CN114423762A - Macrocyclic derivative and preparation method and application thereof - Google Patents

Macrocyclic derivative and preparation method and application thereof Download PDF

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CN114423762A
CN114423762A CN202080063779.5A CN202080063779A CN114423762A CN 114423762 A CN114423762 A CN 114423762A CN 202080063779 A CN202080063779 A CN 202080063779A CN 114423762 A CN114423762 A CN 114423762A
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pharmaceutically acceptable
cancer
acceptable salt
tautomer
stereoisomer
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CN114423762B (en
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陈友喜
毛文涛
龚亮
向清
赵雯雯
胡泰山
陈磊
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Zhejiang Hisun Pharmaceutical Co Ltd
Shanghai Aryl Pharmtech Co Ltd
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Zhejiang Hisun Pharmaceutical Co Ltd
Shanghai Aryl Pharmtech 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
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/504—Pyridazines; Hydrogenated pyridazines forming part of bridged ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07D471/22—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 systems contains four or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains three hetero rings
    • C07D498/18—Bridged systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/22—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains four or more hetero rings

Abstract

The invention relates to macrocyclic derivatives, a preparation method thereof and application thereof in medicines. Specifically, the invention relates to a macrocyclic derivative shown in a general formula (I), a preparation method thereof, a pharmaceutically acceptable salt thereof and application thereof as a therapeutic agent, in particular to SRC, MET and/or CSF-1R inhibitor, wherein the definition of each substituent in the general formula (I) is the same as that in the specification.

Description

Macrocyclic derivative and preparation method and application thereof Technical Field
The present invention relates to a novel macrocyclic derivative, a process for its preparation, a pharmaceutical composition containing the derivative and its use as a therapeutic agent, in particular as an inhibitor of SRC, MET and/or CSF-1R kinase.
Background
Protein kinases are key regulators of cell growth, proliferation and survival, and in recent years, researches on protein kinase-targeted anticancer drugs are well-established, and although good effects of the inhibitors bring hopes to tumor patients, drug resistance caused by one-way inhibition of protein kinases comes along, so that some researchers begin to pay attention to multi-target inhibitors and hope to solve the problem of increasingly outstanding drug resistance.
c-Met, also known as Met or Hepatocyte Growth Factor Receptor (HGFR), is a high affinity natural ligand for c-Met in humans and plays an important role in humans as a tyrosine kinase. c-Met is generally expressed in epithelial cell tissues, and various activation mechanisms exist, such as overexpression, gene amplification, gene variation, and self-or collateral HGF activation. The c-Met protein is a heterodimer formed by connecting a 50kDa extracellular a subunit and a 145kDa transmembrane catalytic beta subunit through a disulfide bond, and comprises three functionally different domains, HGF catalyzes the phosphorylation of Tyrl234 and Tyr1235 of the catalytic domain in the intracellular region of receptor tyrosine kinase to activate the c-Met kinase through the combination with the subunit of the extracellular region of the c-Met, and further combines with a downstream signal SH2(Src homology 2) to generate a plurality of intracellular signal transduction. Under normal conditions, the HGF/c-Met signal channel is involved in embryonic development, wound healing, tissue regeneration and the like, and in normal cells, the RNA of the c-Met is expressed at a low level and only rises temporarily after tissue damage, and then the normal level is restored. On the contrary, the overexpression of HGF/c-Met exists in tumor cells, for example, the high expression of c-Met is observed in cancer cells such as human ovarian cancer, nasopharyngeal carcinoma, uterine cancer, gastric cancer, non-small cell lung cancer, renal cancer and the like, and after the abnormal activation of HGF/c-Met signal pathway in the tumor cells is blocked, the tumor cells can have a series of changes such as slow proliferation, reduced tumor formation rate, reduced invasive ability and the like, so the overexpression of c-Met is closely related to the formation and prognosis of various tumors.
