WO2019238088A1 - 吡啶并吡啶酮衍生物的盐型及晶型 - Google Patents

吡啶并吡啶酮衍生物的盐型及晶型 Download PDF

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WO2019238088A1
WO2019238088A1 PCT/CN2019/091073 CN2019091073W WO2019238088A1 WO 2019238088 A1 WO2019238088 A1 WO 2019238088A1 CN 2019091073 W CN2019091073 W CN 2019091073W WO 2019238088 A1 WO2019238088 A1 WO 2019238088A1
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crystal form
compound
formula
reaction
hydrochloride
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PCT/CN2019/091073
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French (fr)
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徐招兵
蔡进峰
刘迎春
宋宝慧
施沈一
姚婷
胡利红
丁照中
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基石药业
南京明德新药研发有限公司
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Priority to CN201980040138.5A priority Critical patent/CN112292379B/zh
Publication of WO2019238088A1 publication Critical patent/WO2019238088A1/zh

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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
    • A61K31/4965—Non-condensed pyrazines
    • 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
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

  • the invention relates to a salt form, a crystal form of a novel pyridopyridone derivative and a preparation method thereof, and particularly relates to a hydrochloride salt of a compound of the formula (I) and a crystal form of the hydrochloride salt. It also includes the application of the crystal form and salt form in the preparation of medicines for treating lung cancer and other cancers.
  • the cell cycle refers to the continuous dynamic process that normally and continuously divides cells from the end of the previous mitosis to the end of the next mitosis.
  • the mammalian cell cycle consists of four phases: G1 (pre-DNA synthesis), S (DNA synthesis), G2 (late DNA synthesis), and M (mitotic) stages.
  • the M phase is immediately followed by cytokinesis, forming two daughter cells.
  • the restriction point or R point the cell cycle regulation mechanism will determine the ultimate fate of the cell: continue to participate in the cell cycle cycle , Or exit the active proliferative state and enter the stationary (G0) state.
  • the regulation of the cell cycle is mainly affected by a series of serine / threonine kinases.
  • serine / threonine kinases are also known as cyclin-dependent kinases (CDKs). They regulate cyclins through their corresponding subunits. (cyclins) to achieve the purpose of regulating the cell cycle.
  • CDKs cyclin-dependent kinases
  • At least 10 cyclin-dependent kinases (CDKs) and 15 cyclins have been identified.
  • CDK1 is paired with cyclin A or B; CDK2 is paired with cyclin A or E; CDK3 is paired with an unknown cyclin; CDK4 is paired with cyclin D (1-3); CDK5 is paired with Cyclin D or p35Nck5A is paired; CDK6 is paired with cyclin D; CDK7 is paired with cyclin H; CDK8 is paired with cyclin C; CDK9 is paired with cyclin T.
  • Cancer cell abnormal proliferation and normal cell cycle disorders are common features of all types of cancer. Because of this, inhibitors of key regulators of the cell cycle have become an attractive new anti-tumor target.
  • the complex formed by CDK4 / 6 and cyclin D is activated by extracellular growth factors.
  • This activated complex can phosphorylate retinoblastoma protein (RB), thereby releasing its In the non-phosphorylated state, the tightly bound transcription factor E2F, E2F activation further transcription pushes the cell cycle across the R point and progresses from the G1 phase to the S phase. After crossing the R point, other cyclins are activated in order to regulate the progress of the entire cell cycle.
  • cyclin E and CDK2 combine to control the cell into the S phase; cyclin A and CDK2 combine to control the S phase, and then In the G2 phase, cyclin A binds to CDK1; finally, cyclin B binds to CDK1 to control cells into the mitotic phase.
  • the complex formed by CDK4 / 6 and cyclin D is a key "total switch" in cell cycle regulation. Inhibiting CDK4 / 6 prevents it from forming Cyclin D-CDK4 / 6 complex, which can block the cell cycle from G1 to The progress of S phase, thus achieving the purpose of inhibiting tumor proliferation, CDK4 / 6 has therefore become an important anti-cancer target.
  • These small molecule heterocyclic compounds can also be used clinically for the treatment of many other cancers.
  • These patents include WO2014128588, WO2012018540, WO2012129344, WO2011101409, WO2011130232, WO2010075074, WO2009126584, WO2008032157, WO2005094830, WO2005117980, WO2003062236.
  • Palbociclib has been approved for marketing, it has been reported in the literature that its permeability is poor and it is difficult to penetrate the blood-brain barrier, which is not conducive to the treatment of brain metastatic cancer. Palbociclib also has more severe myelosuppressive toxicity; although Abemaciclib's bone marrow Inhibition is less toxic, but its gastrointestinal side effects are more obvious; compared with Palbociclib and Abemaciclib, Ribociclib is less effective, resulting in higher clinical doses.
  • hydrochloride salt of the compound of formula (I) and its crystal form of the present invention are simple in preparation process, relatively stable, relatively little affected by light, heat and humidity, no or almost no hygroscopicity, good water solubility, convenient formulation generation, and good pharmaceutical prospects .
  • the invention provides a hydrochloride salt of a compound of formula (I),
  • the hydrochloride salt has a structure represented by formula (I-1):
  • n 0.9 to 1.1.
  • n is 0.9, 1.0, or 1.1.
  • the hydrochloride salt has a structure represented by formula (I-2):
  • the X-ray powder diffraction pattern of the crystal form of the hydrochloride includes characteristic peaks with 2 ⁇ values of 9.94 ° ⁇ 0.2 °, 12.43 ° ⁇ 0.2 °, and 22.76 ° ⁇ 0.2 °.
  • the X-ray powder diffraction pattern of the crystal form of the hydrochloride includes 2 ⁇ values of 9.94 ° ⁇ 0.2 °, 12.43 ° ⁇ 0.2 °, 13.03 ° ⁇ 0.2 °, 18.63 ° ⁇ 0.2 °, Characteristic peaks of 19.21 ° ⁇ 0.2 °, 22.76 ° ⁇ 0.2 °, 28.08 ° ⁇ 0.2 °, and 31.40 ° ⁇ 0.2 °.
  • the X-ray powder diffraction pattern of the crystal form of the hydrochloride includes 2 ⁇ values of 9.94 ° ⁇ 0.2 °, 12.43 ° ⁇ 0.2 °, 13.03 ° ⁇ 0.2 °, 18.63 ° ⁇ 0.2 °, Characteristic peaks of 19.21 ° ⁇ 0.2 °, 20.38 ° ⁇ 0.2 °, 21.11 ° ⁇ 0.2 °, 22.76 ° ⁇ 0.2 °, 24.41 ° ⁇ 0.2 °, 28.08 ° ⁇ 0.2 °, and 31.40 ° ⁇ 0.2 °.
  • the X-ray powder diffraction pattern of the crystal form of the hydrochloride includes 2 ⁇ values of 9.94 °, 12.43 °, 13.03 °, 15.22 °, 15.97 °, 16.72 °, 18.63 °, 19.21 °, 19.60 °, 20.38 °, 21.11 °, 22.76 °, 23.08 °, 23.79 °, 24.41 °, 25.08 °, 27.21 °, 28.08 °, 28.63 °, 30.04 °, 30.60 °, 31.40 °, 34.20 °, 34.74 °, 35.51 ° And 38.45 ° characteristic peaks.
  • the X-ray powder diffraction pattern of the crystal form of the hydrochloride is shown in FIG. 1.
  • the X-ray powder diffraction pattern of the crystal form of the hydrochloride is shown in Table 1-1.
  • the DSC curve of the crystal form of the hydrochloride shows that the starting point of the endothermic peak is 263.38 ° C ⁇ 2 ° C, and the peak temperature is 266.16 ° C ⁇ 2 ° C.
  • the DSC curve of the crystal form of the hydrochloride is shown in FIG. 2.
  • the TGA curve of the crystal form of the above-mentioned hydrochloride salt begins to lose weight obviously at 211.67 ° C ⁇ 3 ° C, and the weight is reduced by 23.59% in total.
  • the TGA curve of the crystal form of the hydrochloride is shown in FIG. 3.
  • the present invention provides the application of the above-mentioned hydrochloride salt and crystal form in the preparation of a medicament for treating cancer; in some embodiments, the cancer is non-small cell lung cancer.
  • the intermediate compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by combining them with other chemical synthesis methods, and those skilled in the art.
  • Well-known equivalent alternatives, preferred embodiments include, but are not limited to, the embodiments of the present invention.
  • the solvent used in the present invention is commercially available.
  • the present invention uses the following abbreviations:
  • DMF stands for N, N-dimethylformamide
  • 2-MeTHF stands for 2-methyltetrahydrofuran
  • THF stands for tetrahydrofuran
  • EtOAc stands for ethyl acetate
  • EtOH stands for ethanol
  • MTBE stands for methyl tert-butyl ether
  • Ac 2 O stands for acetic acid Anhydride
  • NBS stands for N-bromosuccinimide
  • DIPEA stands for diisopropylethylamine
  • KI stands for potassium iodide
  • KOAc stands for potassium acetate
  • Na 2 CO 3 stands for sodium carbonate
  • K 2 CO 3 stands for potassium carbonate
  • Pd ( PhCN) 2 Cl 2 stands for bis (cyanobenzene) palladium dichloride
  • Pd (OAc) 2 stands for palladium acetate
  • DPEPhos stands for bis 2-diphenylphosphine phenyl ether
  • Xphos stands for 2-dic
  • the compounds of the invention are based on conventional nomenclature in the art or used Software naming. Commercially available compounds use supplier catalog names.
