CN114230524A

CN114230524A - (5-fluoro-2-anilinopyrimidin-4-yl) amino-N-hydroxybenzamide derivative and preparation method and application thereof

Info

Publication number: CN114230524A
Application number: CN202111625354.1A
Authority: CN
Inventors: 冉凡胜; 凌勇; 许中原; 刘云; 吴红梅; 谢旭东; 张雨婷; 王怡晨; 郭李婷
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2021-12-28
Filing date: 2021-12-28
Publication date: 2022-03-25

Abstract

The invention provides a (5-fluoro-2-anilinopyrimidine-4-yl) amino-N-hydroxybenzamide derivative, a preparation method and application thereof. The structure of the (5-fluoro-2-aniline pyrimidine-4-yl) amino-N-hydroxy benzamide derivative is shown as a general formula I,

wherein R is₁Selected from morpholinyl, piperidinyl, C_1～6Alkyl substituted piperazinyl, C1-6 straight chain or branched chain alkyl; r₂Is selected from hydrogen, halogen, methoxy, trifluoromethyl, cyano, C1-6 straight chain or branched chain alkyl. The preparation method comprises the following steps: compound (I)

Reacting the product of the reaction with methyl aminobenzoate compound with substituted aniline, and dissolving the reaction product in NH₂OK solution is continuously reacted to obtain the compound of the invention. The compound has certain BTK/FLT3 double-inhibition activity and anti-tumor activity.

Description

(5-fluoro-2-anilinopyrimidin-4-yl) amino-N-hydroxybenzamide derivative and preparation method and application thereof

Technical Field

The invention relates to the technical field of organic compound synthesis and medical application, in particular to a (5-fluoro-2-anilinopyrimidine-4-yl) amino-N-hydroxybenzamide derivative, and a preparation method and application thereof.

Background

Bruton's Tyrosine Kinase (BTK) is a membrane-bound protein, belongs to the non-receptor tyrosine kinase Tec family, is a signal transduction molecule of a B cell antigen receptor (BCR) and cytokine receptor pathway, can play a role in the signals of the transportation, chemotaxis and adhesion pathways of B cells by activating B cell surface receptors, and has important significance for the proliferation, differentiation and apoptosis of cells. BTK regulates the survival and biological functions of B cells through BCR signaling pathway and becomes a drug action target for treating B cell malignant tumors (see: Current oncology 2019; 26(2): e233-e 240). FMS-like tyrosine kinase 3 (FLT 3) is a transmembrane tyrosine receptor expressed by early myeloid hematopoietic cells (see: Oncogene 2000; 19(49):5548 and 5557) and belongs to the type III Receptor Tyrosine Kinase (RTK) family. Mutation of FLT3 gene is an important cause of Acute Myeloid Leukemia (AML), and after activation, FLT3 receptor dimerizes, binds to corresponding protein and activates downstream multiple signaling pathways (Nat. Rev. cancer 2012:12(11):753-766), inducing abnormal proliferation and growth of hematopoietic cells, leading to cancer. BTK and FLT3 are important targets for treating malignant tumors, and preclinical and clinical studies show that the dual inhibition BTK and FLT3 have synergistic antitumor effect (see: Blood 2019; 134(Supplement _1): 5477-5477). Therefore, the design and synthesis of the novel BTK/FLT3 double-target-point drug with novel structure and drug property have important significance for treating malignant tumors.

Disclosure of Invention

The invention aims to provide a (5-fluoro-2-anilinopyrimidine-4-yl) amino-N-hydroxybenzamide derivative which has a BTK/FLT3 dual-inhibition effect and good anti-tumor activity.

In order to achieve the purpose, the technical scheme of the disclosure is as follows:

in a first aspect of the present invention, the present invention provides a compound which is a (5-fluoro-2-anilinopyrimidin-4-yl) amino-N-hydroxybenzamide derivative having the structure represented by general formula I:

wherein R is₁Represents morpholinyl, piperidinyl, C_1～6Alkyl-substituted piperazinyl, C_1～6A linear or branched alkyl group; r₂Represents hydrogen, halogen, methoxy, trifluoromethyl, cyano, C_1～6Straight or branched chain alkyl.

Preferably, R₁Selected from morpholinyl, piperidinyl, N-methylpiperazinyl or N-ethylpiperazinyl; r₂Represents hydrogen, fluorine or methoxy.

