CN111961035B - Compound containing hydroxyisoquinoline structure, pharmaceutical composition and application thereof - Google Patents

Compound containing hydroxyisoquinoline structure, pharmaceutical composition and application thereof Download PDF

Info

Publication number
CN111961035B
CN111961035B CN201910419225.3A CN201910419225A CN111961035B CN 111961035 B CN111961035 B CN 111961035B CN 201910419225 A CN201910419225 A CN 201910419225A CN 111961035 B CN111961035 B CN 111961035B
Authority
CN
China
Prior art keywords
compound
amino
ring
oxo
pyrrolidin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201910419225.3A
Other languages
Chinese (zh)
Other versions
CN111961035A (en
Inventor
孙海燕
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nanjing Shuohui Pharmaceutical Technology Co ltd
Original Assignee
Nanjing Polytechnic Institute
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nanjing Polytechnic Institute filed Critical Nanjing Polytechnic Institute
Priority to CN201910419225.3A priority Critical patent/CN111961035B/en
Publication of CN111961035A publication Critical patent/CN111961035A/en
Application granted granted Critical
Publication of CN111961035B publication Critical patent/CN111961035B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Rheumatology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Transplantation (AREA)
  • Pain & Pain Management (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Plural Heterocyclic Compounds (AREA)

Abstract

The invention relates to a compound containing a hydroxyisoquinoline structure, a pharmaceutical composition and application thereof, wherein the compound has a structure shown in a formula (I). The compound with the structure shown as the formula (I) has good inhibition effect on the activity of Bruton kinase, and the half inhibition concentration of the compound is generally 10‑7mol.L‑1The following. Meanwhile, the compound with the structure of the formula (I) prepared in the embodiment of the invention shows specific anti-inflammatory activity on different animal models.

