Substituted zoritinib derivative as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicines, in particular to a substituted zoritinib derivative as well as a preparation method and application thereof. Specifically, the invention provides a substituted zoritinib derivative as shown in a general formula (I) or a pharmaceutically acceptable salt or solvate thereof. Wherein R1, R2 and R3 are independently selected from methyl, methoxyl, halogen substituted methyl, halogen substituted methoxyl, deuterium atom substituted methyl and deuterium atom substituted methoxyl. According to the present invention, the structure of the prototype drug is modified to obtain the substituted zoritinib derivative, the stability and the drug effect of the obtained substituted zoritinib derivative are superior to those of AZD3759, and the in-vivo generation concentration of the main active metabolite AZ' 1168 causing the adverse reaction can be reduced. The invention also accidentally finds that when the substituted zoritinib derivative is combined with a CYP3A inhibitor, the inhibition rate of inhibiting prototype drug metabolism can be obviously improved. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a substituted zolitinib derivative and a preparation method and application thereof. Background Art

[0002] EGFR (Epidermal Growth Factor Receptor, referred to as EGFR, ErbB-1, HER1) is a member of the epidermal growth factor receptor (HER) family. Patients with non-small-cell lung cancer (NSCLC) with EGFR mutations respond to treatment with EGFR tyrosine kinase inhibitors (TKIs). However, patients often develop central nervous system (CNS) metastases during treatment. In the absence of effective options, clinicians have tried higher doses of EGFR TKIs to achieve sufficiently high drug concentrations in the central nervous system. Although this approach can achieve a certain degree of anti-tumor response, it results in too high toxicity outside the central nervous system, so there is an urgent need for safer new treatment options for brain metastases.

[0003] AZD3759 (Zorifertinib, Chinese generic name: Zolitinib, chemical structure shown below) is an epidermal growth factor receptor tyrosine kinase inhibitor developed by AstraZeneca specifically for brain metastasis of lung cancer. AZD3759 is a selective EGFR inhibitor that can completely penetrate the blood-brain barrier (BBB) ​​and has the same free concentration in blood, cerebrospinal fluid and brain tissue. AZD3759 caused tumor regression in subcutaneous transplanted tumors, meningeal light metastases and brain metastases lung cancer models, and prevented the occurrence of brain metastases in nude mice. The blood-brain permeability of AZD3759 reached 100%, which can continuously and efficiently inhibit the growth of tumor tissue in organs throughout the body, including brain tissue. Therefore, AZD3759 is an orally available inhibitor that can inhibit the activity of epidermal growth factor receptor tyrosine kinase with activating mutations, and is currently used to treat non-small cell lung cancer with central nervous system metastasis.

[0004] Preclinical and clinical studies have shown that after oral administration, AZD3759 is metabolized to AZ'1168 by CYP450 enzymes due to the first-pass effect in the body, and the exposure in the body is higher than that of the prototype drug AZD3759. AZ'1168 is a metabolite with similar activity generated by the demethylation metabolism of AZD3759, but its pharmacological activity and ability to penetrate the blood-brain barrier are lower than those of the prototype drug, and a large amount of AZ'1168 exposure may affect the clinical efficacy of AZD3759 and increase adverse reactions. Summary of the invention

[0005] In order to improve the metabolic stability of the prototype drug AZD3759, the present invention structurally transforms the prototype drug, and the obtained substituted zolitinib derivative has better stability and efficacy than AZD3759, and can reduce the concentration of the main active metabolite AZ'1168 that causes adverse reactions generated in the body. The present invention also unexpectedly found that the substituted zolitinib derivative can significantly improve the inhibition rate of the prototype drug metabolism when used in combination with a CYP3A inhibitor. The present invention mainly achieves the above purpose through the following technical solutions:

[0006] In a first aspect, the present invention provides a substituted zolitinib derivative represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof: in: R 1 Selected from methyl, methoxy, methyl substituted by halogen (including fluorine, chlorine, bromine, iodine), methoxy substituted by halogen (including fluorine, chlorine, bromine, iodine), methyl substituted by deuterium atom, methoxy substituted by deuterium atom; R 2 Selected from methyl, methoxy, methyl substituted by halogen (including fluorine, chlorine, bromine, iodine), methoxy substituted by halogen (including fluorine, chlorine, bromine, iodine), methyl substituted by deuterium atom, methoxy substituted by deuterium atom; R 3 Selected from methyl, methoxy, methyl substituted by halogen (including fluorine, chlorine, bromine, iodine), methoxy substituted by halogen (including fluorine, chlorine, bromine, iodine), methyl substituted by deuterium atom, methoxy substituted by deuterium atom.

[0007] Preferably, R 1 Selected from methyl, methoxy, methyl substituted with 1 to 3 deuterium atoms, methyl substituted with 1 to 3 fluorine atoms, methoxy substituted with 1 to 3 deuterium atoms, methoxy substituted with 1 to 3 fluorine atoms; R 2Selected from methyl, methoxy, methyl substituted with 1 to 3 deuterium atoms, methyl substituted with 1 to 3 fluorine atoms, methoxy substituted with 1 to 3 deuterium atoms, methoxy substituted with 1 to 3 fluorine atoms; R 3 Selected from methyl, methoxy, methyl substituted with 1 to 3 deuterium atoms, methyl substituted with 1 to 3 fluorine atoms, methoxy substituted with 1 to 3 deuterium atoms, methoxy substituted with 1 to 3 fluorine atoms.

[0008] Preferably, R 1 Selected from methyl, methoxy, trideuteriomethyl, trifluoromethyl, trideuteriomethoxy (methoxy completely substituted by deuterium atoms), trifluoromethoxy (methoxy completely substituted by fluorine atoms); R 2 is selected from methyl, methoxy, trideuteriomethyl, trifluoromethyl, trideuteriomethoxy, trifluoromethoxy; R 3 Selected from methyl, methoxy, trideuteriomethyl, trifluoromethyl, trideuteriomethoxy, trifluoromethoxy.

[0009] Preferably, R 1 is selected from methyl, trideuteriomethyl, trifluoromethyl; R 2 is selected from methyl or trideuterated methyl; R 3 Selected from methoxy or trideuterated methoxy.

[0010] Preferably, the substituted zolitinib derivative is selected from the following compounds:

[0011] In a second aspect, the present invention provides a method for preparing a substituted zolitinib derivative represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof. The preparation method comprises:

[0012] Technical Solution I:

[0013] Among them, R 4 and R 5 is independently selected from methyl, methoxy, methyl substituted by halogen (including fluorine, chlorine, bromine, iodine), methoxy substituted by halogen (including fluorine, chlorine, bromine, iodine), methyl substituted by deuterium atom, methoxy substituted by deuterium atom. Preferably, R 4 and R 5 are independently selected from methyl, methoxy, methyl substituted with 1 to 3 deuterium atoms, methyl substituted with 1 to 3 fluorine atoms, methoxy substituted with 1 to 3 deuterium atoms, and methoxy substituted with 1 to 3 fluorine atoms. Preferably, R 4 and R5 are independently selected from methyl, methoxy, trideuteriomethyl, trifluoromethyl, trideuteriomethoxy (methoxy completely substituted by deuterium atoms), trifluoromethoxy (methoxy completely substituted by fluorine atoms). Preferably, R 4 and R 5 is independently selected from methyl or trideuterated methyl. 4 and R 5 Not the same.

[0014] Preferably, in technical solution I, the molar ratio of compound A to compound B is 0.5:2 to 1:5.

[0015] Preferably, in technical scheme I, the reaction temperature is 15 to 30° C., and the reaction time is 5 to 15 hours.

[0016] Technical Solution II:

[0017] Preferably, in step 1 of technical scheme II, the molar ratio of compound C and CS2 is 0.5-2:1-5; the molar ratio of compound C and CH3I is 0.5-5:2-10; the reaction temperature is 10-30°C, and the reaction time is 0.5-5 hours.

[0018] Preferably, in step 2 of technical scheme II, the molar ratio of compound D to antimony trichloride (SbCl3) is 1:0.01 to 1:1; the molar ratio of compound D to BAST (bis(2-methoxyethyl)aminosulfur trifluoride) is 0.5 to 2:1 to 5; the reaction temperature is 10 to 30°C, and the reaction time is 12 to 36 hours.

[0019] Technical Solution III:

[0020] Preferably, in step 1 of the technical scheme III, the molar ratio of the compound E and BBr3 is 0.5-5:1-10; the reaction temperature is 30-60°C, and the reaction time is 0.5-2 hours.

[0021] Preferably, in step 2 of the technical scheme III, the molar ratio of the compound F and trideuterium 4-methylbenzenesulfonate is 0.5-5:0.5-5; the reaction temperature is 10-30° C., and the reaction time is 12-36 hours.