The C-Src protein, a non-receptor tyrosine kinase, also known as Src, is composed of 6 parts, an N-terminal myristoylation sequence, SH2 domain, SH3 domain, a unique sequence, a kinase domain, and a C-terminal regulatory domain, which exert different biological effects. The N-terminal myristoyl anchored inside the plasma membrane allows the c-Src protein to be closer to cytokine receptors; an SH2 domain consisting of about 100 amino acid residues, which transduces signals by specifically recognizing a phosphotyrosine-containing short peptide and binding thereto; the SH3 domain is formed by about 50 amino acid residues, can mediate the connection between proteins, is combined with target proteins through proline and hydrophobic amino acid residues, and has important significance in the aspects of cytoskeleton recombination and c-Src protein transportation; the two phosphorylation sites of the kinase domain, Y416 and Y527, regulate the biological activity of the kinase itself; the C-terminal negative regulatory region is a characteristic region of C-Src protein, and v-Src protein does not have the regulatory region. The c-Src plays an important role in maintaining normal physiological functions of the body, and in normal cells and tissues, the c-Src exists in an inactive form, a tyrosine 530 site (Tyr530) is bound to the SH2 domain, and a proline linking region is bound to the SH3 domain to maintain the inactive state of the c-Src. Normally, its activation is transient and precisely regulated, whereas in many tumor cells, due to the loss of precise negative regulation, c-Src is not only highly expressed, but also continuously activated in tumor cells or stroma, thus leading to the development and progression of tumors. In some tumors, increased SRC kinase activity is detected, including breast, lung, pancreatic, gastric, ovarian, bladder, head and neck, esophageal, melanoma, kaposi's sarcoma, lymphoproliferative disorders, and the like. c-Src is a c-Met-driven key downstream transducer, and inhibition of c-Src increases the sensitivity of cells to c-Met inhibitors, so that dual inhibition of c-Met and c-Src kinase activity can be a strategy for tumor-targeted therapy.
Colony stimulating factor-1 (CSF-1), also known as macrophage colony stimulating factor, has a receptor (CSF-1R) encoded by a c-fms proto-oncogene, and CSF-1R is a single-chain transmembrane glycoprotein consisting of 972 amino acid residues, belongs to a tyrosine kinase receptor, and is expressed in monocytes, macrophages, precursors of bone marrow cells, fetal membrane trophoblasts, and chorioma cells. CSF-1R has 5 immunoglobulin-like domains in the extracellular ligand-binding portion and a single transmembrane domain and a mitokinase domain in the intracellular portion. Binding of CSF-1R to CSF-1 induces receptor dimerization, resulting in conformational changes that result in phosphorylation of tyrosine residues in the intracellular domain. Most of these phosphorylated residues can attract effector molecules, and activate a series of signal transduction pathways, such as survival, proliferation and differentiation of macrophages. CSF-1 and its receptor are closely related to the occurrence and metastasis of various malignant tumors, such as breast cancer, ovarian cancer, nasopharyngeal cancer, colorectal cancer, pancreatic cancer, Hodgkin lymphoma and other tumors, and the co-expression of CSF-1R and CSF-1 can be detected.
Abnormal activation of the HGF/MET pathway due to Mf7 mutation, gene amplification and translocation, HGF paracrine or autocrine upregulation, etc., is found in a variety of tumors, abnormal HGF/MET signaling leads not only to cancer but also to the development of synergistic drug resistance, SRC is a key downstream converter of MET, and it is also involved in the formation, metastasis and resistance of malignancies. In the tumor microenvironment, CSF-1R plays a role in tumor-associated macrophage regulation, tumor progression and angiogenesis. Thus, multi-target inhibition of MET, SRC and/or CSF-1R may serve as an effective strategy to treat tumors, effectively targeting aberrant HGF/MET signaling, while targeting tumor intrinsic signaling and tumor microenvironment.