Preferably, the compound is any one of the following compounds:

2- ((5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide (I-1)

2- ((5-fluoro-2- ((4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide (I-2)

2- ((5-fluoro-2- ((4- (piperidin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide (I-3)

2- ((2- ((4- (4-ethylpiperazin-1-yl) phenyl) amino) -5-fluoropyrimidin-4-yl) amino) -N-hydroxybenzamide (I-4)

2- ((5-fluoro-2- ((3-fluoro-4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide (I-5)

2- ((5-fluoro-2- ((2-methoxy-4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide (I-6)

3- ((5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide (I-7)

3- ((5-fluoro-2- ((4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide (I-8)

3- ((5-fluoro-2- ((4- (piperidin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide (I-9)

3- ((2- ((4- (4-ethylpiperazin-1-yl) phenyl) amino) -5-fluoropyrimidin-4-yl) amino) -N-hydroxybenzamide (I-10)

3- ((5-fluoro-2- ((3-fluoro-4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide (I-11)

3- ((5-fluoro-2- ((2-methoxy-4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide (I-12)

4- ((5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide (I-13)

4- ((5-fluoro-2- ((4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide (I-14)

4- ((5-fluoro-2- ((4- (piperidin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide (I-15)

4- ((2- ((4- (4-ethylpiperazin-1-yl) phenyl) amino) -5-fluoropyrimidin-4-yl) amino) -N-hydroxybenzamide (I-16)

4- ((5-fluoro-2- ((3-fluoro-4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide (I-17)

The corresponding reference numbers are shown in parentheses after the names of the 17 compounds, and for the convenience of description and the simplicity of expression, the reference numbers in parentheses will be directly used in the following description of the present specification.

The compounds of the present invention may be present in free form or further in the form of salts in order to improve water solubility and increase bioavailability.

The term "pharmaceutically acceptable salt" as used herein refers to conventional non-toxic salts, and includes salts formed from the basic amino groups of the compounds of the present application. These salts are well known to those skilled in the art and the skilled artisan can prepare any pharmaceutically acceptable salt provided by the knowledge in the art. In addition, the skilled artisan may choose one salt and leave out another salt depending on solubility, stability, ease of formulation, etc. The determination and optimization of these salts is within the experience of the skilled artisan.

The invention also provides a method for preparing the compound with the structure shown in the general formula I, which comprises the following reaction route:

wherein R is₁Selected from morpholinyl, piperidinyl, C_1～6Alkyl substituted piperazinyl, C1-6 straight chain or branched chain alkyl; r₂Is selected from hydrogen, halogen, methoxy, trifluoromethyl, cyano, C1-6 straight chain or branched chain alkyl.

Reagents and conditions: (a) methyl aminobenzoate compound, N, N-Diisopropylethylamine (DIPEA), isopropanol, 85 ℃ and 4 h; (b) aniline compounds, trifluoroacetic acid, n-butanol, 110 ℃ and 12 h; (c) potassium hydroxide, hydroxylamine hydrochloride, anhydrous methanol, 0-r.t., 2 h.

The method comprises the following specific steps:

(i) dissolving the compound 1 and methyl aminobenzoate compound in isopropanol, adding DIPEA, and reacting at 85 ℃ for 4 hours. TLC detection, complete reaction, cooling to room temperature, separating out a large amount of solid, filtering, and recrystallizing a filter cake with ethyl acetate to obtain an intermediate 2;

(ii) dissolving the intermediate 2 in n-butanol, adding substituted aniline, dropwise adding trifluoroacetic acid into the solution, and reacting at 110 ℃ for 12 h. TLC detection, complete reaction, cooling to room temperature, reduced pressure evaporation to remove solvent, silica gel column chromatography to obtain intermediate 3.

(iii) Preparation of NH from potassium hydroxide, hydroxylamine hydrochloride and anhydrous methanol₂OK solution. Dissolving intermediate 3 in NH₂In OK solution, react for 2h at room temperature. TLC detection, reaction is completed, the solvent is evaporated under reduced pressure, water is added, the pH value is adjusted to 6-7 by dilute hydrochloric acid, solid is separated out, filtration is carried out, and a filter cake is recrystallized by methanol or ethyl acetate to obtain a compound with a structure shown in a general formula I, namely the (5-fluoro-2-anilinopyrimidine-4-yl) amino-N-hydroxybenzamide derivative.

The invention also provides a pharmaceutical composition containing the compound or the pharmaceutically acceptable salt thereof.

Pharmaceutical compositions of the compounds of the invention may be administered in any manner selected from: oral, aerosol inhalation, rectal, nasal, vaginal, topical, parenteral such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal or intracranial injection or infusion, or by means of an explanted reservoir, with oral, intramuscular, intraperitoneal or intravenous administration being preferred.

In a fourth aspect of the present invention, the present invention also provides a pharmaceutical preparation, which comprises the above compound or a pharmaceutically acceptable salt thereof or a composition containing the above compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable adjuvant and/or carrier.

The compounds of the present invention or pharmaceutical compositions containing them may be administered in unit dosage form. The administration dosage form can be liquid dosage form or solid dosage form. The liquid dosage form can be true solution, colloid, microparticle, emulsion, or mixed suspension. Other dosage forms such as tablet, capsule, dripping pill, aerosol, pill, powder, solution, suspension, emulsion, granule, suppository, lyophilized powder for injection, clathrate, landfill, patch, liniment, etc.