  • X-ray generator Cu, LF type
  • Test conditions equilibrium at 20 ° C, heating up to 280 ° C at 10 ° C / min.
  • Test conditions Equilibrate at 20 ° C and heat up to 350 ° C at a rate of 10 ° C / min.
  • Test conditions 1) The standard solution of chloride ion was prepared into standard solutions of 5, 8, 10, 20, and 40 mg / mL, respectively.
  • Assay represents the content
  • C Target Ion represents the chloride ion concentration in the sample solution
  • C s is the concentration of the sample
  • FIG. 1 is an XRPD spectrum of a crystalline form of a compound of formula (I-2).
  • Fig. 2 is a DSC spectrum of a crystalline form of a compound of formula (I-2).
  • Fig. 3 is a TGA spectrum of a crystalline form of a compound of formula (I-2).
  • FIG. 4 is an XRPD superimposed spectrum diagram of crystal form changes of a compound of formula (I-2) in a stability test under different conditions.
  • step 1
  • the aqueous phase was extracted with dichloromethane (5 liters * 2), and the combined organic phases were washed with saturated brine (10 liters * 2), separated and dried.
  • the combined organic phases were concentrated, when the volume was concentrated to about 5 liters, transferred to a 5 liter measuring cup, and then transferred to two 3 liter single-necked flasks, and the concentration was continued.
  • an equal volume of EtOAc was added and the concentration was continued.
  • an additional volume of EtOAc was added and the concentration was continued.
  • the mixture was filtered, and the filtrate was concentrated and treated in the same way. The obtained solids were mixed together.
  • the reaction mixture was stirred at room temperature (15 ° C) for 16 hours and then filtered, and the filter cake was washed with water (5 liters).
  • the filter cake was transferred to a reaction kettle and EtOAc (25 liters) was added.
  • the resulting mixture was heated to 50-60 ° C and stirred for 16.5 hours.
  • the reaction mixture was filtered while hot at 50-60 ° C.
  • the filtrate was concentrated to a volume of about 2.5 liters, the concentration was stopped and the mixture was cooled to room temperature (15 ° C).
  • the mixture was filtered, and the filter cake was dried in a vacuum drying box at 45 ° C and -0.1 MPa for 24 hours to obtain Compound 4.
  • HPLC showed that about 8% of the starting material remained.
  • Vinyl n-butyl ether (289.7 g, 2.9 moles, 0.5 equivalent) was added to the reaction kettle, and the reaction was continued for 6 hours.
  • HPLC showed about 5% of the starting material remaining.
  • Vinyl n-butyl ether (289.7 g, 2.9 mol, 0.5 equivalent) was added to the reaction kettle, and the reaction was continued for 3 hours.
  • HPLC showed that about 4% of the starting material remained. Turn off the heat to room temperature and stir for 14.5 hours. The mixture was filtered, the filter cake was washed with EtOAc (8 L), and the filtrate was concentrated.
  • the filtrate was concentrated to dryness to give a crude oil (2.7 kg).
  • step 1
  • the reaction system was cooled to 5 ° C, and a large amount of solids were precipitated. Stop stirring and aspirate the supernatant using the siphon principle.
  • 2-MeTHF (5 liters) was added to the reaction kettle to dissolve the remaining product in the kettle, and the supernatant was sucked out again using the siphon principle.
  • the two precipitated 2-MeTHF solutions were mixed and concentrated.
  • petroleum ether (1 liter) was added to the rotary flask and filtered to remove the precipitated piperazine. The filtrate was spin-dried, and as much piperazine as possible was removed with an oil pump to obtain a residue (2.68 kg).
  • the residue was subjected to conventional vacuum distillation (pressure: 19.0 to 32 Torr, fraction temperature ⁇ 70 ° C) to obtain a crude product.
  • the crude product was subjected to molecular distillation (pressure: 1.5 to 2.5 bar, rotation speed 320 to 325 rpm / min, temperature: 70 to 75 ° C, -17 to -13 ° C, and injection speed: 200 mL / h) to obtain compound 7.
  • the reaction mixture was heated to 100 to 110 ° C (outside temperature) under the protection of nitrogen, and the internal temperature was 90 to 100 ° C, and stirred for 17 hours. HPLC showed that compound 5 was complete.
  • the reaction mixture was filtered through diatomaceous earth, the filter cake was washed with 2-MeTHF (1 liter), and the collected filtrate was evaporated to dryness.
  • the obtained residue was dissolved with 2-MeTHF (2 liter) and water (500 ml) and separated.
  • the aqueous phase was extracted with 2-MeTHF (250 ml * 2).
  • the combined organic phases were washed with saturated brine (500 ml * 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated.
  • the resulting residue was slurried with EtOAc (750 ml) at room temperature (15 ° C) and stirred for 16 hours. .
  • the mixture was filtered and the collected filter cake was evaporated to dryness.
  • Example 1 Study on the hygroscopicity of the crystalline form of the compound of formula (I-2)
  • Test conditions Take samples (10-15mg) and place them in the DVS sample tray for testing.
  • the hygroscopic evaluation criteria are shown in Table 1-2.
  • Hygroscopic classification Hygroscopic weight gain * ( ⁇ W%) deliquescence Absorb a sufficient amount of water to form a liquid Very hygroscopic ⁇ W% ⁇ 15% Hygroscopic 15%> ⁇ W% ⁇ 2% Slightly hygroscopic 2%> ⁇ W% ⁇ 0.2% No or almost no hygroscopicity ⁇ W% ⁇ 0.2%
  • the crystalline form of the compound of formula (I-2) has a moisture absorption weight gain of 0.11% at 25 ° C and 80% RH.
  • the crystal form of the compound of formula (I-2) is stable without significant change under the conditions of ethanol, acetonitrile, acetone, ethyl acetate, and tetrahydrofuran.
  • FaSSIF Simulates the intestinal fluid in the small intestine under the condition of pre-meal hunger in humans
  • FeSSIF Simulates the intestinal fluid in the small intestine in a satiety state after human meals
  • SGF Simulate gastric juice when fasting in human hunger
  • the crystal form of the compound of formula (I-2) has very good solubility in water.
  • the crystal form 10mg was accurately weighed, placed in duplicate at the bottom of a 40mL glass sample bottle, spread into a thin layer, and placed under appropriate conditions. In open conditions, make small holes in the aluminum foil to ensure that the sample can fully contact the ambient air. In addition, take a small amount of sample and place it in a 40mL glass sample bottle. Under the same conditions, the crystal form state to be determined. At the time of the inspection, the corresponding test samples were taken out and covered with bottle caps. The samples from day 0 were taken out of the refrigerator and analyzed after the samples returned to room temperature. About 10 mg of the test product is used for XRPD detection. The compounds under investigation were placed under the following conditions and samples were taken at different time points to detect properties, XRPD, content and related substances. The research conditions and testing items are shown in Table 1-5.
  • Test item X includes: traits, XRPD, content and related substances, the 0 day sample is the initial sample.
  • the light test is set out according to ICH option 2 and dark control samples (that is, foil wrapped samples)
  • LOQ refers to the detection limit of the HPLC instrument.
  • RRT is the relative retention time
  • LOQ refers to the detection limit of the HPLC instrument
  • the content of the crystalline form and the related substances after the stability test sampling show that the crystalline form of the compound of formula (I-2) is relatively stable, and the XRPD (see Figure 4) test results show that the crystalline form of the compound of formula (I-2) The crystal form only changed under the condition of 92.5% relative humidity (10 days), and the crystal form of the compound of formula (I-2) was very stable under other test conditions.
  • the compounds involved in the present invention are CDK4 / 6 inhibitors.
  • the following experimental results confirm that the compounds listed in this patent are indeed CDK4 / 6 inhibitors and can be used as potential anticancer drugs.
  • the IC 50 used here refers to the concentration of the corresponding reagent when a certain reagent is used to produce 50% maximum inhibition.
  • CDK4 / cyclin D1, CDK6 / cyclin D1 (Life technology).
  • the tritium-labeled anti-myelin protein antibody and the tritium-labeled rabbit-derived antibody (PerkinElmer) were detected by an Envision multi-label analyzer (PerkinElmer).
  • the compound to be detected is diluted three-fold, including 10 concentration gradients, and the final concentration range is 5 ⁇ M to 0.25 nM.
  • the standard Lance Ultra method is performed by an enzyme reaction system of 10 ⁇ L, including 0.3 nM CDK4 / cyclin D1 protein, 50 nM ULight-4E-BP1 polypeptide, and 350 ⁇ M ATP. Dissolve them separately in an enzyme buffer.
  • the components of the buffer include: pH 7.5 hydroxyethylpiperazine ethanesulfuric acid solution 50mM, ethylenediamine tetraacetic acid 1mM, magnesium chloride 10mM, 0.01% Brij-35, dithiothreo Sugar alcohol 2mM.
  • the standard Lance Ultra method is performed by an enzyme reaction system of 10 ⁇ L, including 0.8 nM CDK6 / cyclin D1 protein, 50 nM ULight-4E-BP1 polypeptide, and 250 ⁇ M ATP. Dissolve them separately in an enzyme buffer.
  • the components of the buffer include a solution of hydroxyethylpiperazine ethanesulfuric acid at pH 7.5, 50 mM, ethylene diamine tetraacetic acid, 1 mM, magnesium chloride, 10 mM, 0.01% Brij-35, and dithiothreose. Alcohol 2 mM. After starting the reaction, seal the OptiPlate 384-well plate with TopSeal-A and incubate at room temperature for 180 minutes.
  • the compounds of the present invention have significant inhibitory activity on CDK4 and CDK6 kinase.