A series of SRC, MET and/or CSF-1R multi-target inhibitor patent applications have been disclosed, including WO2019023417 and the like, while TPX-0022, as a multi-target inhibitor of MET, SRC and CSF-1R, has entered into clinical stage at present, produces significant tumor growth inhibition without significant side effects such as weight loss, has good drug development and preclinical safety, and is yet to be further developed, so that the development of other SRC, MET and/or CSF-1R multi-target inhibitors has great prospects and necessities to solve the unmet clinical needs.
Disclosure of Invention
In view of the above technical problems, the present invention provides a new class of macrocyclic compounds represented by the general formula (I) or stereoisomers, tautomers or pharmaceutically acceptable salts thereof:
Figure PCTCN2020117985-APPB-000001
wherein:
L 1selected from a chemical bond or-C (═ O) -; preferably a chemical bond;
R 1selected from a hydrogen atom or an amino group;
R 2～R 8identical or different, each independently selected from a hydrogen atom or a deuterium atom.
In a preferred embodiment of the present invention, a compound represented by the general formula (I) or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereofSalts thereof, wherein L1Selected from chemical bonds.
In a preferred embodiment of the present invention, a compound represented by the general formula (I) or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof, wherein R is1Selected from hydrogen atoms.
In a preferred embodiment of the present invention, a compound represented by the general formula (I) or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof, wherein R is2～R 8Selected from hydrogen atoms.
Typical compounds of the invention include, but are not limited to:
Figure PCTCN2020117985-APPB-000002
or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof.
Further, the present invention provides a pharmaceutical composition comprising an effective amount of a compound of formula (I) or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, or a combination thereof.
The invention provides an application of a compound shown in a general formula (I) or a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof in preparing SRC, MET and/or CSF-1R inhibitors.
The invention provides an application of a compound shown in a general formula (I) or a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof in preparing a medicament for treating diseases mediated by SRC, MET and/or CSF-1R, wherein the diseases mediated by SRC, MET and/or CSF-1R are preferably cancer; wherein the cancer is preferably gastric cancer, colon cancer, renal cancer, liver cancer, lung cancer, glioblastoma or head and neck tumors.
The invention provides an application of a compound shown in a general formula (I) or a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof in preparing a medicament for treating cancers, wherein the cancers are preferably gastric cancer, colon cancer, renal cancer, liver cancer, lung cancer, glioblastoma or head and neck tumors.
Detailed description of the invention
Unless stated to the contrary, some of the terms used in the specification and claims of the present invention are defined as follows:
"amino" means-NH2。
"pharmaceutically acceptable salts" refers to certain salts of the above compounds which retain their biological activity and are suitable for pharmaceutical use. The pharmaceutically acceptable salts of the compounds of formula (I) may be metal salts, amine salts with suitable acids.
"pharmaceutical composition" means a mixture containing one or more compounds described herein, or a physiologically acceptable salt or prodrug thereof, in admixture with other chemical components, as well as other components such as physiologically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.
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.
Examples
The examples show the preparation of representative compounds represented by formula (I) and the associated structural identification data. It must be noted that the following examples are intended to illustrate the invention and are not intended to limit the invention.1The H NMR spectrum was obtained using a Bruker instrument (400MHz) and the chemical shifts were expressed in ppm. Tetramethylsilane internal standard (0.00ppm) was used.1Method for H NMR expression: s is singlet, d is doublet, t is triplet, m is multiplet, br is broadened, dd is doublet of doublet, dt is doublet of triplet. If a coupling constant is provided, it is in Hz.
The mass spectrum is measured by an LC/MS instrument, and the ionization mode can be ESI or APCI.
The thin layer chromatography silica gel plate adopts HSGF254 of tobacco yellow sea or GF254 of Qingdao, the specification of the silica gel plate used by Thin Layer Chromatography (TLC) is 0.15 mm-0.2 mm, and the specification of the thin layer chromatography separation and purification product is 0.4 mm-0.5 mm.
The column chromatography generally uses 200-300 mesh silica gel of the Tibet Huanghai silica gel as a carrier.