The pharmaceutical combination or pharmaceutical preparation of the present invention may further comprise a conventional carrier, wherein the pharmaceutically acceptable carrier includes but is not limited to: ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycerol, sorbates, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, beeswax, lanolin and the like. The carrier may be present in the pharmaceutical composition in an amount of 1% to 98% by weight, typically about 80% by weight. For convenience, the local anesthetic, preservative, buffer, etc. may be dissolved directly in the vehicle.

Oral tablets and capsules may contain excipients such as binding agents, for example syrup, acacia, sorbitol, tragacanth, or polyvinylpyrrolidone, fillers such as lactose, sucrose, corn starch, calcium phosphate, sorbitol, glycine, lubricants such as magnesium stearate, talc, polyethylene glycol, silica, disintegrants such as potato starch, or acceptable wetting agents such as sodium lauryl sulfate. The tablets may be coated by methods known in the art of pharmacy.

The oral liquid can be made into water and oil suspension, solution, emulsion, syrup, or dried product, and supplemented with water or other suitable medium before use. Such liquid preparations may contain conventional additives such as suspending agents, sorbitol, cellulose methyl ether, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gelatin, hydrogenated edible fats and oils, emulsifying agents such as lecithin, sorbitan monooleate, gum arabic; or a non-aqueous carrier (which may comprise an edible oil), such as almond oil, an oil such as glycerol, ethylene glycol, or ethanol; preservatives, e.g. methyl or propyl p-hydroxybenzoates, sorbic acid. Flavoring or coloring agents may be added if desired.

Suppositories may contain conventional suppository bases such as cocoa butter or other glycerides.

For parenteral administration, liquid dosage forms are generally prepared from the compound and a sterile carrier. The carrier is preferably water. The compound can be dissolved in the carrier or made into suspension solution according to the concentration of the carrier and the drug, and the compound is firstly dissolved in water when made into the solution for injection, filtered and sterilized and then filled into a sealed bottle or ampoule.

It will be appreciated that the optimum dosage and interval for administration of a compound of formula I will be determined by the nature of the compound and external conditions, such as the form, route and site of administration and the particular mammal being treated, and that such optimum dosage may be determined by conventional techniques. It will also be appreciated that the optimal course of treatment, i.e. the daily dosage of a compound of formula I over a nominal period of time, may be determined by methods well known in the art.

The invention also provides application of the compound or the compound obtained by the preparation method in preparing BTK and/or FLT3 inhibitors.

The invention also provides application of the compound or the pharmaceutically acceptable salt thereof in preparing a medicament for treating malignant tumors.

Preferably, the tumor is one of mantle cell lymphoma, acute myeloid leukemia and breast cancer.

The invention also provides a medicinal composition which comprises the compound or the compound prepared by the preparation method.

Compared with the prior art, the invention provides the (5-fluoro-2-aniline pyrimidine-4-yl) amino-N-hydroxy benzamide derivative and the preparation method thereof; different from the prior art that the compounds show kinase inhibition on BTK and FLT3, wherein partial compounds show strong double inhibition on BTK and FLT3, and representative compounds have IC (integrated Circuit) of BTK and FLT3₅₀At low nanomolar level (IC)₅₀<20 nM); compared with the marketed drug Sorafenib, the compound shows equivalent or even better antiproliferative activity in malignant tumor cells of a blood system, and the IC of a representative compound on a plurality of malignant tumor cells of the blood system₅₀At low nanomolar level (IC)₅₀<100nM), the antiproliferative activity of the compound is 12-1000 times better than that of the marketed drug Sorafenib; in addition, representative compounds also showed potent antitumor effects against breast cancer.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers. The following experimental examples are only for illustrating the technical effects of the present invention, but the experimental examples are not intended to limit the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

Example 1: preparation of intermediate 2

The starting materials 2, 4-dichloro-5-fluoropyrimidine (12mmol, 1.2eq), methyl aminobenzoate (10mmol, 1.0eq) and DIPEA (15mmol, 1.5eq) were dissolved in 20mL of isopropanol and reacted by heating at 85 ℃ for 4 hours. After the reaction is finished, the reaction liquid is cooled to room temperature, a large amount of solid is separated out, the solid is filtered, and a filter cake is recrystallized by ethyl acetate to obtain an intermediate 2.

Example 2: preparation of intermediate 3

Dissolving the intermediate 2(1mmol, 1eq) in 30mL of n-butanol, adding different substituted anilines (1.1mmol, 1.1eq), adding 5 drops of trifluoroacetic acid dropwise into the solution, and heating at 110 ℃ for reaction for 12 h. After the reaction, the reaction mixture was cooled to room temperature, the solvent was distilled off under reduced pressure, and silica gel column chromatography (dichloromethane/methanol 200:1 to 20:1) was performed to obtain intermediate 3.