[0022] In a third aspect, the present invention provides use of a substituted zolitinib derivative represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof in the preparation of an epidermal growth factor receptor tyrosine kinase activity inhibitor drug.

[0023] In a fourth aspect, the present invention provides use of a substituted zolitinib derivative represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicament for preventing and / or treating non-small cell lung cancer, glioblastoma, and Parkinson's disease.

[0024] In a fifth aspect, the present invention provides a pharmaceutical composition comprising a substituted zolitinib derivative represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof, and a CYP3A inhibitor.

[0025] Preferably, the molar ratio of the substituted zolitinib derivative or a pharmaceutically acceptable salt or solvate thereof: the CYP3A inhibitor is 1:(0.1-10).

[0026] Preferably, the pharmaceutical composition further comprises the prototype drug AZD3759 or a pharmaceutically acceptable salt or solvate thereof.

[0027] Preferably, the molar ratio of the substituted zolitinib derivative or a pharmaceutically acceptable salt or solvate thereof: the prototype drug AZD3759 or a pharmaceutically acceptable salt or solvate thereof: the CYP3A inhibitor is 1:(0.1-10):(0.1-10).

[0028] CYP3A is the most important type of CYP enzyme, accounting for about 30% of human liver microsomes. Preferably, the CYP3A inhibitor includes but is not limited to a mixture of one or more of ketoconazole, ritonavir, schisandrin and silybin. Preferably, the CYP3A inhibitor is ketoconazole.

[0029] Preferably, the pharmaceutically acceptable salt is selected from sulfate, phosphate, citrate, tartrate, fumarate, benzoate, adipate, succinate, methanesulfonate and maleate, etc.

[0030] In a sixth aspect, the present invention provides use of the pharmaceutical composition in the preparation of an epidermal growth factor receptor tyrosine kinase activity inhibitor drug.

[0031] In a seventh aspect, the present invention provides use of the pharmaceutical composition in the preparation of a drug for preventing and / or treating non-small cell lung cancer, glioblastoma, and Parkinson's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the H NMR spectrum of compound cpds1; Figure 2 This is the H NMR spectrum of compound cpds4; Figure 3 This is the deuterium NMR spectrum of compound cpds4; Figure 4This is the H NMR spectrum of compound cpds2; Figure 5 This is the deuterium NMR spectrum of compound cpds2; Figure 6 This is the H NMR spectrum of compound cpds5; Figure 7 This is the deuterium NMR spectrum of compound cpds5; Figure 8 is the standard curve of compound cpds1 in human liver microsomes; Fig. 9 is the standard curve of compound cpds4 in human liver microsomes; Fig.10 is the standard curve of compound cpds5 in human liver microsomes; Fig.11 is the standard curve of compound cpds6 in human liver microsomes; Fig.12 is the standard curve of compound cpds7 in human liver microsomes; Fig.13 This is the standard curve of verapamil in human liver microsomes. DETAILED DESCRIPTION

[0033] The present invention is further described by the following embodiments. It should be understood that the following embodiments are only used to illustrate the present invention, but not to limit the present invention.

[0034] In order to improve the metabolic stability of the prototype drug AZD3759 and optimize the pharmacokinetic properties, the present invention transforms the molecular structure of the original drug AZD3759.

[0035] In some embodiments, the present invention replaces the N-methyl group of the piperazine ring, the C-methyl group of the piperazine ring, and the 7-methoxy group of the quinazoline ring of the prototype drug AZD3759. Preferably, the hydrogen atoms of the methyl or methoxy groups are replaced by deuterium atoms or fluorine atoms. The present invention can change the metabolic characteristics of the prototype drug by completely deuterating or fluorinating the methyl or methoxy groups of the prototype drug AZD3759. AZ'1168 is mainly produced by metabolic transformation of AZD3759 through the N-position demethylation pathway. The inventors have found that the stability and efficacy of the substituted zolitinib derivatives obtained by the present invention are far superior to those of the prototype drug AZD3759, and can reduce the concentration of the main active metabolite AZ'1168 generated in the body.

[0036] Among them, the derivatives obtained by deuterating the N-methyl group of the piperazine ring, deuterating the C-methyl group of the piperazine ring, fluorinating the N-methyl group of the piperazine ring, and deuterating the 7-methoxy group of the quinazoline ring of the prototype drug AZD3759 have quite superior stability and efficacy.

[0037] The present invention also provides a preparation method of the substituted zolitinib derivative or a pharmaceutically acceptable salt or solvate thereof.

[0038] Technical Solution I:

[0039] Among them, R 4 and R 5 is independently selected from methyl, methoxy, methyl substituted by halogen (including fluorine, chlorine, bromine, iodine), methoxy substituted by halogen (including fluorine, chlorine, bromine, iodine), methyl substituted by deuterium atom, methoxy substituted by deuterium atom. Preferably, R 4 and R 5 are independently selected from methyl, methoxy, methyl substituted with 1 to 3 deuterium atoms, methyl substituted with 1 to 3 fluorine atoms, methoxy substituted with 1 to 3 deuterium atoms, and methoxy substituted with 1 to 3 fluorine atoms. Preferably, R 4 and R 5 are independently selected from methyl, methoxy, trideuteriomethyl, trifluoromethyl, trideuteriomethoxy (methoxy completely substituted by deuterium atoms), trifluoromethoxy (methoxy completely substituted by fluorine atoms). Preferably, R 4 and R 5 is independently selected from methyl or trideuterated methyl. 4 and R 5 Not the same.

[0040] In the preparation process of technical scheme I, the molar ratio of compound A to compound B may be 0.5:2 to 1:5. For example, the molar ratio of compound A to compound B may be 1:1.5. As an example, compound A, compound B and potassium carbonate are dissolved in a solvent and stirred at 15 to 30°C for 5 to 15 hours. The molar ratio of compound A to potassium carbonate may be 0.5:2 to 1:5. For example, the molar ratio of compound A to potassium carbonate may be 1:2. The solvent may be DMF. After the reaction is completed, water is added to the mixture, extracted with EtOAc, and the combined organic layers are dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product is purified to obtain the target compound. The purification may be reverse phase HPLC purification.

[0041] As one of the specific examples, the substituted zolitinib derivative can be prepared according to the following synthetic route:

[0042] As one of the specific examples, the substituted zolitinib derivative can be prepared according to the following synthetic route:

[0043] Technical Solution II:

[0044] Preferably, in the technical scheme II, the molar ratio of compound C to CS2 is 0.5-2:1-5. For example, the molar ratio of compound C to CS2 is 1:2. The molar ratio of compound C to CH3I(MeI) is 0.5-5:2-10. For example, the molar ratio of compound C to CH3I is 1:7. The reaction temperature is 10-30°C, and the reaction time is 0.5-5 hours.

[0045] As an example, an aqueous solution of sodium hydroxide is added to a mixture of compound C in EtOAc at 0°C. Preferably, the molar ratio of compound C to sodium hydroxide is 0.5-2:1-5. For example, the molar ratio of compound C to sodium hydroxide is 1:1.5. CS2 is then added, and the mixture is stirred at 0°C for 1 hour, and then CH3I is added. The mixture is stirred at 25°C for 1 hour. After the reaction is completed, the mixture is extracted with EtOAc, and the combined organic layers are washed with brine, washed with anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. The crude product is purified by silica gel column chromatography to obtain the target compound.

[0046] Preferably, in the technical scheme II, the molar ratio of compound D to antimony trichloride is 1:0.01-1. For example, the molar ratio of compound D to antimony trichloride is 1:0.05. The molar ratio of compound D to BAST (bis(2-methoxyethyl)aminosulfur trifluoride) is 0.5-2:1-5. For example, the molar ratio of compound D to BAST is 1:4. The reaction temperature is 10-30°C, and the reaction time is 12-36 hours.

[0047] As an example, antimony trichloride and BAST were added to a mixture of compound D in CH2Cl2. The mixture was stirred at 20°C for 24 hours. The mixture was quenched by adding saturated NaHCO3, extracted with EtOAc, and the combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated to give a crude product. The crude product was purified by silica gel column chromatography to give the target compound.

[0048] As a specific example, the substituted zolitinib derivative is prepared according to the following synthetic route:

[0049] Technical Solution III:

[0050] Preferably, in step 1 of the technical scheme III, the molar ratio of the compound E to BBr3 is 0.5-5:1-10. For example, the molar ratio of the compound E to BBr3 is 1:4. The reaction temperature is 30-60°C, and the reaction time is 0.5-2 hours. For example, the reaction temperature is 40°C, and the reaction time is 50 minutes.