In the following examples, all temperatures are in degrees Celsius unless otherwise indicated, and unless otherwise indicated, the various starting materials and reagents are commercially available or synthesized according to known methods, and none of the commercially available materials and reagents are used without further purification, and unless otherwise indicated, commercially available manufacturers include, but are not limited to, Aldrich Chemical Company, ABCR GmbH & Co. KG, Acros Organics, Prov Chemical science Inc. and Sci Chemical science Inc., and the like.
CD 3OD: deuterated methanol.
CDCl 3: deuterated chloroform.
DMSO-d 6: deuterated dimethyl sulfoxide.
The argon atmosphere means that the reaction flask is connected with an argon balloon having a volume of about 1L.
In the examples, the solution in the reaction is an aqueous solution unless otherwise specified.
Purifying the compound by silica gel column chromatography using an eluent system selected from the group consisting of: a: petroleum ether and ethyl acetate systems; b: dichloromethane and methanol systems; the volume ratio of the solvent is different according to the polarity of the compound, and a small amount of acidic or basic reagent such as acetic acid or triethylamine can be added for adjustment. For the preparative HPLC separation of the compounds, the specific conditions are as follows: AKZONOBEL Kromasil, 250X 21.2mm I.D., 5 μm, 20 mL/min; fluidity A: 0.05% TFA + H2O, flowability B: CH (CH)3CN, setting the proportion gradient elution of different mobile phases.
Example 1
(11R)-2-ethyl-6-fluoro-11-methyl-14-oxo-10-oxa-2,13,17,21,22-pentaazatetracyclo[13.5.2..0 4,9.0 18,22]docosa-1(21),4,6,8,15,17,19-heptaene-5-carbonitrile
(11R) -2-Ethyl-6-fluoro-11-methyl-14-oxo-10-oxa-2, 13,17,21, 22-pentaazatetracyclo [13.5.2.04,9.0 18,22]Docosane-1 (21),4,6,8,15,17, 19-heptene-5-carbonitrile
Figure PCTCN2020117985-APPB-000003
First step of
2-bromo-3-fluoro-6-hydroxybenzaldehyde
2-bromo-3-fluorobenzaldehyde 1a (8g,40mmol), 2-amino-4-chlorobenzoic acid (3.4g,20mmol), 1-fluoro-2, 4, 6-trimethylpyridine trifluoromethanesulfonate (17g,60mmol), palladium acetate (898mg,4mmol) and p-toluenesulfonic acid (13.8g,80mmol) were added to 50mL of acetic acid, stirred at room temperature for 10 minutes, heated to 120 ℃ and reacted for 24 hours. After completion of the reaction, the reaction mixture was cooled to room temperature, filtered through celite, washed with 50mL of ethyl acetate, and the filtrate was concentrated under reduced pressure to obtain a residue, which was further purified by silica gel column chromatography (eluent: A system) to obtain 2-bromo-3-fluoro-6-hydroxybenzaldehyde 1b (5 g).
1H NMR(400MHz,DMSO-d6)δppm 11.77(s,1H),10.33(s,1H),7.26-7.32(dd,J＝8.0Hz,J＝16.0Hz,1H),6.92-6.96(dd,J＝4.0Hz,J＝12.0Hz,1H).
Second step of
(S) - (2- (3-bromo-4-fluoro-2-formylphenoxy) propyl) carbamic acid tert-butyl ester
2-bromo-3-fluoro-6-hydroxybenzaldehyde 1b (219mg,1mmol), (R) -5-methyl-1, 2, 3-oxathiazolidine-3-carboxylic acid tert-butyl ester 2, 2-dioxide 1c (261mg,1.1mmol) and potassium carbonate (414mg,3mmol) were added to 5mL of N, N-dimethylformamide and reacted at room temperature for 3 hours. After completion of the reaction, 10mL of water was added to the reaction mixture, and the mixture was extracted with ethyl acetate (10 mL. times.3), the organic phases were combined, washed with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a residue, which was further separated and purified by silica gel column chromatography (eluent: A system) to obtain tert-butyl (S) - (2- (3-bromo-4-fluoro-2-formylphenoxy) propyl) carbamate 1d (376 mg).