3-1: methyl 2- ((5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) benzoate

¹H NMR(400MHz,DMSO-d₆)δ10.94(d,J＝2.4Hz,1H),9.17(s,1H),8.93(d,J＝8.5Hz,1H),8.19(d,J＝3.2Hz,1H),8.03(dd,J＝8.0,1.6Hz,1H),7.69–7.57(m,1H),7.50(d,J＝8.9Hz,2H),7.23–7.10(m,1H),6.91(d,J＝9.1Hz,2H),3.90(s,3H),3.80–3.68(m,4H),3.10–3.00(m,4H).¹³C NMR(101MHz,DMSO-d₆)δ168.79,156.30,149.31(d,J＝10.5Hz),146.83,141.77,141.62(d,J＝18Hz),141.14(d,J＝245Hz),134.86,133.37,131.34,122.25,121.30,120.91,116.01,115.48,66.66,53.09,49.77.HRMS(ESI)m/z calcd for C₂₂H₂₃FN₅O₃[M+H]⁺424.1779,found 424.1781.

3-2: methyl 2- ((5-fluoro-2- ((4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) benzoate

¹H NMR(400MHz,DMSO-d₆)δ10.94(d,J＝2.3Hz,1H),9.15(s,1H),8.93(d,J＝8.5Hz,1H),8.19(d,J＝3.2Hz,1H),8.03(dd,J＝8.0,1.6Hz,1H),7.62(dt,J＝8.0,1.2Hz,1H),7.48(d,J＝8.9Hz,2H),7.16(dt,J＝8.0,0.8Hz,1H),6.89(d,J＝9.1Hz,2H),3.90(s,3H),3.07(t,J＝4.8Hz,4H),2.46(t,J＝4.8Hz,4H),2.22(s,3H).¹³C NMR(101MHz,DMSO-d₆)δ168.79,156.31(d,J＝2.6Hz),149.27(d,J＝9.4Hz),146.84,141.80,141.91(d,J＝19Hz),141.11(d,J＝244Hz),134.84,133.04,131.32,122.19,121.33,120.85,116.23,115.37,55.19,53.07,49.36,46.27.HRMS(ESI)m/z calcd for C₂₃H₂₆FN₆O₂[M+H]⁺437.2096,found 437.2095.

3-3: methyl 2- ((5-fluoro-2- ((4- (piperidin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) benzoate

¹H NMR(400MHz,DMSO-d₆)δ10.93(d,J＝2.3Hz,1H),9.13(s,1H),8.93(d,J＝8.5Hz,1H),8.18(d,J＝3.2Hz,1H),8.03(dd,J＝8.0,1.5Hz,1H),7.66–7.57(m,1H),7.46(d,J＝8.9Hz,2H),7.19–7.12(m,1H),6.88(d,J＝9.0Hz,2H),3.90(s,3H),3.11–3.00(m,4H),1.69–1.59(m,4H),1.54-1.49(m,2H).¹³C NMR(101MHz,DMSO-d₆)δ168.79,156.32(d,J＝3.1Hz),149.27(d,J＝9.4Hz),147.72,142.32,141.80,141.61(d,J＝19Hz),141.09(d,J＝245Hz),134.82,132.84,131.32,122.19,121.34,120.87,116.88,115.37,53.07,51.01,25.93,24.35.HRMS(ESI)m/z calcd for C₂₃H₂₅FN₅O₂[M+H]⁺422.1987,found 422.1986.

3-4: 2- ((2- ((4- (4-ethylpiperazin-1-yl) phenyl) amino) -5-fluoropyrimidin-4-yl) amino) benzoic acid methyl ester

¹H NMR(400MHz,DMSO-d₆)δ9.65(s,1H),9.04(s,1H),8.14(d,J＝3.6Hz,1H),8.01(d,J＝8.8Hz,2H),7.87(d,J＝8.8Hz,2H),7.45(d,J＝8.9Hz,2H),6.87(d,J＝9.0Hz,2H),3.83(s,3H),3.12–3.01(m,4H),2.37(q,J＝7.2Hz,2H),1.04(t,J＝7.2Hz,3H).¹³C NMR(101MHz,DMSO-d₆)δ166.39,156.41(d,J＝2.6Hz),149.44(d,J＝10.7Hz),146.87,144.42,141.93(d,J＝19.4Hz),140.80(d,J＝245Hz),133.13,130.30,123.40,121.57,120.10,116.22,52.91,52.34,52.13,49.52,12.51.HRMS(ESI)m/z calcd for C₂₄H₂₈FN₆O₂[M+H]⁺451.2252,found 451.2249.

3-5: methyl 2- ((5-fluoro-2- ((3-fluoro-4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) benzoate

¹H NMR(400MHz,DMSO-d₆)δ10.90(d,J＝1.9Hz,1H),9.42(s,1H),8.86(d,J＝8.3Hz,1H),8.25(d,J＝3.2Hz,1H),8.04(dd,J＝8.0,1.5Hz,1H),7.72–7.60(m,2H),7.27(dd,J＝8.7,1.8Hz,1H),7.22–7.15(m,1H),7.00–6.92(m,1H),3.89(s,3H),3.01–2.89(m,4H),2.50-2.42(m,4H),2.23(s,3H).¹³C NMR(101MHz,DMSO-d₆)δ168.69,155.66(d,J＝3.0Hz),155.17(d,J＝241Hz),149.46(d,J＝10Hz),141.49(d,J＝19Hz),141.48(d,J＝254Hz),141.45,136.36(d,J＝10.9Hz),134.77,134.18(d,J＝9.3Hz),131.37,122.58,121.17,119.60(d,J＝4.4Hz),116.10,115.38,107.60(d,J＝25Hz),55.17,53.08,50.77,46.12.HRMS(ESI)m/z calcd for C₂₃H₂₅F₂N₆O₂[M+H]⁺455.2002,found 455.2001.