  • RPMI 1640 medium (Invitrogen-22400089), fetal bovine serum (Gibco-10099141), penicillin / streptomycin antibiotic (Hyclone-SV30010), L-glutamine (Invitrogen-35050079).
  • the NCI-H358 cell line is derived from the cell bank of WuXi AppTec Biosciences. Envision Multi-Label Analyzer (PerkinElmer).
  • the compound of the present invention has better proliferation inhibitory activity on NCI-H358 lung cancer cells than the reference compound Palbociclib.
  • Caco-2 cell is an in vitro model widely used to study the absorption of the small intestine. It is a human colon cancer cell.
  • the monolayer Caco-2 cell model has been widely used to evaluate passive and active transport processes during small intestinal absorption. This experiment was used to determine the bidirectional permeability of the compound of the present invention and the reference compound Palbociclib through the Caco-2 cell model.
  • -Transport buffer HBSS, pH 7.4;
  • the sample solution in the administration well and the receiving well was immediately mixed with a cold acetonitrile solution containing an internal standard.
  • the LC / MS / MS method was used to analyze the concentration of the test compound in all samples (including the initial dosing solution, the supernatant of the dosing well, and the receiving solution). And calculate the apparent permeability coefficient, efflux ratio and other parameters.
  • Table 2-2 lists the permeability coefficients of the compound of the present invention and the reference compound Palbociclib in Caco-2 monolayer cells.
  • the compound of the present invention has higher permeability and is less likely to be absorbed and transported in vivo by efflux transporters. Better permeability can make the compounds of the present invention more distributed in tissues in the body (such as the lungs), resulting in better antitumor efficacy in vivo. At the same time, better permeability makes it possible for the compounds of the present invention to penetrate the blood-brain barrier and achieve the purpose of treating brain metastatic lung cancer.
  • This test is used to test the metabolic stability of the test substance in rat, mouse and human liver microsomes
  • test compound with a concentration of 1 ⁇ M was incubated with liver microsomes with a protein concentration of 0.5 mg / mL under a reducing coenzyme II regeneration system in a 37-degree water bath.
  • Positive controls include: testosterone (3A4 substrate), amphetamine (2D6 substrate), diclofenac (2C9 substrate). Positive control incubation conditions were consistent with compound incubation conditions.
  • reaction time points are: 0, 5, 10, 20, 30, and 60 minutes. At the corresponding time points, the reaction is terminated using a stop solution containing an internal standard.
  • the compounds were also incubated with microsomes for 60 minutes and served as a negative control.
  • the sample is determined by LC / MS / MS, and the compound concentration is shown as the ratio of the compound peak area to the internal standard peak area (non-standard curve).
  • Rat, mouse and human parameters are 40g / kg, 88g / kg and 20g / kg respectively
  • microsomes microsomes
  • liver liver
  • liver microsomes of the compound of the present invention in human, rat, and mouse is obviously better than that of the reference compound Palbociclib.
  • Non-small cell lung cancer NCI-H358 model BALB / c nude mice were implanted subcutaneously for in vivo drug efficacy experiments.
  • mice Per cage mice Per cage
  • All cages, bedding and water are disinfected before use. All animals have free access to standard certified commercial laboratory diets.
  • a total of 56 mice purchased from Shanghai Lingchang Biotechnology Co., Ltd. were used for research.
  • Each mouse was implanted with NCI-H358 tumor cells subcutaneously on the right back for tumor growth. Dosing was started when the average tumor volume reached approximately 100-200 cubic millimeters.
  • the test compound is administered orally daily, and the dosages are shown in Table 2-4.
  • Antitumor efficacy is determined by dividing the average tumor increase volume of animals treated with a compound by the average tumor increase volume of untreated animals, and the safety of a compound is determined by the weight change of animals treated with the compound .
  • the compound of the present invention exhibits significant antitumor activity on a non-small cell lung cancer NCI-H358 model and has good safety.
  • the antitumor effect of the compounds of the present invention has a dose-dependent tendency.
  • TGI Tumor Growth Inhibition.
  • TGI (%) [(1- (average tumor volume at the end of administration in a treatment group-average tumor volume at the start of administration in this treatment group)) / (average tumor volume at the end of treatment in a solvent control group-treatment in a solvent control group Mean tumor volume)] ⁇ 100%.
  • the in vivo efficacy test was performed on nude mice with colorectal cancer HCT-116 model BALB / c.
  • mice BALB / c nude mice, female, 6-8 weeks, weighing about 18-22 grams, keep the mice in a special pathogen-free environment and in a single ventilated cage (3-5 mice per cage) . All cages, bedding and water are disinfected before use. All animals have free access to standard certified commercial laboratory diets. A total of 48 mice purchased from Shanghai Lingchang Experimental Animal Co., Ltd. were used for research. 0.2 mL of 5 ⁇ 106 HCT-116 cells were subcutaneously inoculated on the right back of each nude mouse. Subgroup administration was started when the average tumor volume reached 132 mm 3 . The test compound is administered orally daily, and the dosages are shown in Table 2-5.
  • Antitumor efficacy is determined by dividing the average tumor increase volume of animals treated with a compound by the average tumor increase volume of untreated animals.
  • the compound of the present invention shows better antitumor activity and higher safety on colorectal cancer HCT-116 model.
  • the in vivo efficacy test was performed on nude mice with colon cancer HCT-116 model BALB / c.
  • mice BALB / c nude mice, female, ⁇ 8 weeks, weighing about 18-21 grams, kept the mice in a special pathogen-free environment and in a single ventilated cage (4 mice per cage). All cages, bedding and water are disinfected before use. All animals have free access to standard certified commercial laboratory diets. A total of 40 mice purchased from Shanghai Lingchang Biotechnology Co., Ltd. were used for research. 0.2 mL of 5 ⁇ 10 6 HCT-116 cells were subcutaneously inoculated into the right back of each nude mouse. Group administration was started when the average tumor volume reached 139 mm 3 . The test compound is administered orally daily, and the dosages are shown in Table 2-6. Tumor volume is measured twice a week with a two-dimensional caliper.
  • Antitumor efficacy is determined by dividing the average tumor increase volume of animals treated with a compound by the average tumor increase volume of untreated animals.
  • the compound of the present invention significantly inhibits the growth of HCT116 human colon cancer cell subcutaneously transplanted tumors in nude mice, the inhibitory effect is dose-dependent, and the safety is good.
  • mice Female, 6-8 weeks, kept the mice in a special pathogen-free environment and in a single ventilated cage (4 mice per cage). All cages, bedding and water are disinfected before use. All animals have free access to standard certified commercial laboratory diets. A total of 64 mice purchased from Shanghai Slark Laboratory Animal Co., Ltd. were used for research. 0.1 mL of 3 ⁇ 10 5 CT-26 cells were subcutaneously inoculated into the right back of each nude mouse. Group administration was started when the average tumor volume reached 75 mm 3 . The test compound is administered orally daily, and the dosages are shown in Table 2-7. Tumor volume is measured twice a week with a two-dimensional caliper.
  • Antitumor efficacy is determined by dividing the average tumor increase volume of animals treated with a compound by the average tumor increase volume of untreated animals.
  • the single-drug treatment of the compound of the invention can significantly inhibit the growth of subcutaneous transplanted tumors of colon cancer cells of CT26 mice, and the inhibitory effect is dose-dependent.
  • the compound of the present invention and the anti-PD-1 antibody CS1003 have a tendency to enhance the efficacy of a single drug.