[0051] As an example, BBr3 was added to a mixture of compound E in DCE at 0°C, and the mixture was then stirred at 40°C for 50 minutes. The mixture was quenched by adding saturated NaHCO3 solution, extracted with EtOAc, and the combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated to give a crude product. The crude product was purified by pre-HPLC to give a yellow solid.

[0052] Preferably, in step 2 of the technical scheme III, the molar ratio of the compound F to trideuterium methyl 4-methylbenzenesulfonate is 0.5-5:0.5-5. For example, the molar ratio of the compound F to trideuterium methyl 4-methylbenzenesulfonate is 1:1.1. The reaction temperature is 10-30°C, and the reaction time is 12-36 hours. For example, the reaction temperature is 20°C, and the reaction time is 24 hours.

[0053] As an example, a mixture of compound F, potassium carbonate and trideuterium methyl 4-methylbenzenesulfonate in DMF is stirred at 20°C for 24 hours. The molar ratio of compound F to potassium carbonate is 0.5-5:0.5-5. For example, the molar ratio of compound F to potassium carbonate is 1:2. The mixture is quenched by adding saturated NaHCO3 solution, extracted with EtOAc, and the combined organic layers are washed with brine, dried with Na2SO4, filtered and concentrated to give a crude product. The crude product is purified by SFC to give the target compound.

[0054] As a specific example, the substituted zolitinib derivative can be prepared according to the following synthetic route:

[0055] The present invention also provides the use of the above-mentioned substituted zolitinib derivatives or their pharmaceutically acceptable salts or solvates in the preparation of epidermal growth factor receptor tyrosine kinase activity inhibitor drugs. The epidermal growth factor receptor tyrosine kinase activity inhibitor drugs can be used to prevent and / or treat non-small cell lung cancer, glioblastoma, Parkinson's disease, etc.

[0056] The present invention also provides a pharmaceutical composition. The pharmaceutical composition comprises the above-mentioned substituted zolitinib derivative or a pharmaceutically acceptable salt or solvate thereof. A single substituted zolitinib derivative can be used. Multiple substituted zolitinib derivatives can also be used.

[0057] Preferably, the pharmaceutical composition may also include the prototype drug AZD3759 or a pharmaceutically acceptable salt or solvate thereof.

[0058] The pharmaceutically acceptable salts include, but are not limited to, sulfates, phosphates, citrates, tartrates, fumarates, benzoates, adipates, succinates, methanesulfonates, maleates and the like.

[0059] Preferably, the pharmaceutical composition further comprises a CYP3A inhibitor. CYP3A is the most important type of CYP enzyme, accounting for about 30% of human liver microsomes. The CYP3A inhibitor includes but is not limited to a combination of one or more of ketoconazole, schisandrin and silybin.

[0060] The applicant previously studied the use of the prototype drug AZD3759 or its salt in combination with a variety of CYP450 enzyme inhibitors, and found that schisandrin A has a significant inhibitory effect on human liver microsomal cytochrome CYP450 enzymes compared to other CYP450 inhibitors, and the degree of inhibition is moderate, and the metabolism of the prototype drug can be reduced after combined use. The present invention further found that by examining different substituted zolitinib derivatives and adding CYP3A inhibitors (ketoconazole, schisandrin A and silibinin) when incubating human liver microsomes, the prototype drug AZD3759, substituted zolitinib derivatives, and metabolites (AZ'1168) in human liver microsomes were quantitatively analyzed by liquid-mass spectrometry. The results showed that the inhibition rate of the substituted zolitinib derivatives combined with ketoconazole to assist in inhibiting the metabolism of the prototype drug AZD3759 was the highest.

[0061] In some embodiments, the molar ratio of the substituted zolitinib derivative or its pharmaceutically acceptable salt or solvate to the CYP3A inhibitor is 1:(0.1-10). Optionally, the substituted zolitinib derivative is deuterated zolitinib cpds 1. Optionally, the CYP3A inhibitor is ketoconazole. For example, the molar ratio of the substituted zolitinib derivative or its pharmaceutically acceptable salt or solvate to the CYP3A inhibitor is 1:1.

[0062] The pharmaceutical composition may also include the prototype drug AZD3759 or a pharmaceutically acceptable salt or solvate thereof. Optionally, the molar ratio of the substituted zolitinib derivative or a pharmaceutically acceptable salt or solvate thereof: the prototype drug AZD3759 or a pharmaceutically acceptable salt or solvate thereof: the CYP3A inhibitor is 1:(0.1-10):(0.1-10). For example, the molar ratio of the substituted zolitinib derivative or a pharmaceutically acceptable salt or solvate thereof: the prototype drug AZD3759 or a pharmaceutically acceptable salt or solvate thereof: the CYP3A inhibitor is 1:1:2.

[0063] The pharmaceutical composition may also include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and delayed absorption agents that are physiologically compatible. The carrier may be one or more of water, saline, phosphate buffered saline, glucose, glycerol, ethanol, etc. and combinations thereof. Isotonic agents may also be included in the pharmaceutical composition, such as sugars, polyols, sodium chloride, etc. Of course, pharmaceutically acceptable carriers may also include trace amounts of auxiliary substances, such as wetting agents or emulsifiers, preservatives or buffers, to extend the shelf life or efficacy of the antibody.

[0064] The pharmaceutical composition can be a type of oral preparation (oral preparation), injection preparation, pill, sustained-release preparation, implant or aerosol, etc., and is not particularly limited. The pharmaceutical composition can also be incorporated into a pharmaceutical composition suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). Typical pharmaceutical compositions are in the form of oral preparations, injection solutions or infusion solutions. For administration, the pharmaceutical composition can be administered orally, intravenously or by injection, or intramuscularly or subcutaneously.

[0065] These pharmaceutical compositions can be prepared in various forms, such as liquid, semisolid and solid dosage forms, including but not limited to liquid solutions (e.g., injection solutions and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories.

[0066] The present invention also provides the use of the above-mentioned pharmaceutical composition in the preparation of epidermal growth factor receptor tyrosine kinase activity inhibitor drugs. The epidermal growth factor receptor tyrosine kinase activity inhibitor drugs can be used to prevent and / or treat non-small cell lung cancer, glioblastoma, Parkinson's disease, etc.

[0067] The following examples are further listed to illustrate the present invention in detail. It should also be understood that the following examples are only used to further illustrate the present invention and cannot be construed as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention all belong to the scope of protection of the present invention. The specific process parameters of the following examples are also only examples within a suitable range, that is, those skilled in the art can make selections within a suitable range through the description of this article, and are not limited to the specific values ​​​​exemplified below. In the case where the present invention is not specifically described, the ratio of the eluent refers to the volume ratio.

[0068] Preparation of substituted zolitinib derivatives Cpds1

[0069] Preparation of compound 3B

[0070] To a mixture of compound 3A (2.98 g, 14.90 mmol, 1 eq) and K2CO3 (2.06 g, 14.9 mmol, 1 eq) in MeOH (20 mL) was added triiodomethane (2.14 g, 15.05 mmol, 917.02 μL, 1.01 eq) at 20°C. The mixture was stirred at 25°C for 3 hours. TLC (CH2Cl2 / MeOH=10 / 1) showed that compound 3A was consumed and a new main spot was observed. The mixture was concentrated to give a crude product. The crude product was purified by silica gel column chromatography (CH2Cl2 / MeOH=100 / 1-10 / 1) to give a yellow oily compound 3B (3.00 g, 13.80 mmol, yield 92.62%). 1 H NMR (400MHz, DMSO-d6): δ4.26-4.14(m,1H),3.81(br d,J=13.3Hz,1H),3.09(dt,J=3.3,12.8Hz,1H),2.72(br dd,J=1.4,11.1Hz,1H),2.58(d,J=11.3Hz,1H),2.09(dd,J=4.1,11.3Hz,1H),1.46(s,9H),1.24(d,J=6.9Hz,3H).

[0071] Preparation of compound 3C

[0072] A mixture of compound 3B (3.35 g, 15.41 mmol, 1 eq) in HCl / EtOAc (4 M, 1.33 mL, 3.44 e-1 eq) was stirred at 25 ° C for 2 hours. TLC (CH2Cl2 / MeOH=10 / 1) showed that compound 3B was completely consumed. The mixture was filtered and the filter cake was concentrated to give a yellow solid compound 3C (1.9 g, 12.36 mmol, yield 80.21%, hydrochloride). The yellow solid was used directly in the next step without further purification. 1 H NMR (400MHz, DMSO-d6): δ3.87-3.58(m,4H), 3.50-3.39(m,1H), 3.38-3.27(m,1H), 3.18(br t,J=12.5Hz,1H), 1.38(d,J=6.5Hz,3H).