MS m/z(ESI):397.9[M+Na]
The third step
(S) - (2- (3-bromo-2- ((ethylamino) methyl) -4-fluorophenoxy) propyl) carbamic acid tert-butyl ester
(S) - (2- (3-bromo-4-fluoro-2-formylphenoxy) propyl) carbamic acid tert-butyl ester 1d (376mg,1mmol) and ethylamine (90mg,2mmol) were added to 5mL of methanol, and reacted at room temperature for 30 minutes, followed by addition of sodium borohydride (76mg,2mmol) and reaction at room temperature for 2 hours. After the reaction, 10mL of water was added to the reaction mixture, and the mixture was extracted with ethyl acetate (15 mL. times.3), the organic phases were combined, washed with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure, and the obtained residue was further separated and purified by silica gel column chromatography (eluent: A system) to obtain tert-butyl (S) - (2- (3-bromo-2- ((ethylamino) methyl) -4-fluorophenoxy) propyl) carbamate 1e (290 mg).
MS m/z(ESI):404.9[M+H]
The fourth step
(S) -6- ((2-bromo-6- ((1- ((tert-butoxycarbonyl) amino) propan-2-yl) oxy) -3-fluorobenzyl) (ethyl) amino) imidazo [1,2] -b ] pyridazine-3-carboxylic acid ethyl ester
(S) - (tert-butyl 2- (3-bromo-2- ((ethylamino) methyl) -4-fluorophenoxy) propyl) carbamate 1e (240mg,0.592mmol), ethyl 6-chloroimidazo [1,2-b ] pyridazine-3-carboxylate 1f (133mg,0.592mmol), and potassium fluoride (172mg,2.96mmol) were added to 5mL of dimethyl sulfoxide, heated to 120 ℃ and reacted overnight. After completion of the reaction, the reaction mixture was cooled to room temperature, 10mL of water was added to the reaction mixture, extraction was performed with ethyl acetate (10 mL. times.3), the organic phases were combined, washed with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure, and the obtained residue was further separated and purified by silica gel column chromatography (eluent: A system) to obtain 1g (175mg) of ethyl (S) -6- ((2-bromo-6- ((1- ((tert-butoxycarbonyl) amino) propan-2-yl) oxy) -3-fluorobenzyl) (ethyl) amino) imidazo [1,2] -b ] pyridazine-3-carboxylate.
MS m/z(ESI):593.9[M+H]
The fifth step
(S) -6- ((2-bromo-6- ((1- ((tert-butoxycarbonyl) amino) prop-2-yl) oxy) -3-fluorobenzyl) (ethyl) amino) imidazo [1,2] -b ] pyridazine-3-carboxylic acid
Reacting (S) -6- ((2-bromo-6- ((1- ((tert-butoxycarbonyl) amino) prop-2-yl) oxy) -3-fluorobenzyl) (ethyl) amino) imidazo [1,2]-b]1g (175mg,0.29mmol) of ethyl pyridazine-3-carboxylate and lithium hydroxide monohydrate (98mg,2.32mmol) were added to 2.5mL of a mixed solution (V)Tetrahydrofuran (THF):V Ethanol:V Water (W)2:2:1), heated to 75 ℃, and reacted for 5 hours. After the reaction, the reaction mixture was cooled to room temperature, 10mL of ethyl acetate was added to the reaction mixture, the mixture was washed with a 10% citric acid aqueous solution, a saturated sodium chloride solution and dried over anhydrous sodium sulfate, and the mixture was concentrated under reduced pressure to obtain (S) -6- ((2-bromo-6- ((1- ((tert-butoxycarbonyl) amino) propan-2-yl) oxy) -3-fluorobenzyl) (ethyl) amino) imidazo [1,2]-b]The crude pyridazine-3-carboxylic acid was taken as 1h (164mg) and the product was directly used in the next reaction without purification.