Description

Compound containing hydroxyisoquinoline structure, pharmaceutical composition and application thereof
Technical Field
The invention relates to the field of medicinal chemistry, in particular to a compound containing a hydroxyisoquinoline structure, a medicinal composition and application thereof.
Background
Protein kinases constitute one of the largest families of human enzymes and regulate many different signaling processes by adding phosphate groups to proteins (t. Hunter, ce11 1987 823-829. In particular, tyrosine kinases phosphorylate proteins on the phenol portion of tyrosine residues. The tyrosine kinase family includes members that control cell growth, migration, and differentiation. Abnormal kinase activity has been implicated in a number of human diseases including cancer, autoimmune and inflammatory diseases. Since protein kinases belong to key regulators of cell signaling, they provide the goal of regulating cellular function with small molecule kinase inhibitors and are therefore good drug design targets. In addition to the treatment of kinase-mediated disease processes, selective and potent inhibitors of kinase activity may also be useful in studying cellular signaling processes and in identifying other therapeutically significant cellular targets.
There is good evidence for the critical role of B cells in the pathogenesis of autoimmune and/or inflammatory diseases. Protein-based therapeutics that deplete B cells, such as Rituxan, are effective against autoantibody-induced inflammatory diseases such as rheumatoid arthritis (rasetter et al, annu Rev Med 2004 55. Therefore, inhibitors of protein kinases that play a role in B cell activation should be useful therapeutic agents for B cell mediated disease pathologies such as autoantibody production.
Signaling through the B Cell Receptor (BCR) controls a range of B cell responses, including proliferation and differentiation into mature antibody producing cells. BCR is a key regulatory point for B cell activity and aberrant signaling can lead to deregulated B cell proliferation and the formation of pathogenic autoantibodies that lead to a variety of autoimmune and/or inflammatory diseases. Bruton's tyrosine protein kinase (Btk) is a non-BCR-associated kinase proximal to and immediately downstream of the membrane of BCR. Deficiency of Btk has been shown to block BCR signaling, and thus inhibition of Btk can be an effective therapeutic approach to block B-cell mediated disease processes.
Btk is a member of the Tec family of tyrosine kinases and has been shown to be a key regulator of early B cell formation and mature B cell activation and survival (Khan et al, immunity 1995 3, 283, ej 1meier et al, j.exp.med.2000 1611. Human Btk mutations lead to disorder X-linked agammaglobulinemia (XLA) (reviewed in Rosen et al New eng.j.med.1995 333 and Lindvall et al immunol. Rev.2005 203. These patients are immunocompromised and show impaired B cell maturation, reduced immunoglobulin and external B cell levels, reduced T cell-independent immune responses and reduced calcium mobilization following BCR stimulation.
Evidence for a role for Btk in autoimmune and inflammatory diseases has been provided via Btk-deficient mouse models. In a preclinical murine model of Systemic Lupus Erythematosus (SLE), btk-deficient mice show significant improvement in disease progression. Furthermore, btk-deficient mice are resistant to collagen-induced arthritis (Jansson and Holmdahl clin. Exp. Immunol.1993 94. Dose-dependent efficacy of selective Btk inhibitors in mouse arthritis models has been demonstrated (z.pan et al, chem.med chem.2007 2.
Btk is also expressed by cells other than B cells that may be involved in disease processes. For example, btk is expressed by mast cells and Btk deficient bone marrow derived mast cells show impaired antigen-induced degranulation (Iwaki et al j.biol.chem.2005 280. This shows that Btk can be used to treat pathological mast cell responses such as allergy and asthma. Furthermore, monocytes from XLA patients, where Btk activity is absent, show reduced TNF α production following stimulation (Horwood et al J Exp Med 197, 1603, 2003). Thus, TNF α -mediated inflammation can be modulated by small molecule Btk inhibitors. Furthermore, it has been reported that Btk plays a role in apoptosis in Oslam and Smith immunol.rev.2000178: 49), and therefore Btk inhibitors would be effective for treating certain B-cell lymphomas and leukemias (Feldhahn et al J exp.med.2005 183.
Although a series of inhibitors of bruton's kinase have been disclosed, there is still a need to develop compounds with more abundant structural types, and new compounds with high activity and better pharmaceutical properties.
Disclosure of Invention
Through continuous efforts, the present inventors designed compounds having a structure represented by general formula (I), and found that compounds having such a structure exhibit excellent effects and actions.
The present application provides Btk inhibitor compounds of the structure shown in formula (I), methods of use thereof, as described herein below:
a compound having the structure of formula (I) or a pharmaceutically acceptable salt thereof:
Figure BDA0002065446960000031
wherein,
x, Y, Z, W is independently selected from CH or N;
ring A is C6-C10Aryl radical, C6-C10A heteroaryl group;
ring B is C3-C6Cycloalkyl radical, C3-C6A heterocycloalkyl group;
R1、R2selected from H, halogen, cyano, C1-C4Alkyl radical, C1-C3Alkoxy radical, C3-C6Cycloalkyl radical, C3-C6Heterocycloalkyl, said alkyl, alkoxy, cycloalkyl, heterocycloalkyl being optionally substituted by one or more halogens, C1-C6Alkyl radical, C1-C6Alkoxy, halo C1-C6Alkyl radical, C3-C6Cycloalkyl radical, C3-C6Heterocycloalkyl substitution;
R3is selected from
Figure BDA0002065446960000032
Further, Z is selected from CH, W is selected from N or CH; or Z is selected from N and W is selected from CH.
Further, ring A is a benzene ring or a pyridine ring.
Further, ring B is cyclohexane, cyclopentane, piperidine, morpholine, pyrrolidine.
Further, the compound shown in the formula (I) or the pharmaceutically acceptable salt thereof, at least one compound shown in the formula (II),
Figure BDA0002065446960000033
z is selected from CH, W is selected from N or CH; or Z is selected from N and W is selected from CH;
ring A is benzene ring or pyridine ring;
ring B is cyclohexane, cyclopentane, piperidine, morpholine, pyrrolidine;
R1、R2selected from H, halogen, cyano, C1-C4Alkyl radical, C1-C3Alkoxy radical, C3-C6Cycloalkyl radical, C3-C6Heterocycloalkyl, said alkyl, alkoxy, cycloalkyl, heterocycloalkyl being optionally substituted by one or moreHalogen, C1-C6Alkyl radical, C1-C6Alkoxy, halo C1-C6Alkyl radical, C3-C6Cycloalkyl radical, C3-C6Heterocycloalkyl substitution;
R3is selected from
Figure BDA0002065446960000041
Further, the compound is selected from:
Figure BDA0002065446960000042
the use of a compound as described above or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for use as a BTK inhibitor.