[0073] Preparation of compound 3

[0074] Pyridine (1.82 g, 23.04 mmol, 1.86 mL, 3 eq) was added dropwise to a solution of triphosgene (2.35 g, 7.92 mmol, 1.03 eq) in CH2Cl2 (20 mL) at 0°C, and then compound 3C (900 mg, 7.68 mmol, 1 eq) was slowly added at 0°C, and the resulting mixture was stirred at 20°C for 3 hours. TLC (CH2Cl2:MeOH=10:1) showed that compound 3C was completely consumed. The mixture was concentrated to give a brown solid compound 3 (1.39 g, crude product). The brown solid was used directly in the next step without further purification.

[0075] Preparation of compound 2

[0076] A mixture of compound 1 (15 g, 41.46 mmol, 1 eq) and K2CO3 (12.61 g, 91.22 mmol, 2.2 eq) in MeOH (300 mL) was stirred at 15° C. for 12 hours. LCMS showed that compound 1 had been consumed and the expected molecular weight was observed (RT=0.852 minutes). The mixture was concentrated to give yellow solid compound 2 (23.5 g, crude product).

[0077] Preparation of target compound cpds 1

[0078] A mixture of compound 2 (1.60 g, 5.00 mmol, 1 eq), compound 3 (1.35 g, 7.51 mmol, 1.5 eq) and K2CO3 (1.38 g, 10.01 mmol, 2 eq) in DMF (20 mL) was stirred at 20 ° C for 12 hours. LCMS showed that compound 2 had been consumed and the desired molecular weight was observed (RT = 0.548 minutes). Water (60 mL) was added to the mixture, extracted with EtOAc (20 mL x 2), and the combined organic layers were dried with Na2SO4, filtered, and concentrated to give a crude product. The crude product was purified by reverse phase HPLC (YMCTriart C18 250*50mm*7um; water (0.05% HCl)-ACN) to give a yellow solid compound cpds 1 (1.50 g, 3.24 mmol, 64.75% yield).

[0079] Figure 1 This is the H NMR spectrum of compound cpds1. 1H NMR (400MHz, DMSO-d6): δ8.75(s,1H),8.44(br d,J=9.8Hz,1H),7.60-7.50(m,2H),7.42-7.37(m,1H),7.33-7.27(m,1H),4.91-4.88(m,1H),4.69(br s,1H),4.46(br s,1H),4.29(br s,1H),4.11(s,3H),3.79-3.37(m,4H),3.29-3.16(m,1H),1.65-1.41(m,3H),1.32(t,J=7.3Hz,1H).

[0080] Preparation of substituted zolitinib derivatives cpds4

[0081] Preparation of compound 5

[0082] A solution of compound 4 (45.5 g, 375.48 mmol, 47.79 mL, 1 eq) and compound 4A (100 g, 412.98 mmol, 1.1 eq) in DIEA (107.18 g, 829.29 mmol, 144.44 mL, 2.21 eq) was stirred at 120 ° C for 16 hours. TLC (petroleum ether: EtOAc = 1: 1) showed that the starting material (Rf = 0.27) was consumed, and the desired point (Rf = 1.12) was observed. MeOH (100 mL) was added to the mixture, which was then stirred at 25 ° C for 15 minutes. The mixture was concentrated to obtain a residue. The residue was purified by silica gel column chromatography (petroleum ether: EtOAc = 1: 0 ~ 3: 1) to obtain a yellow oily compound 5 (14.5 g, 49.93 mmol, yield 13.30%).

[0083] Preparation of compound 6

[0084] To a solution of compound 5 (10.0 g, 34.44 mmol, 1 eq) and TMEDA (5.20 g, 44.77 mmol, 6.76 mL, 1.3 eq) in Et2O (200 mL) was added s-BuLi (1.3 M, 79.47 mL, 3.0 eq) at -68°C, and the mixture was stirred at -68°C for 1 hour. Triiodomethane (10.24 g, 70.7 mmol, 4.40 mL, 2.05 eq) was added to the resulting mixture. The mixture was then heated to 25°C and stirred at 25°C for 16 hours. TLC (petroleum ether: EtOAc = 10: 1) showed that the starting material (Rf = 0.25) had been consumed, and two new spots (Rf = 4.40, 0.45) were observed. LCMS showed that the starting material had been consumed, and the expected molecular weight was detected (Rt1 = 4.703 min and Rt2 = 4.860 min). Saturated NaHCO3 solution (5 mL) was added to the mixture and extracted with EtOAc (100 mL x 3). The combined organic layer was washed with H2O (50 mL x 3), dried with Na2SO4, filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 0-10 / 1) to obtain a colorless oil. The colorless oil was purified by SFC (0.1% NH3H2O-ETOH, (s, s) WHELK-O1 (250mm*30mm, 5um)) to obtain a colorless oily compound 6 (2.40 g, 7.81 mmol, yield 22.68%). LCMS: Rt1 = 4.703 minutes, MS (ESI) m / z = 308.2 [M+H] + . SFC: Rt = 2.315 min, ee% = 100%. 1 H NMR (400MHz, CDCl3): δ1.34(d,J=6.80Hz,3H),1.44(s,9H),1.95-2.06(m,2H),2.50(br d,J=11.6Hz,1H),2.99(br d,J=10.8Hz,1H),3.12(td,J=12.6,3.40Hz,1H),3.28-3.34(m,1H),3.84(br d,J=12.8Hz,1H),4.06(br s,1H),7.21-7.25(m,1H),7.29-7.33(m,4H).

[0085] Preparation of compound 7

[0086] Pd / C (900 mg, 849.06 μmol, 10% purity, 7.91e-2 eq) was added to a solution of compound 14 (3.30 g, 10.73 mmol, 1 eq) in MeOH (50 mL) under N2. The resulting suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred at 25 °C under H2 (15 psi) for 16 hours. TLC (petroleum ether: EtOAc = 10: 1) showed that the starting material (Rf = 0.56) was completely consumed, and the desired point (Rf = 0.01) was observed. The reaction mixture was filtered and concentrated to give a colorless oily compound 15 (2.10 g, 10.28 mmol, yield 95.76%). The colorless oil was used directly in the next step without further purification. 1 H NMR (400MHz, CDCl3): δ1.22(d,J=6.80Hz,3H),1.46(s,9H),2.64-2.71(m,1H),2.75-2.78(m,1H),2.88-3.01(m,3H),3.76-3.80(m,1H),4.16(br s,1H).

[0087] Preparation of compound 8

[0088] A mixture of compound 15 (2.10 g, 10.28 mmol, 1 eq), formaldehyde (1.67 g, 20.56 mmol, 1.53 mL, purity 37%, 2 eq) and NaBH3CN (1.29 g, 20.56 mmol, 2 eq) in MeOH (20 mL) was stirred at 25 ° C for 2 hours. TLC (petroleum ether: EtOAc = 1: 1) showed that the starting material (Rf = 0.03) was consumed and a new spot (Rf = 0.36) was observed. The mixture was concentrated to obtain a residue, EtOAc (30 mL) and water (10 mL) were added to the residue, the organic layer was separated, washed with brine (10 ml), dried over Na2SO4, filtered, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (petroleum ether: EtOAc = 100: 1 to 5: 1) to obtain colorless oily compound 16 (1.70 g, 7.79 mmol, yield 75.75%). 1H NMR (400MHz, CDCl3): δ1.46 (s, 9H), 1.87-1.94 (m, 1H), 2.10 (dd, J = 5.60, 4.00Hz, 1H), 2.24 (s, 3H), 2 .59(dt,J=11.4,1.60Hz,1H),2.73(dt,J=11.2,1.52Hz,1H),3.10(td,J=12.8,3.60Hz,1H),3.81(br d,J=13.2Hz,1H),4.19(br s,1H).

[0089] Preparation of compound 9

[0090] A solution of compound 8 (1.70 g, 7.79 mmol, 1 eq) in HCl / EtOAc (4 M, 11.68 mL, 6 eq) was stirred at 25 ° C for 2 hours. TLC (petroleum ether: EtOAc = 1: 1) showed that the starting material had been consumed. The mixture was concentrated to give a white solid compound 9 (1.60 g, crude product, 2HCl). The white solid was used directly in the next step without further purification. 1 H NMR (400MHz, D2O): δ2.88-3.79 (m, 10H).

