reaction solution is slowly quenched by water, and the extraction concentration is carried out, thus obtaining compound DI603.
Example 8: synthesis of Compound DI302
According to "example 7: synthesis of compound DI603 "proceeds, and in the seventh step only, compound DI201 is changed to compound DI402, resulting in compound DI302.
Example 9: synthesis of Compound DI303
According to "example 7: synthesis of compound DI603 "proceeds, and in the seventh step only, compound DI201 is changed to compound DI403 to obtain compound DI303.
Example 10: synthesis of Compound DI304
According to "example 7: synthesis of Compound DI603 "proceeds, and in the seventh step, compound DI201 is changed to Compound DI404, to Compound DI304.
Example 11: synthesis of Compound DI105
According to "example 3: synthesis of Compound DI102 "was performed, and in the fourth step, only the tridentate acryloyl chloride (Compound DI 608) was replaced with acryloyl chloride (Compound DI 609), thus obtaining Compound DI105. The nuclear magnetic resonance hydrogen spectrum of the compound DI105 is ：1H-NMR(DMSO-d6)δ8.3(1H),7.7(2H),7.4(2H),7.0-7.3(2H),6.7-6.9(1H),6.1(1H),5.7(1H),4.7(1H),4.5(0.5H),4.2(1H),4.1(0.5H),3.7(0.5H),3.2(1H),3.0(0.5H),2.3(1H),2.1(1H),1.9(1H),1.6(1H).
Example 12: synthesis of Compound DI106
According to "example 4: synthesis of Compound DI103 "was performed, and in the fourth step, only the tridentate acryloyl chloride (Compound DI 608) was replaced with acryloyl chloride (Compound DI 609), thus obtaining Compound DI106. The nuclear magnetic resonance hydrogen spectrum of the compound DI106 is ：1H-NMR(DMSO-d6)δ8.3(1H),7.7(2H),7.0-7.3(4H),6.7-6.9(1H),6.1(1H),5.7(1H),4.7(1H),4.5(0.5H),4.2(1H),4.1(0.5H),3.7(0.5H),3.2(1H),3.0(0.5H),2.3(1H),2.1(1H),1.9(1H),1.6(1H).
Example 13: synthesis of Compound DI107
According to "example 1: synthesis of Compound DI104 "was performed, and in the fourth step, only the tridentate acryloyl chloride (Compound DI 608) was replaced with acryloyl chloride (Compound DI 609), thus obtaining Compound DI107. The nuclear magnetic resonance hydrogen spectrum of the compound DI107 is ：1H-NMR(DMSO-d6)δ8.3(1H),7.7(2H),7.4(2H),7.0-7.3(4H),6.7-6.9(1H),6.1(1H),5.7(1H),4.7(1H),4.5(0.5H),4.2(1H),4.1(0.5H),3.7(0.5H),3.2(1H),3.0(0.5H),2.3(1H),2.1(1H),1.9(1H),1.6(1H).
Example 14: comparative study of rat pharmacokinetics
48 Male Sprague-Dawley rats, 6-9 weeks old, weighing about 220g, were divided into 8 groups (group I/B101, group I/B102, group I/B103, group I/B104, group I/B105, group I/B106 and group I/B107) of 6. Ibutinib, compound DI101, compound DI102, compound DI103, compound DI104, compound DI105, compound DI106 and compound DI107 were administered in groups in a single bolus respectively.
Rats began fasted 12 hours prior to dosing. The dosing solution was formulated with 0.5% sodium carboxymethyl cellulose (CMC-Na). Orbital vein Cong Caixie, blood collection time points were 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, and 24 hours post-administration. After separation of the plasma from the blood sample, the plasma was stored in a-80℃freezer for further use. The LC-MS/MS analysis method is established to measure the plasma sample.
From the test results, it was found that the elimination half-lives T 1/2 and/or the area under the curve AUC and/or the maximum blood concentration C max of the compounds DI101, DI102, DI103, DI104, DI105, DI106 and DI107 were increased by 40% or more as compared to that of ibutinib.
From the results, it can be seen that the compounds DI101, DI102, DI103, DI104, DI105, DI106 and/or DI107 of the present invention have better pharmacokinetic properties in rats compared to ibutinib, indicating better pharmacodynamics and therapeutic effects.
Example 15: determination of inhibitory Activity of BTK
The effect of the compounds of the invention on the activity of BTK was determined using the ADP-Glo TM kit. The experimental method is as follows:
ADP is a product of a kinase reaction, and kinase activity can be detected by detecting the amount of ADP produced. The ADP-Glo TM kit developed by Promega corporation is to measure the in vitro activity of BTK by detecting the ADP level produced in the kinase reaction. In kinase assay experiments, kinase consumes ATP to phosphorylate substrates while producing ADP. The kinase reaction was then stopped by adding ADP-Glo reagent and the remaining ATP was completely consumed. And then adding a kinase detection reagent to convert the generated ADP into new ATP, wherein luciferase in the detection reagent can catalyze luciferin under the participation of ATP and O 2 to generate an optical signal, so that a chemical signal is converted into an optical signal, and the intensity of the optical signal is positively correlated with the amount of ADP generated in a kinase reaction, thereby quantitatively detecting the activity of kinase BTK.