3-6: methyl 2- ((5-fluoro-2- ((2-methoxy-4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) benzoate

¹H NMR(400MHz,DMSO-d₆)δ9.59(s,1H),8.03(d,J＝3.6Hz,1H),7.89(s,1H),7.83(d,J＝8.7Hz,2H),7.77(d,J＝8.8Hz,2H),7.43(d,J＝8.2Hz,1H),6.64(d,J＝2.4Hz,1H),6.47(dd,J＝8.7,2.4Hz,1H),3.81(s,3H),3.75(s,3H),3.19–3.12(m,4H),2.24(s,3H).¹³C NMR(101MHz,DMSO-d6)δ166.37,157.44(d,J＝2.5Hz),153.30,149.50,149.36(d,J＝10Hz),144.56,142.08(d,J＝19Hz),140.72(d,J＝245Hz),130.21,125.93,123.11,121.06,119.84,107.18,100.54,55.84,55.21,52.27,49.25,46.29.HRMS(ESI)m/z calcd for C₂₄H₂₈FN₆O₃[M+H]⁺467.2201,found 467.2203.

3-7: 3- ((5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) benzoic acid methyl ester

¹H NMR(400MHz,DMSO-d₆)δ9.53(s,1H),9.01(s,1H),8.20(d,J＝10.5Hz,2H),8.10(d,J＝3.6Hz,1H),7.68–7.62(m,1H),7.51–7.43(m,3H),6.79(d,J＝9.1Hz,2H),3.83(s,3H),3.76–3.69(m,4H),3.04–2.96(m,4H).¹³C NMR(101MHz,DMSO-d₆)δ166.66,156.21(d,J＝2.7Hz),149.87(d,J＝10.7Hz),146.38,141.64(d,J＝19Hz),140.66(d,J＝245Hz),139.87,133.67,130.39,129.41,126.21,123.94,122.39,120.67,115.95,66.65,52.62,49.79.HRMS(ESI)m/z calcd for C₂₂H₂₃FN₅O₃[M+H]⁺424.1779,found 424.1779.

3-8: 3- ((5-fluoro-2- ((4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) benzoic acid methyl ester

¹H NMR(400MHz,DMSO-d₆)δ9.52(s,1H),8.99(s,1H),8.26–8.15(m,2H),8.09(d,J＝3.6Hz,1H),7.69–7.61(m,1H),7.55–7.41(m,3H),6.78(d,J＝9.1Hz,2H),3.83(s,3H),3.08–2.96(m,4H),2.47–2.40(m,4H),2.22(s,3H).¹³C NMR(101MHz,DMSO-d₆)δ166.66,156.23(d,J＝2.7Hz),149.86(d,J＝10.7Hz),146.41,141.65(d,J＝19Hz),140.64(d,J＝245Hz),139.87,133.34,130.38,129.41,126.21,123.93,122.40,120.68,116.19,55.19,52.61,49.39,46.26.HRMS(ESI)m/z calcd for C₂₃H₂₆FN₆O₂[M+H]⁺437.2096,found437.2097.

3-9: 3- ((5-fluoro-2- ((4- (piperidin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) benzoic acid methyl ester

¹H NMR(400MHz,DMSO-d₆)δ9.46(s,1H),8.92(s,1H),8.17(s,1H),8.16–8.09(m,1H),8.03(d,J＝3.6Hz,1H),7.60(d,J＝7.7Hz,1H),7.43-7.37(m,3H),6.71(d,J＝9.0Hz,2H),3.77(s,3H),2.99–2.90(m,4H),1.61–1.51(m,4H),1.46-1.42(m,2H).¹³C NMR(101MHz,DMSO-d₆)δ166.66,156.28(d,J＝2.6Hz),149.88(d,J＝10.7Hz),147.32,141.64(d,J＝19Hz),140.63(d,J＝244Hz),139.88,133.16,130.39,129.38,126.23,123.93,122.45,120.73,116.85,52.59,51.07,25.94,24.33.HRMS(ESI)m/z calcd for C₂₃H₂₅FN₅O₂[M+H]⁺422.1987,found 422.1984.