MS m/z(ESI):566.3[M+H]
The sixth step
(S) -6- ((6- ((1-aminopropyl-2-yl) oxy) -2-bromo-3-fluorobenzyl) (ethyl) amino) imidazo [1,2-b ] pyridazine-3-carboxylic acid
Crude 1h (164mg,0.29mmol) of (S) -6- ((2-bromo-6- ((1- ((tert-butoxycarbonyl) amino) prop-2-yl) oxy) -3-fluorobenzyl) (ethyl) amino) imidazo [1,2] -b ] pyridazine-3-carboxylic acid is added to 3mL of dichloromethane, and 0.5mL of a 4M solution of hydrochloric acid in dioxane is added. The reaction was carried out at room temperature for 3 hours. After the reaction, the reaction solution was concentrated under reduced pressure to obtain crude (S) -6- ((6- ((1-aminopropyl-2-yl) oxy) -2-bromo-3-fluorobenzyl) (ethyl) amino) imidazo [1,2-b ] pyridazine-3-carboxylic acid 1i (135mg), and the product was directly subjected to the next reaction without purification.
MS m/z(ESI):466.3[M+H]
Seventh step
(11R) -5-bromo-2-ethyl-6-fluoro-11-methyl-10-oxa-2, 13,17,21, 22-pentaazatetracyclo [13.5.2.04,9.0 18,22]Docosane-1 (21),4,6,8,15,17, 19-hepten-14-one
Reacting (S) -6- ((6- ((1-aminopropyl-2-yl) oxy) -2-bromo-3-fluorobenzyl) (ethyl) amino) imidazo [1,2-b]Pyridazine-3-carboxylic acid 1i (135mg, 0)29mmol), pentafluorophenyl diphenyl phosphate (134mg,0.35mmol) and N, N-diisopropylethylamine (0.4mL,2.32mmol) were dissolved in 6mL of the mixed solution (V)Methylene dichloride:V N, N-dimethylformamide1:1), at room temperature for 16 hours. After completion of the reaction, 10mL of water was added to the reaction mixture, followed by extraction with methylene chloride (10 mL. times.3), combination of the organic phases, washing with a saturated sodium chloride solution, drying over anhydrous sodium sulfate, and concentration under reduced pressure to obtain a residue which was further separated and purified by silica gel column chromatography (eluent: B system) to obtain (11R) -5-bromo-2-ethyl-6-fluoro-11-methyl-10-oxa-2, 13,17,21, 22-pentaazatetracyclo [13.5.2.0 ]4,9.0 18,22]Docosane-1 (21),4,6,8,15,17, 19-hepten-14-one 1j (65 mg).
MS m/z(ESI):447.9[M+H]
Eighth step
(11R) -2-Ethyl-6-fluoro-11-methyl-14-oxo-10-oxa-2, 13,17,21, 22-pentaazatetracyclo [13.5.2.04,9.0 18,22]Docosane-1 (21),4,6,8,15,17, 19-heptene-5-carbonitrile
Under the protection of argon, (11R) -5-bromo-2-ethyl-6-fluoro-11-methyl-10-oxa-2, 13,17,21, 22-pentaazatetracyclo [13.5.2.04,9.0 18,22]Docosahexane-1 (21),4,6,8,15,17, 19-hepten-14-one 1j (130mg,0.29mmol), zinc cyanide (170mg,1.45mmol), zinc (2mg,0.029mmol), 1' -bisdiphenylphosphinoferrocene (80mg,0.145mmol) and tris (dibenzylideneacetone) dipalladium (53mg,0.058mmol) were added to 5mL of N, N-dimethylacetamide, heated to 130 ℃ and reacted for 3 hours. After the reaction, the reaction mixture was cooled to room temperature, filtered through celite, washed with 20mL of ethyl acetate, the filtrate was washed with water and saturated sodium chloride solution in this order, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a residue, which was subjected to preparative liquid phase separation (AKZONOBEL Kromasil, 250X 21.2mm I.D., 5 μm, 20 mL/min; fluidity A: 0.05% TFA + H2O, flowability B: CH (CH)3CN) to give (11R) -2-ethyl-6-fluoro-11-methyl-14-oxo-10-oxa-2, 13,17,21, 22-pentaazatetracyclo [13.5.2.04,9.0 18,22]Docosane-1 (21),4,6,8,15,17, 19-heptene-5-carbonitrile 1(25 mg).
