CN114573591A - Substituted pyrrolopyrimidine compound and application thereof - Google Patents
Substituted pyrrolopyrimidine compound and application thereof Download PDFInfo
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- C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
- C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
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Abstract
The invention discloses a pyrrolopyrimidine compound shown as a formula (I), a stereoisomer or pharmaceutically acceptable salt thereof. The invention also provides a preparation method of the compound, a stereoisomer or pharmaceutically acceptable salt thereof, and application of the compound in preparing a medicament for preventing or treating C-Kit and/or PDGFR, particularly mutant C-Kit and/or mutant PDGFR mediated diseases. The compound of the invention has better pharmacokinetic property and stronger clinical medication compliance.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to a substituted pyrrolopyrimidine compound, a preparation method thereof, and application thereof in preventing and treating C-Kit and/or PDGFR mediated diseases.
Background
KIT belongs to the group III receptor tyrosine kinase family members encoded by the proto-oncogene C-KIT. The KIT protein coded by the C-KIT gene consists of an intracellular tyrosine kinase region, a transmembrane region and an extracellular region with a ligand binding site, and after the KIT receptor is combined with a ligand SCF (stem cell factor), transcription factors in cells are finally activated by forming dimers and activating downstream signals comprising Ras, Raf, MAPK pathways and the like, so that the gene expression is regulated, and the cell growth and proliferation are controlled.
Imatinib is a tyrosine protease inhibitor that blocks the function of the tyrosine protein kinase KIT receptor, thereby inhibiting tumor formation. It has been shown that the position of the KIT mutation can affect the response of tumor patients to imatinib. The KIT mutation is positioned in a regulation region, namely a non-tyrosinase structural region, so that an inhibitor can effectively block enzyme sites, the partial remission rate of imatinib is obviously improved, the average life cycle is prolonged, and the disease progress is slow.
Such as: gastrointestinal stromal tumors are a more common malignant gastrointestinal tumor. Pathological studies prove that the kinase c-Kit is an effective target for treating gastrointestinal stromal tumors, plays an important role in the process of cell metastasis and differentiation, and the overexpression of the kinase c-Kit is closely related to the gastrointestinal stromal tumors. Currently, imatinib is the first-line clinical drug for treating gastrointestinal stromal tumors, but after long-term administration, nearly 80-85% of patients develop drug resistance, wherein the main drug resistance factor is the drug resistance mutation of C-Kit kinase, so that the development of a compound for inhibiting wild-type C-Kit and mutant C-Kit is very urgent for clinical treatment.
The patent CN201810040590 discloses a C-Kit inhibitor and application thereof, and the disclosed compound III of the embodiment has strong inhibition effect on wild C-Kit (WT), mutant C-Kit (D861V) and PDGFR alpha (D842V) kinase; not only has therapeutic effect on the diseases mediated by the wild type c-Kit, but also has the same effect and better effect on the diseases mediated by the mutant c-Kit. However, this compound is metabolized faster in vivo and longer acting C-Kit inhibitors are clinically desirable.
Disclosure of Invention
In view of the deficiencies of the prior art, it is an object of the present invention to provide deuterated pyrrolopyrimidine compounds having a longer half-life useful for the prevention and treatment of C-Kit and/or PDGFR mediated diseases including gastrointestinal stromal tumors, lupus, leukemia, mast cell disease, melanoma, seminoma, rheumatoid arthritis, multiple sclerosis, frailty, multiple myeloma and pancreatic cancer, etc.
The invention provides a compound shown as a formula (I), a stereoisomer or a pharmaceutically acceptable salt thereof:
wherein R1, R2, R3, R4, R5, R6 are independently hydrogen or deuterium, and R1, R2, R3, R4, R5, R6 are not all hydrogen.
Further, the compound represented by the formula (I), a stereoisomer or a pharmaceutically acceptable salt thereof has the following structure:
wherein at least three of R1, R2, R3, R4, R5 and R6 are deuterium, and the rest are hydrogen.
