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CN111718350B - Pyrazole-substituted pyrazolopyrimidine compounds, pharmaceutical compositions and uses thereof - Google Patents

Pyrazole-substituted pyrazolopyrimidine compounds, pharmaceutical compositions and uses thereof Download PDF

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CN111718350B
CN111718350B CN202010183822.3A CN202010183822A CN111718350B CN 111718350 B CN111718350 B CN 111718350B CN 202010183822 A CN202010183822 A CN 202010183822A CN 111718350 B CN111718350 B CN 111718350B
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黄伟
杨光富
王明书
卓林胜
徐红闯
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Central China Normal University
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Abstract

The invention relates to the field of biomedicine, and discloses a pyrazole substituted pyrazolopyrimidine compound, a pharmaceutical composition and application thereof. The pyrazole-substituted pyrazolopyrimidine compound having the structure shown in formula (I) or a pharmaceutically acceptable salt thereof, or a stereoisomer, a geometric isomer, a tautomer, a nitrogen oxide, a hydrate, a solvate, a metabolite or a prodrug thereof shows excellent inhibitory activity on TRK kinase, and simultaneously shows good antitumor activity on an animal level.

Description

Pyrazole-substituted pyrazolopyrimidine compounds, pharmaceutical compositions and uses thereof
Technical Field
The invention relates to the field of biomedicine, and particularly relates to a pyrazole-substituted pyrazolopyrimidine compound, a pharmaceutical composition containing the pyrazole-substituted pyrazolopyrimidine compound, and application of the pyrazole-substituted pyrazolopyrimidine compound and the pharmaceutical composition.
Background
NTRK/TRK (Tropomosin receptor kinase) is neurotrophic factor and belongs to receptor tyrosine kinase family. The TRK family mainly comprises 3 members, NTRK1/TRKA, NTRK2/TRKB and NTRK 3/TRKC. The complete TRK kinase comprises three parts, namely an extracellular region, a transmembrane region and an intracellular region. The extracellular region of TRK kinase can cause the change of kinase configuration after being combined with corresponding ligand to form dimer. The intracellular region of TRK kinase is autophosphorylated to activate the kinase activity of the TRK kinase, and further activate the downstream signal transduction pathway (such as MAPK, AKT, PKC and the like) to generate corresponding biological functions; wherein NGF (nerve growth factor) binds TRKA, BDNF (derived neurotrophic factor) binds TRKB, and NT3 (neurotrophic factor 3) binds TRKC.
TRK kinases play important physiological roles in the development of nerves, including the growth and functional maintenance of neuronal axons, the development of memory, and the protection of neurons from injury, among others. Meanwhile, a large number of researches show that activation of a TRK signal transduction pathway is closely related to generation and development of tumors, and activated TRK signal proteins are found in neurocytoma, prostatic cancer, breast cancer and the like.
In recent years, the discovery of various TRK fusion proteins shows that the TRK fusion proteins can promote the biological function of tumorigenesis. The earliest TPM3-TRKA fusion protein was found in colon cancer cells, with an incidence of about 1.5% in the clinical patients tested. Later, different types of TRK fusion proteins were found in different types of clinical tumor patient samples, such as lung cancer, head and neck cancer, breast cancer, thyroid cancer, glioma and the like, such as CD74-NTRK1, MPRIP-NTRK1, QKI-NTRK2, ETV6-NTRK3, BTB1-NTRK3 and the like. Under the condition that ligand binding is not needed, the different NTRK fusion proteins are in a highly activated kinase activity state, so that downstream signal pathways can be continuously phosphorylated, cell proliferation is induced, and generation and development of tumors are promoted.
Therefore, in recent years, TRK fusion proteins have become a potent anticancer target and a hotspot for research, for example, WO2010048314, WO2012116217, WO2011146336, W02010033941, WO2018077246 and the like all disclose TRK kinase inhibitors with different structural types.
In addition, target mutations occurring after continuous administration are important causes of tumor resistance, and recent clinical cases of TRK mutations, such as TRKA G595R, G667C, G667S and F589L (Russo M et al; Cancer Discovery,2016,6(1), 36-44), TRKC G623R and G696A (Drilon A. et al Annals of Oncology 2016,27(5), 920-.
In addition, nitrogen-containing aromatic heterocycles are generally preferred for their potency, a typical example being the ALK kinase inhibitor crizotinib (Cui J. et al. J. Med. chem.2011,54, 6342-. WO2007147647 and WO2007025540 also disclose pyrazole substituted pyrazolopyridine compounds and pyrazole substituted imidazopyridazine compounds, respectively, as ALK kinase inhibitors and their use in the treatment of disease.
Figure BDA0002413469320000021
Disclosure of Invention
An object of the present invention is to provide a novel pyrazole-substituted pyrazolopyrimidine compound having excellent antitumor activity.
Although the typical compound a and the typical compound B provided in the prior art have good inhibitory activity on ALK kinase, the inhibitory effect of structural analogs represented by the typical compound a and the typical compound B on TRK kinase is not good. Through a large number of scientific researches, the inventor of the invention finds that the pyrazole substituted pyrazolopyrimidine compound having the structure shown in formula (I) of the invention has excellent inhibitory activity on TRK kinase, and the inhibitory activity is obviously superior to that of the typical compound A and the typical compound B in the prior art. More importantly, the pyrazole substituted pyrazolopyrimidine compound containing the phenoxy structure has the antitumor activity on the animal level which is also obviously superior to that of the typical compound A and the typical compound B, so that the compound shows more excellent tumor treatment effect than the prior art.
In order to achieve the above objects, a first aspect of the present invention provides a pyrazole-substituted pyrazolopyrimidine compound having a structure represented by formula (I) or a pharmaceutically acceptable salt thereof, or a stereoisomer, geometric isomer, tautomer, nitrogen oxide, hydrate, solvate, metabolite, or prodrug thereof,
Figure BDA0002413469320000031
wherein, in the formula (I),
R1、R2、R3and R4Each independently selected from H, halogen, C1-12Alkyl of (2), C substituted by 1-6 halogens1-12Alkyl groups of (a);
R5selected from H, C1-12Alkyl of (2), C substituted by 1-6 halogens1-12Alkyl, hydroxy-substituted C of1-12Alkyl, alkoxy-substituted C2-12Alkyl, cyano-substituted C2-12Alkyl of (a), C containing 1-3 heteroatoms selected from N, O and S2-12Cycloalkyl groups of (a);
R6selected from H, C1-12Alkyl, hydroxy-substituted C of1-12Alkyl and halogen.
A second aspect of the present invention provides a pyrazole-substituted pyrazolopyrimidine compound having a structure represented by formula (I) or a pharmaceutically acceptable salt thereof, or a stereoisomer, a geometric isomer, a tautomer, a nitrogen oxide, a hydrate, a solvate, a metabolite, or a prodrug thereof, described in the first aspect, for use in the preparation of a medicament for the prevention and/or treatment of a TRK kinase receptor-mediated disease.
A third aspect of the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient or diluent, and, as an active ingredient, a pyrazole-substituted pyrazolopyrimidine compound having a structure represented by formula (I) according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof, or a stereoisomer, geometric isomer, tautomer, nitrogen oxide, hydrate, solvate, metabolite, or prodrug thereof.
A fourth aspect of the invention provides the use of a pharmaceutical composition according to the third aspect of the invention in the manufacture of a medicament for the prevention and/or treatment of a TRK kinase receptor mediated disease.
A fifth aspect of the present invention provides a pyrazole-substituted pyrazolopyrimidine compound having a structure represented by formula (I) described in the first aspect of the present invention, or a pharmaceutically acceptable salt thereof, or a stereoisomer, a geometric isomer, a tautomer, a nitrogen oxide, a hydrate, a solvate, a metabolite, or a prodrug thereof, or a pharmaceutical composition described in the third aspect of the present invention for use in the preparation of a medicament for the prevention and/or treatment of tumors.