MS m/z(ESI):395.0[M+H]
1H NMR(400MHz,CDCl 3)δppm 9.68(s,1H),8.46(s,1H),8.20(s,1H),7.26-7.31(m,1H), 7.10-7.12(m,2H),5.58-5.64(m,1H),3.86-4.62(m,5H),3.39-3.41(m,1H),1.54-1.61(m,3H),1.30-1.35(m,3H).
Biological evaluation
Test example 1 test of Src/Met/CSF-1R kinase inhibitory Activity of Compounds of the present invention
The following method was used to determine the degree of inhibition of the recombinant human Src/Met/CSF-1R kinase activity of the compounds of the invention under in vitro conditions. The method uses Cisbio
Figure PCTCN2020117985-APPB-000004
KinEASE-TK tyrosine kinase kit (cat # 62TK0PEB), whose principle is based on time-resolved fluorescence energy resonance transfer (TF-FRET), reflects the strength of the inhibition of SRC/MET/CSF-1R kinase activity by compounds by measuring the extent of SRC/MET/CSF-1R kinase mediated phosphorylation of biotinylated polypeptide substrates. The detailed experimental procedures can be referred to the kit instructions. Recombinant human SRC/MET/CSF-1R proteins were purchased from SignalChem (MET # M52-18G-10, SRC # S19-18G-10, CSF-1R # R14-11G-10, respectively)
The experimental procedure is briefly described as follows: test compounds were first dissolved in DMSO to prepare stock solutions, followed by preparation of compound dilution buffer (Src: HEPES 50mM, MgCl)25mM，Na 3VO 40.1mM, DTT 1mM, 0.01% BSA; met and CSF-1R: HEPES 50mM, MgCl25mM，MnCl 2 1mM，Na 3VO 40.1mM, DTT 1mM, 0.01% BSA) was diluted in a gradient, and the final concentration of the test compound in the reaction system ranged from 1000nM to 0.004 nM. ATP Km value concentration of Src/Met/CSF-1R protein was measured using ATP solution (Bio-engineering, Shanghai, Ltd., A600311) diluted in a gradient, and the ATP concentration in the corresponding kinase reaction system was set based on the obtained Km value. The reaction was carried out in 384-well microplatesFirst, the compound and an amount of the corresponding SRC/MET/CSF-1R protein are added to the wells and incubated at room temperature for 5-10 minutes, then the ATP solution and biotinylated polypeptide substrate solution are added to the reaction solution and incubated at room temperature for 40-60 minutes with shaking. Then, an anti-phosphotyrosine antibody conjugated with a europium-based element compound and streptavidin conjugated with modified allophycocyanin XL665 were added to the reaction, and incubation was continued for 1 hour with shaking at room temperature. After the incubation, the fluorescence intensity values of each well at an excitation wavelength of 304nM, an emission wavelength of 620nM and 665nM are measured in a microplate reader in TF-FRET mode. Percent inhibition of compound at each concentration was calculated by comparison with the fluorescence intensity ratio of the control (0.1% DMSO), and non-linear regression analysis was performed by GraphPad Prism 5 software at compound concentration log-inhibition, or IC of compound50Values, see table 1.
TABLE 1 IC inhibition of SRC/MET/CSF-1R kinase Activity by Compounds of the invention50Value of
Figure PCTCN2020117985-APPB-000005
As can be seen from Table 1, Compound 1 of the present invention has significant inhibitory effects on SRC, MET and CSF-1R kinases, and has significantly better inhibitory activity on MET kinase than SRC and CSF-1R.