All assays were performed at 23℃and constant room temperature using Corning 3674 white 384-well assay plates, kinase BTK (Invitrogen), kinase substrate polypeptide (4:l Glu, tyr) (SIGNAL CHEM) and ATP (Sigma), and optical signals were read using a microplate reader EnVision (Perkin Elmer). The detection buffer included 40mM Tris-HCl (pH 7.5), 10mM MgCl 2(Sigma)、2mM MnCl2 (Sigma), 0.05mM DTT (Sigma), and 0.01% BSA (Sigma); preparing kinase BTK into kinase reaction solution with concentration of 1.3 ng/. Mu.L by using detection buffer; the substrate reaction solution included 0.25mg/mL of polypeptide substrate and 60. Mu.M ATP.
Diluting a compound to be tested (a compound, ibutinib, a compound DI901 or a compound DI 902) into a solution of 0.167mM by using DMSO, then carrying out three-fold gradient dilution to the minimum concentration of 0.0085 mu M by using DMSO, adding a 50nL series concentration of compound solution and 2.5 mu L kinase reaction solution into a 384-well plate by using Echo555, uniformly mixing, and incubating for 30 minutes at room temperature in a dark place; subsequently, 2.5. Mu.L of a substrate reaction solution was added thereto, the total reaction volume was 5.05. Mu.L, and the reaction mixture was allowed to react at room temperature for 60 minutes in the absence of light; then 5 mu L of ADP-Glo TM reagent is added to stop the reaction, and the mixture is placed for 40 minutes at room temperature after uniform mixing; finally, 10. Mu.L of kinase assay reagent was added, left at room temperature for 30 minutes in the dark, and the values were read on Envision.
Percent inhibition was calculated as follows:
Inhibition% = [1- (RLU Compounds of formula (I) -RLUmin)/(RLUmax-RLUmin) ] ×100
Where RLU Compounds of formula (I) is the reading at a given concentration of test compound, RLU min is the reading without addition of kinase BTK, and RLU max is the reading without addition of test compound. IC 50 values for compounds were calculated by using XLfit program in Excel.
Table 1: the IC 50 values for the compounds of the invention are shown in the following table:
Compounds of formula (I) IC50(nM) Compounds of formula (I) IC50(nM)
DI101 0.79 DI104 0.63
DI105 0.18 DI106 0.71
DI107 0.34 DI901 1.76
DI902 0.89 Ibutenib 0.93
From the results, the compounds of the present invention have a remarkable inhibitory effect on BTK.
Example 16: comparison study of mouse liver toxicity
(1) Experimental animal
Adult male ICR mice were selected for 48, body weight (25±2 g), all allowed free water intake and maintenance of feed, and were cycled alternately day and night at a temperature of 25±2 ℃,50±10% relative humidity.
(2) Grouping and administration of animals
The 48 male ICR mice were divided into six groups of 8, each of which was a normal control group, a model group, a model+example compound group, a model+ibutinib group, a model+compound DI901 group, and a model+compound DI902 group, respectively. Model + example compound group example compound was administered once daily by intragastric administration at doses (50 mg/kg); the model + ibutinib group was dosed (50 mg/kg) daily with ibutinib by intragastric administration; model + compound DI901 group compound DI901 was dosed (50 mg/kg) daily by intragastric administration; model + compound DI902 group compound DI902 was dosed (50 mg/kg) once daily by intragastric administration for 8-16 weeks, respectively, with normal control and model groups each being filled with equal volumes of purified water. Starting run out of grain after the last administration, performing intraperitoneal injection of 250mg/kg of acetaminophen (APAP) physiological saline solution on a model group, a model+example compound group, a model+ibutinib group mouse, a model+compound DI901 group and a model+compound DI902 group after 1h, performing eyeball blood collection on each group mouse after molding for 24h, centrifuging for 10min at 3000r/min, separating serum, and preserving at 4 ℃ for later use; liver and spleen were dissected rapidly. Washing with 4deg.C physiological saline, drying with filter paper, weighing, fixing part of liver in 10% formaldehyde solution, slicing, and preserving the rest liver in-80deg.C refrigerator.
(3) Determination of biochemical indicators in liver:
Weighing part of liver, adding 9 times of ice physiological saline, homogenizing with a tissue homogenizer to obtain 10% liver tissue homogenate, and centrifuging to obtain supernatant. The plates were spotted according to the kit method, OD values were measured at 450nm, and MDA content and GSH activity in the liver were calculated according to the formula.