Still further, the compound represented by the above formula (I), a stereoisomer or a pharmaceutically acceptable salt thereof, has the following structure:
Still further, the compound of formula (I) above, a stereoisomer or a pharmaceutically acceptable salt thereof, has the structure:
Still further, the compound of formula (I) above, a stereoisomer or a pharmaceutically acceptable salt thereof, has the structure:
Further, the above compound, its stereoisomer or pharmaceutically acceptable salt thereof, is characterized in that the salt includes, but is not limited to, acetate, ascorbate, benzoate, benzenesulfonate, citrate, fumarate, hydrochloride, hydrobromide, maleate, methanesulfonate, nitrate, oxalate, phosphate, succinate or sulfate.
Further, the above compounds also include nitrogen oxides, hydrates, solvent compounds or metabolites thereof.
Further, the invention provides a pharmaceutical composition containing the compound, the stereoisomer, the nitrogen oxide, the hydrate, the solvent compound, the metabolite or the pharmaceutically acceptable salt thereof, and the composition further comprises a pharmaceutically acceptable carrier and/or an auxiliary material.
Furthermore, the pharmaceutically acceptable carriers or adjuvants include, but are not limited to, oral preparation adjuvants or parenteral administration adjuvants, and the administration route can be oral administration, injection, etc.; the administration dosage form can be liquid dosage form, solid dosage form, the liquid dosage form can be syrup, injection solution, non-aqueous solution, suspension or emulsion, the solid dosage form can be tablet, lozenge, capsule, dripping pill, granule, powder, cream, solution, suppository, dispersible powder such as lyophilized powder for injection, aerosol, etc.; the adjuvants used include, but are not limited to: lactose, calcium carbonate, calcium phosphate, sodium phosphate, starch, cyclodextrin, sucrose, mannitol, microcrystalline cellulose sodium, calcium sulfate, water, ethanol, propanol, glycerol, propylene glycol, isopropanol, glucose, sodium carboxymethylcellulose, potassium phosphate, dried starch, agar powder, calcium carbonate, sodium bicarbonate, sodium dodecyl sulfate, methyl cellulose, glyceryl tristearate, hydrogenated oil, talc, triethylamine magnesium stearate, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin.
Further, the invention provides application of the compound, the stereoisomer, the nitric oxide, the hydrate, the solvent compound, the metabolite or the pharmaceutically acceptable salt thereof in preparing a medicament for preventing or treating C-Kit and/or PDGFR mediated diseases.
Further, the above-mentioned C-Kit and/or PDGFR mediated diseases include gastrointestinal stromal tumors, lupus, leukemia, mastocytosis, melanoma, seminoma, rheumatoid arthritis, multiple sclerosis, gradually freezing, multiple myeloma or pancreatic cancer.
Furthermore, the C-Kit and/or PDGFR are mutant C-Kit and/or PDGFR.
Has the advantages that: the compound of the invention has better pharmacokinetic property, slower metabolism, longer half-life and longer time for exerting the drug effect, is more suitable for clinical medication and provides a better choice for tumor patients.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are provided for illustration only and are not intended to limit the scope of the present invention, and any equivalent replacement in the field made in the light of the present disclosure is included in the scope of the present invention.
The compounds of the present invention, stereoisomers or pharmaceutically acceptable salts thereof can be prepared by selecting the synthetic routes of the examples, and the conventional conditions of the reaction raw materials and the reaction solvent are adjusted according to the requirements of substituents or salt formation, which can be realized by those skilled in the art based on the present disclosure. In addition, the column chromatography of the present invention refers to silica gel column chromatography without specific description, and the elution solvent without specific description may be combined with a reaction solvent and common knowledge or common means of those skilled in the art to determine a single or mixed elution solvent.
The structure of the compound is determined by nuclear magnetic resonance1H NMR) or liquid chromatography-mass spectrometry (LC-MS).