3-10: 3- ((2- ((4- (4-ethylpiperazin-1-yl) phenyl) amino) -5-fluoropyrimidin-4-yl) amino) benzoic acid methyl ester

¹H NMR(400MHz,DMSO-d₆)δ9.53(s,1H),8.99(s,1H),8.22(t,J＝1.7Hz,1H),8.18(d,J＝7.8Hz,1H),8.09(d,J＝3.6Hz,1H),7.66(d,J＝7.8Hz,1H),7.50–7.41(m,3H),6.78(d,J＝9.0Hz,2H),3.83(s,3H),3.07–2.98(m,4H),2.48(d,J＝5.2Hz,4H),2.36(q,J＝7.2Hz,2H),1.03(t,J＝7.2Hz,3H).¹³C NMR(101MHz,DMSO-d₆)δ166.67,156.25(d,J＝2.7Hz),149.87(d,J＝10.8Hz),146.42,141.63(d,J＝19Hz),140.65(d,J＝245Hz),139.88,133.36,130.40,129.39,126.21,123.93,122.41,120.74,116.17,52.85,52.60,52.09,49.44,12.39.HRMS(ESI)m/z calcd for C₂₆H₃₂FN₅O₂[M+H]⁺451.2252,found 451.2254.

3-11: methyl 3- ((5-fluoro-2- ((3-fluoro-4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) benzoate

¹H NMR(400MHz,DMSO-d₆)δ9.61(s,1H),9.29(s,1H),8.24(s,1H),8.15(d,J＝3.6Hz,1H),8.12(d,J＝8.4Hz,1H),7.69(d,J＝7.8Hz,1H),7.61(dd,J＝15.6,2.2Hz,1H),7.49(t,J＝7.9Hz,1H),7.22(dd,J＝8.7,1.8Hz,1H),6.91–6.81(m,1H),3.81(s,3H),2.91(s,4H),2.45(s,4H),2.21(s,3H).¹³C NMR(101MHz,DMSO-d6)δ166.54,155.68(d,J＝3.3Hz),155.16(d,J＝241Hz),149.98(d,J＝11.3Hz),141.66(d,J＝19.9Hz),140.93(d,J＝242Hz),139.66,136.51(d,J＝10.9Hz),133.85(d,J＝9Hz),130.51,129.49,126.51,124.24,122.70,119.43(d,J＝5.3Hz),114.86,107.12(d,J＝26Hz),55.27,52.54,50.89,46.29.HRMS(ESI)m/z calcd for C₂₃H₂₅F₂N₆O₂[M+H]⁺455.2002,found 455.2002.

3-12: 3- ((5-fluoro-2- ((2-methoxy-4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) benzoic acid methyl ester

¹H NMR(400MHz,DMSO-d₆)δ9.52(s,1H),8.26–8.22(m,1H),8.12(d,J＝8.1Hz,1H),8.06(d,J＝3.7Hz,1H),7.67(s,1H),7.61(t,J＝8.0Hz,2H),7.40(t,J＝7.9Hz,1H),6.61(d,J＝2.5Hz,1H),6.36(dd,J＝8.8,2.5Hz,1H),3.84(s,3H),3.78(s,3H),3.14–3.05(m,4H),2.48–2.43(m,4H),2.23(s,3H).13C NMR(101MHz,DMSO-d6)δ166.64,156.64(d,J＝2.6Hz),151.42,149.86(d,J＝10.8Hz),148.41,141.61(d,J＝19.6Hz),140.73(d,J＝244Hz),139.95,130.32,129.31,125.84,123.74,123.23,121.98,121.37,107.06,100.52,55.97,55.18,52.61,49.28,46.25.HRMS(ESI)m/z calcd for C24H28FN6O3[M+H]+467.2201,found467.2196.

3-13: 4- ((5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) benzoic acid methyl ester

¹H NMR(400MHz,DMSO-d₆)δ9.66(s,1H),9.06(s,1H),8.14(d,J＝3.6Hz,1H),8.01(d,J＝8.7Hz,2H),7.87(d,J＝8.8Hz,2H),7.48(d,J＝8.9Hz,2H),6.89(d,J＝9.1Hz,2H),3.83(s,3H),3.79–3.71(m,4H),3.08–2.99(m,4H).¹³C NMR(101MHz,DMSO-d₆)δ166.40,156.37(d,J＝2.7Hz),149.45(d,J＝10.6Hz),146.78,144.41,141.90(d,J＝20.7Hz),140.82(d,J＝245Hz),133.49,130.31,123.41,121.50,120.10,116.03,66.64,52.34,49.79.MS(ESI)m/z calcd for C₂₂H₂₃FN₅O₃[M+H]⁺424.1779,found 424.1781.

3-14: 4- ((5-fluoro-2- ((4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) benzoic acid methyl ester

¹H NMR(400MHz,DMSO-d₆)δ9.65(s,1H),9.04(s,1H),8.14(d,J＝3.6Hz,1H),8.01(d,J＝8.7Hz,2H),7.87(d,J＝8.8Hz,2H),7.45(d,J＝8.9Hz,2H),6.87(d,J＝9.1Hz,2H),3.83(s,3H),3.11–3.01(m,4H),2.49–2.42(m,4H),2.23(s,3H).¹³C NMR(101MHz,DMSO-d₆)δ166.40,156.42(d,J＝2.2Hz),149.45(d,J＝10.6Hz),146.83,144.41,141.93(d,J＝21.9Hz),140.81(d,J＝245Hz),133.14,130.29,123.42,121.57,120.11,116.26,55.18,52.33,49.40,46.27.HRMS(ESI)m/z calcd for C₂₃H₂₆FN₆O₂[M+H]⁺437.2096,found437.2096.