Test example 2 ICR mouse pharmacokinetics study of Compound of the present invention
1. Purpose of experiment
An ICR mouse is taken as a test animal, TPX-0022 and the compound are measured by an LC/MS/MS method when the mouse is subjected to intravenous injection or gastric lavage, the drug concentration in plasma at different moments is measured, and the pharmacokinetic characteristics of the compound in the mouse are researched.
2. Experimental protocol
2.1 Experimental drugs and animals
TPX-0022 and the compound of example 1;
ICR mice, male, 18, 28.8-37.0 g, purchased from Beijing Wittingle laboratory animal technology, Inc. Animal quality certification number: 1909270043.
2.2 pharmaceutical preparation
Intravenous group: weighing a proper amount of medicine, adding DMA (30% Solutol HS-15) and Saline (10: 10: 80) (v/v/v), and preparing the final preparation concentration to be 0.2 mg/mL;
oral gavage group: weighing appropriate amount of medicine, adding appropriate amount of sodium carboxymethylcellulose (CMC-Na, containing 0.5% Tween 80), vortex oscillating, and ultrasonically preparing 0.5mg/mL suspension;
2.3 administration of drugs
18 ICR mice, on average divided into 2 groups, were fasted overnight and then administered intravenously (dose 1mg/kg, dose volume 5mL/kg) and gavage (dose 5mg/kg, dose volume 10mL/kg), respectively, 4 hours after administration and then fed.
3. Operation of
80 μ L of blood was collected periorbital at 0.083, 0.25, 0.5, 1,2, 4, 8, 12, and 24 hours before and after administration, and was anticoagulated with EDTA-K2. Blood samples were collected and placed on ice, and plasma was centrifuged (centrifugation conditions: 1500g, 10 minutes). The collected plasma was stored at-40 to-20 ℃ before analysis.
The LC-MS/MS is used for measuring the content of the compound to be measured in the plasma of the mice after the intravenous injection and the gavage administration of different compounds.
4. Pharmacokinetic parameter results
The pharmacokinetic parameters of the compounds of the invention and the positive control TPX-0022 are shown in the table below.
Figure PCTCN2020117985-APPB-000006
Remarking: N/A denotes no correlation results
And (4) conclusion: compared with TPX-0022, the compound of the embodiment 1 of the invention has obviously increased Cmax, good drug absorption, long half life, low clearance rate, obviously improved bioavailability and better pharmacokinetic property.
TPX-0022 is prepared according to the method disclosed in patent application WO2019023417, and has the following specific structure:
Figure PCTCN2020117985-APPB-000007

Claims (9)

  1. a compound of formula (I) or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof:
    Figure PCTCN2020117985-APPB-100001
    wherein:
    L 1selected from a chemical bond or-C (═ O) -; preferably a chemical bond;
    R 1selected from a hydrogen atom or an amino group;
    R 2～R 8identical or different, each independently selected from a hydrogen atom or a deuterium atom.
  2. A compound according to claim 1, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein L1Selected from chemical bonds.
  3. A compound according to claim 1, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein R1Selected from hydrogen atoms.
  4. A compound according to claim 1, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein R2～R 8Selected from hydrogen atoms.
  5. A compound according to any one of claims 1 to 4, or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof, wherein said compound is selected from the group consisting of:
    Figure PCTCN2020117985-APPB-100002
  6. a pharmaceutical composition comprising an effective amount of a compound according to any one of claims 1-5, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, or combination thereof.
  7. Use of a compound according to any one of claims 1 to 5, or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 6, for the preparation of a SRC, MET, and/or CSF-1R inhibitor.
  8. Use of a compound according to any one of claims 1 to 5, or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 6, for the manufacture of a medicament for the treatment of a disease mediated by SRC, MET, and/or CSF-1R, preferably a cancer; wherein the cancer is preferably gastric cancer, colon cancer, renal cancer, liver cancer, lung cancer, glioblastoma or head and neck tumors.
  9. Use of a compound according to any one of claims 1 to 5, or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 6, for the manufacture of a medicament for the treatment of a cancer, preferably of the stomach, colon, kidney, liver, lung, glioblastoma or head and neck.
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