The pyrazole-substituted pyrazolopyrimidine compound having the structure shown in formula (I) or a pharmaceutically acceptable salt thereof, or a stereoisomer, a geometric isomer, a tautomer, a nitrogen oxide, a hydrate, a solvate, a metabolite or a prodrug thereof shows excellent inhibitory activity on TRK kinase, and simultaneously shows good antitumor activity on an animal level.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
As previously mentioned, the first aspect of the present invention provides a pyrazole-substituted pyrazolopyrimidine compound or a pharmaceutically acceptable salt thereof, or a stereoisomer, geometric isomer, tautomer, nitrogen oxide, hydrate, solvate, metabolite, or prodrug thereof.
Some of the terms involved in the present invention are explained below:
“C1-12the "alkyl group" represents an alkyl group having a total number of carbon atoms of 1 to 12, inclusiveThe alkyl group, branched alkyl group or cycloalkyl group may be, for example, a linear alkyl group, branched alkyl group or cycloalkyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms in total, and may be, for example, a methyl group, an ethyl group, a n-propyl group, an isopropyl group, a n-butyl group, an isobutyl group, a tert-butyl group, a n-pentyl group, an isopentyl group, a n-hexyl group, a cyclopropyl group, a methylcyclopropyl group, an ethylcyclopropyl group, a cyclopentyl group, a methylcyclopentyl group, a cyclohexyl group or the like. For "C1-8Alkyl of (2), "" C1-6The "alkyl group" has a similar explanation except that the number of carbon atoms is different.
"C substituted by 1-6 halogens1-12The "alkyl group" of (A) represents an alkyl group having a total number of carbon atoms of 1 to 12, including a straight-chain alkyl group, a branched-chain alkyl group and a cycloalkyl group, and the C1-121-6H in the alkyl group of (A) are substituted by halogen atoms selected from halogen, e.g. C1-12In the alkyl group of (1), (2), (3), (4), (5) or (6) H is substituted by any one or more halogen atoms selected from fluorine, chlorine, bromine and iodine, and may be, for example, trifluoromethyl, difluoromethyl, monofluoromethyl, monofluoroethyl, difluoroethyl, trifluoroethyl, etc. For "C substituted by 1-6 halogen1-8Alkyl of (2), "" C substituted by 1 to 6 halogens1-6The "alkyl group" has a similar explanation except that the number of carbon atoms is different.
"hydroxy-substituted C1-12The "alkyl group" of (A) represents an alkyl group having a total number of carbon atoms of 1 to 12, including a straight-chain alkyl group, a branched-chain alkyl group or a cyclic alkyl group, and the C1-12At least one H in the alkyl group of (a) is substituted by a hydroxyl group.
"alkoxy-substituted C2-12The alkyl group of (1) represents a group having 2 to 12 carbon atoms in total, and the structural formula of the group may be represented by-R1OR2Wherein R is1And R2Wherein the sum of the number of carbon atoms is 2 to 12, and R1Directly linked with the phenoxy group in the pyrazole-substituted pyrazolopyrimidine compound of the structure represented by formula (I) of the invention.
"cyano-substituted C2-12The "alkyl group" of (A) represents an alkyl group having 2 to 12 carbon atoms in total, including a straight-chain alkyl group, a branched-chain alkyl group or a cyclic alkyl group, and the C2-12At least one H in the alkyl group of (a) is substituted with a cyano group, and the carbon atoms in the "cyano" group count the total number of carbon atoms in the group.
"C containing 1-3 heteroatoms selected from N, O and S2-12The "cycloalkyl group" of (a) represents a cycloalkyl group having 2 to 12 carbon atoms in total, and 1 to 3 atoms of the atoms forming the ring are hetero atoms selected from N, O and S, and the atoms forming the ring may have an alkyl substituent group having a carbon atom number included in the aforementioned range of the total carbon atom number. "C containing 1-3 heteroatoms selected from N, O and S2-12The "cycloalkyl group" of (a) may be, for example, a three-membered ring, a four-membered ring, a five-membered ring, a six-membered ring, a seven-membered ring, an eight-membered ring, a nine-membered ring, a ten-membered ring, an eleven-membered ring or a twelve-membered ring, and H in the cycloalkyl group may be optionally substituted with, if substituted, at least one substituent independently selected from the group consisting of halogen, hydroxyl, nitro and mercapto.
Several preferred embodiments of the pyrazole-substituted pyrazolopyrimidine compounds of the structure of formula (I) according to the invention are provided below:
embodiment mode 1:
in the formula (I), the compound represented by the formula (I),
R1、R2、R3and R4Each independently selected from H, fluorine, chlorine, bromine, C1-8Alkyl of (2), C substituted by 1-6 halogens selected from fluorine, chlorine and bromine1-8Alkyl groups of (a);
R5selected from H, C1-8Alkyl of (2), C substituted by 1-6 halogens selected from fluorine, chlorine and bromine1-8Alkyl, hydroxy-substituted C of1-8Alkyl, alkoxy-substituted C2-8Alkyl, cyano-substituted C2-8Alkyl of (a), C containing 1-3 heteroatoms selected from N, O and S2-10Cycloalkyl groups of (a);
R6selected from H, C1-8Alkyl, hydroxy-substituted C of1-8Alkyl and halogen.
Embodiment mode 2:
in the formula (I), the compound represented by the formula (I),
R1、R2、R3and R4Each independently selected from H, fluorine, chlorine, bromine, C1-6Alkyl of (2), C substituted by 1 to 4 halogens selected from fluorine, chlorine and bromine1-6Alkyl groups of (a);
R5selected from H, C1-6Alkyl of (2), C substituted by 1 to 4 halogens selected from fluorine, chlorine and bromine1-6Alkyl, hydroxy-substituted C of1-6Alkyl, alkoxy-substituted C2-8Alkyl, cyano-substituted C2-6Alkyl of (a), C containing 1-3 heteroatoms selected from N, O and S2-8Cycloalkyl groups of (a);
R6selected from H, C1-6Alkyl, hydroxy-substituted C of1-6Alkyl and halogen.
In particular, the inventors of the present invention found that when said R is2When the compound is halogen, the pyrazole substituted pyrazolopyrimidine compound with the structure shown in the formula (I) has higher inhibitory activity on TRK kinase, particularly mutant TRK kinase; and simultaneously, the compound has more reasonable pharmacokinetic property and more excellent in-vivo anti-tumor activity. Therefore, the following embodiment 3, embodiment 4, embodiment 5 and embodiment 6 are provided to illustrate more excellent effects of the compound provided by the present invention.
Embodiment mode 3:
in the formula (I), the compound represented by the formula (I),
R1、R3and R4Each independently selected from H, halogen, C1-12Alkyl of (2), C substituted by 1-6 halogens1-12Alkyl groups of (a);
R2is halogen;
R5selected from H, C1-12Alkyl of (2), C substituted by 1-6 halogens1-12Alkyl, hydroxy-substituted C of1-12Alkyl, alkoxy-substituted C2-12Alkyl, cyano-substituted C2-12Alkyl of (a), C containing 1-3 heteroatoms selected from N, O and S2-12Cycloalkyl groups of (a);
R6is selected from C1-12Alkyl, hydroxy-substitutedC1-12Alkyl and halogen.
Embodiment 4:
in the formula (I), the compound represented by the formula (I),
R1、R3and R4Each independently selected from H, fluorine, chlorine, bromine, C1-8Alkyl of (2), C substituted by 1-6 halogens selected from fluorine, chlorine and bromine1-8Alkyl groups of (a);
R2is F;
R5selected from H, C1-8Alkyl of (2), C substituted by 1-6 halogens selected from fluorine, chlorine and bromine1-8Alkyl, hydroxy-substituted C of1-8Alkyl, alkoxy-substituted C2-8Alkyl, cyano-substituted C2-8Alkyl of (a), C containing 1-3 heteroatoms selected from N, O and S2-10Cycloalkyl groups of (a);
R6is selected from C1-8Alkyl, hydroxy-substituted C of1-8Alkyl and halogen.