[0091] Preparation of compound 10

[0092] Pyridine (2.90 g, 36.62 mmol, 2.96 mL, 5 eq) was added dropwise to a CH2Cl2 (40 mL) solution at 0°C, and then compound 9 (1.40 g, 7.32 mmol, 1 eq, 2HCl) was slowly added at 0°C, and then triphosgene (1.44 g, 4.85 mmol, 6.63 e-1 eq) was added to the above solution, and the mixture was stirred at 10°C for 3 hours. TLC (CH2Cl2: MeOH = 20: 1) showed that the starting material (Rf = 0.035) was completely consumed. The mixture was concentrated under reduced pressure to obtain a dark brown oily compound 10 (1.32 g, crude product). The dark brown oily compound was used directly in the next step without further purification.

[0093] Preparation of compound cpds4

[0094] A mixture of compound 10 (1.32 g, 7.31 mmol, 1.51 eq), compound 2 (1.55 g, 4.85 mmol, 1 eq) and K2CO3 (1.34 g, 9.70 mmol, 2 eq) in DMF (10 mL) was stirred at 25°C for 12 hours. LCMS showed that 3.5% of the starting material was retained (RT = 1.109 minutes), and the desired molecular weight was detected (RT = 1.021 minutes). H2O (60 mL) was added to the mixture, extracted with EtOAc (20 mL x 2), and the combined organic layers were dried over Na2SO4, filtered, and concentrated to give a crude product. The crude product was purified by reverse phase HPLC (water (0.05% HCl)-ACN, Xtimate C18 150*40mm*10um) to obtain an off-white solid compound cpds 4 (540 mg, 1.17 mmol, yield 24.04%, purity 99.9%). LCMS: RT = 1.02 min, MS (ESI) m / z = 463.3 [M+H] + . HPLC: RT=2.392min, purity: 99.8%.

[0095] Figure 2 This is the H NMR spectrum of compound cpds4. Figure 3 This is the deuterium NMR spectrum of compound cpds4. 1 H NMR (400MHz, MeOD): δ3.01(s,3H),3.14-3.27(m,1H),3.40-3.70(m,4H),4.12(s,3H),4.25-4.50(m, 1H),4.66-4.77(m,1H),7.30-7.34(m,1H),7.39(s,1H),7.51-7.59(m,2H),8.43(s,1H),8.78(s,1H).

[0096] Preparation of substituted zolitinib derivatives cpds2

[0097] Preparation of compound 2

[0098] To a mixture of compound 1 (4.00 g, 8.97 mmol, 1 eq) in EtOAc (40 mL) was added a solution of NaOH (538.23 mg, 13.46 mmol, 1.5 eq) in H2O (10 mL) at 0°C, CS2 (1.37 g, 17.94 mmol, 1.08 mL, 2 eq) was added, and the mixture was stirred at 0°C for 1 hour. CH3I (8.91 g, 62.80 mmol, 3.91 mL, 7 eq) was then added. The mixture was stirred at 25°C for 1 hour. TLC (petroleum ether / EtOAc=1 / 1) showed that compound 1 was consumed, and a new major spot (Rf=0.37) was observed. The reaction mixture was extracted with EtOAc (100 mL x 2), the combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered and concentrated to give a crude product. The crude product was purified by silica gel column chromatography (petroleum ether / EtOAc=100 / 1-50 / 50) to obtain yellow solid Compound 2 (3.00 g, 5.60 mmol, yield 62.38%).

[0099] Preparation of compound cpds2

[0100] Antimony trichloride (63.83 mg, 279.83 μmol, 0.05 eq) and BAST (4.95 g, 22.39 mmol, 4.90 mL, 4 eq) were added to a mixture of compound 2 (3.00 g, 5.60 mmol, 1 eq) in CH Cl (20 mL). The mixture was stirred at 20 ° C for 24 hours. TLC (CH Cl / MeOH = 20 / 1) showed that compound 2 remained, and new main points were observed. The mixture was quenched by adding saturated NaHCO solution (30 mL), extracted with EtOAc (20 mL x2), washed with salt water (20 mL) for the combined organic layer, dried over Na SO, filtered and concentrated to give a crude product. The crude product was purified by silica gel column chromatography (CH2Cl2 / MeOH=100 / 1-20 / 1) to obtain a white solid compound cpds 2 (900 mg, 1.73 mmol, yield 30.86%, purity 98.6%).

[0101] Figure 4 This is the H NMR spectrum of compound cpds2. Figure 5 This is the deuterium NMR spectrum of compound cpds2. 1HNMR (400MHz, CDCl3): δ8.73 (s, 1H), 8.46 (dt, J = 3.4, 6.8Hz, 1H), 7.66 (s, 1H), 7.61-7.42(m,1H),7.33(s,1H),7.20-7.11(m,2H),4.77-4.42(m,1H),4.15(br dd,J=2.9,10.1Hz,1H),3.95(s,3H),3.49-3.26(m,2H),3.15(br d,J=11.6Hz,1H),2.91(br d,J=9.8Hz,1H),2.74(br t,J=11.0Hz,1H),1.80(br s,3H),1.45(br s,3H).

[0102] Preparation of substituted zolitinib derivatives cpds5

[0103] Preparation of compound 4

[0104] At 0°C, BBr (21.79 g, 86.98 mmol, 8.38 mL, 4 eq) was added to a mixture of compound 3 (10.0 g, 21.74 mmol, 1 eq) in DCE (200 mL), and the mixture was then stirred at 40°C for 50 minutes. LCMS showed that compound 3 was retained (RT = 2.503 minutes), and the desired molecular weight (RT = 1.236 minutes) was observed. The mixture was quenched by adding saturated NaHCO solution (500 mL), extracted with EtOAc (1000 mL x 2), and the combined organic layers were washed with brine (500 mL), dried over NaSO, filtered and concentrated to give a crude product. The crude product was purified by pre-HPLC (column: YMC-Triart C18 250*50mm*7um; mobile phase: [water (0.05% NH3H2O+10mM NH4HCO3)-ACN]; B%: 5%-35%, 20 minutes) to obtain a yellow solid. The yellow solid was purified by pre-HPLC (column: YMC Triart C18 250*50mm*7um; mobile phase: [water (0.05% NH3H2O+10mM NH4HCO3)-ACN]; B%: 5%-35%, 20 minutes) to obtain a yellow solid compound 4 (1.60 g, 3.57 mmol, yield 16.44%, purity 99.6%). LCMS: RT = 1.236 minutes, MS m / z = 446.1 [M+H] + . 1HNMR (400MHz, DMSO-d6): δ11.31-10.63(m,1H),9.64(s,1H),8.38(s,1H),8.16(s,1H),7.63-7.38( m,2H),7.32-6.96(m,2H),4.56-4.11(m,1H),3.98-3.70(m,1H),2.80(brd,J=11.0Hz,1H),2.68(br d,J=11.4Hz,1H),2.20(s,3H),2.10(br d,J=8.4Hz,1H),1.96-1.85(m,1H),1.34(br s,3H).

[0105] Preparation of compound cpds5

[0106] A mixture of compound 4 (1.4 g, 3.14 mmol, 1 eq), K2CO3 (867.91 mg, 6.28 mmol, 2 eq) and trideuterium methyl 4-methylbenzenesulfonate (653.64 mg, 3.45 mmol, 1.1 eq) in DMF (10 mL) was stirred at 20° C. for 24 hours. SFC showed that the desired molecular weight was observed (RT=2.601 minutes). The mixture was quenched by adding saturated NaHCO3 solution (20 mL), extracted with EtOAc (20 mL x 2), and the combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated to give a crude product. The crude product was purified by SFC (column: DAICEL CHIRALPAK AD (250 mm*30 mm, 10 um); mobile phase: [0.1% NH3H2O-IPA]; B%: 40%-40%, 10 minutes) to obtain cpds 5 (670 mg, 1.42 mmol, yield 45.27%, purity 98.2%) as an off-white solid. SFC: RT = 2.601 min, MS m / z = 463.2 [M+H] + .

[0107] Figure 6 This is the H NMR spectrum of compound cpds5. Figure 7 This is the deuterium NMR spectrum of compound cpds5. 1HNMR (400MHz, DMSO-d6): δ9.73(s,1H),8.47(s,1H),8.22(s,1H),7.61-7.42(m,2H),7.33(s,1H) ),7.28(dt,J=1.2,8.1Hz,1H),4.51-4.12(m,1H),4.00-3.70(m,1H),3.29-3.14(m,1H),2.80(br d,J=11.1Hz,1H),2.72-2.66(m,1H),2.21(s,3H),2.14-2.08(m,1H),2.07(s,1H),1.97-1.82(m,1H),1.34(br s,3H).