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Abstract

一种吡啶并吡啶酮衍生物的盐型、晶型及其制备方法，具体公开了式(I)化合物的盐酸盐及其盐酸盐的晶型，还包括所述晶型和盐型在制备治疗肺癌与其他癌症药物中的应用。

Description

吡啶并吡啶酮衍生物的盐型及晶型
相关申请的交叉引用
本申请主张如下优先权：CN201810609544.6，申请日2018年06月13日。
技术领域
本发明涉及一种新型吡啶并吡啶酮衍生物的盐型、晶型及其制备方法，具体涉及式(I)化合物的盐酸盐及其盐酸盐的晶型。还包括所述晶型和盐型在制备治疗肺癌与其他癌症药物中的应用。
背景技术
细胞周期是指正常连续分裂的细胞从前一次有丝分裂结束后到下一次有丝分裂结束所经历的连续动态过程。哺乳动物的细胞周期由四个阶段组成：分别分G1期(DNA合成前期)，S期(DNA合成期)，G2期(DNA合成后期)和M期(有丝分裂期)。M期之后紧接着产生胞质分裂，形成两个子细胞。虽然经细胞周期分裂产生的新生细胞会重新进入细胞周期，但是在G1晚期的某个时间点(称为限制点或R点)，细胞周期调控机制会决定细胞的最终命运：继续参与细胞周期循环，或者退出活跃的增殖状态转而进入静止(G0)的状态。细胞周期的调控主要受一系列丝氨酸/苏氨酸激酶的影响，这类丝氨酸/苏氨酸激酶又被称作细胞周期蛋白依赖性激酶(CDK)，他们通过与其相对应的调节亚基周期素(cyclins)相结合从而达到调控细胞周期的目的。到现在为止，已经鉴定出至少10种细胞周期蛋白依赖性激酶(CDK)和15种周期素(cyclins)。它们可以形成如下配对复合物：CDK1与周期素A或B配对；CDK2与周期素A或E配对；CDK3与一种未知的周期素配对；CDK4与周期素D(1-3)配对；CDK5与周期素D或p35Nck5A配对；CDK6与周期素D配对；CDK7与周期素H配对；CDK8与周期素C配对；CDK9与周期素T配对。
癌症细胞异常增殖与正常细胞周期失调是所有类型癌症共有的特征。正因为如此，细胞周期关键调节因子的抑制剂已成为一个有吸引力的新颖的抗肿瘤靶点。在细胞周期的G1期早期，CDK4/6与周期素D形成的复合物被细胞外生长因子激活，这种被激活的复合物可以使视网膜母细胞瘤蛋白(RB)磷酸化，从而释放其在未被磷酸化的状态下紧密结合的转录因子E2F，E2F激活进一步转录推动细胞周期跨过R点并从G1期进展到S期。跨过R点后，其它的细胞周期素依次被激活从而可以调控整个细胞周期的进行，例如：周期素E与CDK2结合控制细胞进入S期；周期素A与CDK2结合控制S期的进行，然后在G2期周期素A与CDK1结合；最后是周期素B结合CDK1控制细胞进入有丝分裂期。CDK4/6与周期素D形成的复合物是细胞周期调控的一个关键“总开关”，抑制CDK4/6使之无法形成Cyclin D-CDK4/6复合物，就能够阻滞细胞周期自G1期向S期的进程，从而达到抑制肿瘤增殖的目的，CDK4/6因此成为一个重要的抗癌靶点。
近年来，有几个CDK4/6小分子抑制剂已经进入临床研究阶段用于癌症的治疗，它们或者是单独给药，或者是联合用药。基于II期临床试验PALOMA-1的中期数据，FDA于2015年2月批准了Palbociclib的上市请求，并与来曲唑联合应用作为治疗雌激素受体(ER)阳性/人表皮生长因子受体2(HER2)阴性绝经后转移性乳腺癌的一线治疗，而Palbociclib治疗非小细胞肺癌的研究也正处于Ⅲ期临床阶段。基于Ⅲ期临床试验MONALEESA-2研究结果，美国FDA于2016年8月授予CDK4/6抑制剂Ribociclib(LEE-011)突破性疗法认定，随后Ribociclib于2017年3月被美国FDA批准上市，联合来曲唑用于一线治疗晚期或 转移性激素受体(HR)阳性/人表皮生长因子受体2(HER2)阴性乳腺癌。此外基于MONARCH 2的Ⅲ期临床试验结果，美国礼来公司的CDK4/6抑制剂Abemaciclib(LY2835219)于2017年9月被美国FDA批准上市，用于激素受体(HR)阳性/人表皮生长因子受体2(HER2)阴性晚期或复发乳腺癌的治疗。这些小分子的杂环化合物在临床上还可以用于其它多种癌症的治疗。这些专利包括WO2014128588，WO2012018540，WO2012129344，WO2011101409，WO2011130232，WO2010075074，WO2009126584，WO2008032157，WO2005094830，WO2005117980，WO2003062236。
Figure PCTCN2019091073-appb-000001
虽然在开发用于癌症和其他疾病治疗的CDK4/6抑制剂的道路上已经做了很多努力，但是到目前为止只有三个针对该靶点的药物(Palbociclib、Ribociclib和Abemaciclib)上市，且适应症只是HR阳性/HER2阴性绝经后转移性的乳腺癌。尽管CDK4/6抑制剂的肺癌临床研究已进展到三期临床，但是暂时还没有药物上市，因此仍然迫切需要开发新颖的、更加安全有效的、能够治疗多种癌症(包括肺癌)的CDK4/6抑制剂。另外一个方面，虽然Palbociclib已经获批上市，但是已有文献报道其渗透性差，难以透过血脑屏障，不利于治疗脑转移的癌症，而且Palbociclib还存在较严重的骨髓抑制毒性；虽然Abemaciclib的骨髓抑制毒性较轻，但是其胃肠道副作用比较明显；而与Palbociclib和Abemaciclib相比，Ribociclib的药效较差，导致临床用药剂量更大。
技术效果
本发明的式(I)化合物的盐酸盐及其晶型制备工艺简单，并且相对比较稳定、受光热湿度影响较小，无或几乎无吸湿性，水溶性好，便于制剂生成，成药前景良好。
发明内容
一方面，本发明提供了式(I)化合物的盐酸盐，
Figure PCTCN2019091073-appb-000002
本发明的一些方案中，上述盐酸盐具有式(I-1)所示结构：
Figure PCTCN2019091073-appb-000003
其中，n为0.9～1.1。
本发明的一些方案中，上述n为0.9、1.0或1.1。
本发明的一些方案中，上述盐酸盐具有式(I-2)所示结构：
Figure PCTCN2019091073-appb-000004
本发明的一些方案中，上述盐酸盐的晶型的X-射线粉末衍射图谱包含在2θ值为9.94°±0.2°、12.43°±0.2°和22.76°±0.2°的特征峰。
本发明的一些方案中，上述盐酸盐的晶型的X-射线粉末衍射图谱包含在2θ值为9.94°±0.2°、12.43°±0.2°、13.03°±0.2°、18.63°±0.2°、19.21°±0.2°、22.76°±0.2°、28.08°±0.2°和31.40°±0.2°的特征峰。
本发明的一些方案中，上述盐酸盐的晶型的X-射线粉末衍射图谱包含在2θ值为9.94°±0.2°、12.43°±0.2°、13.03°±0.2°、18.63°±0.2°、19.21°±0.2°、20.38°±0.2°、21.11°±0.2°、22.76°±0.2°、24.41°±0.2°、28.08°±0.2°和31.40°±0.2°的特征峰。
本发明的一些方案中，上述盐酸盐的晶型的X-射线粉末衍射图谱包含在2θ值为9.94°、12.43°、13.03°、15.22°、15.97°、16.72°、18.63°、19.21°、19.60°、20.38°、21.11°、22.76°、23.08°、23.79°、24.41°、25.08°、27.21°、28.08°、28.63°、30.04°、30.60°、31.40°、34.20°、34.74°、35.51°和38.45°的特征峰。
本发明的一些方案中，上述盐酸盐的晶型的X-射线粉末衍射图谱如图1所示。
本发明的一些方案中，上述盐酸盐的晶型的X-射线粉末衍射图谱如表1-1所示。
表1-1式(I-2)化合物的晶型的XRPD衍射数据
Figure PCTCN2019091073-appb-000005
Figure PCTCN2019091073-appb-000006
本发明的一些方案中，上述盐酸盐的晶型的DSC曲线显示吸热峰的起始点为263.38℃±2℃，峰值温度为266.16℃±2℃。
本发明的一些方案中，上述盐酸盐的晶型的DSC曲线如图2所示。
本发明的一些方案中，上述盐酸盐的晶型的TGA曲线在211.67℃±3℃时开始明显失重，重量共减少了23.59％。
本发明的一些方案中，上述盐酸盐的晶型的TGA曲线如图3所示。
另一方面，本发明提供了上述盐酸盐及晶型在制备治疗癌症药物中的应用；在一些方案中，所述癌症为非小细胞肺癌。
定义和说明
除非另有说明，本文所用的下列术语和短语旨在含有下列含义。一个特定的短语或术语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。当本文出现商品名时，旨在指代其对应的商品或其活性成分。
本发明的中间体化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施例。
本发明具体实施方式的化学反应是在合适的溶剂中完成的，所述的溶剂须适合于本发明的化学变化及其所需的试剂和物料。为了获得本发明的化合物，有时需要本领域技术人员在已有实施方式的基础上对合成步骤或者反应流程进行修改或选择。
下面会通过实施例具体描述本发明，这些实施例并不意味着对本发明的任何限制。