The LC-MS is Agilent G6120B (matched with liquid phase Agilent 1260); nuclear magnetic resonance apparatus (1H NMR is Bruker AVANCE-300, nuclear magnetic resonance (C)1H NMR) shifts (δ) Given in parts per million (ppm), the assay solvent is DMSO, the internal standard is Tetramethylsilane (TMS), and the chemical shifts are given as 10~6(ppm) is given as a unit.
The term "room temperature" in the present invention means a temperature of 10 to 25 ℃.
Example 1: preparation of Compound 1
Step 1: synthesis of Compound c
A single vial was charged with compound a (2.86 g, 10 mmol) and anhydrous THF (200 mL) and stirred to dissolve, compound b (2.42 g, 20 mmol) and tetraethyltitanate (6.83 g, 30 mmol) were added and the mixture was allowed to warm to 70 ℃ under nitrogen and stirred overnight. After TLC monitoring of the reaction completion, water (100 mL) was added, ethyl acetate was extracted (300 mL) three times, the organic phases were combined, washed twice with water, twice with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered and concentrated. The product was isolated by column chromatography to give 2.43 g of a yellow solid. Yield: 62.5 percent.
MS：m/z (ES): 390.5[M+1]。
Step 2: synthesis of Compound D
Magnesium powder (280 mg, 11.6 mmol) was charged into a 50mL two-necked flask, evacuated and purged with nitrogen, diethyl ether (10mL) was added, deuterated iodomethane CD3I (1.44 g, 10 mmol) was added dropwise, after completion of the dropwise addition, the temperature was raised to reflux, and the reaction was maintained under stirring for 2 hours. And cooling to room temperature.
Adding the compound c (778 mg, 2.0 mmol) and anhydrous THF (30 mL) into another 50mL two-necked flask, stirring for clarification, vacuumizing, protecting with nitrogen, cooling to 0 ℃, slowly adding the prepared CD3MgI ether solution dropwise, continuing stirring, and reacting at 0 ℃ for 2 hours. The reaction was quenched by the addition of saturated aqueous ammonium chloride (25mL), extracted with ethyl acetate (50mLx3), and the organic phases combined, washed with water (50mL), saturated brine (50mL), dried over anhydrous sodium sulfate, filtered, concentrated and passed through a column to give 511 mg of a white solid in 62.6% yield.
MS：m/z (ES):409.5[M+1]。
And step 3: synthesis of Compound E
Compound D (408 mg, 1.0 mmol) and methanol (15mL) were added to a 50mL magnetically stirred two-necked flask, stirred until clear, added with a solution of hydrogen chloride in dioxane (12 mL, 8M), and then stirred at room temperature under nitrogen for 1 hour. The solvent was evaporated under reduced pressure, dichloromethane (30 mL) and saturated aqueous sodium bicarbonate (20 mL) were added, stirring was carried out for 2 minutes, the organic layer was separated, the aqueous phase was extracted with dichloromethane (30mL × 2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give 212mg of a white solid with a yield of 69.7%.
MS：m/z (ES):305.5[M+1]。
And 4, step 4: synthesis of Compound 1
Compound E (200 mg, 0.66 mmol) and 1, 4-dioxane (10mL) were added to a 50mL single neck flask equipped with a magnetic stirrer, stirred to clear, DIPEA (131 mg, 1 mmol) and compound f (128mg, 0.55mmol) were added, and the reaction was stirred at room temperature under nitrogen overnight. The solvent was distilled off under reduced pressure, and the residue was passed through a silica gel column to give 255 mg of a white solid in a yield of 92.8%.
MS：m/z (ES): 502.5[M+1]。
1H NMR(300MHz, DMSO-d6): 8.58 (m,2H), 8.22 (m,1H),7.91-7.96 (m,2H), 7.19-7.28 (m,4H), 6.41(s,1H), 5.10 (t,2H), 5.01(s,1H)，3.95(m,3H)，3.26 (m,4H)，3.18-3.22（(m,4H)。
Example 2: preparation of Compound 2
100mg of compound 1 was taken and separated by chiral HPLC to give 40mg of a white solid as isomer compound 2.