Embodiment 5:
in the formula (I), the compound represented by the formula (I),
R1、R3and R4Are all H; r2Is F;
R5selected from H, C1-8Alkyl of (2), C substituted by 1-6 halogens selected from fluorine, chlorine and bromine1-8Alkyl, hydroxy-substituted C of1-8Alkyl, alkoxy-substituted C2-8Alkyl, cyano-substituted C2-8Alkyl of (a), C containing 1-3 heteroatoms selected from N, O and S2-10Cycloalkyl groups of (a);
R6is selected from C1-8Alkyl, hydroxy-substituted C of1-8Alkyl and halogen.
Embodiment 6:
the pyrazole-substituted pyrazolopyrimidine compound is at least one of the compounds listed in claim 5, or a pharmaceutically acceptable salt thereof, or a stereoisomer, geometric isomer, tautomer, nitrogen oxide, hydrate, solvate, metabolite, or prodrug thereof.
Among the compounds according to claim 5, those not having a specific configuration of a chiral center are represented as racemates.
The process for producing a pyrazole-substituted pyrazolopyrimidine compound having the structure represented by formula (I) is not particularly limited in the present invention, and for example, the compound can be produced by the following production process:
Figure BDA0002413469320000081
the preparation method involves Suzuki coupling reaction, the reaction conditions of the coupling reaction are not particularly limited, and those skilled in the art can obtain appropriate reaction conditions according to common general knowledge in the field of organic synthesis and specific examples provided in the examples section of the present invention.
As described above, the second aspect of the present invention provides the use of the pyrazole-substituted pyrazolopyrimidine compound having the structure represented by formula (I) or a pharmaceutically acceptable salt thereof, or a stereoisomer, a geometric isomer, a tautomer, a nitrogen oxide, a hydrate, a solvate, a metabolite, or a prodrug thereof, described in the first aspect of the present invention, for the preparation of a medicament for the prevention and/or treatment of a TRK kinase receptor-mediated disease.
As described above, the third aspect of the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient or diluent, and, as an active ingredient, a pyrazole-substituted pyrazolopyrimidine compound having a structure represented by formula (I) according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof, or a stereoisomer, a geometric isomer, a tautomer, a nitrogen oxide, a hydrate, a solvate, a metabolite, or a prodrug thereof.
As mentioned above, a fourth aspect of the present invention provides the use of a pharmaceutical composition as described in the third aspect of the present invention in the manufacture of a medicament for the prevention and/or treatment of a TRK kinase receptor mediated disease.
As described above, the fifth aspect of the present invention provides the pyrazole-substituted pyrazolopyrimidine compound having the structure represented by formula (I) or a pharmaceutically acceptable salt thereof, or a stereoisomer, a geometric isomer, a tautomer, a nitrogen oxide, a hydrate, a solvate, a metabolite, or a prodrug thereof, according to the first aspect of the present invention, or the use of a pharmaceutical composition according to the third aspect of the present invention in the preparation of a medicament for the prevention and/or treatment of tumors.
Preferably, the tumor is at least one of breast cancer, large intestine cancer, lung cancer, thyroid cancer, skin cancer, bone cancer, melanoma, leukemia, salivary gland tumor, neuroendocrine tumor, lymphoma, brain tumor, neuroblastoma, ovarian cancer, pancreatic cancer, mesothelioma, esophageal cancer, pulmonary sarcoma, medulloblastoma, glioblastoma, colon cancer, liver cancer, retinoblastoma, kidney cancer, bladder cancer, osteosarcoma, stomach cancer, uterine cancer, vulval cancer, small intestine cancer, prostate cancer, bile duct cancer, ureteral cancer, adrenocortical cancer, or head and neck cancer.
The present invention will be described in detail below by way of examples. In the following examples, the various starting materials used are commercially available and are of analytical purity, unless otherwise specified.
Example 1: preparation of 2- (1- ((3- (1H-pyrazol-4-yl) pyrazolo [1,5-a ] pyrimidin-5-yl) amino) ethyl) -4-fluorophenol (compound b1-1)
Figure BDA0002413469320000101
Step 1): 1- (5-fluoro-2-hydroxyphenyl) ethanone (32.4mmol), 4-methoxybenzyl bromide (38.9mmol), cesium carbonate (48.6mmol) were added to a 200mL pear-shaped vial, to which was added acetonitrile (50 mL). Heated overnight in an oil bath (80 ℃). The reaction was cooled to ambient temperature and the crude product was purified by column chromatography to give a white solid with 78% yield.
Step 2): 1- (5-fluoro-2- ((4-methoxybenzyl) oxy) phenyl) ethanone (25.3mmol), hydroxylamine hydrochloride (30.4mmol), cesium carbonate (38.0mmol) were added to a 200mL pear-shaped bottle, to which methanol (50mL) was added. The reaction was carried out for 4h at room temperature with magnetic stirring. The reaction solution was poured into water, filtered with suction, and dried to obtain a white solid with a yield of 98%.
Step 3): 1- (5-fluoro-2- ((4-methoxybenzyl) oxy) phenyl) ethanone oxime (24.8mmol), zinc powder (372mmol), ammonium chloride (372mmol), and acetic acid (372mmol) were charged to a 200mL pear-shaped bottle, to which methanol (50mL) was added. Heated overnight in an oil bath (80 ℃). The reaction was cooled to ambient temperature, neutralized, filtered through celite, and the filtrate was extracted with water and ethyl acetate. The organic phase was washed twice with saturated brine, dried over anhydrous sodium sulfate and concentrated to give a yellow liquid with a yield of 77%.
Step 4): 5-Chloropyrazolo [1,5-a ] pyrimidine (19.1mmol), 1- (5-fluoro-2- ((4-methoxybenzyl) oxy) phenyl) ethylamine (19.1mmol), anhydrous N-butanol (50mL) and N, N-diisopropylethylamine (DIPEA, 38.2mmol) were added to a 200mL pear-shaped vial. The mixture was heated overnight in an oil bath (140 ℃). The reaction was cooled to ambient temperature and concentrated under reduced pressure to remove N-butanol and N, N-Diisopropylethylamine (DIPEA) as much as possible to give a crude yellow oil which was purified by column chromatography to give a pale yellow solid with a yield of 68%.
Step 5): n- (1- (5-fluoro-2- ((4-methoxybenzyl) oxy) phenyl) ethyl) pyrazolo [1,5-a ] pyrimidin-5-amine (13.0mmol) was added to a 200mL pear-shaped vial, to which was added acetonitrile (50 mL). N-iodosuccinimide (NIS, 14.3mmol) was added under magnetic stirring at room temperature. The reaction was carried out at room temperature for 1h and TLC monitored for completion. After removing acetonitrile under reduced pressure as much as possible, the mixture was diluted with 250mL of ethyl acetate and transferred to a separatory funnel. Washing with 1mol/L NaOH for 3 times, washing with saturated salt for two times, drying with anhydrous sodium sulfate, concentrating to obtain red oily crude product, and purifying by column chromatography to obtain light yellow solid with yield of 67%.
Step 6): n- (1- (5-fluoro-2- ((4-methoxybenzyl) oxy) phenyl) ethyl) -3-iodopyrazolo [1,5-a ] pyrimidin-5-amine (0.77mmol), 1-Boc-pyrazole-4-boronic acid pinacol ester (1.16mmol), anhydrous potassium carbonate (2.00mmol), tetrakis (triphenylphosphine) palladium (0.08mmol) were added to a 100ml reaction tube, replaced with argon for 3 times, and 10ml anhydrous DMF,2ml water were added. The reaction was carried out at 100 ℃ for 2h under argon atmosphere and the completion of the reaction was monitored by TLC. Cooled to 50 ℃, filtered through celite, and the filtrate was extracted with water and ethyl acetate. The organic phase was washed twice with saturated brine, dried over anhydrous sodium sulfate, concentrated to give a crude product as a black oil, which was purified by column chromatography to give a pale yellow solid with a yield of 50%.