本发明所使用的所有溶剂是市售的，无需进一步纯化即可使用。
本发明所使用的溶剂可经市售获得。
本发明采用下述缩略词：
DMF代表N,N-二甲基甲酰胺；2-MeTHF代表2-甲基四氢呋喃；THF代表四氢呋喃；EtOAc代表乙酸乙酯；EtOH代表乙醇；MTBE代表甲基叔丁基醚；Ac 2O代表醋酸酐；NBS代表N-溴代丁二酰亚胺；DIPEA代表二异丙基乙胺；KI代表碘化钾；KOAc代表醋酸钾；Na 2CO 3代表碳酸钠；K 2CO 3代表碳酸钾；Pd(PhCN) 2Cl 2代表二(氰基苯)二氯化钯；Pd(OAc) 2代表醋酸钯；DPEPhos代表双2-二苯基膦苯基醚；Xphos代表2-二环己基磷-2',4',6'-三异丙基联苯；PO代表口服；QD代表一天一次。
本发明化合物依据本领域常规命名原则或者使用
Figure PCTCN2019091073-appb-000007
软件命名，市售化合物采用供应商目录名称。
仪器及分析方法
1.1X-射线粉末衍射(X-ray powder diffractometer,XRPD)
仪器型号：布鲁克D8 advance X-射线衍射仪
测试条件：
X-ray发生器：Cu，LF型；
工作电压：40kV；
工作电流：40mA；
扫描角度：3°～50°；
扫描角度：2°/min；
步进角度：0.02°；
探测器：NaI闪烁探测器；
发散狭缝(DS)：1deg；
散射狭缝(SS)：1deg；
接收狭缝(RS)：0.3mm。
1.2差式扫描量热法(Differential Scanning Calorimeter,DSC)
仪器型号：TA Instruments DSC Q2000
测试条件：20℃平衡，10℃/min升温至280℃。
1.3热重分析(Thermal Gravimetric Analyzer,TGA)
仪器型号：TA Instruments TGA Q500
测试条件：20℃平衡，以10℃/min的速度升温至350℃。
1.4稳定性实验有关物质分析及含量方法
表A
Figure PCTCN2019091073-appb-000008
Figure PCTCN2019091073-appb-000009
表B
Figure PCTCN2019091073-appb-000010
1.5氯离子含量检测方法
仪器型号：Thermo ICS-1000离子色谱仪
测试条件：1)将氯离子标准溶液分别配制成5、8、10、20、40mg/mL的标准溶液。
2)平行配制两份约0.2mg/mL的式(I-2)化合物的晶型溶液。
3)用Thermo ICS-1000离子色谱仪检测。
4)离子色谱仪仪器参数见表C：
表C
Figure PCTCN2019091073-appb-000011
5)计算方法：
Figure PCTCN2019091073-appb-000012
其中：Assay表示含量；C Target Ion表示样品溶液中氯离子浓度；C s是样品的浓度；
附图说明
图1为式(I-2)化合物的晶型的XRPD谱图。
图2为式(I-2)化合物的晶型的DSC谱图。
图3为式(I-2)化合物的晶型的TGA谱图。
图4为式(I-2)化合物的晶型在不同条件的稳定性试验中晶型变化的XRPD叠加谱图。
具体实施方式
下面通过实施例对本发明进行详细描述，但并不意味着对本发明任何不利限制。本文已经详细地描述了本发明，其中也公开了其具体实施例方式，对本领域的技术人员而言，在不脱离本发明精神和范围的情况下针对本发明具体实施方式进行各种变化和改进将是显而易见的。
制备实施例
制备流程1：
Figure PCTCN2019091073-appb-000013
第1步：
在15℃下，依次将DMF(20升)、化合物1(2.0千克，8.81摩尔，1.0当量)、K 2CO 3(1.34千克，9.7摩尔，1.1当量)和环戊胺(975.75克，11.46摩尔，1.3当量)加入到反应釜中，将反应混合物加热至65～75℃，搅拌17小时。HPLC显示原料剩余约2.04％，停止反应。反应液不做任何处理直接用于下一步。
第2步：
在20℃下，依次将DMF(20升)、巴豆酸(835.05克，9.70摩尔，1.1当量)和DIPEA(4.56千克，35.27摩尔，4.0当量)加入到反应釜中。向反应釜中鼓入氮气30分钟后，依次加入三邻甲氧基磷(268.39克，881.8毫摩尔，0.1当量)和Pd(PhCN) 2Cl 2(169.12克，440.90毫摩尔，0.05当量)。在氮气保护下将反应混合物加热至80～85℃，搅拌17小时。HPLC显示反应完全。向上述反应液中加入Ac 2O(1.80千克，17.62摩尔，2当量)，将反应液继续在80～85℃搅拌0.5小时。HPLC显示反应完全。将反应液冷却至15℃，将反应液取一半置于另一个反应釜中，向每个反应釜中加入水(20升)，各搅拌19小时。分别将两个反应 釜中的混合物过滤，每个滤饼用水(5升)洗涤两次。将两个滤饼分别溶于二氯甲烷(10升)中，各加入水(20升)，搅拌后静置，分液。水相用二氯甲烷(5升*2)萃取，将合并的有机相用饱和食盐水(10升*2)洗涤，分液，干燥。将合并的有机相浓缩，当体积浓缩至5升左右时，转移至5升量杯中，再转移至两个3升单口瓶中，继续浓缩。当体积减少一半时，加入等体积的EtOAc，继续浓缩。当体积再减少一半时，再加入等体积的EtOAc，继续浓缩。当体积减少一半时，将混合物过滤，滤液浓缩，同样的方法处理，得到的固体混合在一起。将两个单口瓶做同样处理，得到的固体混合在一起，置于50℃真空干燥箱中干燥16小时，得到化合物3。 1H NMR(400MHz,CDCl 3)δ8.59(s,1H),7.26(s,1H),6.45(s,1H),5.35-5.44(m,1H),2.40(s,3H),2.17-2.08(m,2H),2.06-1.99(m,2H),1.97-1.89(m,2H),1.74-1.68(m,2H)。
第3步：
在20℃下，依次将DMF(13.4升)、化合物3(1.341千克，4.77摩尔，1.0当量)和NBS(2.12千克，11.94摩尔，2.5当量)加入到反应釜中，将得到的混合物加热至50～60℃，搅拌2小时。HPLC显示反应完全。将反应混合物冷却至20℃。将亚硫酸钠(902.65克，7.16摩尔，1.5当量)配制成水溶液(26.8升)，然后缓慢加入反应釜中，有大量固体析出。将反应混合物在室温(15℃)下搅拌16小时后过滤，滤饼用水(5升)洗涤。将滤饼转移至反应釜中并加入EtOAc(25升)，得到的混合物加热至50～60℃，搅拌16.5小时。将反应混合物在50～60℃下趁热过滤，滤液浓缩至体积约2.5升时，停止浓缩，冷却至室温(15℃)。将混合物过滤，滤饼用真空干燥箱在45℃，-0.1MPa条件下干燥24小时，得到化合物4。 1H NMR(400MHz,CDCl 3)δ8.79(s,1H),7.39(s,1H),5.39-5.29(m,1H),2.73(s,3H),2.29-2.20(m,2H),2.18-2.10(m,2H),2.06-1.98(m,2H),1.81-1.74(m,2H)。
第4步：
在15℃下，依次向反应釜中加入正丁醇(12升)、化合物4(2.0千克，5.79摩尔，1当量)和KOAc(1.14千克，11.57摩尔，2.0当量)，向反应釜中鼓入氮气20分钟后，向反应釜中依次加入DPEPhos(149.6克，277.69毫摩尔，0.05当量)、Pd(PhCN) 2Cl 2(88.8克，231.41毫摩尔，0.04当量)，继续鼓入氮气5分钟，再加入乙烯基正丁基醚(1.74千克，17.36摩尔，3.0当量)。在氮气保护下将反应混合物加热至90～100℃，搅拌17小时。HPLC显示原料剩余约8％。向反应釜中补加乙烯基正丁基醚(289.7克，2.9摩尔，0.5当量)，继续反应6小时。HPLC显示原料剩余约5％。向反应釜中补加乙烯基正丁基醚(289.7克，2.9摩尔，0.5当量)，继续反应3小时。HPLC显示原料剩余约4％。关闭加热降至室温，搅拌14.5小时。将混合物过滤，滤饼用EtOAc(8升)洗涤，滤液浓缩，得到的油状物在15℃下用乙酸乙酯：正庚烷＝1:3的混合溶剂(16升)搅拌打浆16小时。将混合物通过100～200目硅胶(2.0千克)过滤，滤饼用乙酸乙酯：正庚烷＝1:3的混合溶剂(12升)洗涤。滤液浓缩至干，得到棕色油状粗品(2.7千克)。
用量筒量取浓盐酸(1.35升)加入到去离子水(6.75升)中充分搅拌混合均匀，配制成2M盐酸(8.1升)备用。
在15℃下，将上述棕色油状粗品(2.7千克，粗品)和乙醇(5.4升)依次加入到反应釜中，在搅拌下滴加配置好的2M盐酸(8.1升)，有大量固体析出。将反应混合物在室温(15℃)下搅拌16小时。HPLC显示反应完全。将反应混合物过滤，滤饼用去离子水(4L)洗涤，得到的滤饼用真空干燥箱在50℃，-0.1MPa条件下干燥66小时，得到灰白色固体(1290千克，粗品)。将上述粗品转移至反应釜中，加入乙腈(6450 毫升)，将反应混合物在50℃下搅拌2小时。将反应混合物降至室温，过滤，滤液浓缩。向得到的残余物中加入EtOAc(6升)，继续浓缩。当EtOAc基本蒸干(瓶中无明显溶剂)时，向旋蒸瓶中加入正庚烷(6升)，继续旋蒸。当溶剂剩余约3升时，过滤，滤液蒸干后与滤饼混合。将混合后的固体用真空干燥箱在50℃，-0.1MPa条件下干燥16小时，得到化合物5。 1H NMR(400MHz,CDCl 3)δ8.77(s,1H),7.36(s,1H),5.41(quin,J＝8.9Hz,1H),2.56(s,3H),2.44(s,3H),2.26-2.18(m,2H),2.15-2.07(m,2H),2.05-1.97(m,2H),1.84-1.75(m,2H)。