MS m/z (ES): 502.5[M+1]。
1H NMR(300MHz, DMSO-d6): 8.59 (m,2H), 8.21 (m,1H),7.91-7.96 (m,2H), 7.19-7.28 (m,4H), 6.41(s,1H), 5.10 (t,2H), 5.01(s,1H)，3.95(m,3H)，3.26 (m,4H)，3.18-3.21（(m,4H)）。
Example 3: preparation of Compound 3
100mg of compound 1 was taken and separated by chiral HPLC to give 40mg of a white solid as isomer compound 3.
MS m/z (ES): 502.5[M+1]。
1H NMR(300MHz, DMSO-d6): 8.59 (m,2H), 8.21 (m,1H),7.91-7.96 (m,2H), 7.19-7.28 (m,4H), 6.41(s,1H), 5.10 (t,2H), 5.01(s,1H)，3.95(m,3H)，3.26 (m,4H)，3.18-3.21(m,4H)。
Example 4: preparation of Compound 4
Step 1: synthesis of Compound G
Compound h (10.0g, 51.5mmol) and anhydrous THF (50mL) were dissolved with stirring in a 150mL two-necked flask equipped with a magnetic stirrer, NaH (4.5g, 103.0mmol, 55% w/w) was added, then stirred under nitrogen for 10 min, CD3I (14.9 g, 103.0mmol) was added dropwise, the ice bath was removed after the addition, and the reaction was stirred under nitrogen at room temperature overnight. The reaction was quenched by addition of methanol (10mL), the reaction was diluted with ethyl acetate (50mL), the insoluble material was filtered off, the filtrate was concentrated and passed through a silica gel column to give 6.6g of a colorless oil in 60.7% yield.
MS：m/z (ES): 212.1[M+1]。
Step 2: synthesis of Compound K
Compound j (2.14G, 10 mmol), compound G (4.22G, 20 mmol), cesium carbonate (9.77G, 30 mmol), Pd (dppf) Cl2. CH2 Cl2 (816 mg, 1 mmol) was placed in 100mL equipped with magnetic stirringIn a one-necked flask containing 1, 4-dioxane (80mL), ethanol (15mL) and water (10mL), vacuum was applied and replaced with nitrogen gas 3 times, the mixture was heated to 110 ℃ overnight under nitrogen atmosphere, cooled to room temperature, concentrated to dryness under reduced pressure, and subjected to column chromatography to obtain 1.36 g of a yellow solid with a yield of 62.3%.
MS：m/z (ES):219.2[M+1]。
And step 3: synthesis of Compound F
Compound K (1.36 g, 6.23 mmol) and phosphorus oxychloride (13 mL) were charged to a 50mL single-necked flask, heated to 95 ℃ under nitrogen, and the reaction was stirred for 5 hours. After cooling to room temperature, the residual phosphorus oxychloride was evaporated under reduced pressure, methylene chloride (30 mL) and a saturated aqueous sodium bicarbonate solution (10mL) were added to separate an organic layer, and an aqueous layer was extracted with methylene chloride (20 mL) to obtain an organic phase, which was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography to obtain 476 mg of a white solid with a yield of 31.3%.
MS：m/z (ES): 237.5[M+1]。
And 4, step 4: synthesis of Compound d
Magnesium powder (280 mg, 11.5 mmol) was charged to a 50mL two-necked flask, evacuated and purged with nitrogen, ether (10mL) was added under high pressure, and methyl iodide (1.42 g, 10 mmol) was added dropwise. After the addition, the temperature was raised to reflux and the reaction was maintained under stirring for 2 hours. And cooling to room temperature.