The BTK inhibitor can be applied to the medicines for treating inflammatory and/or autoimmune diseases, in particular to rheumatoid arthritis.
To examine the level of action of the compounds provided herein on protein kinases, biochemical level enzyme activity assays and cellular level enzyme activity assays were used to determine the activity and level of action of various compounds of the invention on one or more PKs. Similar experiments can be designed in the same way for any kinase using methods well known in the art.
In biochemical level enzyme activity assays, tyrosine kinase activity is measured using the HTRF technique, a time-resolved fluorescence resonance energy transfer technique, which can be performed according to known protocols or literature procedures, see Kolb et al, "Tyrosine kinase assays adapted to homologous time-resolved fluorescence determination". Drug Discovery Today, vol.3: pp 333-342.HTRF (homogeneous time-resolved fluorescence) is one of the most commonly used methods for detecting analytes in homogeneous systems, combining Fluorescence Resonance Energy Transfer (FRET) and time-resolved Techniques (TR), and has been widely used in various stages of drug development based on cellular and biochemical experiments. According to the determination principle of an HTRF method, after pure enzyme, biotinylated substrate and ATP are incubated and reacted, avidin labeled XL-665 and Eu labeled antibody for recognizing phosphorylation of the substrate are added, when the substrate is phosphorylated by Btk, the Eu labeled antibody can recognize the phosphorylation product, time-resolved Fluorescence Resonance Energy Transfer (FRET) is formed with the avidin labeled XL665, and non-phosphorylated substrate cannot form FRET signal due to the fact that the non-phosphorylated substrate cannot be recognized by the antibody, and the inhibition activity of the object to be detected on Btk tyrosine kinase under different concentrations is determined by determining the difference value of fluorescence signals of 665nm and 620 nm. Thus, the effect of the compounds of the invention on the biochemical level of activity of the Btk tyrosine kinase can be determined using this method, while similar assays can be used for other protein kinases using methods well known in the art.
The determination of the enzymatic activity at the cellular level is carried out by measuring the calcium flux. Fluo-4Direct was used for this experimentTMCalcium Assay Kits. The dye mainly used in the kit is Fluo 4-AM. Fluo 4-AM is an acetyl methyl ester derivative of Fluo 4, which can be easily introduced into cells by culture. AM is hydrolyzed by intracellular esterase after entering cells, the generated Fluo 4 is then combined with calcium ions and emits fluorescence, and the change of the concentration of the calcium ions in the cells can be detected by using instruments such as a laser confocal microscope or a flow cytometer.
By adopting a general testing method of a rat in-vivo drug-induced experiment, the pharmacodynamic property of the compound in the rat can be investigated.
The in vivo efficacy of compounds in mice or rats can be examined using the universal mouse Arthus Reaction model or the rat collagen-induced arthritis (rCIA) model.
The compound prepared by the invention and having the structure shown in the formula (I) has good inhibition effect on the activity of Bruton kinase, and the half Inhibition Concentration (IC) of the compound50) Is commonly used at 10-7mol/L is less than. Meanwhile, the compound with the structure of formula I prepared in the embodiment of the invention has good oral pharmacokinetic properties, and shows clear in vivo efficacy on an Arthus Reaction model or a rat collagen-induced arthritis (rCIA) model. It is presumed that the compounds of the present invention having the structure of formula (I) can be used for the preparation of a medicament for the treatment of Bruton's kinase-associated conditions in an organismAnd/or autoimmune disorders.
Unless stated to the contrary, the following terms used in the specification and claims have the following meanings.
"alkyl" refers to a saturated aliphatic hydrocarbon group. Including straight or branched chain groups of 1 to 20 carbon atoms. Medium size alkyl groups containing 1 to 6 carbon atoms are preferred, such as methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl, t-butyl, pentyl, and the like. More preferred is a lower alkyl group having 1 to 4 carbon atoms, such as methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, or the like. Alkyl groups may be substituted or unsubstituted, and when substituted, preferred groups are: halogen, C2-C6Alkenyl radical, C6-C10Aryl radical, C5-C10Heteroaryl, halo C1-C6Alkyl, 4-to 8-membered heteroalicyclic, hydroxy, C1-C6Alkoxy radical, C6-C10An aryloxy group.
"alkylene" means a divalent saturated straight-chain hydrocarbon group of 1 to 10 carbon atoms (e.g., (CH)2)n) Or a branched saturated divalent hydrocarbon radical of 2 to 10 carbon atoms (e.g., -CHMe), unless otherwise specified. Except in the case of methylene, the open states of the alkylene groups are not attached to the same atom. Examples of alkylene groups include, but are not limited to: methylene, ethylene, propylene, 2-methyl-propylene, 1,1-dimethyl-ethylene, butylene, 2-ethylbutylene.
"cycloalkyl" refers to a 3 to 8 membered all carbon monocyclic, all carbon 5/6 or 6/6 membered fused ring or multiple fused ring (by "fused" ring is meant that each ring in the system shares an adjacent pair of carbon atoms with other rings in the system) group in which one or more rings have a fully attached pi-electron system, examples of cycloalkyl are (without limitation) cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, adamantane, cyclohexadiene, cycloheptane and cycloheptatriene. Cycloalkyl groups are substitutable and are substituted. When substituted, the substituents are preferably one or more groups each selected from the group consisting of: hydrogen, hydroxy, mercapto, oxo, lower alkyl, lower alkoxy, lower cycloalkyl, lower heterocycloalkyl, lower haloalkoxy, alkylthio, halogen, lower haloalkyl, lower hydroxyalkyl, lower cycloalkylalkylene, lower heterocycloalkylalkylene, aryl, heteroaryl, alkoxycarbonyl, amino, alkylamino, alkylsulfonyl, arylsulfonyl, alkylaminosulfonyl, arylaminosulfonyl, alkylsulfonylamino, arylsulfonylamino, alkylaminocarbonyl, arylaminocarbonyl, alkylcarbonylamino, arylcarbonylamino.
"aryl" means an all-carbon monocyclic or fused polycyclic group of 6 to 14 carbon atoms having a completely conjugated pi-electron system. "aryl" includes:
six-membered carbon aromatic rings, such as benzene;
bicyclic rings in which at least one of the rings is a carbon aromatic ring, e.g., naphthalene, indene, and 1,2,3,4-tetrahydroquinoline; and
tricyclic rings in which at least one ring is a carbocyclic aromatic ring, e.g., fluorene.