Step 7): n- (1- (5-fluoro-2- ((4-methoxybenzyl) oxy) phenyl) ethyl) pyrazolo [1,5-a ] pyrimidin-5-amine (0.22mmol) was added to a 200mL pear-shaped bottle, dichloromethane (10mL) was added thereto, and trifluoroacetic acid (3.3mmol) was added dropwise. The reaction was carried out for 4h at room temperature with magnetic stirring. Neutralized and extracted with ethyl acetate. The organic phase was washed twice with saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography to obtain a white solid with a yield of 85%.
1H NMR(400MHz,DMSO-d6)δ12.63(s,1H),9.66–9.56(m,1H),8.44(d,J＝7.6Hz,1H),8.05(s,1H),7.97–7.82(m,3H),7.08–6.98(m,1H),6.89–6.76(m,2H),6.32(d,J＝7.6Hz,1H),5.57–5.46(m,1H),1.43(d,J＝6.8Hz,3H).
Example 2: preparation of N- (1- (5-fluoro-2-methoxyphenyl) ethyl) -3- (1H-pyrazol-4-yl) pyrazolo [1,5-a ] pyrimidin-5-amine (compound b2-5)
Figure BDA0002413469320000121
Step 1): n- (1- (5-fluoro-2- ((4-methoxybenzyl) oxy) phenyl) ethyl) pyrazolo [1,5-a ] pyrimidin-5-amine (13.0mmol) was added to a 200mL pear-shaped bottle, dichloromethane (50mL) was added thereto, and trifluoroacetic acid (195mmol) was added dropwise. The reaction was carried out for 4h at room temperature with magnetic stirring. Neutralized and extracted with ethyl acetate. The organic phase was washed twice with saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography to obtain a white solid with a yield of 85%.
Step 2): 4-fluoro-2- (1- (pyrazolo [1,5-a ] pyrimidin-5-ylamino) ethyl) phenol (11.0mmol), methyl iodide (16.5mmol), and cesium carbonate (22mmol) were added to a 200mL pear-shaped flask, to which DMF (50mL) was added. Heated overnight in an oil bath (100 ℃). The reaction was cooled to ambient temperature and the crude product was purified by column chromatography to give a white solid with a yield of 90%.
Step 3): n- (1- (5-fluoro-2-methoxyphenyl) ethyl) pyrazolo [1,5-a ] pyrimidin-5-amine (9.8mmol) was added to a 200mL pear-shaped bottle, and acetonitrile (50mL) was added thereto. N-iodosuccinimide (NIS, 14.85mmol) was added under magnetic stirring at room temperature. The reaction was carried out at room temperature for 1h and TLC monitored for completion. After removing acetonitrile under reduced pressure as much as possible, the mixture was diluted with 250mL of ethyl acetate and transferred to a separatory funnel. Washing with 1mol/L NaOH for 3 times, washing with saturated salt for two times, drying with anhydrous sodium sulfate, concentrating to obtain red oily crude product, and purifying by column chromatography to obtain light yellow solid with yield of 67%.
Step 4): n- (1- (5-fluoro-2-methoxyphenyl) ethyl) -3-iodopyrazolo [1,5-a ] pyrimidin-5-amine (0.50mmol), 1-Boc-pyrazole-4-boronic acid pinacol ester (0.75mmol), anhydrous potassium carbonate (2.00mmol), tetrakis (triphenylphosphine) palladium (0.05mmol) were added to a 100ml reaction tube, replaced with argon 3 times, and 10ml anhydrous DMF,2ml water were added. The reaction was carried out at 100 ℃ for 2h under argon atmosphere and the completion of the reaction was monitored by TLC. Cooled to 50 ℃, filtered through celite, and the filtrate was extracted with water and ethyl acetate. The organic phase was washed twice with saturated brine, dried over anhydrous sodium sulfate, concentrated to give a crude product as a black oil, which was purified by column chromatography to give a pale yellow solid with a yield of 50%.
1H NMR(400MHz,DMSO-d6)δ12.70(s,1H),8.44(d,J＝7.6Hz,1H),8.14–7.91(m,2H),7.82(s,2H),7.16–6.87(m,3H),6.32(d,J＝8.0Hz,1H),5.57–5.40(m,1H),3.95(s,3H),1.40(d,J＝7.2Hz,3H).
Example 3: preparation of N- (1- (5-fluoro-2- (fluoromethoxy) phenyl) ethyl) -3- (1H-pyrazol-4-yl) pyrazolo [1,5-a ] pyrimidin-5-amine (compound b3-6)
Figure BDA0002413469320000131
Step 1): the procedure of step 2 in example 2 was followed except that iodomethane was replaced with fluoroiodomethane.
Step 2): the procedure of step 3 in example 2 was used.
Step 3): the synthesis procedure used step 4 of example 2.
1H NMR(400MHz,DMSO-d6)δ12.68(s,1H),8.46(d,J＝7.6Hz,1H),8.04(d,J＝3.2Hz,1H),8.02(s,1H),7.86(s,1H),7.79(s,1H),7.27–7.02(m,3H),6.33(d,J＝7.6Hz,1H),6.07(s,1H),5.93(s,1H),5.58–5.47(m,1H),1.45(d,J＝6.8Hz,3H).
Example 4: preparation of N- (1- (2- (difluoromethoxy) -5-fluorophenyl) ethyl) -3- (1H-pyrazol-4-yl) pyrazolo [1,5-a ] pyrimidin-5-amine (compound b4-7)
Figure BDA0002413469320000132
Step 1): the procedure of step 2 in example 2 was used except that iodomethane was replaced with difluoromethonoiodomethane.
Step 2): the procedure of step 3 in example 2 was used.
Step 3): the synthesis procedure used step 4 of example 2.
1H NMR(400MHz,DMSO-d6)δ12.70(s,1H),8.50(d,J＝7.6Hz,1H),8.08(d,J＝1.6Hz,1H),8.03(d,J＝6.8Hz,1H),7.85(s,2H),7.52–7.07(m,4H),6.36(d,J＝7.6Hz,1H),5.50(t,J＝7.2Hz,1H),1.50(d,J＝6.8Hz,3H).
Example 5: preparation of N- (1- (2-ethoxy-5-fluorophenyl) ethyl) -3- (1H-pyrazol-4-yl) pyrazolo [1,5-a ] pyrimidin-5-amine (compound b5-11)
Figure BDA0002413469320000141
Step 1): the process of step 2 in example 2 was employed except that methyl iodide was replaced with ethyl iodide.
Step 2): the procedure of step 3 in example 2 was used.
Step 3): the synthesis procedure used step 4 of example 2.
1H NMR(400MHz,DMSO-d6)δ12.70(s,1H),8.47(d,J＝7.2Hz,1H),8.06(s,1H),8.00(d,J＝7.2Hz,1H),7.87(s,2H),7.09(dd,J＝9.6,3.0Hz,1H),7.04–6.91(m,2H),6.35(d,J＝7.2Hz,1H),5.65–5.53(m,1H),4.27–4.10(m,2H),1.51–1.41(m,6H).
Example 6: preparation of N- (1- (5-fluoro-2- (2-methoxyethoxy) phenyl) ethyl) -3- (1H-pyrazol-4-yl) pyrazolo [1,5-a ] pyrimidin-5-amine (compound b6-15)
Step 1): the procedure of step 2 in example 2 was followed except that methyl iodide was replaced with 2-iodoethyl methyl ether.