(4) Data processing
Experimental data are expressed as mean ± standard deviation (±s), analyzed using SPSS22.0 statistical software, and differences compared between groups using one-way analysis of variance. P <0.05 bit was significantly different.
(5) Effects of the Compounds of the examples of the application on lipid peroxidation of liver tissue of APAP liver injury mice
Compared with the normal control group, the MDA content in the liver tissue homogenate of the mice in the model group is obviously increased, the GSH level is obviously reduced (P < 0.05), so that lipid peroxidation products in the mice are accumulated, and the antioxidant metabolism level is reduced; no significant change (P > 0.05) in MDA content and GSH level was observed for the model + example compound group compared to the model group; the MDA content of the model + ibutenib group was significantly increased (P < 0.05) and GSH levels were significantly decreased (P < 0.05) compared to the model group; MDA content of model + compound DI901 group was significantly increased (P < 0.05) and GSH level was significantly decreased (P < 0.05) compared to model group; the MDA content of the model + compound DI902 group was significantly increased (P < 0.05) and GSH levels were significantly decreased (P < 0.05) compared to the model group. The compounds of the examples of the present application (50 mg/kg) were shown to have no significant effect on lipid peroxidation induced by APAP, whereas ibutinib (50 mg/kg), compound DI901 (50 mg/kg) and compound DI902 (50 mg/kg) had an effect on lipid peroxidation induced by APAP, suggesting that the compounds of the examples of the present application have less hepatotoxicity in mice than ibutinib, compound DI901 and compound DI902. The results are shown in Table 2.
TABLE 2 lipid peroxidation effects on APAP liver injury mice liver tissue
Grouping MDA(nmol/mg) GSH(μmol/L)
Normal control group A- B-
Model group A+ B+
Model + example compound group A+ B+
Model + ibutenib group A++ B++
Model + Compound DI901 group A++ B++
Model + Compound DI902 group A++ B++
Labeling (grade): a+ is 2.8-3.6; a-is 0.8-2.6; a++ is 3.8-5.5; b+ is 28-37; b-is 39-57; b++ is 10-28.
Conclusion: the compounds of the examples of the present application (50 mg/kg) had no significant effect on lipid peroxidation by APAP, whereas ibutinib (50 mg/kg), compound DI901 (50 mg/kg) and compound DI902 (50 mg/kg) had an effect on lipid peroxidation by APAP, suggesting that the compounds of the examples of the present application have less hepatotoxicity than ibutinib, compound DI901 and compound DI902.
Finally, it should be noted that the above describes in detail specific embodiments of the invention, but is only exemplary and the invention is not limited to the above described specific embodiments. Any equivalent modifications and substitutions for the present invention will occur to those skilled in the art, and are also within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.

Claims (7)

1. The following compounds or their tautomers, their mixture forms and their pharmaceutically acceptable crystalline forms, salts, hydrates or solvates:
2. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to claim 1 or a tautomer thereof, a mixture of same, and pharmaceutically acceptable crystalline forms, salts, hydrates or solvates thereof, and a pharmaceutically acceptable carrier and/or excipient.
3. A compound according to claim 1 or a tautomer thereof, a mixture of forms thereof, and pharmaceutically acceptable crystalline forms, salts, hydrates or solvates thereof, or a pharmaceutical composition according to claim 2, which is a prophylactic and/or therapeutic agent for BTK-related diseases.
4. A compound according to claim 1 or a tautomer, mixture of forms thereof, and pharmaceutically acceptable crystalline forms, salts, hydrates or solvates thereof, or a pharmaceutical composition according to claim 2, for the prevention and/or treatment of a BTK-related disease being an allergic disorder, an autoimmune disease, an inflammatory disease, a thromboembolic disease or cancer.
5. A compound according to claim 1 or a tautomer thereof, a mixture of forms thereof, and pharmaceutically acceptable crystalline forms, salts, hydrates or solvates thereof, or a pharmaceutical composition according to claim 2, for the prophylaxis and/or treatment of a BTK-related disease being an autoimmune disease.
6. A compound according to claim 1 or a tautomer thereof, a mixture of forms thereof, and pharmaceutically acceptable crystalline forms, salts, hydrates or solvates thereof, or a pharmaceutical composition according to claim 2, for the prophylaxis and/or treatment of a BTK-related disease being cancer.
7. The compound according to claim 1 or a tautomer, mixture of forms thereof, and pharmaceutically acceptable crystalline forms, salts, hydrates or solvates thereof, or the pharmaceutical composition according to claim 2, for preventing and/or treating BTK-related cancers selected from chronic lymphocytic lymphomas, non-hodgkin's lymphomas, diffuse large B-cell lymphomas, mantle cell lymphomas, follicular lymphomas and chronic lymphocytic leukemias.
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