Adding the compound c (778 mg, 2.0 mmol) and anhydrous THF (30 mL) into another 50mL two-necked flask, stirring for clarification, vacuumizing, protecting with nitrogen, cooling to 0 ℃, slowly adding the prepared CH3MgI ether solution dropwise, continuing stirring, and reacting at 0 ℃ for 2 hours. The reaction was quenched by the addition of saturated aqueous ammonium chloride (25mL), extracted with ethyl acetate (50mL x3), and the organic phases combined, washed with water (50mL), saturated brine (50mL), dried over anhydrous sodium sulfate, filtered, concentrated, and column chromatographed to give 511 mg of white solid in 63.1% yield.
MS：m/z (ES):406.5[M+1]。
And 5: synthesis of Compound e
Compound d (408 mg, 1.0 mmol) and methanol (15mL) were added to a 50mL magnetically stirred two-necked flask, stirred until clear, added with a solution of hydrogen chloride in dioxane (12 mL, 8M), and then stirred at room temperature under nitrogen for 1 hour. The solvent was evaporated under reduced pressure, dichloromethane (30 mL) and saturated aqueous sodium bicarbonate (20 mL) were added, stirring was carried out for 2 minutes, the organic layer was separated, the aqueous phase was extracted with dichloromethane (30mL × 2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give 212mg of a white solid with a yield of 70.3%.
MS：m/z (ES):302.3[M+1]。
Step 6: synthesis of Compound 4
Compound e (200 mg, 0.66 mmol) and 1, 4-dioxane (10mL) were added to a 50mL single-necked flask equipped with a magnetic stirrer, stirred to clear, DIPEA (131 mg, 1 mmol) and compound F (130 mg, 0.55mmol) were added, and the reaction was stirred at room temperature under nitrogen overnight. The solvent was distilled off under reduced pressure, and the residue was passed through a silica gel column to give 252 mg of a white solid in a yield of 91.5%.
MS：m/z (ES): 502.2[M+1]。
1H NMR：(300MHz，DMSO-d6):8.59(m，2H)，8.21(m，1H)，7.91-7.96(m，2H)，7.19-7.28(m，4H)，6.41(s，1H)，5.10(t，2H)，5.01(s，1H)，3.26(m，4H)，3.18-3.21(m，4H)，1.76(s，3H)。
Example 5: preparation of Compound 5
100mg of compound 4 was taken and separated by chiral HPLC to give 40mg of a white solid as isomer compound 5.
MS m/z (ES): 502.2[M+1]。
1H NMR：(300MHz，DMSO-d6):8.59(m，2H)，8.21(m，1H)，7.91-7.96(m，2H)，7.19-7.28(m，4H)，6.41(s，1H)，5.10(t，2H)，5.01(s，1H)，3.26(m，4H)，3.18-3.21(m，4H)，1.76(s，3H)。
Example 6: preparation of Compound 6
100mg of compound 4 was taken and separated by chiral HPLC to give 40mg of a white solid as isomer compound 6.
MS m/z (ES): 502.2[M+1]。
1H NMR：(300MHz，DMSO-d6):8.59(m，2H)，8.21(m，1H)，7.91-7.96(m，2H)，7.19-7.28(m，4H)，6.41(s，1H)，5.10(t，2H)，5.01(s，1H)，3.26(m，4H)，3.18-3.21(m，4H)，1.76(s，3H)。
Example 7: preparation of Compound 7
Compound E (200 mg, 0.66 mmol) and 1, 4-dioxane (10mL) were added to a 50mL single neck flask equipped with a magnetic stirrer, stirred to clear, DIPEA (131 mg, 1 mmol) and compound F (128mg, 0.54 mmol) were added, and the reaction was stirred at room temperature under nitrogen overnight. The solvent was distilled off under reduced pressure, and the residue was passed through a silica gel column to give 255 mg of a white solid in a yield of 93.4%.