Step 2): the procedure of step 3 in example 2 was used.
Step 3): the synthesis procedure used step 4 of example 2.
1H NMR(400MHz,DMSO-d6)δ12.67(s,1H),8.45(d,J＝7.6Hz,1H),8.05(s,1H),7.94(d,J＝7.2Hz,1H),7.86(s,2H),7.16–6.87(m,3H),6.34(d,J＝7.6Hz,1H),5.63–5.46(m,1H),4.33–4.12(m,2H),3.85–3.68(m,2H),3.44–3.38(m,3H),1.44(d,J＝6.8Hz,3H).
Example 7: preparation of (R) -N- (1- (5-fluoro-2- (2-fluoroethoxy) phenyl) ethyl) -3- (1H-pyrazol-4-yl) pyrazolo [1,5-a ] pyrimidin-5-amine (compound b7-16)
Figure BDA0002413469320000151
Step 1): 5-Chloropyrazolo [1,5-a ] pyrimidine (19.1mmol), (R) -1- (5-fluoro-2- ((4-methoxybenzyl) oxy) phenyl) ethylamine (19.1mmol), anhydrous N-butanol (50mL) and N, N-diisopropylethylamine (DIPEA, 38.2mmol) were added to a 200mL pear-shaped vial. The mixture was heated overnight in an oil bath (140 ℃). Cooling the reaction to ambient temperature, concentrating under reduced pressure to remove N-butanol and N, N-Diisopropylethylamine (DIPEA) to obtain yellow oily crude product, purifying by column chromatography to obtain light yellow solid with yield of 68%
Step 2): (R) -N- (1- (5-fluoro-2- ((4-methoxybenzyl) oxy) phenyl) ethyl) pyrazolo [1,5-a ] pyrimidin-5-amine (13.0mmol) was added to a 200mL pear-shaped bottle, dichloromethane (50mL) was added thereto, and trifluoroacetic acid (195mmol) was added dropwise. The reaction was carried out for 4h at room temperature with magnetic stirring. Neutralized and extracted with ethyl acetate. The organic phase was washed twice with saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography to obtain a white solid with a yield of 85%.
Step 3): the procedure of step 2 in example 2 was employed except that methyl iodide was replaced with 1-fluoro-2-iodoethane.
Step 4): the procedure of step 3 in example 2 was used.
Step 5): the synthesis method is the same as step 4 in example 2.
1H NMR(400MHz,DMSO-d6)δ12.68(s,1H),8.48(d,J＝7.2Hz,1H),8.07(s,1H),8.02(d,J＝7.2Hz,1H),7.86(s,2H),7.16–6.95(m,3H),6.36(d,J＝7.6Hz,1H),5.64–5.54(m,1H),4.94(t,J＝3.6Hz,1H),4.82(t,J＝3.6Hz,1H),4.48–4.44(m,1H),4.41–4.36(m,1H),1.46(d,J＝7.2Hz,3H).
Example 8: preparation of (R) -N- (1- (5-fluoro-2- (2,2, 2-trifluoroethoxy) phenyl) ethyl) -3- (1H-pyrazol-4-yl) pyrazolo [1,5-a ] pyrimidin-5-amine (compound b8-30)
Figure BDA0002413469320000161
Step 1): the procedure of step 2 in example 2 was employed except that methyl iodide was replaced with 2-iodo-1, 1, 1-trifluoroethane.
Step 2): the procedure of step 3 in example 2 was used.
Step 3): the synthesis method is the same as step 4 in example 2.
1H NMR(400MHz,DMSO-d6)δ12.67(s,1H),8.46(d,J＝7.6Hz,1H),8.04(s,1H),8.02(d,J＝7.2Hz,1H),7.80(s,2H),7.08(m,3H),6.34(d,J＝7.6Hz,1H),5.52–5.44(m,1H),5.04–4.70(m,2H),1.43(d,J＝6.4Hz,3H).
Example 9: preparation of N- (1- (5-fluoro-2-propoxyphenyl) ethyl) -3- (1H-pyrazol-4-yl) pyrazolo [1,5-a ] pyrimidin-5-amine (compound b9-33)
Figure BDA0002413469320000162
Step 1): the process of step 2 in example 2 was employed except that methyl iodide was replaced with iodopropane.
Step 2): the procedure of step 3 in example 2 was used.
Step 3): the synthesis method is the same as step 4 in example 2.
1H NMR(400MHz,DMSO-d6)δ12.67(s,1H),8.45(d,J＝7.6Hz,1H),8.04(s,1H),7.98(d,J＝7.2Hz,1H),7.83(s,2H),7.10–6.90(m,3H),6.33(d,J＝7.6Hz,1H),5.61–5.48(m,1H),4.13(dt,J＝9.2,6.0Hz,1H),4.03(dt,J＝9.2,6.4Hz,1H),1.84(q,J＝6.8Hz,2H),1.42(d,J＝6.8Hz,3H),1.05(t,J＝7.6Hz,3H).
Example 10: preparation of N- (1- (5-fluoro-2- (3-fluoropropoxy) phenyl) ethyl) -3- (1H-pyrazol-4-yl) pyrazolo [1,5-a ] pyrimidin-5-amine (compound b10-34)
Figure BDA0002413469320000171
Step 1): the method of step 2 in example 2 was employed except that methyl iodide was replaced with 3-fluoro-1-iodopropane.
Step 2): the procedure of step 3 in example 2 was used.
Step 3): the synthesis method is the same as step 4 in example 2.
1H NMR(400MHz,DMSO-d6)δ12.70(s,1H),8.46(d,J＝7.6Hz,1H),8.06(s,1H),7.98(d,J＝7.2Hz,1H),7.86(s,2H),7.15–6.90(m,3H),6.35(d,J＝7.6Hz,1H),5.60–5.46(m,1H),4.74(tt,J＝5.6,2.8Hz,1H),4.62(td,J＝6.0,2.4Hz,1H),4.29–4.15(m,2H),2.30–2.17(m,2H),1.44(d,J＝6.8Hz,3H).
Example 11: preparation of 2- (2- (1- ((3- (1H-pyrazol-4-yl) pyrazolo [1,5-a ] pyrimidin-5-yl) amino) ethyl) -4-fluorophenoxy) acetonitrile (compound b11-36)
Figure BDA0002413469320000172
Step 1): the process of step 2 in example 2 was used except that iodomethane was replaced with iodoacetonitrile.
Step 2): the procedure of step 3 in example 2 was used.
Step 3): the synthesis procedure used step 4 of example 2.
1H NMR(400MHz,DMSO-d6)δ12.64(s,1H),8.48(d,J＝7.2Hz,1H),8.06(s,1H),7.86(s,1H),7.50(s,1H),7.41(s,1H),7.12(d,J＝9.0Hz,1H),7.09–6.92(m,2H),6.36(d,J＝7.2Hz,1H),5.66–5.49(m,1H),4.76–4.50(m,2H),1.51(d,J＝6.6Hz,3H).
Example 12: preparation of N- (1- (2- (cyclopropylmethoxy) -5-fluorophenyl) ethyl) -3- (1H-pyrazol-4-yl) pyrazolo [1,5-a ] pyrimidin-5-amine (compound b12-37)
Figure BDA0002413469320000181
Step 1): the procedure of step 2 in example 2 was employed except that methyl iodide was replaced with (iodomethyl) cyclopropane.
Step 2): the procedure of step 3 in example 2 was used.
Step 3): the synthesis method is the same as step 4 in example 2.
1H NMR(400MHz,DMSO-d6)δ12.68(s,1H),8.47(d,J＝7.6Hz,1H),8.06(s,1H),7.98(d,J＝7.2Hz,1H),7.85(s,2H),7.12–6.91(m,3H),6.36(d,J＝7.6Hz,1H),5.66–5.50(m,1H),4.01(qd,J＝10.0,6.8Hz,2H),1.46(d,J＝6.8Hz,3H),1.40–1.31(m,1H),0.65–0.54(m,2H),0.47–0.41(m,2H).