制备流程2：
Figure PCTCN2019091073-appb-000014
第1步：
在22℃下，向反应釜内依次加入2-MeTHF(25升)、化合物6(3000.00克，20.83摩尔，1.0当量)，然后分批加入KOH(2340.01克，41.65摩尔，2.0当量)。在40℃下，向反应釜内加入KI(345.76克，2.08摩尔，0.1当量)，然后再加入哌嗪(2150.03克，24.99摩尔，1.2当量)，最后再加入2-MeTHF(5升)。将反应体系加热至80℃，搅拌16小时。GC显示产物约60.74％，副产物约23.94％。将反应体系降温至5℃，有大量固体析出。停止搅拌，利用虹吸原理将上清液吸出。向反应釜内加入2-MeTHF(5升)，溶解出釜内残留的产物，再次利用虹吸原理将上清液吸出。将两次析出的2-MeTHF溶液混合后浓缩，当大部分溶剂被蒸出时，向旋蒸瓶中加入石油醚(1升)，过滤，除掉析出的哌嗪。滤液再旋干，并用油泵旋出尽可能多的哌嗪，得到残余物(2.68千克)。该残余物经过常规减压蒸馏(压力：19.0～32Torr，馏分温度≤70℃)，得到粗品。该粗品通过分子精馏(压力：1.5～2.5bar，转速320～325rpm/min，温度：70～75℃，-17～-13℃，进样速度：200mL/h)，得到化合物7。
第2步：
在21℃下，向反应釜内依次加入2-MeTHF(5升)、2,5-二溴吡嗪(750.00克，3.15摩尔，1.0当量)、Na 2CO 3(668.33克，6.31摩尔，2.0当量，1.4当量)，然后在搅拌下加入化合物7(749.68克，4.41摩尔)，再加入2-MeTHF(2.5升)。将反应体系加热至80℃，搅拌16小时。HPLC显示反应完全。将反应体系降 温至21℃，将反应混合物通过硅藻土过滤。向反应釜中加入2-MeTHF(1.5升)，清洗釜壁上残留的产物，然后过滤。将合并后的滤液置于分液器中，加入10％食盐水(750毫升)洗涤有机相，共洗涤两次。有机相浓缩后置于托盘内自然凝固成固体。在室温(22℃)下，向反应釜中加入水(3升)和上述固体，然后再加入水(250毫升)，将反应体系加热至50℃，搅拌22小时。将温度降至5℃，搅拌1小时。将反应混合物过滤，滤饼用真空干燥箱在50℃，-0.1MPa条件下干燥约18小时，得到化合物8。
第3步：
向一个5升高压釜中加入化合物8(300.01克，930.89毫摩尔，1.00当量)、铜粉(8.87克，139.63毫摩尔，0.15当量)和氨水(3000毫升)，密封高压釜，开启搅拌和加热，外温设为95℃。向另一个反应釜中加入化合物8(300.00克，930.86毫摩尔，1.00当量)、铜粉(8.87克，139.63毫摩尔，0.15当量)和氨水(3000毫升)，密封高压釜，开启搅拌和加热，外温设为95℃。将两个反应混合物在95℃搅拌18小时。分别取样送LCMS检测，LCMS显示原料反应完全。将反应体系降温至50℃，分别向两个反应釜中加入氯化钠固体(600克)，并在室温(15℃)下搅拌1.5小时。分别将反应混合物过滤，滤饼用真空干燥箱在55℃条件下干燥约17小时。将两个滤饼合并后平分为四份，投入四个3升反应瓶中，并向每个反应瓶中加入无水THF(2.5升)，在25℃下搅拌1.5小时。将混合物过滤，滤液合并后浓缩，共浓缩出约8升溶剂。向旋蒸瓶中加入MTBE(500毫升)，浓缩置换剩余的THF，共置换三次，将混合物过滤，分别将反应混合物过滤，滤饼用真空干燥箱在55℃条件下干燥约12小时，得到化合物9(305.79克)。
第4步：
在室温(15℃)下，依次将1,4-二氧六环(2.5升)、化合物5(250.08克，772.71毫摩尔，1.0当量)、化合物9(240.13克，927.25毫摩尔，1.2当量)、K 2CO 3(213.59克，1.55摩尔，2.0当量)和水(250毫升)加入到反应釜中，向反应釜中鼓入氮气，在氮气保护下将Xphos(73.67克，154.54毫摩尔，0.2当量)和Pd(OAc) 2(17.35克，77.27毫摩尔，0.1当量)。在氮气保护下将反应混合物加热至100～110℃(外温)，內温为90～100℃，搅拌17小时。HPLC显示化合物5反应完全。将反应混合物通过硅藻土过滤，滤饼用2-MeTHF(1升)洗涤，将收集的滤液蒸干，得到的残余物用2-MeTHF(2升)和水(500毫升)溶解并分液，水相用2-MeTHF(250毫升*2)萃取。将合并的有机相用饱和食盐水(500毫升*2)洗涤，经无水硫酸钠干燥后过滤，滤液浓缩，得到的残余物用EtOAc(750毫升)在室温(15℃)下打浆搅拌16小时。将该混合物过滤，将收集的滤饼蒸干。得到的残余物用正庚烷：乙酸乙酯＝3:1的混合溶剂(7.8升)在室温(15℃)下打浆搅拌16小时。将该混合物过滤，滤饼用正庚烷：乙酸乙酯＝3:1的混合溶剂(600毫升)洗涤，将滤饼蒸干，得到的固体(322.59克)。取上述固体(321.59克)置于三口烧瓶中，然后加入EtOH(1600毫升)，将反应体系加热至100℃(外温)，剧烈回流，使固体全部溶解。关闭加热，使体系自然降温，并搅拌16小时。将化合物过滤，滤饼用EtOH(200毫升)洗涤，将滤饼用真空干燥箱在55℃，-0.1MPa条件下干燥7小时，得到粗品(286.44克)。 1H NMR(400MHz,CD 3OD)δ8.69(s,1H),8.33(s,1H),7.89(s,1H),7.77(s,1H),5.56-5.65(m,1H),3.53-3.46(m,4H),2.68-2.62(m,4H),2.60-2.55(m,4H),2.51-2.45(m,3H),2.38(s,3H),2.31(s,6H),2.30-2.22(m,2H),2.15-2.18(m,2H),2.02-1.92(m,2H),1.84-1.74(m,2H)。
在20℃下，依次将上述粗品(282.50克)和THF(2.8升)加入到三口烧瓶中，开启搅拌，体系变澄清。然后向反应瓶中加入硫脲树脂LS2000(113.06克)，将混合物加热至40℃，搅拌16小时。将混合物 通过硅藻土过滤，滤饼用THF(200毫升)洗涤，收集滤液。将滤液蒸干，得到式(I)化合物。
式(I-2)化合物的晶型的制备
在18℃下，依次将THF(2.8升)和式(I)化合物(288.25克，528.09毫摩尔，1.0当量)加入到反应釜中，并加热到40℃，体系少许浑浊。将混合物趁热过滤，将滤液重新转移至反应釜中，并向反应釜中缓慢滴加浓盐酸(39.60毫升，0.9当量)，再加入THF(280毫升)，并在40～45℃下搅拌16小时。将反应混合物过滤，滤饼用THF(280毫升)洗涤，将滤饼置于真空干燥箱中于50℃下干燥4小时，得到粗品。
在室温12℃下，依次将上述粗品(257.16克)、EtOH(2.9升)和水(145毫升)加入到反应釜中，将反应混合物加热至80℃(剧烈回流)，反应体系变澄清。停止加热并缓慢降温，当温度降至55℃时，有大量固体析出，继续降温。将反应混合物降至室温，搅拌16.5小时。将混合物过滤，滤饼用EtOH(300毫升)洗涤，将滤饼用真空干燥箱在50℃，-0.1MPa条件下干燥至恒重，得到式(I-2)化合物的晶型。 1H NMR(400MHz,CD 3OD)δ8.73(s,1H),8.37(s,1H),7.93(s,1H),7.78(s,1H),5.57-5.66(m,1H),3.59-3.53(m,4H),3.39-3.34(m,2H),2.97(s,6H),2.81(t,J＝5.8Hz,2H),2.75-2.70(m,4H),2.51(s,3H),2.41(s,3H),2.37-2.27(m,2H),2.23-2.13(m,2H),2.04-1.94(m,2H),1.85-1.76(m,2H)；依据氯离子含量检测方法测出其氯离子含量为6.7％。
表征实施例
实施例1：式(I-2)化合物的晶型的吸湿性研究
实验条件：
仪器型号：SMSDVS Advantage动态蒸汽吸附仪
测试条件：取样品(10～15mg)置于DVS样品盘内进行测试。
DVS参数：
温度：25℃
平衡：dm/dt＝0.01％/min(最短：10min，最长：180min)
干燥：0％RH下干燥120min
RH(％)测试梯级：10％
RH(％)测试梯级范围：0％-90％-0％
吸湿性评价标准见表1-2。
表1-2
吸湿性分类 吸湿增重*(ΔW％)
潮解 吸收足量水分形成液体
极具吸湿性 ΔW％≥15％
有吸湿性 15％>ΔW％≥2％
略有吸湿性 2％>ΔW％≥0.2％
无或几乎无吸湿性 ΔW％<0.2％
注：*在25±1℃和80±2％RH下的吸湿增重
实验结果：式(I-2)化合物的晶型在25℃、80％RH下的吸湿增重为0.11％。
实验结论：式(I-2)化合物的晶型无或几乎无吸湿性。
实施例2：式(I-2)化合物的晶型研究
称取50毫克左右的式(I)化合物晶型加入到不同的1.5mL玻璃小瓶中，加入适量的溶剂或溶剂混合物(见下表1-3)使其成悬浊液或溶液。加入磁子后，将上述样品置于磁力加热搅拌器上(40℃)进行2天试验。
表1-3
编号 溶剂 晶型
1 乙醇 式(I-2)化合物的晶型
2 乙腈 式(I-2)化合物的晶型
3 丙酮 式(I-2)化合物的晶型
4 乙酸乙酯 式(I-2)化合物的晶型
5 四氢呋喃 式(I-2)化合物的晶型