[0108] Study on metabolic stability of human liver microsomes

[0109] Reagents Cpds1, purity 98.16%, molecular weight 499.38, homemade; Cpds4, purity 99.8%, molecular weight 462.92, homemade; Cpds5, purity 99.3%, molecular weight 462.92, homemade; Cpds6 (AZ'1168), purity 99.63%, molecular weight 445.87, self-made; Cpds7 (AZD3759), purity 99.9%, molecular weight 496.36, self-made; Ketoconazole, batch number: LR70R89, source: Bailingwei Technology Co., Ltd.; Schisandrin A, batch number: WK920022108, from Sichuan Weikeqi Biotechnology Co., Ltd.; Silybin, batch number: LP20U54, from Bailingwei Technology Co., Ltd.; NADPH (reduced coenzyme II, scientific name reduced nicotinamide adenine dinucleotide phosphate), batch number: L9C0T54, source: J&K Technology Co., Ltd.; Verapamil hydrochloride, batch number: 10223-200102, source: China Inspection and Quarantine Institute; Human liver microsomes, batch number: NJT, source: Red Liver Disease Research (Shanghai) Co., Ltd.; concentration 20 mg / mL.

[0110] instrument Liquid chromatography system, model: Waters Acquity I-Class UPLC, source: Waters Company; Tandem mass spectrometry (MS / MS system), model TRIPLE QUAD 6500, source: SCIEX; Data processing software: Microsoft Excel software, sourced from Microsoft Corporation; Analysis 1.6.3 quantitative processing software, sourced from SCIEX Corporation.

[0111] Preparation of stock solution. Accurately weigh a certain amount of Cpds1, Cpds4, Cpds5, Cpds6 (AZ'1168), Cpds7 (AZD3759), ketoconazole, schisandrin, silybin, and verapamil hydrochloride standard, and dissolve them in dimethyl sulfoxide to obtain a 10 mmol / L stock solution.

[0112] Preparation of phosphate buffer solution PBS (10mmoL / L): Accurately weigh 8.00g NaCl, 3.63g Na2HPO4·12H20, 0.24g KH2PO4, and 0.20g KCl, add distilled water to 1000mL, adjust the pH to 7.4, filter with a 0.45μm filter membrane, and store at 4°C.

[0113] Preparation of NADPH working solution: The final concentration of NADPH in the incubation system is 1mmol / L, so use TRISbuffer or water to prepare a 5mmol / L (250 / 50) NADPH working solution. Prepare the NADPH working solution and use it immediately.

[0114] Preparation of reaction termination solution: Prepare a reaction termination solution with a volume ratio of methanol:acetonitrile (1:1).

[0115] Preparation of standard curve samples: Accurately pipette Cpds1, Cpds4, Cpds5, Cpds6, Cpds7, and verapamil stock solutions (10 mmol / L), and use methanol:water (1:1) and PBS solution as diluents to prepare standard curve working solutions (0.03125 μmol / L, 0.0625 μmol / L, 0.125 μmol / L, 0.25 μmol / L, 0.5 μmol / L, 1.25 μmol / L, 2 μmol / L, 2.5 μmol / L).

[0116] Table 1 Preparation of standard curve working solution

[0117] Take 100 μL of each of the above standard curve working solutions, add 100 μL of human liver microsome working solution and 50 μL of PBS, take out 20 μL, precipitate with 180 μL of reaction termination solution to obtain standard curve samples of different concentrations of Cpds1 / Cpds4 / Cpds5 / Cpds6 / Cpds7 / verapamil (0.0125 μmol / L, 0.025 μmol / L, 0.05 μmol / L, 0.1 μmol / L, 0.2 μmol / L, 0.5 μmol / L, 0.8 μmol / L, 1 μmol / L), vortex for about 1 min, centrifuge for 10 min (15200 rpm), take 100 μL of supernatant, and perform LC-MS / MS analysis.

[0118] Preparation of quality control samples and LLOQ samples: Accurately pipette Cpds1, Cpds4, Cpds5, Cpds6, Cpds7, and verapamil stock solutions (10 mmol / L), and use methanol: water (1:1) and PBS as diluents to prepare LLOQ working solutions and quality control working solutions (0.03125 μmol / L, 0.075 μmol / L, 0.375 μmol / L, 1.875 μmol / L).

[0119] Table 2 Preparation of QC working solution

[0120] Take 100 μL of the above-mentioned quality control working solution and LLOQ working solution, add 100 μL of human liver microsome working solution and 50 μL of PBS, take out 20 μL, and precipitate with 180 μL of reaction termination solution to obtain low, medium and high concentrations of Cpds1 / Cpds4 / Cpds5 / Cpds6 / Cpds7 / verapamil quality control samples (0.03 μmol / L, 0.15 μmol / L, 0.75 μmol / L) and LLOQ sample (0.0125 μmol / L), vortex for about 1 min, centrifuge for 10 min (15200 rpm), take 100 μL of supernatant, and perform LC-MS / MS analysis.

[0121] Preparation of human liver microsome working solution: Frozen human liver microsomes (HLM) should be thawed quickly in a 37°C water bath. Thawed HLM cannot be stored at room temperature for a long time and should be kept within 5 minutes. The concentration of HLM in the final incubation system is 0.2 mg / mL, and the dilution factor from the working solution to the final incubation system is 2.5 (250 / 100), so a HLM working solution with a concentration of 0.5 mg / mL needs to be prepared.

[0122] Chromatographic conditions: Chromatographic column: SHIMADZU Shim-pack XR-ODS (75*3.0m, 2.2μm); Mobile phase: Phase A: 10mM ammonium acetate aqueous solution (containing 0.1% formic acid); Phase B: acetonitrile; Sealwash: 10% methanol aqueous solution; Needle washing solution: methanol; Injection volume: 4μL, flow rate: 0.4mL / min, automatic injection chamber temperature: 15℃±5℃; Gradient elution method: Time 0min, Phase A 80%, Phase B 20%; Time 0.5min, Phase A 80%, Phase B 20%; Time 1.5min, Phase A 5%, Phase B 95%; Time 3min, Phase A 5%, Phase B 95%; Time 3.01min, Phase A 80%, Phase B 20%; Time 4min, Phase A 80%, Phase B 20%. 0min~1.2min into waste liquid, 3.0min~4.0min into mass spectrometer. Mass spectrometry conditions: ion source is Turbo Spray; positive ion detection; Curtain Gas: 20psi; Collision: 4; lon Spray Voltage: 4500V; Temperature: 500℃; Ion Source Gas1 (GS1): 30psi; lon Source Gas2 (GS2): 30psi; scanning mode is multiple reaction monitoring (MRM); scanning time is 100ms.

[0123] In this experiment, human liver microsomes were used for in vitro metabolism study. The study was divided into 12 groups (n=3), and samples were taken at 0min, 15min, 30min, 1h, 2h, and 4h, respectively, and methanol: acetonitrile (1:1) precipitation was performed, centrifuged, and the supernatant was taken for determination. LC / MS / MS was used to detect the prototype and metabolites of each compound, and the Ln residual percentage-time curve was drawn to compare the clearance rate of the chemically modified drug and the reference drug. The specific grouping is shown in Table 3, and the preparation of the compound incubation working solution is shown in Table 4, so that the final concentration of the compound in the incubation system is 1uM and the final concentration of the inhibitor is 10uM.

[0124] Table 3 Experimental groups

[0125] Table 4 Preparation of compound incubation working solution

[0126] Diluent 1 was methanol: water (1:1); diluent 2 was phosphate buffer solution PBS (10 mmol / L).

[0127] Human liver microsome incubation system

[0128] Experimental group: Take 100 μL HLM working solution (0.5 mg / mL) and 100 μL incubation working solution (2.5 μmol / L) of experimental group 1 to experimental group 10 in Table 4 into 1.1 mL micro-incubation tubes, shake gently and vortex. Pre-incubate it with NADPH (5 mmol / L) in a 37°C water bath for 5 min. After that, take 50 μL NADPH working solution into micro-incubation tubes, shake gently and vortex to start the reaction. After 0 min, 15 min, 30 min, 1 h, 2 h, and 4 h of reaction, take 20 μL of the above reaction solution into 180 μL of reaction termination solution (methanol: acetonitrile = 1:1). Vortex for about 1 min, centrifuge for 10 min (4°C, 15200 rpm), take 120 μL supernatant, and perform LC-MS / MS analysis.

[0129] Positive control group: Take 100 μL of HLM working solution (0.5 mg / mL) and 100 μL of experimental group 11 working solution (2.5 μmol / L) in Table 4 into 1.1 mL micro-incubation tubes, shake gently, and vortex. Pre-incubate it with NADPH (5 mmol / L) in a 37℃ water bath for 5 min. After that, take 50 μL of NADPH working solution into micro-incubation tubes, shake gently, and vortex to start the reaction. After 0 min, 15 min, 30 min, 1 h, 2 h, and 4 h of the reaction, take 20 μL of the above reaction solution into 180 μL of reaction termination solution (methanol: acetonitrile = 1:1). Vortex for about 1 min, centrifuge for 10 min (4℃, 15200 rpm), take 100 μL of supernatant, and perform LC-MS / MS analysis.