For example, an aryl group includes a six-membered carbocyclic aromatic ring and a six-membered heterocyclic ring containing one or more heteroatoms selected from nitrogen, oxygen and sulfur, provided that the point of attachment is on the carbocyclic aromatic ring. However, aryl does not include, nor overlap in any way with, the heterocyclic aryl groups respectively defined below. Thus, as defined herein, if one or more carbocyclic rings are fused to a heteroaromatic ring, the resulting ring system is heteroaryl, not aryl. Non-limiting examples of aryl groups are phenyl, naphthyl. The aryl group may be substituted or unsubstituted. When substituted, preferred groups are: hydrogen, hydroxy, nitro, cyano, oxo, lower alkyl, lower alkoxy, lower cycloalkyl, lower heterocycloalkyl, lower haloalkoxy, alkylthio, halogen, lower haloalkyl, lower hydroxyalkyl, lower cycloalkylalkylene, lower heterocycloalkylalkylene, aryl, heteroaryl, alkoxycarbonyl, amino, alkylamino, alkylsulfonyl, arylsulfonyl, alkylaminosulfonyl, arylaminosulfonyl, alkylsulfonylamino, arylsulfonylamino, alkylaminocarbonyl, arylaminocarbonyl, alkylcarbonylamino, arylcarbonylamino.
"heteroaryl" means a monocyclic or fused ring group of 5 to 14 ring atoms containing one, two, three or four ring heteroatoms selected from N, O or S, the remaining ring atoms being C, and additionally having a completely conjugated pi-electron system. Heteroaryl refers to:
5-8 membered monocyclic aromatic hydrocarbons containing one or more heteroatoms selected from N, O and S, such as 1-4 heteroatoms, and in some embodiments 1-3 heteroatoms, with the other atoms in the ring being carbon atoms;
8-12 membered bicyclic aromatic hydrocarbons containing one or more heteroatoms selected from N, O and S, such as 1-4 heteroatoms, and in some embodiments 1-3 heteroatoms, with the other atoms in the ring being carbon atoms; wherein at least one ring is aromatic; and
11-14 membered tricyclic aromatic hydrocarbons containing one or more heteroatoms selected from N, O and S, such as 1-4 heteroatoms, and in some embodiments 1-3 heteroatoms, with the other atoms in the ring being carbon atoms; wherein at least one ring is aromatic.
For example, heteroaryl includes a 5-6 membered heteroaromatic ring and a 5-6 membered cycloalkyl. For such bicyclic fused heteroaryl groups, only one ring contains one or more heteroatoms and the attachment site is on the heteroaromatic ring.
When the total number of sulfur and oxygen atoms on the heteroaryl group exceeds 1, these heteroatoms are not adjacent one to another. In some embodiments, the total number of sulfur and oxygen atoms in the heteroaryl group is no more than 2. In some embodiments, the total number of sulfur and oxygen atoms in the heteroaryl group is no more than 1.
Examples of heteroaryl groups include, but are not limited to, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, triazole, pyrimidine, pyridine, pyridone, imidazopyridine, pyrazine, pyridazine, indole, azaindole, benzimidazole, benzotriazole, indoline, indolone, quinoline, isoquinoline, quinazoline, thienopyridine, thienopyrimidine, and the like. Preferred examples of such radicals are pyrrolyl, pyrazolyl, imidazolyl, triazolyl, furyl, oxazolyl, thienyl, thiazolyl, benzimidazolyl, benzotriazolyl. One or all of the hydrogen atoms in the heteroaryl group may be substituted by: hydrogen, hydroxy, nitro, cyano, oxo, lower alkyl, lower alkoxy, lower cycloalkyl, lower heterocycloalkyl, lower haloalkoxy, alkylthio, halogen, lower haloalkyl, lower hydroxyalkyl, lower cycloalkylalkylene, lower heterocycloalkylalkylene, aryl, heteroaryl, alkoxycarbonyl, amino, alkylamino, alkylsulfonyl, arylsulfonyl, alkylaminosulfonyl, arylaminosulfonyl, alkylsulfonylamino, arylsulfonylamino, alkylaminocarbonyl, arylaminocarbonyl, alkylcarbonylamino, arylcarbonylamino. "Heterocycloalkyl" means a monovalent saturated cyclic group consisting of one or more rings, preferably 1 to 2 rings (including spiro ring systems), 3 to 8 atoms per ring, to which one or more ring heteroatoms (selected from N, O or S (O) are bonded0-2) And which may be optionally independently substituted by one or more, preferably 1 or 2, substituents selected from the group consisting of hydrogen, hydroxy, mercapto, oxo, lower alkyl, lower alkoxy, lower cycloalkyl, lower heterocycloalkyl, lower haloalkoxy, alkylthio, halogen, lower haloalkyl, lower hydroxyalkyl, lower cycloalkylalkylene, lower heterocycloalkylalkylene, aryl, heteroaryl, alkoxycarbonyl, amino, alkylamino, alkylsulfonyl, arylsulfonyl, alkylaminosulfonyl, arylaminosulfonyl, alkylsulfonylamino, arylsulfonylamino, alkylaminocarbonyl, arylaminocarbonyl, alkylcarbonylamino, arylcarbonylamino. Unless otherwise indicated. Examples of heterocycloalkyl include, but are not limited to, morpholinyl, piperazinyl, piperidinyl, azetidinyl, pyrrolidinyl, hexahydroazepinyl, oxetanyl, tetrahydrofuranyl, tetrahydrothienyl, oxazolidinyl, thiazolidinyl, isoxazolidinyl, tetrahydropyranyl, thiomorpholinyl, quinuclidinyl, and imidazolinyl, preferably
Figure BDA0002065446960000081
Figure BDA0002065446960000082
Figure BDA0002065446960000083
W is O, S or NR12Examples may also be bicyclic, such as, for example, 3,8-diaza-bicyclo [3.2.1]Octane, 2,5-diazabicyclo [2.2.2]Octane or octahydro-pyrazino [2,1-c][1,4]Oxazines. The heterocycloalkyl (and derivatives) thereof include ionic forms thereof.
"alkoxy" means-O- (unsubstituted alkyl) and-O (unsubstituted cycloalkyl). Representative examples include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy, and the like.
"aryloxy" means-O-aryl and-O-heteroaryl. Representative examples include, but are not limited to, phenoxy, pyridyloxy, furyloxy, thiophenyloxy, pyrimidyloxy, pyrazinyloxy, and the like, and derivatives thereof.
"arylalkylene" denotes alkyl, preferably lower alkyl as defined above, which is substituted by aryl as defined above, e.g. -CH2Phenyl, - (CH)2)2Phenyl, - (CH)2)3Phenyl radical, CH3CH(CH3)CH2Phenyl and its derivatives.
"Heteroarylalkylene" denotes alkyl, preferably lower alkyl as defined above, substituted by heteroaryl as defined above, e.g. -CH2Pyridyl, - (CH)2)2Pyrimidinyl, - (CH)2)3Imidazolyl and the like and derivatives thereof.
"hydroxy" means an-OH group.
"mercapto" means an-SH group.
"halogen" means fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine.
"haloalkyl" denotes alkyl, preferably lower alkyl as defined above, substituted by one or more identical or different halogen atoms, e.g. -CH2Cl、-CF3、-CCl3、-CH2CF3、-CH2CCl3And so on.
"cyano" means a-CN group.
"amino" means-NH2A group.
"nitro" means-NO2A group. By "optionally," it is meant that the subsequently described event or circumstance may or may not occur, and that the description includes instances where it does or does not occur.
In some embodiments, "substituted with one or more groups" means that the same or different groups selected from the group in which the indicated atom or group has one, two, three, or four hydrogen atoms in the group, respectively, indicated for the range are replaced.
The wavy line indicates the attachment site;
"pharmaceutically acceptable salts" refers to those salts that retain the biological effectiveness and properties of the parent compound. Such salts include:
(1) Salts with acids obtained by reacting the free base of the parent compound with inorganic acids or organic acids, the inorganic acids including hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, metaphosphoric acid, sulfuric acid, sulfurous acid, perchloric acid, and the like, and the organic acids including acetic acid, propionic acid, acrylic acid, oxalic acid, (D) or (L) malic acid, fumaric acid, maleic acid, hydroxybenzoic acid, gamma-hydroxybutyric acid, methoxybenzoic acid, phthalic acid, methanesulfonic acid, ethanesulfonic acid, naphthalene-1-sulfonic acid, naphthalene-2-sulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, lactic acid, mandelic acid, succinic acid, malonic acid, and the like.
(2) The acidic proton present in the parent compound is replaced by a metal ion such as an alkali metal ion, an alkaline earth metal ion or an aluminum ion, or is complexed with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, etc.
"pharmaceutical composition" refers to the combination of one or more of the compounds of the present invention, or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof, with another chemical ingredient, such as a pharmaceutically acceptable carrier. The purpose of the pharmaceutical composition is to facilitate the administration process to the animal.
By "pharmaceutically acceptable carrier" is meant an inactive ingredient in a pharmaceutical composition that does not cause significant irritation to an organism and does not interfere with the biological activity and properties of the administered compound, such as, but not limited to: calcium carbonate, calcium phosphate, various sugars (e.g., lactose, mannitol, etc.), starch, cyclodextrin, magnesium stearate, cellulose, magnesium carbonate, acrylic or methacrylic polymers, gelatin, water, polyethylene glycol, propylene glycol, ethylene glycol, castor oil or hydrogenated or polyethoxylated hydrogenated castor oil, sesame oil, corn oil, peanut oil, and the like.
The aforementioned pharmaceutical compositions may contain, in addition to pharmaceutically acceptable carriers, adjuvants commonly used in pharmacology, such as: antibacterial agents, antifungal agents, antimicrobial agents, shelf-stable agents, hueing agents, solubilizing agents, thickening agents, surfactants, complexing agents, proteins, amino acids, fats, sugars, vitamins, minerals, trace elements, sweeteners, pigments, flavors or combinations thereof, and the like.
Detailed Description
The present invention will be further described with reference to the following examples, which are not intended to limit the scope of the present invention.
Example 1: preparation of N- (1- (8- ((5- (4-methylpiperazin-1-yl) pyridin-2-yl) amino) -1-oxo-1,2-dihydroisoquinolin-6-yl) piperidin-3-yl) acrylamide (EXP 1)
Figure BDA0002065446960000101
Figure BDA0002065446960000111
1) Preparation of 6-chloro-8- ((5- (4-methylpiperazin-1-yl) pyridin-2-yl) amino) isoquinolin-1 (2H) -one (Compound C)
8-bromo-6-chloroisoquinolin-1 (2H) -one (2590mg, 10mmol), 5- (4-methylpiperazin-1-yl) pyridin-2-amine (1923mg, 1)0mmol)、Pd(dppf)2Cl2·CH2Cl2(2340mg, 2.3mmol) was placed in a 100mL three-necked flask, and after replacement with argon gas three times, 1,4-dioxane (50 mL) was added thereto, and the mixture was stirred for 5 minutes, followed by addition of 2M Na2CO3(2700mg, 25.5mmol), replacing with argon gas for three times, heating to 100 deg.C, reacting for 2 hours, stopping reaction, cooling to room temperature, suction-filtering with diatomaceous earth, extracting the filtrate with water and ethyl acetate (100mL x 3), washing with saturated NaCl (25 mL) once, and finally washing the ester layer with anhydrous Na2SO4After drying, column chromatography separation was carried out to obtain compound C (925mg, 2.5mmol) with a yield of 25%. MS (ESI) M/z: [ M + H ]]+＝370.5。1H-NMR(DMSO-d6，400MHz)：δ11.84(s,1H),10.05(s,1H),7.71(m,1H),7.46(s,1H),7.13-7.18(m,2H),6.79(d,1H),6.61(d, 1H),6.21(d,1H),3.10-3.17(m,4H),2.30-2.37(m,4H),2.21(s,3H)ppm。
2) Preparation of 1- (8- ((5- (4-methylpiperazin-1-yl) pyridin-2-yl) amino) -1-oxo-1,2-dihydroisoquinolin-6-yl) piperidin-3-yl tert-butyl) carbamate (Compound D)
Coupling 6-chloro-8- ((5- (4-methylpiperazin-1-yl) pyridin-2-yl) amino) isoquinolin-1 (2H) -one (738 mg,2 mmol), piperidin-3-ylcarbamic acid tert-butyl ester (1001mg, 5mmol), pd (OAc)2(100 mg, 0.44 mmol) and potassium acetate (980mg, 100mmol) were dissolved in 100mL of anhydrous 1,4-dioxane, and the reaction was carried out under nitrogen protection and heating to reflux for 12 hours. Cooling to room temperature after reaction, filtering with celite, extracting the filtrate with water and ethyl acetate (150mL x 3), washing with saturated NaCl (25 mL) once, and collecting the ester layer with anhydrous Na2SO4After drying, column chromatography was carried out to obtain Compound D (960mg, 1.8mmol).
3) Preparation of N- (1- (8- ((5- (4-methylpiperazin-1-yl) pyridin-2-yl) amino) -1-oxo-1,2-dihydroisoquinolin-6-yl) piperidin-3-yl) acrylamide (EXP 1)
1- (8- ((5- (4-methylpiperazin-1-yl) pyridin-2-yl) amino) -1-oxo-1,2-dihydroisoquinolin-6-yl) piperidin-3-yl tert-butyl) carbamate (compound D) (534mg, 1mmol), 0.1mL of hydrochloric acid, 50mL of dichloromethane were added to a 100mL eggplant-shaped bottle, and reacted at room temperature for 12 hours under nitrogen protection. After the reaction was complete the aqueous phase was extracted with dichloromethane (50ml × 3). The organic phases were combined and washed 3 times with pure water. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and used directly in the next reaction.
To the above concentrated solution were added 50mL of anhydrous dichloromethane, triethylamine (303mg, 3mmol), acryloyl chloride (180mg, 2mmol), and reacted at 45 ℃ for 4 hours under nitrogen protection. After the reaction is finished, concentrating the reaction liquid to be dry, and performing column chromatography to obtain N- (1- (8- ((5- (4-methylpiperazin-1-yl) pyridine-2-yl) amino) -1-oxo-1,2-dihydroisoquinoline-6-yl) piperidine-3-yl) acrylamide (146mg, 0.3mmol), wherein the yield is as follows: 30 percent.
MS(ESI)m/z:[M+H]+＝488.4。1H-NMR(DMSO-d6，400MHz)：δ12.01(s,1H),10.04(s,1H),8.45(m,1H),7.69-7.72(m,1H),7.13-7.18(m,1H),6.65-6.79(m ,2H),6.42-6.51(m,2H),6.04-6.17(m,2H),5.74(m,1H),3.54(m,1H),3.42(m,1H),3.08-3.1 9(m,3H),2.32-2.37(m,4H),2.18(s,3H),1.42-1.57(m,4H),ppm。
Example 2: preparation of N- (1- (8-oxo-1- ((4- (2- (pyrrolidin-1-yl) ethyl) phenyl) amino) -7,8-dihydro-2,7-naphthyridin-3-yl) pyrrolidin-3-yl) acrylamide (EXP 2)
Figure BDA0002065446960000121
Referring to the procedure of example 1, N- (1- (8-oxo-1- ((4- (2- (pyrrolidin-1-yl) ethyl) phenyl) amino) -7,8-dihydro-2,7-naphthyridin-3-yl) pyrrolidin-3-yl) acrylamide (EXP 2) 35mg, yield: 25 percent. MS (ESI) M/z: [ M + H ]]+＝473.50.1H-NMR(DMSO-d6，400MHz)：δ11.78(s,1H),10.52(s,1H),8.34(s,1H),7.82(d,1H),7.45(d,2H),7.05(d,2H),6.54(t,1H),6.2 8(s,1H),6.12(m,1H),5.85(m,1H),5.72(m,1H),3.65(m,1H),3.13-3.52(m,4H),2.65-2.69( m,2H),2.45-2.51(m,4H),1.65-1.94(m,4H)ppm。