MS：m/z (ES): 505.5[M+1]。
1H NMR：(300MHz，DMSO-d6):8.59(m，2H)，8.21(m，1H)，7.91-7.96(m，2H)，7.19-7.28(m，4H)，6.41(s，1H)，5.10(t，2H)，5.01(s，1H)，3.26(m，4H)，3.18-3.21(m，4H)。
Example 8: preparation of Compound 8
100mg of compound 7 was taken and separated by chiral HPLC to give 42mg of a white solid as isomer compound 8.
MS m/z (ES): 505.5[M+1]。
1H NMR：(300MHz，DMSO-d6):8.59(m，2H)，8.21(m，1H)，7.91-7.96(m，2H)，7.19-7.28(m，4H)，6.41(s，1H)，5.10(t，2H)，5.01(s，1H)，3.26(m，4H)，3.18-3.21(m，4H)。
Example 9: preparation of Compound 9
100mg of compound 7 was taken and separated by chiral HPLC to give 42mg of a white solid as the isomer compound 9.
MS m/z (ES): 505.5[M+1]。
1H NMR：(300MHz，DMSO-d6):8.59(m，2H)，8.21(m，1H)，7.91-7.96(m，2H)，7.19-7.28(m，4H)，6.41(s，1H)，5.10(t，2H)，5.01(s，1H)，3.26(m，4H)，3.18-3.21(m，4H)。
Comparative example: synthesized according to a preparation method disclosed in patent CN201810040590
Test example 1: in vitro enzyme inhibition Activity assay
The test principle is as follows:
detection of chemiluminescence in Assay plates by Envision as IC of the compound50Values were used as indices to evaluate the inhibition of c-Kit (WT), c-Kit mutation D861V and PDGFR alpha mutation D842V kinase by the compounds.
The test method comprises the following steps:
comparative and example compounds 2, 3, 5, 8 were diluted to 1mM in DMSO until use. Before use, each test compound is diluted 25 times with ultrapure water and equilibrated at room temperature for 30min for use. In the Assay plate, 3-fold gradient dilution is carried out on each compound to be detected, and 11 concentration gradients with final concentration from 1uM to 0.017nM are obtained; adding a c-Kit (WT) or c-Kit (D861V) or PDGFR alpha (D842V) kinase mixture into the Assay plate, incubating at room temperature for 30min, adding a substrate ATP mixture to start the reaction, incubating at room temperature for 90min, adding a stop solution to stop the reaction, incubating at room temperature for 1 h, reading by Envision, analyzing the data, and calculating the IC of the compound50The value is obtained. It is composed ofWherein A represents IC50 <10 nM, B10 nM< IC50 < 100 nM。
As can be seen from Table 1, the comparative example compounds and the example compounds 2, 3, 5 and 8 all had strong inhibitory effects on wild-type c-Kit (WT), mutant c-Kit (D861V) and PDGFR alpha (D842V) kinases.
Test example 2: in vitro liver microsome stability test
Test materials
Medicine preparation: comparative and example compounds 1, 2, 3, 5, 8;
reagent: methanol, acetonitrile, formic acid as chromatographically pure, purchased from MERCK corporation; other reagents are all commercially available analytical reagents; the experimental water is ultrapure water;
materials: human liver microsomes were purchased from Celsis/red liver disease research ltd (shanghai); NADPH was purchased from Toronto Research Chemicals Inc (Ontario, Canada).
Test operation
Comparative and example compounds 1, 2, 3, 5, 8 metabolic stability test in human liver microparticles
(1) Preparation of stock solution
A) Liver microparticles: 20 mg/mL;
B) sodium phosphate buffer: 200 mM, pH 7.4;
C) testosterone (yang ginseng): 5 mM;
D)NADPH：10 mM；
E) stopping liquid: acetonitrile (with internal standard 40 ng/mL bromobutenol);
F) test article: 2 mM.