Example 13: preparation of N- (1- (2- (cyclopentyloxy) -5-fluorophenyl) ethyl) -3- (1H-pyrazol-4-yl) pyrazolo [1,5-a ] pyrimidin-5-amine (compound b13-39)
Figure BDA0002413469320000182
Step 1): the method of step 2 in example 2 was used except that methyl iodide was replaced with iodocyclopentane.
Step 2): the procedure of step 3 in example 2 was used.
Step 3): the synthesis method is the same as step 4 in example 2.
1H NMR(600MHz,DMSO-d6)δ12.66(s,1H),8.45(d,J＝7.2Hz,1H),8.05(s,1H),7.91(s,1H),7.85(s,2H),7.05–6.96(m,3H),6.37–6.28(m,1H),5.55–5.48(m,1H),4.99–4.89(m,1H),2.05–1.55(m,8H),1.40(d,J＝6.6Hz,3H).
Example 14: preparation of N- (1- (5-fluoro-2- ((tetrahydrofuran-3-yl) oxy) phenyl) ethyl) -3- (1H-pyrazol-4-yl) pyrazolo [1,5-a ] pyrimidin-5-amine (compound b14-40)
Figure BDA0002413469320000191
Step 1): the procedure of step 2 in example 2 was followed except that methyl iodide was replaced with 3-iodotetrahydrofuran.
Step 2): the procedure of step 3 in example 2 was used.
Step 3): the synthesis method is the same as step 4 in example 2.
1H NMR(400MHz,DMSO-d6)δ12.68(s,1H),8.47(d,J＝7.6Hz,1H),8.06(s,1H),7.96(t,J＝6.0Hz,1H),7.85(s,1H),7.84(s,1H),7.17–6.95(m,3H),6.36(d,J＝7.6Hz,1H),5.53–5.46(m,1H),5.21–5.16(m,1H),4.14–3.76(m,4H),2.37–2.00(m,2H),1.43(d,J＝6.8Hz,3H).
Example 15: preparation of N- (1- (5-fluoro-2- ((tetrahydro-2H-pyran-4-yl) methoxy) phenyl) ethyl) -3- (1H-pyrazol-4-yl) pyrazolo [1,5-a ] pyrimidin-5-amine (compound b15-42)
Figure BDA0002413469320000201
Step 1): the procedure of step 2 in example 2 was used except that methyl iodide was replaced with 4-iodomethyltetrahydropyran.
Step 2): the procedure of step 3 in example 2 was used.
Step 3): the synthesis method is the same as step 4 in example 2.
1H NMR(400MHz,DMSO-d6)δ12.65(s,1H),8.47(d,J＝7.6Hz,1H),8.06(s,1H),7.98(d,J＝6.8Hz,1H),7.84(s,2H),7.13–6.94(m,3H),6.35(d,J＝7.6Hz,1H),5.60–5.51(m,1H),4.11–3.99(m,1H),3.99–3.79(m,3H),3.32–3.30(m,2H),2.20–2.05(m,1H),1.77(t,J＝12.4Hz,2H),1.50–1.41(m,5H).
Example 16: preparation of N- (1- (5-fluoro-2- (2-morpholinoethoxy) phenyl) ethyl) -3- (1H-pyrazol-4-yl) pyrazolo [1,5-a ] pyrimidin-5-amine (compound b16-44)
Figure BDA0002413469320000202
Step 1): the procedure of step 2 in example 2 was followed except that methyl iodide was replaced with 2- (4-morpholine) ethyl bromide.
Step 2): the procedure of step 3 in example 2 was used.
Step 3): the synthesis method is the same as step 4 in example 2.
1H NMR(600MHz,DMSO-d6)δ12.68(s,1H),8.46(d,J＝7.8Hz,1H),8.04(s,1H),8.00–7.90(m,1H),7.83(s,2H),7.13–6.91(m,3H),6.33(d,J＝7.8Hz,1H),5.60–5.45(m,1H),4.32–4.13(m,2H),3.59–3.44(m,4H),2.81(t,J＝6.0Hz,2H),2.56–2.47(m,4H),1.43(d,J＝6.6Hz,3H).
Test example 1: in vitro biochemical level inhibition Protein Kinase (PK) activity experiment
The material and the method are as follows: wild type kinases such as TRKA, TRKB and TRKC, and mutant kinases such as TRKA-G595R, TRKA-G667C, TRKA-F589L, TRKC-G623R and TRKC-G696A, which are derived from Carna Biosciences 08-186, 08-187, 08-197; HTRF KinEASE TKkit (Cisbio 62TK0 PEC); 384 well plates (Greiner corporation); ATP (Life technologies PV3227), MgCl2(sigma) corporation; PHERAstar FS multifunctional microplate reader (BM)Company G); low speed centrifuge (StaiteXiangyi corporation); incubator (Binder Co.).
The control compounds selected were typical compound B disclosed in typical compound A, WO2007025540 disclosed in WO2007147647, and comparative compound C, comparative compound D, wherein comparative compound C (nuclear magnetic data is:1H NMR(400MHz,DMSO-d6) δ 12.78-12.66 (m,1H),8.45(D, J ═ 7.6Hz,1H),8.05(s,1H), 8.01-7.80 (m,3H),6.94(s,1H),6.90(D, J ═ 9.2Hz,1H),6.77(dt, J ═ 10.8,2.4Hz,1H), 6.56-6.21 (m,2H),5.20(D, J ═ 6.0Hz,1H),4.31(tt, J ═ 14.4,3.6Hz,2H),1.48(D, J ═ 6.8Hz,3H)) and comparative compound D (data:1H NMR(400MHz,DMSO-d6) δ 12.70(br,1H),8.44(d, J ═ 7.6Hz,1H),8.05(s,1H),7.94(d, J ═ 6.8Hz,2H),7.85(s,1H),7.35 to 7.14(m,3H),6.54 to 6.20(m,2H),5.28 to 5.11(m,1H),4.39 to 4.30(m,2H),1.48(d, J ═ 6.8Hz,3H)) the procedure used for the preparation of example 1 is as follows, except that the starting materials are different:
Figure BDA0002413469320000211
compound dissolution and preservation: preparing a test compound into a mother solution of 10mmol/L by using dimethyl sulfoxide (DMSO) according to the solubility, subpackaging and storing at-20 ℃;
preparing a compound working solution: before testing, the dispensed compound was removed from the freezer and diluted to 100 × the desired concentration with pure DMSO; then the compound was diluted to 4 x the desired concentration with deionized water;
1.33 Xpreparation of enzyme buffer (enzymic buffer): the 5 x enzyme buffer (from HTRF kit) was diluted 1.33 x with deionized water and 1.33 x final concentration of the corresponding ingredients was added: 1.33mmol/L Dithiothreitol (DTT) and 1.33mmol/L MnCl2、6.65mmol/L MgCl2And 39.9nmol/L SEB;
preparation of a kinase working solution: TRKA, TRKB and TRKC were diluted to 2 Xthe desired concentrations of 0.404 ng/. mu.L, 0.304 ng/. mu.L and 0.236 ng/. mu.L with 1.33 Xenzyme buffer;
preparing a substrate working solution: TK Substrate-biotin (from HTRF kit) and ATP (10mM) were diluted to 4X the desired final concentration in 1.33 Xenzyme buffer; the final ATP concentrations for TRKA, TRKB, and TRKC are: 3.727. mu. mol/L, 2.56. mu. mol/L and 2.526. mu. mol/L. TK Substrate-biotin (from HTRF KinEASE TKkit) final concentrations were: 0.2. mu. mol/L.