Example 3: preparation of N- (1- (5-oxo-4- ((4- (2- (pyrrolidin-1-yl) ethyl) phenyl) amino) -5,6-dihydro-1,6-naphthyridin-2-yl) pyrrolidin-3-yl) acrylamide (EXP 3)
Figure BDA0002065446960000131
Referring to the procedure of example 1, N- (1- (5-oxo-4- ((4- (2- (pyrrolidin-1-yl) ethyl) phenyl) amino) -5,6-dihydro-1,6-naphthyridin-2-yl) pyrrolidin-3-yl) acrylamide (EXP 3) 65mg was prepared in yield: 35 percent. MS (ESI) M/z: [ M + H ]]+＝473.2.1H-NMR(DMSO-d6，400MHz)：δ11.82(s,1H),11.55(s,1H),8.35(s,1H),7.84(d,1H),7.47(d,2H),7.08(d,2H),6.47(m,1H),6. 08-6.15(s,2H),5.76(m,1H),3.67(m,1H),3.08-3.45(m,4H),2.65(m,2H),2.52-2.55(m,6H), 1.67-1.69(m,6H)ppm。
Example 4: preparation of N- (1- (5-oxo-4- ((4- (2- (pyrrolidin-1-yl) ethyl) phenyl) amino) -5,6-dihydropyrido [4,3-d ] pyrimidin-2-yl) pyrrolidin-3-yl) acrylamide (EXP 4)
Figure BDA0002065446960000132
By referring to the procedure of example 1, N- (1- (5-oxo-4- ((4- (2- (pyrrolidin-1-yl) ethyl) phenyl) amino) -5,6-dihydropyrido [4,3-d) was prepared]Pyrimidin-2-yl) pyrrolidin-3-yl) acrylamide (EXP 4) 28mg, yield: 29 percent. MS (ESI) M/z: [ M + H ]]+＝474.3.1H-NMR(DMSO-d6，400MHz)：δ11.78(s,1H),8.83(s,1H),8.34(s,1H),7.54(d,1H),7.38(d,2H),7.05(d,2H),6.45(m,1H),6.0 8(m,1H),5.93(d,1H),5.75(m,1H),3.69(m,1H),3.45(m,1H),3.14-3.19(m,2H),3.05(m,1H ),2.68(m,2H),2.53(m,4H),1.64-1.69(m,6H)ppm。
Example 5: preparation of N- (1- (8- ((4- (2-methoxyethoxy) phenyl) amino) -1-oxo-1,2-dihydroisoquinolin-6-yl) pyrrolidin-3-yl) acrylamide (EXP 5)
Figure BDA0002065446960000141
Referring to the procedure of example 1, N- (1- (8- ((4- (2-methoxyethoxy) phenyl) amino) -1-oxo-1,2-dihydroisoquinolin-6-yl) pyrrolidin-3-yl) acrylamide (EXP 5) was prepared in 65mg yield: 12 percent. MS (ESI) m/z:[M+H]+＝448.6.1H-NMR(DMSO-d6，400MHz)：δ12.36(s,1H),11.85(s,1H),8.33(s,1H),7.68(d,1H),7.42(d,2H),6.95(d,2H),6.43-6.45(m, 3H),6.08-6.18(m,2H),5.75(d,1H),4.32(m,2H),3.78(m,2H),3.40-3.44(m,4H),3.05-3.19( m,3H),1.64-1.69(m,2H)ppm。
example 6: preparation of N- (1- (8- ((5- (4-methylpiperazin-1-yl) pyridin-2-yl) amino) -1-oxo-1,2-dihydroisoquinolin-6-yl) pyrrolidin-3-yl) acrylamide (EXP 6)
Figure BDA0002065446960000142
Referring to the procedure of example 1, N- (1- (8- ((5- (4-methylpiperazin-1-yl) pyridin-2-yl) amino) -1-oxo-1,2-dihydroisoquinolin-6-yl) pyrrolidin-3-yl) acrylamide (EXP 6) 74mg was prepared in yield: 12 percent. MS (ESI) M/z: [ M + H ]]+＝474.3。1H-NMR(DMSO-d6，400MHz)：δ12.03(s,1H),10.07(s,1H),8.37(m,1H),7.74(m,1H),7.19(m,1H),6.79(m,1H),6.67(m,1H ),6.45(m,3H),6.07-6.16(m,2H),5.78(m,1H),3.68(m,1H),3.05-3.44(m,4H),2.38(m,4H), 2.18(m,3H),1.67-1.92(m,2H)ppm。
Example 7: preparation of N- (1- (8- ((5- ((S) -3-methyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-yl) amino) -1-oxo-1,2-dihydroisoquinolin-6-yl) pyrrolidin-3-yl) but-2-ynamide (EXP 7)
Figure BDA0002065446960000151
Referring to the procedure of example 1, N- (1- (8- ((5- ((S) -3-methyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-yl) amino) -1-oxo-1,2-dihydroisoquinolin-6-yl) pyrrolidin-3-yl) but-2-ynylamide (EXP 7) 82mg was prepared in yield: 35 percent. MS (ESI) M/z: [ M + H ]]+＝542.5。1H-NMR(DMSO-d6， 400MHz)：δ11.91(s,1H),10.01(s,1H),8.09(s,1H),7.68(d,1H),7.18(s,1H),6.79(d,1H),6.68(d,1H),6. 44(m,2H),6.18(m,1H),4.68-4.82(m,4H),3.67-3.85(m,2H),2.80-3.41(m,11H),1.69-1.80 (m,5H),1.08(d,3H)ppm。
Example 8: preparation of (N-1- (8- ((4- (morpholine-4-carbonyl) phenyl) amino) -1-oxo-1,2-dihydroisoquinolin-6-yl) pyrrolidin-3-yl) acrylamide (EXP 8)
Figure BDA0002065446960000152
Referring to the procedure of example 1, N-1- (8- ((4- (morpholine-4-carbonyl) phenyl) amino) -1-oxo-1,2-dihydroisoquinolin-6-yl) pyrrolidin-3-yl) acrylamide (EXP 8) 71mg was prepared in yield: 25 percent.
1H-NMR(DMSO-d6，400MHz)：δ12.23(br,1H),11.87(br,1H),8.32(br,1H),7.68-7.70(m,5H),6.45-6.90(m,2H),6.10-6.19( m,2H),5.74(d,1H),3.50-3.61(m,4H),3.08-3.16(m,4H),1.68-1.90(m,2H)ppm。
Example 9: preparation of N- (1- (1-oxo-8- ((4- (2- (pyrrolidin-1-yl) ethyl) phenyl) amino) -1,2-dihydroisoquinolin-6-yl) pyrrolidin-3-yl) acrylamide (EXP 9)
Figure BDA0002065446960000161
Referring to the procedure of example 1, N- (1- (1-oxo-8- ((4- (2- (pyrrolidin-1-yl) ethyl) phenyl) amino) -1,2-dihydroisoquinolin-6-yl) pyrrolidin-3-yl) acrylamide (EXP 9) was prepared in 34mg, yield: 32 percent. MS (ESI) M/z: [ M + H ]]+＝472.2。1H-NMR(DMSO-d6，400MHz)：δ12.31(br,1H),11.91(br,1H),8.34(br,1H),8.71(m,1H),7.38(d,2H),7.05(d,2H),6.41-6.47( m,3H),6.10-6.19(m,2H),5.74(d,1H),3.71(m,1H),3.05-3.44(m,4H),2.74(t,2H),2.52-2.5 5(m,6H),1.67-1.91(m,6H)ppm。
Example 10: preparation of N- (1- (8- ((2-methoxy-4- (4-methylpiperazin-1-yl) phenyl) amino) -1-oxo-1,2-dihydroisoquinolin-6-yl) pyrrolidin-3-yl) acrylamide (EXP 10)
Figure BDA0002065446960000162
Referring to the procedure of example 1, N- (1- (8- ((2-methoxy-4- (4-methylpiperazin-1-yl) phenyl) amino) -1-oxo-1,2-dihydroisoquinolin-6-yl) pyrrolidin-3-yl) acrylamide (EXP 10) 23mg was prepared in yield: 19 percent. MS (ESI) M/z: [ M + H ]]+＝503.5。1H-NMR(DMSO-d6，400MHz)：δ11.98(br,1H),9.98(br,1H),8.36(m,1H),7.75(d,1H),7.69(d,1H),6.44-6.48(m,3H),6.37(s ,1H),5.75(m,1H),3.88(s,3H),3.72(m,1H),3.05-3.44(m,4H),2.36(t,4H),2.18(s,3H),1.67- 1.90(m,2H)ppm。
Example 11: (E) Preparation of (E) -3- (dimethylamino) -N- (1- (8- ((2-fluoro-4- (4-methylpiperazin-1-yl) phenyl) amino) -1-oxo-1,2-dihydroisoquinolin-6-yl) pyrrolidin-3-yl) acrylamide (EXP 11)
Figure BDA0002065446960000171
Referring to the procedure of example 1, there was prepared (E) -3- (dimethylamino) -N- (1- (8- ((2-fluoro-4- (4-methylpiperazin-1-yl) phenyl) amino) -1-oxo-1,2-dihydroisoquinolin-6-yl) pyrrolidin-3-yl) acrylamide (EXP 11) 83mg, yield: and 47 percent. MS (ESI) M/z: [ M + H ]]+＝534.5。1H-NMR(DMSO-d6，400MHz)：δ11.95(br,1H),10.02(br,1H),8.93(d,1H),8.33(s,1H),7.73(d,1H),6.98(d,1H),6.82(d,1H), 6.53(m,1H),6.44(m,2H),6.21(d,1H),5.25(m,1H),3.73(m,1H),3.15-3.44(m,7H),3.06(m, 1H),2.86(s,6H),2.38(t,6H),2.18(s,3H),1.65-1.90(m,2H)ppm。
Example 12: preparation of N- (1- (8- ((6- ((4-methylpiperazin-1-yl) methyl) pyridin-3-yl) amino) -1-oxo-1,2-dihydroisoquinolin-6-yl) pyrrolidin-3-yl) acrylamide (EXP 12)
Figure BDA0002065446960000172
With reference to the procedure of example 1, N- (1- (8- ((6- ((4-methylpiperazin-1-yl) methyl) pyridin-3-yl) amino) -1-oxo-1,2-dihydroisoquinolin-6-yl) pyrrolidin-3-yl) acrylamide (EXP 12) was prepared in 78mg with yield： 20％。MS(ESI)m/z:[M+H]+＝488.2。1H-NMR(DMSO-d6，400MHz)：δ11.98(br,1H),9.25(s,1H),8.33(s,1H),7.70(m,2H),7.45(d,1H),7.19(d,1H),6.45-6.52(m, 3H),6.10-6.22(m,2H),5.75(m,1H),3.98(s,2H),3.59(m,1H),3.05-3.45(m,4H),2.45(t,4H), 2.35(t,4H),2.13(s,3H),1.71-1.87(m,2H)ppm。
Example 13: in vitro biochemical level inhibition protein kinase activity experiment
The material and the method are as follows: BTK kinase, from Invitrogen; HTRF KinEASE; TKkit (Cisbio Co.); 384 well plates (Greiner corporation); ATP (Sigma Co.), mgCl2(sigma) Corp; PHERAstar FS multifunctional microplate reader (BMG company); low speed centrifuge (StaiteXiangyi corporation); incubator (Binder Co.). The selected positive medicine is Reference compound, and the structure is as follows:
Figure BDA0002065446960000181
compound dissolution and preservation: preparing a test compound into a mother solution of 0.5-10mmol/L by DMSO according to solubility, subpackaging and storing at-20 ℃;
preparing a compound working solution: before testing, the dispensed compound was removed from the freezer and diluted to 50 × the desired concentration with pure DMSO; then the compound was diluted to 4 x the desired concentration with deionized water;
1.33 × preparation of enzyme buffer: 5 × enzymic buffer from HTRF kit) was diluted 1.33 × with deionized water and 1.33 × final concentration of the corresponding ingredients were added: 1.33mmol/L DTT and 1.33mmol/L MgCl2;
preparation of a kinase working solution: btk was diluted to 2X with 1.33X Enzymatic buffer to the desired final concentration of 0.2 ng/. Mu.L;
preparing a substrate working solution: substrate-biotin (from HTRF kit) and ATP (10 mM) were diluted with 1.33 × Enzymatic buffer to 4 × the desired final concentration of the mixture;
preparation of detection working solution: 16.67. Mu. Mol/L of Streptavidin-XL665 were diluted to 4 Xthe desired final concentration with HTRF detection buffer and then mixed with an equal volume of Antibody-Cryptate (both from HTRF kit).
An enzyme reaction step: mu.l of kinase working solution was added to each well of a low volume 384 microwell plate, together with 4. Mu.l of 1.33 × Enzymatic buffer as Negative control (Negative); add 2. Mu.l of compound working solution to the wells, while adding 2. Mu.l of 8% aqueous DMSO solution as a zero compound concentration control (i.e., positive control, positive); incubating at 25 deg.C (or 30 deg.C) for 5-10min; add 2. Mu.L of substrate working solution to the wells to start the enzymatic reaction, shake the reaction at 25 deg.C (or 30 deg.C) for 15-60min.
HTRF reagent detection step: adding 8 mu L of detection working solution into the hole to terminate the reaction; reacting for 1h at 25 ℃;
reading of HTRF signal: the PHERAStar FS reading is adopted to detect signals, and the corresponding settings of the instrument are as follows:
Optic module
Figure BDA0002065446960000182
Integration delay(lag time)50μs
Integration time 400μs
Number of flashes 200
for the raw data read out per well, the ratio =665nm/620nm;
calculation of inhibition ratio:
Figure BDA0002065446960000191
IC50calculation of the value: taking the logarithm of the compound concentration as abscissa and the inhibition as ordinate, in GraphPad Prism 5, a non-linear curve was fitted: log (inhibitor) vs. response- -Variable slope, and determining the concentration of the compound to be tested, namely IC when the enzyme activity inhibition rate is 50%50。
The experimental results are as follows: half maximal Inhibitory Concentration (IC) of BTK kinase activity50，nM)
The invention provides a half Inhibitory Concentration (IC) of a compound with a structure shown as a formula I on the activity of BTK kinase50)
Reference compound EXP 1 EXP2 EXP3 EXP4 EXP5 EXP6
25.6nM 3.5nM 8.8nM 6.3nM 3.2nM 0.8nM 0.9nM
EXP7 EXP8 EXP9 EXP10 EXP11 EXP12
2.4nM 0.5nM 8.2nM 2.6nM 1.5nM 5.8nM
Example 14: in vitro cell level inhibition protein kinase activity experiment
The material and the method are as follows: fluo-4DirectTMCalcum Assay Kits, invitrogen corporation; RPMI1640 medium: GIBCO corporation; blackboard with 96 holes: CORNING corporation; PHERAstar FS multifunctional microplate reader (BMG); low speed centrifuge (StaiteXiangyi). The positive drug selected was RN486.
Cell treatment: cells were washed with serum-free medium and serum was removed.
Dye preparation: then 2% of the dye was diluted 1 x with serum-free medium.
Cell resuspension: the washed cells were resuspended with the 1 × dye prepared above.
Cell inoculation: 20 ten thousand per well, 40 mul per well, 96 well plate, black panel was required.
And (3) dye incubation: placing into incubator, and incubating for 40min.
Adding medicine: then a series of formulated compounds were added at 10. Mu.l/well and the action was continued for 20min.
And (3) balancing at room temperature: the test plate is removed and allowed to equilibrate at room temperature for 5min.
Determination of baseline: baseline was measured using the PHERStar microplate reader before agonist addition.
Adding an agonist: igM was added to a final concentration of 10. Mu.g/ml, 10. Mu.l/well.
And (3) determination: immediately after the addition of the agonist, the assay was performed with a PHERStarter microplate reader for 8min at 10s intervals.
Data processing: OD (highest) -OD (baseline), and then IC50 values were calculated using GraphPad Prism 5 software.
The experimental results are as follows: half inhibitory concentration of calcium flux of BTK cells: (IC50，nM)
The invention provides half Inhibition Concentration (IC) of a compound shown as a structural formula I on calcium current of BTK cells50)
RN486 EXP1 EXP2 EXP3 EXP4 EXP5 EXP6
45.7nM 3.2nM 3.5nM 10.2nM 8.9nM 5.2nM 6.5nM
EXP7 EXP8 EXP9 EXP10 EXP11 EXP12 Reference compound
12.6nM 1.2nM 5.3nM 11.5nM 20.3nM 13.9nM 185nM
Example 15: pharmacodynamic study on rat collagen-induced arthritis (rCIA) model
The compound of the invention has the function of inhibiting rheumatoid arthritis, DBA/1J mice are selected, 50ug Niu collagen and equal volume of Complete Freund's Adjuvant (CFA) are completely emulsified and then injected subcutaneously. After 21 days, 50ug of the same antigen and incomplete Freund's adjuvant (after IFA is fully emulsified, the immunization is strengthened for 1 time, observation and recording are carried out from the 45 th day, a 1-4 scoring method is adopted, 1 is normal, 2 is normal, 1 is arthrocele, 3 is over 1 is arthrocele, but all the articulations are not accumulated, 4 is serious tumescence or stiffness of the whole paw is obtained, the score of each paw is added, the total score of the arthrocele of the mouse is obtained, the mouse with the total score of the arthrocele larger than 1 is successfully established as a model, the compound is adopted to be infused into the stomach of the mouse after the mouse rheumatoid arthritis model is successfully established, the arthritis of the mouse is scored after the administration for 2 weeks, and the result shows that the compound has obvious treatment effect on the mouse rheumatoid arthritis.
Group of Compounds Arthritis score
Control 1
Physiological saline control group 4
Cyclosporin control group 2.4
EXP8 0.8
Reference compound 2.8
The foregoing invention has been described in some detail by way of illustration and example for purposes of illustration and understanding. It will be apparent to those skilled in the art that changes and modifications may be made within the scope of the appended claims. Accordingly, it is to be understood that the above description is intended to be illustrative, and not restrictive. The scope of the invention should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the following appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims (4)