由上表可知，在乙醇、乙腈、丙酮、乙酸乙酯、四氢呋喃溶剂条件下，式(I-2)化合物的晶型稳定，未发生明显变化。
实施例3：式(I-2)化合物的晶型的溶解度实验
分别称取6毫克式(I-2)化合物的晶型加入到不同的8毫升玻璃小瓶中，分别加入3毫升对应的生物媒介溶液。加入磁子后，将上述样品置于磁力加热搅拌器上(37对应避光)进行试验并观察现象；若溶清，则继续以6毫克为单位进行补加相应化合物，直至不再溶解或者达到10mg/mL为止。于4小时以及24小时取样测试对应溶解度。
表1-4
Figure PCTCN2019091073-appb-000015
注：FaSSIF:模拟人类餐前饥饿状态下小肠内的肠液；
FeSSIF:模拟人类餐后饱食状态下小肠内的肠液；
SGF:模拟人类饥饿状态下空胃时的胃液；
由上表可知，式(I-2)化合物的晶型在水中的溶解度非常好。
实施例4：式(I-2)化合物的晶型的稳定性试验
实验方法：
根据影响因素和加速试验条件，准确称取该晶型10mg，一式两份分别置于40mL玻璃样品瓶的底部，摊成薄薄的一层，放置在合适的条件下。敞口条件在铝箔纸上扎些小孔，保证样品能与环境空气充分接触。另外分别取少量样品放置在40mL玻璃样品瓶中，同样条件下待测定晶型状态。在考察时间点，将相应的供试样品取出，用瓶盖盖好，0天的样品从冰箱中取出，待样品恢复至室温后进行分析。大约10mg供试品用于XRPD检测。考察化合物在以下条件放置并在不同的时间点取样检测性状，XRPD，含量和有关物质。研究条件和检测项目见表1-5。
表1-5固体稳定性放样分析条件
Figure PCTCN2019091073-appb-000016
Figure PCTCN2019091073-appb-000017
注：*测试项目X包括：性状，XRPD，含量及有关物质，0天样品为初始样品。
其中，光照试验按照ICH option 2放样和dark control样品(即锡箔纸包裹样品)
放样(与稳定性试验指导原则一致)。
实验结果：上述稳定性放样条件下受试物性状没有发生明显变化，含量及有关物质结果见表1-6和表1-7。
表1-6影响因素稳定性试验中晶型及有关物质含量的检测结果
Figure PCTCN2019091073-appb-000018
注：LOQ是指HPLC仪器的检测限。
RRT是指相对保留时间。
表1-7加速稳定性试验中晶型及有关物质含量的检测结果
Figure PCTCN2019091073-appb-000019
注：LOQ是指HPLC仪器的检测限
试验结论：
稳定性测试取样后的晶型的含量及有关物质的检测结果显示，式(I-2)化合物的晶型比较稳定，其XRPD(见图4)检测结果显示式(I-2)化合物的晶型只有在92.5％相对湿度(10天)条件下的晶型发生变化，其他测试条件下式(I-2)化合物的晶型都非常稳定。
药理部分
本发明涉及的化合物是CDK4/6抑制剂。以下实验结果证实本专利列举的化合物确实是CDK4/6抑制剂并且可作为潜在的抗癌药。此处用到的IC 50是指使用某种试剂产生50％最大抑制时对应该试剂的浓度。
实验例一：酶活性测试
实验材料：
CDK4/cyclin D1，CDK6/cyclin D1(Life technology)。ULight标记的多肽底物ULight-4E-BP1和ULight-MBP(PerkinElmer)。铕标记的抗髓磷脂碱蛋白抗体和铕标记的兔源抗体(PerkinElmer)，Envision多标记分析仪进行信号的检测(PerkinElmer)。
实验方法：
将待检测的化合物进行三倍稀释，包括10个浓度梯度，最终的浓度范围是5μM到0.25nM。
●CDK4/cyclin D1的酶反应体系
标准的Lance Ultra方法通过10μL的酶反应体系进行，包含0.3nM CDK4/cyclin D1蛋白，50nM ULight-4E-BP1多肽，和350μM ATP。分别将其溶解在酶缓冲液中，缓冲液的成分包括：PH7.5的羟乙基哌嗪乙硫磺酸溶液50mM，乙二胺四乙酸1mM，氯化镁10mM，0.01％Brij-35，二硫苏糖醇2mM。开始反应后，用顶部热封膜TopSeal-A将OptiPlate384孔板封好，室温孵育180分钟。
●CDK6/cyclin D1的酶反应体系
标准的Lance Ultra方法通过10μL的酶反应体系进行，包含0.8nM CDK6/cyclin D1蛋白，50nM ULight-4E-BP1多肽，和250μM ATP。分别将其溶解在酶缓冲液中，缓冲液的成分包括PH7.5的羟乙基哌嗪乙硫磺酸溶液50mM，乙二胺四乙酸1mM，氯化镁10mM，0.01％Brij-35，二硫苏糖醇2mM。开始反应后，用顶部热封膜TopSeal-A将OptiPlate384孔板封好，室温孵育180分钟。
准备酶反应终止缓冲液，用1倍稀释的检测缓冲液溶解EDTA，终止反应在室温进行5分钟。分别在CDK4/cyclin D1和CDK6/cyclin D1反应中加入5μL检测混合液(分别用铕标记的抗髓磷脂碱蛋白抗体和铕标记的兔源抗体配置)。室温孵育60min，根据时间分辨荧光共振能量转移原理利用Envision仪器检测反应信号。
数据分析：
利用方程式(Max-Ratio)/(Max-Min)*100％将原始数据换算成抑制率，IC 50的值即可通过四参数进行曲线拟合得出(XLFIT5中205模式得出，iDBS)。表1提供了本发明的化合物对CDK4/CDK6激酶的抑制活性。
实验结果：见表2-1。
实验结论：
本发明化合物对CDK4和CDK6激酶具有显著的抑制活性。
实验例二：细胞活性测试
实验材料：
RPMI 1640培养基(Invitrogen-22400089)，胎牛血清(Gibco-10099141)，青霉素/链霉素抗生素(Hyclone-SV30010)，L-谷氨酰胺(Invitrogen-35050079)。NCI-H358细胞系来自药明康德生物部细胞库。Envision多标记分析仪(PerkinElmer)。
实验方法：
1)向384微孔板的外围孔中加入100μL磷酸盐缓冲液，分别向其它孔中加40μL NCI-H358细胞悬液，其中包含250个NCI-H358细胞。然后将细胞板放到二氧化碳培养箱中过夜培养。
2)用Echo对待测化合物进行3倍梯度稀释，将每个化合物稀释10个浓度梯度(从25μM稀释至1.27nM)并分别加100nL到细胞板的对应孔中，然后将细胞板放回到二氧化碳培养箱中培养7天。
3)向细胞板中加入每孔20μL的Promega CellTiter-Glo试剂，室温避光震荡10分钟使发光信号稳定。采用PerkinElmer Envision多标记分析仪读数。
数据分析：
利用方程式(Max-Sample)/(Max-Min)*100％将原始数据换算成抑制率，IC 50的值即可通过四参数进行曲线拟合得出(GraphPad Prism中log(inhibitor)vs.response--Variable slope拟合公式计算得出)。表1提供了本发明的化合物对H358细胞增殖的抑制活性。
实验结果：见表2-1。
实验结论：
本发明化合物较参照化合物Palbociclib对NCI-H358肺癌细胞具有更好的增殖抑制活性。
表2-1
Figure PCTCN2019091073-appb-000020
实验例三：Caco-2细胞双向渗透性评估实验
实验目的：
Caco-2细胞是一种广泛用于研究小肠吸收的体外模型，它是一种人的结肠癌细胞。单层Caco-2细胞模型已广泛地应用于评估在小肠吸收过程中的被动和主动转运过程。本实验用于测定本发明化合物及参照化合物Palbociclib穿过Caco-2细胞模型的双向渗透性。
实验操作：
实验标准条件如下：
-测试浓度:2μM(DMSO≤1％)；
-重复：n＝2；
-方向:双向转运，包括A→B(细胞内→细胞外)和B→A(细胞外→细胞内)两个方向；
-孵育时间：单个时间点，2小时；
-转运缓冲液：HBSS，pH 7.4；
-孵育条件:37℃,5％CO 2。
孵育结束后，取给药孔和接收孔内的样品溶液立即与含有内标的冷乙腈溶液混合。采用LC/MS/MS方法分析待测化合物在所有样品(包括起始给药液，给药孔上清液，接收液)中的浓度。并计算表观渗透系数，外排比等参数。
实验结果：
见表2-2。表2-2列出了本发明化合物及参照化合物Palbociclib在Caco-2单层细胞中的渗透系数。
实验结论：
与参照化合物Palbociclib相比，本发明化合物具有较高渗透性，而且体内吸收及转运受到外排转运体 影响的可能性较低。更好的渗透性可使得本发明化合物在体内组织(比如肺部)中分布更多，带来更好的体内抗肿瘤药效。同时，更好的渗透性使得本发明化合物有可能穿透血脑屏障，达到治疗脑转移肺癌的目的。
表2-2
Figure PCTCN2019091073-appb-000021
实验例四：大鼠，小鼠和人肝微粒体代谢稳定性实验
实验目的：
本试验用于测试受试物在大鼠，小鼠和人肝微粒体的代谢稳定性
实验操作：
1)测试化合物浓度为1μM在还原性辅酶Ⅱ再生体系作用下与蛋白浓度为0.5mg/mL的肝微粒体共同孵育在37度水浴条件中。
2)阳性对照包括：睾丸酮(3A4底物)，丙胺苯丙酮(2D6底物)，双氯芬酸(2C9底物)。阳性对照孵育条件同化合物孵育条件保持一致。
3)反应时间点为：0、5、10、20、30和60分钟，在相应时间点时使用含内标的终止液终止反应。
在没有还原性辅酶Ⅱ再生体系作用下，化合物同样与微粒体孵育60分钟并作为阴性对照。