Preparation of detection working solution: 16.67. mu. mol/L of Streptavidin-XL665 (Streptavidin-XL665) were diluted to 4 Xthe desired final concentration with HTRF test buffer and then mixed with an equal volume of Antibody europium Cryptate (Antibody-Cryptate) (both from HTRF kits).
An enzyme reaction step: add 4. mu.L of kinase working solution to each well of a low volume 384 microwell plate, while adding 4. mu.L of 1.33 Xenzyme buffer as a Negative control (Negative); add 2. mu.l of compound working solution to the wells, while adding 2. mu.l of 8% DMSO aqueous solution as a zero compound concentration control (i.e., Positive control); incubating at 25 deg.C for 5 min; add 2. mu.L of substrate working solution to the wells to start the enzymatic reaction, shake the reaction for 30min at 37 ℃.
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 BDA0002413469320000233
integration delay (lag time)50 μ s
Integration time (Integration time) 400. mu.s
Flash Number of flash (Number of flashes)200
For the raw data read out per well, the ratio is 665nm/620 nm;
calculation of inhibition ratio:
Figure BDA0002413469320000231
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 percent50。
The experimental results are as follows: TRKA, TRKB and TRKC kinase activity half Inhibitory Concentration (IC)50,nM)
The invention provides the half Inhibitory Concentrations (IC) of compounds having the structure shown in formula (I) and control compounds on TRKA, TRKB and TRKC50) See table 1:
table 1: TRKA, TRKB and TRKC kinase inhibitory Activity of Compounds
Figure BDA0002413469320000232
Figure BDA0002413469320000241
As shown in table 1, the compounds provided by the present invention all exhibit excellent inhibitory activity against wild TRKA, TRKB and TRKC kinases, which is significantly superior to typical compound a, typical compound B, comparative compound C and comparative compound D.
Test example 2: drug metabolism study in rats
The compounds provided in the previous section of the examples were administered to rats as polyethylene glycol 400 in water (70%). For oral administration, rats were given a dose of 5 mg/kg. Approximately 0.3mL of each blood sample was collected 15, 30, 45min, 1, 2, 4,6, 8, 10, 24h after oral group administration into heparinized Eppendorf tubes, buffered on ice and centrifuged. The whole blood was centrifuged at 8000rpm for 5min and plasma was collected, transferred to a 96 well plate and stored at-20 ℃ until detection by LC-MS/MS.
The pharmacokinetic parameters after administration in rats were calculated using a non-compartmental model of the software WinNonlin software.
Peak concentration Cmax: adopting an actual measurement value;
time curve belowArea AUC0-t value: calculating by adopting a trapezoidal method; AUC0-∞＝AUC0-t+ Ct/ke, where Ct is the blood concentration at the last measurable time point and ke is the elimination rate constant;
elimination of half-life t1/2＝0.693/ke；
Absolute bioavailability of F ═ Doseiv*AUC0-t，ig/Doseig*AUC0-t，iv×100％。
Table 2 lists the pharmacokinetic parameters of the compounds of the invention in rats after intravenous administration. The results indicate that the compounds of the invention have good pharmacokinetic properties, including ideal Clearance (CL), half-life (t)1/2) Peak concentration (C)max) And exposure (AUC)0-t)。
Table 3 lists the pharmacokinetic parameters of the compounds of the invention in rats after oral administration. The results indicate that the compounds of the invention have good pharmacokinetic properties, including ideal Clearance (CL), half-life (t)1/2) Peak concentration (C)max) Exposure (AUC)0-t) And oral bioavailability.
Table 2: primary pharmacokinetic parameters for rat intravenous administration of Compound b8-30
Figure BDA0002413469320000251
Table 3: primary pharmacokinetic parameters for oral rat administration of Compound b8-30
Figure BDA0002413469320000252
Test example 3: inhibitory Activity of Compounds of the present invention against five TRK kinase mutants
This test example was conducted by the same test method as in test example 1.
The results of this test example are shown in Table 4.
TABLE 4
Figure BDA0002413469320000253
As can be seen from Table 4, the inhibitory activity of the compound of the invention on five TRK kinase mutants is superior to that on wild TRK kinase, and the compound is expected to effectively overcome the tumor drug resistance reported in clinic.
Test example 4: antitumor Activity of the Compounds of the invention on nude mouse xenograft tumor model
The efficacy of the compounds of the invention was assessed by a standard murine model of transplanted tumors. Human NSCLC H2228 was cultured, collected, and then subcutaneously inoculated into 5-6 week-old female nude mice (BALB/c, Shanghai Ling Chang Biotech Co., Ltd.) on the posterior flank. When the tumor volume reaches 100-3At this time, animals were randomly divided into a solvent control group (70% PEG-400 in water) and a compound group (6 animals per group). Animals were subsequently gavaged with the compounds of the examples (corresponding doses, dissolved in 70% PEG-400 in water), starting anywhere from 0 to 13 days after tumor cell inoculation, and were performed once or twice daily in the experiment.
The experimental index is to examine the influence of the compound of the embodiment on the growth of the tumor, and the specific index is T/C% or tumor inhibition rate TGI (%).
Tumor diameter was measured twice weekly with a vernier caliper and tumor volume (V) was calculated as:
V＝1/2×a×b2wherein a and b represent length and width, respectively.
T/C(％)＝(T-T0)/(C-C0) X 100 where T, C is the tumor volume at the end of the experiment; t is0、C0Tumor volume at the beginning of the experiment.
Tumor inhibition rate (TGI) (%) 100-T/C (%).
When tumors regress, tumor inhibition rate (TGI) (%) 100- (T-T)0)/T0×100
If the tumor is reduced from the initial volume, i.e. T<T0Or C<C0When, it is defined as partial tumor regression (PR); if swelling, the swelling occursComplete tumor disappearance, defined as complete tumor regression (CR).
Comparison between two groups of tumor volumes was tested using a two-tailed Student's t test, with P <0.05 defined as a statistically significant difference.
The results of this test example are shown in Table 5.
Hereinafter BID means twice-a-day dosing and QD means once-a-day dosing.
TABLE 5
Grouping Administration of drugs Tumor inhibition rate D14 Partial regression
Solvent(s) BID,D0-7 - -
Compound b 8-3025 mg/kg BID,D0-13 127％ 6/6
Compound b 8-3050 mg/kg QD,D0-10 110％ 3/6
As can be seen from table 5, the compounds of the present invention showed excellent antitumor activity on the human non-small cell lung cancer H2228 nude mouse xenograft tumor model. Wherein, the compound b8-30(25mg/kg, BID × 14) obviously inhibits the growth of subcutaneous transplantation tumor of human non-small cell lung cancer H2228 nude mice, when the compound is administered to D7, the tumor inhibition rate is 95 percent, the dose is increased from D8 to 50mg/kg, the tumor is regressed, and when the experiment is finished (D14), the tumor inhibition rate is 127 percent, and the tumor 6/6 is partially regressed; the compound b8-30(50mg/kg, QD, QD × 11) showed a tumor suppression rate of 96% (D7) against subcutaneous transplantable tumors in H2228 nude mice, with a dose increase from D8 to 100mg/kg, with regression of the tumors occurring, to the end of the experiment (D14), with a tumor suppression rate of 110%, with partial regression of 3/6 tumors occurring.