1. A compound having the structure:
Figure FDA0003850488530000011
2. a pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 1 and a pharmaceutically acceptable excipient or diluent.
3. Use of a compound of claim 1 in the preparation of a BTK inhibitor.
4. Use of a compound according to claim 1 for the preparation of a medicament for the treatment of an inflammatory and/or autoimmune condition, wherein the inflammatory condition is rheumatoid arthritis.
CN201910419225.3A 2019-05-20 2019-05-20 Compound containing hydroxyisoquinoline structure, pharmaceutical composition and application thereof Active CN111961035B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910419225.3A CN111961035B (en) 2019-05-20 2019-05-20 Compound containing hydroxyisoquinoline structure, pharmaceutical composition and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910419225.3A CN111961035B (en) 2019-05-20 2019-05-20 Compound containing hydroxyisoquinoline structure, pharmaceutical composition and application thereof

Publications (2)

Publication Number Publication Date
CN111961035A CN111961035A (en) 2020-11-20
CN111961035B true CN111961035B (en) 2022-11-01

Family

ID=73358212

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910419225.3A Active CN111961035B (en) 2019-05-20 2019-05-20 Compound containing hydroxyisoquinoline structure, pharmaceutical composition and application thereof

Country Status (1)

Country Link
CN (1) CN111961035B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2024506909A (en) * 2021-02-12 2024-02-15 ニンバス サターン， インコーポレイテッド HPK1 antagonists and their uses

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101983198A (en) * 2008-02-01 2011-03-02 Irm责任有限公司 Pyrido [4, 3-d] pyrimidinone derivatives as kinase inhibitors
CN102341383A (en) * 2009-03-02 2012-02-01 霍夫曼-拉罗奇有限公司 Inhibitors of Bruton's Tyrosine Kinase
CN102548992A (en) * 2009-07-30 2012-07-04 Irm责任有限公司 2, 7 -naphthyridin- 1 -one derivatives as syk kinase inhibitors
CN103664938A (en) * 2012-09-12 2014-03-26 山东亨利医药科技有限责任公司 Benzopyrimidine-containing SYK inhibitor
CN103974955A (en) * 2011-08-23 2014-08-06 远藤制药公司 Pyrimido- pyridazinone compounds and use thereof
CN108601785A (en) * 2015-12-04 2018-09-28 阿沙纳生物科学公司 Purposes of the pyrimido pyridazinone in treating cancer
CN109180644A (en) * 2018-10-04 2019-01-11 南京先进生物材料与过程装备研究院有限公司 A kind of mesylate of novel B TK kinase inhibitor and preparation method thereof and purposes

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101983198A (en) * 2008-02-01 2011-03-02 Irm责任有限公司 Pyrido [4, 3-d] pyrimidinone derivatives as kinase inhibitors
CN102341383A (en) * 2009-03-02 2012-02-01 霍夫曼-拉罗奇有限公司 Inhibitors of Bruton's Tyrosine Kinase
CN102548992A (en) * 2009-07-30 2012-07-04 Irm责任有限公司 2, 7 -naphthyridin- 1 -one derivatives as syk kinase inhibitors
CN103974955A (en) * 2011-08-23 2014-08-06 远藤制药公司 Pyrimido- pyridazinone compounds and use thereof
CN103664938A (en) * 2012-09-12 2014-03-26 山东亨利医药科技有限责任公司 Benzopyrimidine-containing SYK inhibitor
CN108601785A (en) * 2015-12-04 2018-09-28 阿沙纳生物科学公司 Purposes of the pyrimido pyridazinone in treating cancer
CN109180644A (en) * 2018-10-04 2019-01-11 南京先进生物材料与过程装备研究院有限公司 A kind of mesylate of novel B TK kinase inhibitor and preparation method thereof and purposes

Also Published As

Publication number Publication date
CN111961035A (en) 2020-11-20

Similar Documents

Publication Publication Date Title
CN109963842B (en) Benzimidazole compound kinase inhibitor and preparation method and application thereof
KR102488323B1 (en) Composition for treating or ameliorating Huntington's disease
AU2014324595B2 (en) Substituted nicotinimide inhibitors of BTK and their preparation and use in the treatment of cancer, inflammation and autoimmune disease
KR101965025B1 (en) PYRAZOLO[1,5-a]PYRIMIDINE-BASED COMPOUNDS, COMPOSITIONS COMPRISING THEM, AND METHODS OF THEIR USE
TWI617560B (en) Inhibitors of pi3k-delta and methods of their use and manufacture
CN104211703B (en) A kind of fused heterocyclic compound as Bu Ludun kinase inhibitors
AU2013324681B2 (en) Quinazolinone derivatives as PARP inhibitors
KR20160092991A (en) Irak inhibitors and uses thereof
CN104902959A (en) Irak inhibitors and uses thereof
AU2012310168B2 (en) 6 - substituted 3 - (quinolin- 6 - ylthio) - [1,2,4] triazolo [4, 3 -a] pyradines as tyrosine kinase
CN104177338B (en) A kind of Bu Ludun kinase inhibitors
JP2018504432A (en) Isothiazolopyrimidinone, pyrazolopyrimidinone and pyrrolopyrimidinone as ubiquitin-specific protease 7 inhibitors
CN104039790A (en) Novel purine derivatives and their use in the treatment of disease
WO2020035020A1 (en) Imidazopyridine derivative, preparation method therefor, and application thereof in medicine
CN108658990B (en) Novel imidazo [1,5-a ] pyrazine Bruton kinase inhibitors
AU2014250836A1 (en) Quinazolines and azaquinazolines as dual inhibitors of RAS/RAF/MEK/ERK and PI3K/AKT/PTEN/mTOR pathways
WO2013028818A1 (en) Pyrimido- pyridazinone compounds and use thereof
CN103930408A (en) Morpholinyl benzotriazine for use in cancer therapy
CN111961035B (en) Compound containing hydroxyisoquinoline structure, pharmaceutical composition and application thereof
CN108658976B (en) Novel pyrazolo [4,3-c ] pyridine-4 (5) -ketone Bruton kinase inhibitor
CN110283171A (en) Compound of the one kind containing Pyridopyrimidine -4- amine structure, pharmaceutical composition and its application
KR101897631B1 (en) Urea compounds containing 3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one skeleton as protein kinase inhibitors
CN115785088A (en) Compounds as SOS1 inhibitors and uses thereof
CN109206401B (en) methyl-1H-indazole ROR gamma regulator and application thereof
WO2023115142A1 (en) Arylsulphonamide compounds

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
TR01 Transfer of patent right

Effective date of registration: 20231201

Address after: 210028 Maigaoqiao Entrepreneurship Park Technology Research and Development Base, Qixia District, Nanjing City, Jiangsu Province (No. 18 Yinchun Road - F2507)

Patentee after: Nanjing shuohui Pharmaceutical Technology Co.,Ltd.

Address before: No.188, Xinle Road, Jiangbei new district, Liuhe District, Nanjing City, Jiangsu Province, 210000

Patentee before: NANJING POLYTECHNIC INSTITUTE

TR01 Transfer of patent right