(2) Reaction System (final volume, concentration)
Total incubation volume: 1 mL;
sodium phosphate buffer: 0.1M, pH 7.4;
liver microparticle protein concentration: 0.20 mg/mL;
the concentration of the test substance: 1 mu M;
testosterone (yang ginseng) concentration: 10 mu M;
NADPH concentration: 1 mM;
reaction time: 0, 5, 15, 30 and 50 min.
(3) Reaction and termination
The reaction was preincubated at 37 ℃ for 5 min, and then NADPH was added to start the reaction. The negative control group was added to the system using sodium phosphate buffer instead of NADPH. 100. mu.L of the reaction solution was extracted according to the above-mentioned time and mixed with 300. mu.L of the internal standard bromobutenol-containing frozen stop solution to terminate the reaction. The supernatant was centrifuged at 4 ℃ and LC-MS/MS checked for the amount of compound at each time point. 2 replicates per time point. While a positive control group with testosterone as substrate was set.
Instrument for measuring the position of a moving object
Liquid phase-mass spectrometry analysis system (LC-MS/MS), including the Yangtze company VP series Nanospace S1-23301 binary pump and Nanospace S1-23202 on-line degasser (Japan), Nanospace 51-23133 multipurpose autosampler and AB company API-4000Q-Trap mass spectrometer (containing ESI ion source), column: venusil MP-C18 (2.1X 30 mm, 3 μm).
Chromatographic conditions
Mobile phase a was aqueous, 5% methanol (containing 0.1% formic acid); mobile phase B was an organic phase, 95% methanol (containing 0.1% formic acid). The flow rate was 0.3 mL/min-1Column temperature: at room temperature, a gradient was used as shown in the following table.
Conditions of Mass Spectrometry
Test results
As shown in table 4, compounds 1, 2, 3, 5, and 8 showed much higher percentage of maternal residuals in human liver microsomes for 60min than the comparative compound. Indicating that the example compounds of the invention are metabolized more slowly and potentially act in vivo for a longer period of time than the comparative compounds.
Test example 3: pharmacokinetic testing of rats
Test materials
Compounds 2, 5, 8 and comparative example compounds.
Main instrument equipment
LC Mass spectrometer, manufacturer: waters; the model is as follows: ACQUITY UPLC I-Class + Xevo TQ-S.
Test system
(1) Variety/line/grade
Strain: SD rat; grade: SPF grade.
(2) Sex and number
Number and sex of animals entering acclimation period: 40, female and male halves;
animal numbers and sex were used: 32, female and male.
(3) Body weight and age
Weight: 136.9-148.5 g female, 137.2-158.5 g male when purchased, 186.0-207.8 g female when divided, 267.3-303.9 g male when purchased, and the weight of the individual is within the range of the average weight of the same sex plus or minus 20 percent;
age: 5-6 weeks old when purchased, and 7-9 weeks old when grouped in the test.
The experiment is divided into 4 groups (single groups of example compounds 2, 5 and 8 and comparative compound) 8 animals/group, each of which is a male half and a female half, and in order to prevent the situation that the experiment animals cannot meet the experiment requirements due to unknown reasons, 2 animals/sex are additionally purchased, and 40 SD rats (32 SD rats are actually used) are totally used.
Animal grouping information is given in the following table:
note: the first digit of the animal numbers represents the group (1, 2, 3, 4 represent test group 1, 2, 3 and comparative example group, respectively); the second letter represents sex (F is female, M is male), and the last 3 digits represent the animal serial number.
Administration dose: the administration doses of compounds 2, 5, 8 and the group of comparative compounds were all 20 mg/kg; the administration route is as follows: is administered orally.
Blood sample collection and assay
(1) PK blood sample collection
Compounds 2, 5, 8 and comparative compound groups sampling times: 2 min (+ -1 min), 10 min (+ -1 min), 30min (+ -1 min), 1 h (+ -2 min), 2 h (+ -5 min), 4 h (+ -5 min), 6 h (+ -5 min), 8 h (+ -10 min), 10 h (+ -10 min), and 24 h (+ -10 min) before and after administration;
sampling method and sampling amount: collecting about 0.15 mL of whole blood in jugular vein, and putting EDTA.K into the whole blood after sampling2An anticoagulation tube;
blood sample treatment: placing the whole blood sample in an ice box before centrifugation, transporting the whole blood sample in the ice box, centrifuging for 10 min at the temperature of 2-8 ℃ at 4000 r/min, separating plasma (plasma-1), and storing at the temperature below-60 ℃.