4)每个时间点是单点(n＝1)。
5)样品由LC/MS/MS测定，并且化合物浓度显示为化合物峰面积与内标峰面积的比值(非标曲)。
6)项目报告小结中，将会计算半衰期和清除率。
7)下列公式用于计算清除率：
Figure PCTCN2019091073-appb-000022
Figure PCTCN2019091073-appb-000023
注：
a)microsomal protein in incubation：孵育时蛋白浓度
肝重体重比：大鼠，小鼠和人参数分别为40g/kg，88g/kg和20g/kg
利用
Figure PCTCN2019091073-appb-000024
计算整个肝中的清除率：
Figure PCTCN2019091073-appb-000025
注：
a)microsomes：微粒体；
b)liver：肝；
c)body weight：体重；
实验结果：
实验结果见表2-3。
实验结论：
本发明化合物在人、大鼠、小鼠体内的肝微粒体稳定性明显优于参照化合物Palbociclib。
表2-3
受试物 人/大鼠/小鼠T 1/2(min)
Palbociclib 44.7/47.8/53.3
式(I)化合物(盐酸盐) 43.1/>145/40.2
实验例五：体内药效研究(一)
在皮下植入非小细胞肺癌NCI-H358模型BALB/c裸小鼠上进行体内药效实验。
实验操作：
实验动物信息如下：BALB/c裸鼠，雌性，6-8周，体重约16-18克，将小鼠保持在一个特殊的无病原体的环境中，且在单个通风笼中(4只小鼠每笼)。所有的笼子，铺垫和水在使用前进行消毒。所有的动物都可以自由获取标准认证的商业实验室饮食。共有56只购于上海灵畅生物科技有限公司的小鼠用于研究。
每只小鼠在右后背皮下植入NCI-H358肿瘤细胞，用于肿瘤的生长。当平均肿瘤体积达到约100-200立方毫米时开始给药。将试验化合物每日口服给药，给药剂量如表2-4所示。肿瘤体积每周两次用二维卡尺测量，体积以立方毫米计量，通过以下公式计算：V＝0.5a x b 2，其中a和b分别是肿瘤的长径和短径。抗肿瘤药效是通过用化合物处理过的动物的平均肿瘤增加体积除以未处理过动物的平均肿瘤增加体积来确定，而化合物的安全性是通过用化合物处理过的动物的体重变化来确定的。
实验结果：见表2-4。
实验结论：
本发明化合物在非小细胞肺癌NCI-H358模型上展示了显著的抗肿瘤活性，且具有较好的安全性。此外，在此模型中，本发明化合物的抗肿瘤作用具有量效依赖的趋势。
表2-4
Figure PCTCN2019091073-appb-000026
TGI：Tumor Growth Inhibition(肿瘤增长抑制率)。TGI(％)＝[(1-(某处理组给药结束时平均瘤体积－该处理组开始给药时平均瘤体积))/(溶剂对照组治疗结束时平均瘤体积－溶剂对照组开始治疗时平均瘤体积)]×100％。
实验例六：体内药效研究(二)
在皮下植入结直肠癌HCT-116模型BALB/c裸小鼠上进行体内药效实验。
实验操作：
BALB/c裸鼠，雌性，6-8周，体重约18-22克，将小鼠保持在一个特殊的无病原体的环境中，且在单个通风笼中(3-5只小鼠每笼)。所有的笼子，铺垫和水在使用前进行消毒。所有的动物都可以自由获取标 准认证的商业实验室饮食。共有48只购于上海灵畅实验动物有限公司的小鼠用于研究。将0.2mL 5×106个HCT-116细胞皮下接种于每只裸小鼠的右后背。肿瘤平均体积达到132mm 3时开始分组给药。将试验化合物每日口服给药，给药剂量如表2-5所示。肿瘤体积每周两次用二维卡尺测量，体积以立方毫米计量，通过以下公式计算：V＝0.5a x b 2，其中a和b分别是肿瘤的长径和短径。抗肿瘤药效是通过用化合物处理过的动物的平均肿瘤增加体积除以未处理过动物的平均肿瘤增加体积来确定。
实验结果：见表2-5。
实验结论：
本发明化合物在结直肠癌HCT-116模型上展示出较好的抗肿瘤活性和较高的安全性。
表2-5
Figure PCTCN2019091073-appb-000027
实验例七：体内药效研究(三)
在皮下植入结肠癌HCT-116模型BALB/c裸小鼠上进行体内药效实验。
实验操作：
BALB/c裸鼠，雌性，～8周，体重约18-21克，将小鼠保持在一个特殊的无病原体的环境中，且在单个通风笼中(每笼4只小鼠)。所有的笼子，铺垫和水在使用前进行消毒。所有的动物都可以自由获取标准认证的商业实验室饮食。共有40只购于上海灵畅生物科技有限公司的小鼠用于研究。将0.2mL 5×10 6个HCT-116细胞皮下接种于每只裸小鼠的右后背。肿瘤平均体积达到139mm 3时开始分组给药。将试验化合物每日口服给药，给药剂量如表2-6所示。肿瘤体积每周两次用二维卡尺测量，体积以立方毫米计量，通过以下公式计算：V＝0.5a x b 2，其中a和b分别是肿瘤的长径和短径。抗肿瘤药效是通过用化合物处理过的动物的平均肿瘤增加体积除以未处理过动物的平均肿瘤增加体积来确定。
实验结果：见表2-6。
实验结论：
本发明化合物显著抑制HCT 116人结肠癌细胞裸鼠皮下移植瘤的生长，抑制效果具有剂量依赖性，且安全性良好。
表2-6
Figure PCTCN2019091073-appb-000028
Figure PCTCN2019091073-appb-000029
实验例八：体内药效研究(四)
在皮下植入结肠癌CT-26模型BALB/c裸小鼠上进行体内药效实验。
实验操作：
BALB/c裸鼠，雌性，6-8周，将小鼠保持在一个特殊的无病原体的环境中，且在单个通风笼中(每笼4只小鼠)。所有的笼子，铺垫和水在使用前进行消毒。所有的动物都可以自由获取标准认证的商业实验室饮食。共有64只购于上海斯莱克实验动物有限责任公司的小鼠用于研究。将0.1mL 3×10 5个CT-26细胞皮下接种于每只裸小鼠的右后背。肿瘤平均体积达到75mm 3时开始分组给药。将试验化合物每日口服给药，给药剂量如表2-7所示。肿瘤体积每周两次用二维卡尺测量，体积以立方毫米计量，通过以下公式计算：V＝0.5a x b 2，其中a和b分别是肿瘤的长径和短径。抗肿瘤药效是通过用化合物处理过的动物的平均肿瘤增加体积除以未处理过动物的平均肿瘤增加体积来确定。
实验结果：见表2-7。
实验结论：
本发明化合物单药治疗能显著的抑制CT26小鼠结肠癌细胞皮下移植瘤的生长，抑制效果具有剂量依赖性。本发明化合物与抗PD-1抗体CS1003联用后有增强单药药效的趋势。
表2-7
Figure PCTCN2019091073-appb-000030

Claims (15)

  1. 式(I)化合物的盐酸盐，
    Figure PCTCN2019091073-appb-100001
  2. 根据权利要求1所述的盐酸盐，其具有式(I-1)所示结构：
    Figure PCTCN2019091073-appb-100002
    其中，n为0.9～1.1。
  3. 根据权利要求2所述的盐酸盐，其中，n为0.9、1.0或1.1。
  4. 根据权利要求3所述的盐酸盐，其具有式(I-2)所示结构：
    Figure PCTCN2019091073-appb-100003
  5. 根据权利要求1～4任一项所述的盐酸盐的晶型，其X-射线粉末衍射图谱包含在2θ值为9.94°±0.2°、12.43°±0.2°和22.76°±0.2°的特征峰。
  6. 根据权利要求5所述的晶型，其X-射线粉末衍射图谱包含在2θ值为9.94°±0.2°、12.43°±0.2°、13.03°±0.2°、18.63°±0.2°、19.21°±0.2°、22.76°±0.2°、28.08°±0.2°和31.40°±0.2°的特征峰。
  7. 根据权利要求6所述的晶型，其X-射线粉末衍射图谱包含在2θ值为9.94°±0.2°、12.43°±0.2°、13.03°±0.2°、18.63°±0.2°、19.21°±0.2°、20.38°±0.2°、21.11°±0.2°、22.76°±0.2°、24.41°±0.2°、28.08°±0.2°和31.40°±0.2°的特征峰。
  8. 根据权利要求7所述的晶型，其X-射线粉末衍射图谱包含在2θ值为9.94°、12.43°、13.03°、15.22°、15.97°、16.72°、18.63°、19.21°、19.60°、20.38°、21.11°、22.76°、23.08°、23.79°、24.41°、25.08°、27.21°、28.08°、28.63°、30.04°、30.60°、31.40°、34.20°、34.74°、35.51°和38.45°的特征峰。
  9. 根据权利要求8所述的晶型，其X-射线粉末衍射图谱如图1所示。
  10. 根据权利要求6～9任一项所述的晶型，其DSC曲线显示吸热峰的起始点为263.38℃±2℃，峰值温度为 266.16℃±2℃。
  11. 根据权利要求10所述的晶型，其DSC曲线如图2所示。
  12. 根据权利要求6～9任一项所述的晶型，其TGA曲线在211.67℃±3℃时开始明显失重，重量共减少了23.59％。
  13. 根据权利要求12所述的晶型，其TGA曲线如图3所示。
  14. 根据权利要求1～4任一项所述的盐酸盐或权利要求5～13任一项所述的晶型在制备治疗癌症药物中的应用。
  15. 根据权利要求14所述的应用，其中所述癌症为非小细胞肺癌。
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