3-15: 4- ((5-fluoro-2- ((4- (piperidin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) benzoic acid methyl ester

¹H NMR(400MHz,DMSO-d₆)δ9.64(s,1H),9.01(s,1H),8.13(d,J＝3.6Hz,1H),8.00(d,J＝8.9Hz,2H),7.86(d,J＝8.9Hz,2H),7.43(d,J＝8.8Hz,2H),6.87(d,J＝9.0Hz,2H),3.83(s,3H),3.11–3.00(m,4H),1.67-1.60(m,4H),1.55-1.49(m,2H).¹³C NMR(101MHz,DMSO-d₆)δ166.39,156.48(d,J＝2.6Hz),149.44(d,J＝10.5Hz),147.72,144.42,141.93(d,J＝16Hz),140.80(d,J＝250Hz),132.91,130.28,123.41,121.70,120.12,116.88,52.32,51.05,25.89,24.37.HRMS(ESI)m/z calcd for C₂₃H₂₅FN₅O₂[M+H]⁺422.1987,found 422.1985.

3-16: 4- ((2- ((4- (4-ethylpiperazin-1-yl) phenyl) amino) -5-fluoropyrimidin-4-yl) amino) benzoic acid methyl ester

¹H NMR(400MHz,DMSO-d₆)δ9.64(s,1H),9.01(s,1H),8.12(d,J＝3.6Hz,1H),7.99(d,J＝8.7Hz,2H),7.86(d,J＝8.8Hz,2H),7.45(d,J＝8.9Hz,2H),6.87(d,J＝9.1Hz,2H),3.83(s,3H),3.12–3.00(m,4H),2.37(q,J＝7.2Hz,2H),1.04(t,J＝7.2Hz,3H).¹³C NMR(101MHz,DMSO-d₆)δ166.40,156.43(d,J＝2.7Hz),149.46(d,J＝10.5Hz),146.89,144.44,141.92(d,J＝19Hz),140.82(d,J＝245Hz),133.13,130.29,123.41,121.61,120.12,116.21,52.90,52.32,52.12,49.53,12.48.HRMS(ESI)m/z calcd for C₂₄H₂₈FN₆O₂[M+H]⁺451.2252,found 451.2250.

3-17: 4- ((5-fluoro-2- ((3-fluoro-4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) benzoic acid methyl ester

¹H NMR(400MHz,DMSO-d₆)δ9.73(s,1H),9.32(s,1H),8.19(d,J＝3.5Hz,1H),7.99(d,J＝8.7Hz,2H),7.90(d,J＝8.8Hz,2H),7.67(dd,J＝15.5,2.1Hz,1H),7.23(dd,J＝8.6,1.6Hz,1H),6.94(t,J＝9.4Hz,1H),3.84(s,3H),2.99–2.89(m,4H),2.49–2.40(m,4H),2.22(s,3H).¹³C NMR(101MHz,DMSO-d₆)δ166.39,155.72(d,J＝2.9Hz),155.17(d,J＝241Hz),149.62(d,J＝10.7Hz),144.29,141.78(d,J＝20.4Hz),141.16(d,J＝246Hz),136.43(d,J＝10.9Hz),134.18(d,J＝9.3Hz),130.34,123.68,120.41,119.53(d,J＝4.3Hz),115.40(d,J＝1.8Hz),107.66(d,J＝25.8Hz),55.25,52.35,50.91(d,J＝2.4Hz),46.27.HRMS(ESI)m/z calcd for C₂₃H₂₅F₂N₆O₂[M+H]⁺455.2002,found 455.2000.

Example 3: preparation of the target Compound I

KOH (28.55g, 509mmol) and NH₂OH & HCI (23.84g, 343mmol) was dissolved in 70mL and 120mL of anhydrous methanol, respectively, to give solution A and solution B. Dropwise adding the solution A into the solution B under the ice bath condition, separating out white solid, continuing to react for 1 hour, filtering the precipitate to obtain NH₂OK solution. Intermediate 3(0.50mmol) was dissolved in 10mL NH₂OK solution was stirred at room temperature for 2 h. After the reaction is finished, the solvent is evaporated under reduced pressure, 20mL of water is added, the pH value is adjusted to 6-7 by 1M HCl, solid is separated out, the filtration is carried out, and a filter cake is recrystallized by methanol/ethyl acetate to obtain the target compound I.