The above results indicate that the pyrazole-substituted pyrazolopyrimidine compound having the structure shown in formula (I) or a pharmaceutically acceptable salt thereof, or a stereoisomer, a geometric isomer, a tautomer, a nitrogen oxide, a hydrate, a solvate, a metabolite, or a prodrug thereof, shows excellent inhibitory activity against TRK kinase, and at the same time, can show good antitumor activity at an animal level.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

Claims (12)

1. A pyrazole-substituted pyrazolopyrimidine compound having a structure represented by formula (I) or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof,
Figure 399729DEST_PATH_IMAGE001
，
wherein, in the formula (I),
R1、R2、R3and R4Each independently selected from H, halogen, C1-12Alkyl of (2), C substituted by 1-6 halogens1-12Alkyl groups of (a);
R5selected from H, C1-12Alkyl of (2), C substituted by 1-6 halogens1-12Alkyl, hydroxy-substituted C of1-12Alkyl, alkoxy-substituted C2-12Alkyl, cyano-substituted C2-12Alkyl of (a), C containing 1-3 heteroatoms selected from N, O and S2-12Cycloalkyl groups of (a);
R6selected from H, C1-12Alkyl, hydroxy-substituted C of1-12The alkyl group and the halogen of (a),
wherein,
said alkoxy-substituted C2-12Represents a group having 2 to 12 carbon atoms in total, and the alkoxy group is substituted C2-12The alkyl group of (A) has the formula-R7OR8Wherein, said R7And said R8The sum of the number of carbon atoms in (A) is 2 to 12, and the R7Directly connecting with the phenoxy in the pyrazole substituted pyrazolopyrimidine compound with the structure shown in the formula (I);
said cyano-substituted C2-12Represents an alkyl group having 2 to 12 carbon atoms in total, the cyano group being substituted C2-12Is a straight-chain alkyl group, a branched-chain alkyl group or a cyclic alkyl group, and the cyano group is substituted C2-12Is substituted by cyano, and the carbon atom in said cyano is taken to account for C substituted by said cyano2-12The total number of carbon atoms in the alkyl group of (a);
c containing 1-3 heteroatoms selected from N, O and S2-12Represents cycloalkyl having a total number of carbon atoms of 2 to 12, forming said C containing 1 to 3 heteroatoms selected from N, O and S2-121-3 of the heteroatoms of cycloalkyl of (a) are selected from the group consisting of N, O and S, and form the C containing 1-3 heteroatoms selected from the group consisting of N, O and S2-12Optionally contains an alkyl substituent on the atom, the carbon atom contained in the alkyl substituent being counted in the group containing 1 to 3 substituentsC of a heteroatom from N, O and S2-12And said C containing 1 to 3 hetero atoms selected from N, O and S2-12Any H in the cycloalkyl group of (a) is optionally substituted or unsubstituted with a substituent, if substituted, each independently selected from at least one of halogen, hydroxyl, nitro and mercapto.
2. The compound according to claim 1, wherein, in formula (I),
R1、R2、R3and R4Each independently selected from H, fluorine, chlorine, bromine, C1-8Alkyl of (2), C substituted by 1-6 halogens selected from fluorine, chlorine and bromine1-8Alkyl groups of (a);
R5selected from H, C1-8Alkyl of (2), C substituted by 1-6 halogens selected from fluorine, chlorine and bromine1-8Alkyl, hydroxy-substituted C of1-8Alkyl, alkoxy-substituted C2-8Alkyl, cyano-substituted C2-8Alkyl of (a), C containing 1-3 heteroatoms selected from N, O and S2-10Cycloalkyl groups of (a);
R6selected from H, C1-8Alkyl, hydroxy-substituted C of1-8Alkyl and halogen.
3. The compound according to claim 2, wherein, in formula (I),
R1、R2、R3and R4Each independently selected from H, fluorine, chlorine, bromine, C1-6Alkyl of (2), C substituted by 1 to 4 halogens selected from fluorine, chlorine and bromine1-6Alkyl groups of (a);
R5selected from H, C1-6Alkyl of (2), C substituted by 1 to 4 halogens selected from fluorine, chlorine and bromine1-6Alkyl, hydroxy-substituted C of1-6Alkyl, alkoxy-substituted C2-8Alkyl, cyano-substituted C2-6Alkyl of (a), C containing 1-3 heteroatoms selected from N, O and S2-8Cycloalkyl groups of (a);
R6selected from H, C1-6Alkyl, hydroxy-substituted C of1-6Alkyl and halogen.
4. The compound according to claim 1, wherein, in formula (I),
R1、R3and R4Each independently selected from H, halogen, C1-12Alkyl of (2), C substituted by 1-6 halogens1-12Alkyl groups of (a);
R2is halogen;
R5selected from H, C1-12Alkyl of (2), C substituted by 1-6 halogens1-12Alkyl, hydroxy-substituted C of1-12Alkyl, alkoxy-substituted C2-12Alkyl, cyano-substituted C2-12Alkyl of (a), C containing 1-3 heteroatoms selected from N, O and S2-12Cycloalkyl groups of (a);
R6is selected from C1-12Alkyl, hydroxy-substituted C of1-12Alkyl and halogen.
5. The compound according to claim 4, wherein, in formula (I),
R1、R3and R4Each independently selected from H, fluorine, chlorine, bromine, C1-8Alkyl of (2), C substituted by 1-6 halogens selected from fluorine, chlorine and bromine1-8Alkyl groups of (a);
R2is F;
R5selected from H, C1-8Alkyl of (2), C substituted by 1-6 halogens selected from fluorine, chlorine and bromine1-8Alkyl, hydroxy-substituted C of1-8Alkyl, alkoxy-substituted C2-8Alkyl, cyano-substituted C2-8Alkyl of (a), C containing 1-3 heteroatoms selected from N, O and S2-10Cycloalkyl groups of (a);
R6is selected from C1-8Alkyl, hydroxy-substituted C of1-8Alkyl and halogen.
6. The compound according to claim 4, wherein, in formula (I),
R1、R3and R4Are all H; r2Is F;
R5selected from H, C1-8Alkyl of (2), C substituted by 1-6 halogens selected from fluorine, chlorine and bromine1-8Alkyl, hydroxy-substituted C of1-8Alkyl, alkoxy-substituted C2-8Alkyl, cyano-substituted C2-8Alkyl of (a), C containing 1-3 heteroatoms selected from N, O and S2-10Cycloalkyl groups of (a);
R6is selected from C1-8Alkyl, hydroxy-substituted C of1-8Alkyl and halogen.
7. The compound of claim 1, wherein the pyrazole-substituted pyrazolopyrimidine compound is at least one of the following compounds, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof:
Figure 129788DEST_PATH_IMAGE002
Figure 533087DEST_PATH_IMAGE003
Figure 57610DEST_PATH_IMAGE004
Figure 93830DEST_PATH_IMAGE005
Figure 627579DEST_PATH_IMAGE006
。
8. use of a pyrazole-substituted pyrazolopyrimidine compound having a structure represented by formula (I) or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof according to any one of claims 1 to 7 in the preparation of a medicament for the prevention and/or treatment of a TRK kinase-mediated disease.
9. A pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient or diluent, and as an active ingredient a pyrazole-substituted pyrazolopyrimidine compound having a structure represented by formula (I) or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof according to any one of claims 1 to 7.
10. Use of the pharmaceutical composition according to claim 9 for the preparation of a medicament for the prevention and/or treatment of a TRK kinase receptor mediated disease.
11. Use of a pyrazole-substituted pyrazolopyrimidine compound having a structure represented by formula (I) or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof according to any one of claims 1 to 7, or a pharmaceutical composition according to claim 9 for the preparation of a medicament for the prevention and/or treatment of tumors.
12. The use of claim 11, wherein the tumor is at least one of breast cancer, large intestine cancer, lung cancer, thyroid cancer, skin cancer, bone cancer, melanoma, leukemia, salivary gland tumor, neuroendocrine tumor, lymphoma, brain tumor, neuroblastoma, ovarian cancer, pancreatic cancer, mesothelioma, esophageal cancer, lung sarcoma, medulloblastoma, glioblastoma, colon cancer, liver cancer, retinoblastoma, kidney cancer, bladder cancer, osteosarcoma, stomach cancer, uterine cancer, vulval cancer, small intestine cancer, prostate cancer, bile duct cancer, ureter cancer, adrenocortical cancer, or head and neck cancer.
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