[0130] Negative control group: Take 100 μL of HLM working solution (0.5 mg / mL) and 100 μL of experimental group 12 working solution (2.5 μmol / L) in Table 4 into 1.1 mL micro-incubation tubes, shake gently and vortex. After that, take 50 μL of PBS into micro-incubation tubes, shake gently and vortex. After 0 min, 15 min, 30 min, 1 h, 2 h, and 4 h of reaction, take 20 μL of the above reaction solution into 180 μL of reaction termination solution (methanol: acetonitrile = 1:1). Vortex for about 1 min, centrifuge for 10 min (4 ° C, 15200 rpm), take 100 μL of supernatant, and perform LC-MS / MS analysis.

[0131] Data processing and analysis

[0132] This experiment used Analyst1.6.3 quantitative processing software for data acquisition and automatic integration, and quantification was performed based on the batch standard curve.

[0133] Clearance rate: The concentration of the drug at time zero is taken as 100%, and the concentration after a certain period of incubation is compared with the concentration at time zero to obtain the remaining percentage of the original drug. The remaining percentage of the original drug at each time point is transformed by natural logarithm and plotted against the corresponding incubation time. The slope (-k) is obtained by linear regression, and the clearance rate of the drug metabolism in the microsome is calculated by formula (1). CL (ml / h / mg) =K*V / microparticle volume mg Formula (1) The volume of the incubation system V = 0.25 mL (100 μL HLM working solution, 100 μL incubation working solution, and 50 μL NADPH working solution), and the amount of microparticles mg = 0.5 mg / mL*0.1 mL = 0.05 mg.

[0134] Standard curve: According to the configuration of the above standard curve samples, prepare a series of standard samples of Cpds1, Cpds4, Cpds5, Cpds6, Cpds7, and verapamil (0.0125 μmol / L, 0.025 μmol / L, 0.05 μmol / L, 0.1 μmol / L, 0.2 μmol / L, 0.5 μmol / L, 0.8 μmol / L, 1 μmol / L), and record the chromatogram; take the concentration of the analyte as the abscissa and the peak area of ​​the analyte as the ordinate, and use weighting (W = 1 / x 2 ) The least squares method is used for regression calculation, and the linear regression equation obtained is the standard curve.

[0135] See also Figures 9 to 13 : When the compound was in the range of 0.0125μmol / L, 0.025μmol / L, 0.05μmol / L, 0.1μmol / L, 0.2μmol / L, 0.5μmol / L, 0.8μmol / L and 1μmol / L, the concentration and response ratio showed a good linear relationship, and the lower limit of quantification was 0.0125μmol / L. The correlation coefficient r was: Cpds1 (0.9968); Cpds4 (0.9978,); Cpds5 (0.9987); Cpds6 (0.9996); Cpds7 (0.9986); verapamil (0.9964).

[0136] In addition, according to the preparation of quality control samples and LLOQ samples, quality control samples (0.03 μmol / L, 0.15 μmol / L, 0.75 μmol / L) and LLOQ samples (0.0125 μmol / L) of Cpds1, Cpds4, Cpds5, Cpds6, Cpds7, and verapamil were prepared. Six samples were analyzed for each concentration, and one analysis batch was measured. According to the accompanying standard curve, the measured concentration of the sample was calculated, and the accuracy and precision of the method were calculated. After investigation, the method of the present invention has excellent accuracy and precision.

[0137] Experimental group 1: Cpds1 was added to human liver microsomes for incubation. The stability data after incubation is shown in Table 5.

[0138] Experimental group 2: Cpds4 was added to human liver microsomes for incubation. The stability data after incubation is shown in Table 6.

[0139] Experimental group 3: Cpds5 was added to human liver microsomes for incubation. The stability data after incubation is shown in Table 7.

[0140] Experimental group 4: Cpds7 (AZD3759) was added to human liver microsomes for incubation. The stability data after incubation is shown in Table 8.

[0141] Experimental group 5: Cpds1 and Cpds7 (AZD3759) were added to human liver microsomes for incubation. The stability data after incubation are shown in Table 9.

[0142] Experimental group 6: Cpds4 and Cpds7 (AZD 3759) were added to human liver microsomes for incubation. The stability data after incubation are shown in Table 10.

[0143] Experimental group 7: Cpds5 and Cpds7 (AZD 3759) were added to human liver microsomes for incubation. The stability data after incubation are shown in Table 11.

[0144] Table 5

[0145] Table 6

[0146] The concentration of Cpds6, a possible metabolite of Cpds4, was undetectable at different time points after incubation of human liver microsomes with cpds4.

[0147] Table 7

[0148] The concentration of Cpds6, a possible metabolite of Cpds5, was undetectable at different time points after incubation of cpds5 with human liver microsomes.

[0149] Table 8

[0150] Table 9

[0151] Table 10

[0152] Table 11

[0153] It can be seen from Table 5 that the clearance rate of Cpds1 (deuterated) incubated alone in human liver microsomes is 12.0882. The concentration of the metabolite AZ'1168 reaches Cmax after 15 minutes of incubation in human liver microsomes, and then the concentration gradually decreases.

[0154] It can be seen from Table 6 that the clearance rate of Cpds4 (deuterated) incubated alone in liver microsomes is 14.2358. The concentration of AZ'1168, a possible metabolite of Cpds4, was not detected at different time points after Cpds4 was incubated in human liver microsomes.

[0155] It can be seen from Table 7 that the clearance rate of Cpds5 (deuterated) incubated alone in liver microsomes was 12.5729. The concentration of the possible metabolite AZ'1168 was not detected at different time points after incubation of Cpds5 in human liver microsomes.

[0156] It can be seen from Table 8 that the clearance rate of Cpds7 incubated alone in liver microsomes was 12.7654.

[0157] Therefore, compared with the above-mentioned different single compound incubation systems, Cpds1 (deuterated) improved the metabolic stability of the prototype drug and reduced the generation of the product Cpds6 in the single incubation system.

[0158] As can be seen from Table 9: Cpds1 (deuterated) has a higher metabolic stability in human liver microsomes than Cpds7 (AZD3759), which are 11.7881 and 12.8531 respectively. Compared with the results of experimental group 1, there is no significant difference in the clearance rate of Cpds1 (deuterated) incubated alone and mixed (Cpds1+Cpds7) in human liver microsomes, and there is no metabolic interference between the compounds. The concentration of the total metabolite Cpds6 (AZ'1168) reached Cmax after 15 minutes of incubation in human liver microsomes, and then the concentration gradually decreased.

[0159] As can be seen from Table 10: The metabolic stability of Cpds 4 and Cpds7 (AZD3759) in human liver microsomes is not very different, and the clearance rates are 13.4743 and 14.3201, respectively. There is no significant difference in the clearance rate of single incubation and mixed (Cpds4+Cpds7) in human liver microsomes. The concentration of the total metabolite AZ'1168 reaches Cmax after 15 minutes of incubation in human liver microsomes, and then the concentration gradually decreases.

[0160] As can be seen from Table 11: The metabolic stability of Cpds 5 and Cpds7 (AZD3759) in human liver microsomes is not very different, and the clearance rates are 13.2572 and 13.7638 respectively. There is no significant difference in the clearance rate of single incubation and mixed (Cpds5+Cpds7) in human liver microsomes. The concentration of the total metabolite AZ'1168 reaches Cmax after 15 minutes of incubation in human liver microsomes, and then the concentration gradually decreases.

[0161] Experimental group 8: Cpds1 and Cpds7 (AZD3759) were added to human liver microsomes and a certain concentration of the inhibitor schisandrin was added for incubation. The stability data after incubation are shown in Table 12.

[0162] Experimental group 9: Cpds1 and Cpds7 (AZD3759) were added to human liver microsomes and a certain concentration of the inhibitor silybin was added for incubation. The stability data after incubation are shown in Table 13.

[0163] Experimental group 10: Cpds1 and Cpds7 (AZD3759) were added to human liver microsomes and a certain concentration of inhibitor ketoconazole was added for incubation. The stability data after incubation are shown in Table 14.

[0164] For experimental groups 8 to 10, under the premise that there is no metabolic interference between the compounds, the experiments in the same system can exclude other reasons or inter-batch effects caused by different batches of experiments.