(2) Blood concentration detection
And (3) detecting the blood concentration of animals in each dose group by adopting a verified LC-MS/MS method. The concentration-time curve was plotted using Phoenix WinNonlin 7.0 software and the following pharmacokinetic parameters were calculated according to a non-compartmental model: half life (T)1/2) Maximum blood concentration (C)max) Time to peak (T)max) Area under the time curve (AUC)last) Clearance (CL), etc.
BLOQ (below the lower limit of quantitation) was defined before the concentration data were imported into WinNonlin 7.0: BLOQ is defined as 0 before there is data, and the rest as Missing.
Data collection and analysis
The collection system for collecting and reporting electronic data is as follows:
results
(1) Analysis of drug delivery formulations
Through detection, the system adaptability, the stock solution comparison, the standard curve linear range and the quality control all meet the requirements of an analysis method. The accuracy (ratio of detected concentration to labeled concentration of administered formulation) of compound 2, 5, 8 and comparative compound single groups was between 92.92% and 102.10%. The analysis results all meet the requirements of the test scheme on concentration analysis.
(2) Single dose Pharmacokinetic (PK) results
The mean pharmacokinetic parameters for each group after a single administration of compounds 2, 5, 8 and the comparative compound in SD rats are shown in table 7:
as can be seen from the above table, the half-lives of the compounds 2, 5 and 8 are significantly longer than those of the comparative compounds, and AUC and CmaxThe compound 2, 5 and 8 after deuteration has slower metabolism in vivo, longer action time and better pharmacokinetic property, thereby ensuring better pharmacodynamic action.
Claims (10)
1. A compound of formula (I), a stereoisomer or a pharmaceutically acceptable salt thereof:
wherein R1, R2, R3, R4, R5, R6 are independently hydrogen or deuterium, and R1, R2, R3, R4, R5, R6 are not all hydrogen.
2. The compound, a stereoisomer or pharmaceutically acceptable salt thereof, according to claim 1, wherein the formula (I) is:
wherein at least three of R1, R2, R3, R4, R5 and R6 are deuterium, and the rest are hydrogen.
3. The compound, a stereoisomer, or pharmaceutically acceptable salt thereof, according to claim 2, wherein the compound is:
4. A compound, stereoisomer or pharmaceutically acceptable salt thereof, according to claim 3, wherein the compound is:
5. A compound, stereoisomer or pharmaceutically acceptable salt thereof, according to claim 3, wherein the compound is:
6. A compound, a stereoisomer or pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein the salt is an acetate, ascorbate, benzoate, benzenesulfonate, citrate, fumarate, hydrochloride, hydrobromide, maleate, methanesulfonate, nitrate, oxalate, phosphate, succinate or sulfate salt.
7. A pharmaceutical composition comprising a compound of any one of claims 1 to 5, a stereoisomer or a pharmaceutically acceptable salt thereof, wherein the composition further comprises a pharmaceutically acceptable carrier and/or adjuvant.
8. Use of a compound of any one of claims 1 to 5, a stereoisomer or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention or treatment of a C-Kit and/or PDGFR mediated disease.
9. Use according to claim 8, wherein the C-Kit and/or PDGFR mediated disease comprises gastrointestinal stromal tumor, lupus, leukemia, mastocytosis, melanoma, seminoma, rheumatoid arthritis, multiple sclerosis, frailty, multiple myeloma or pancreatic cancer.
10. Use according to claim 8, wherein the C-Kit and/or PDGFR is a mutant C-Kit and/or PDGFR.
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