[0165] Table 12

[0166] Table 13

[0167] Table 14

[0168] It can be seen from Table 12 that after the addition of schisandrin A, the clearance rates of Cpds1 (deuterated) and Cpds7 (AZD 3759) in human liver microsomes decreased (7.08 / 12.85=0.55), indicating that schisandrin A inhibited the metabolic enzyme activity of liver microsomes, resulting in a decrease in the clearance rates of Cpds1 and Cpds7.

[0169] It can be seen from Table 13 that after the addition of silybin, the clearance rates of Cpds1 (deuterated) and Cpds7 (AZD3759) in human microsomes decreased, indicating that silybin inhibited the metabolic enzyme activity of liver microsomes, resulting in a decrease in the clearance rates of Cpds1 and Cpds7 (10.37 / 12.85=0.81), but did not inhibit the microsomal activity as significantly as silybin.

[0170] As can be seen from Table 14: After adding ketoconazole, the clearance rates of Cpds1 (deuterated) and Cpds7 (AZD3759) in human liver microsomes were significantly reduced (1.54 / 12.85=0.12), indicating that ketoconazole inhibited the metabolic enzyme activity of liver microsomes, resulting in a decrease in the clearance rates of Cpds1 and Cpds7, which was significantly stronger than the inhibition of microsomal activity by Schisandrin A. The generation of Cpds6 metabolites tended to increase gradually, and the Cmax time was delayed to 4h.

[0171] Experimental group 11: Only verapamil was added to human liver microsomes for incubation. The stability data after incubation are shown in Table 15.

[0172] Experimental group 12: Cpds4, Cpds5, Cpds6, Cpds7 and verapamil were added to human liver microsomes for incubation. The stability data after incubation are shown in Table 16.

[0173] Table 15

[0174] Table 16

[0175] It can be seen from Table 15 that the clearance rate of verapamil in liver microsomes is 36.1276. This group of data is used as a control and the results are normal.

[0176] It can be seen from Table 16 that the results of combined incubation and separate incubation are similar, and there is no metabolic interference between the compounds.

[0177] In summary, CYP3A inhibitors ketoconazole, schisandrin and silybin can effectively inhibit the metabolism of compound Cpds7. The inhibitory effect (Cpds7 metabolism inhibition rate: CL inh / CL non-inh ) was, ketoconazole (0.12) had the strongest inhibitory effect, followed by schisandrin (0.55), and silybin (0.81) was the weakest.

Claims

1. A substituted zolitinib derivative represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof: in: R 1 is selected from the group consisting of methyl, methoxy, methyl substituted by halogen, methoxy substituted by halogen, methyl substituted by deuterium atom, and methoxy substituted by deuterium atom; R 2 is selected from the group consisting of methyl, methoxy, methyl substituted by halogen, methoxy substituted by halogen, methyl substituted by deuterium atom, and methoxy substituted by deuterium atom; R 3 It is selected from the group consisting of methyl, methoxy, methyl substituted by halogen, methoxy substituted by halogen, methyl substituted by a deuterium atom, and methoxy substituted by a deuterium atom.

2. The substituted zolitinib derivative or a pharmaceutically acceptable salt or solvate thereof according to claim 1, characterized in that: R 1 Selected from methyl, methoxy, methyl substituted with 1 to 3 deuterium atoms, methyl substituted with 1 to 3 fluorine atoms, methoxy substituted with 1 to 3 deuterium atoms, methoxy substituted with 1 to 3 fluorine atoms; R 2 Selected from methyl, methoxy, methyl substituted with 1 to 3 deuterium atoms, methyl substituted with 1 to 3 fluorine atoms, methoxy substituted with 1 to 3 deuterium atoms, methoxy substituted with 1 to 3 fluorine atoms; R 3 Selected from methyl, methoxy, methyl substituted with 1 to 3 deuterium atoms, methyl substituted with 1 to 3 fluorine atoms, methoxy substituted with 1 to 3 deuterium atoms, methoxy substituted with 1 to 3 fluorine atoms.

3. The substituted zolitinib derivative or a pharmaceutically acceptable salt or solvate thereof according to claim 1 or 2, characterized in that: R 1 is selected from methyl, methoxy, trideuteriomethyl, trifluoromethyl, trideuteriomethoxy, trifluoromethoxy; R 2 is selected from methyl, methoxy, trideuteriomethyl, trifluoromethyl, trideuteriomethoxy, trifluoromethoxy; R 3 is selected from methyl, methoxy, trideuteriomethyl, trifluoromethyl, trideuteriomethoxy, trifluoromethoxy; preferably, R 1 is selected from methyl, trideuteriomethyl, trifluoromethyl; R 2 is selected from methyl or trideuterated methyl; R 3 Selected from methoxy or trideuterated methoxy.

4. The substituted zolitinib derivative or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 3, characterized in that: The substituted zolitinib derivatives are selected from the following compounds:

5. A method for preparing a substituted zolitinib derivative represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof, characterized in that: The preparation method comprises: Technical Solution I: The substituted zolitinib derivative of the general formula (I) is Among them, R 4 and R 5 are independently selected from methyl, methoxy, halogen-substituted methyl, halogen-substituted methoxy, deuterium atom-substituted methyl, deuterium atom-substituted methoxy; preferably, R 4 and R 5 is independently selected from methyl, methoxy, methyl substituted with 1 to 3 deuterium atoms, methyl substituted with 1 to 3 fluorine atoms, methoxy substituted with 1 to 3 deuterium atoms, methoxy substituted with 1 to 3 fluorine atoms; more preferably, R 4 and R 5 are independently selected from methyl, methoxy, trideuteriomethyl, trifluoromethyl, trideuteriomethoxy, trifluoromethoxy; further preferably, R 4 and R 5 is independently selected from methyl or trideuterated methyl; further preferably, R 4 and R 5 are not the same; Technical Solution II: The substituted zolitinib derivative of the general formula (I) is Technical Solution III: The substituted zolitinib derivative of the general formula (I) is 6. The preparation method according to claim 5, characterized in that: The reaction parameters of the preparation method include: Technical solution I: The molar ratio of compound A to compound B is 0.5:2 to 1:5; the reaction temperature is 15 to 30°C, and the reaction time is 5 to 15 hours; Technical scheme II: In step 1, the molar ratio of compound C and CS2 is 0.5-2:1-5; the molar ratio of compound C and CH3I is 0.5-5:2-10; the reaction temperature is 10-30°C, and the reaction time is 0.5-5 hours; in step 2, the molar ratio of compound D and antimony trichloride is 1:0.01-1:1; the molar ratio of compound D and BAST is 0.5-2:1-5; the reaction temperature is 10-30°C, and the reaction time is 12-36 hours; Technical Scheme III: In step 1, the molar ratio of the compound E and BBr3 is 0.5-5:1-10; the reaction temperature is 30-60°C, and the reaction time is 0.5-2 hours; in step 2, the molar ratio of the compound F and trideuteriummethyl 4-methylbenzenesulfonate is 0.5-5:0.5-5; the reaction temperature is 10-30°C, and the reaction time is 12-36 hours.

7. Use of a substituted zolitinib derivative represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof in the preparation of an epidermal growth factor receptor tyrosine kinase activity inhibitor.

8. Use of a substituted zolitinib derivative represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicament for preventing and / or treating non-small cell lung cancer, glioblastoma, and Parkinson's disease.

9. A pharmaceutical composition comprising a substituted zolitinib derivative represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof, and a CYP3A inhibitor.

10. The pharmaceutical composition according to claim 9, characterized in that The molar ratio of the substituted zolitinib derivative or its pharmaceutically acceptable salt or solvate: the CYP3A inhibitor is 1:(0.1-10).

11. The pharmaceutical composition according to claim 9, characterized in that The pharmaceutical composition also includes the prototype drug AZD3759 or a pharmaceutically acceptable salt or solvate thereof.

12. The pharmaceutical composition according to claim 11, characterized in that The molar ratio of the substituted zolitinib derivative or its pharmaceutically acceptable salt or solvate: the prototype drug AZD3759 or its pharmaceutically acceptable salt or solvate: the CYP3A inhibitor is 1:(0.1-10):(0.1-10).

13. The pharmaceutical composition according to any one of claims 9 or 12, characterized in that The CYP3A inhibitor includes a mixture of one or more of ketoconazole, ritonavir, schisandrin and silybin.

14. Use of the pharmaceutical composition according to any one of claims 9 to 13 in the preparation of drugs for epidermal growth factor receptor tyrosine kinase activity inhibitors.

15. Use of the pharmaceutical composition according to any one of claims 9 to 13 in the preparation of a medicament for preventing and / or treating non-small cell lung cancer, glioblastoma, and Parkinson's disease.

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