I-1: 2- ((5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide

¹H NMR(400MHz,DMSO-d₆)δ11.56(s,1H),11.25(s,1H),9.37(s,1H),9.11(s,1H),8.79(d,J＝8.4Hz,1H),8.13(d,J＝3.3Hz,1H),7.63(dd,J＝7.9,1.3Hz,1H),7.57–7.45(m,3H),7.13–7.07(m,1H),6.89(d,J＝9.1Hz,2H),3.74(t,J＝4.8Hz,4H),3.04(t,J＝4.8Hz,4H).HRMS(ESI)m/z calcd for C₂₁H₂₂FN₆O₃[M+H]⁺425.1732,found 425.1729.

I-2: 2- ((5-fluoro-2- ((4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide

¹H NMR(400MHz,DMSO-d₆)δ11.48(s,1H),11.16(s,1H),9.31(s,1H),9.02(s,1H),8.72(d,J＝8.4Hz,1H),8.06(d,J＝3.2Hz,1H),7.56(dd,J＝7.8,1.5Hz,1H),7.42(dd,J＝8.5,6.4Hz,3H),7.03(td,J＝7.6,1.2Hz,1H),6.82(d,J＝9.1Hz,2H),3.00(s,4H),2.17(s,3H).¹³C NMR(101MHz,DMSO-d₆)δ166.03,156.34(d,J＝2.8Hz),149.39(d,J＝10.1Hz),146.68,141.15(d,J＝245Hz),141.00(d,J＝20Hz),139.58,133.28,131.51,128.10,122.01,121.13,120.96,119.80,116.25,55.20,49.40,46.26.HRMS(ESI)m/z calcd for C₂₂H₂₅FN₇O₂[M+H]⁺438.2048,found 438.2045.

I-3: 2- ((5-fluoro-2- ((4- (piperidin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide

¹H NMR(400MHz,DMSO-d₆)δ11.14(s,1H),9.49(s,1H),9.02(s,1H),8.96(s,1H),8.10(d,J＝3.7Hz,1H),7.93(d,J＝8.7Hz,2H),7.70(d,J＝8.8Hz,2H),7.46(d,J＝9.0Hz,2H),6.86(d,J＝9.1Hz,2H),3.10–2.99(m,4H),1.69–1.58(m,4H),1.54-1.48(m,2H).¹³C NMR(101MHz,DMSO-d₆)δ164.51,156.35(d,J＝2.8Hz),149.64(d,J＝10.4Hz),147.55,142.42,141.99,141.54(d,J＝18.8Hz),139.54,133.12,127.81,126.88,121.09,120.16,116.95,51.10,25.93,24.36.HRMS(ESI)m/z calcd for C₂₂H₂₄FN₆O₂[M+H]⁺423.1939,found423.1935.

I-4: 2- ((2- ((4- (4-ethylpiperazin-1-yl) phenyl) amino) -5-fluoropyrimidin-4-yl) amino) -N-hydroxybenzamide

¹H NMR(400MHz,DMSO-d₆)δ11.09(s,1H),9.45(s,1H),8.97(d,J＝24.4Hz,2H),8.04(d,J＝3.6Hz,1H),7.87(d,J＝8.7Hz,2H),7.64(d,J＝8.8Hz,2H),7.43(d,J＝9.0Hz,2H),6.82(d,J＝9.1Hz,2H),3.07(s,4H),2.66(s,4H),2.52(dd,J＝14.2,6.9Hz,2H),1.03(t,J＝7.2Hz,3H).¹³C NMR(101MHz,DMSO-d₆)δ166.36,156.28(d,J＝2.9Hz),149.31(d,J＝10.1Hz),146.20,141.11(d,J＝244Hz),141.19(d,J＝20.3Hz),139.80,133.59,132.23,128.02,122.18,121.21,121.13,118.75,116.48,52.24,51.80,48.71,11.51.HRMS(ESI)m/z calcd for C₂₃H₂₇FN₇O₂[M+H]⁺452.2205,found 452.2200.

I-5: 2- ((5-fluoro-2- ((3-fluoro-4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide

¹H NMR(400MHz,DMSO-d₆)δ11.55(s,1H),11.23(s,1H),9.40(s,2H),8.74(d,J＝8.3Hz,1H),8.20(d,J＝3.2Hz,1H),7.71(dd,J＝15.5,2.1Hz,1H),7.64(d,J＝7.8Hz,1H),7.52(t,J＝7.3Hz,1H),7.28(dd,J＝8.7,1.6Hz,1H),7.14(t,J＝7.6Hz,1H),6.98(t,J＝9.4Hz,1H),3.01(s,4H),2.68(s,4H),2.38(s,3H).¹³C NMR(101MHz,DMSO-d₆)δ166.29,155.66(d,J＝3.1Hz),155.19(d,J＝241Hz),149.41(d,J＝10.0Hz),141.41(d,J＝245Hz),141.10(d,J＝21.5Hz),139.55,136.73(d,J＝11.1Hz),133.63(d,J＝9.7Hz),132.21,128.13,122.46,121.35,119.72(d,J＝3.7Hz),119.19,115.21(d,J＝1.7Hz),107.44(d,J＝25.6Hz),54.63,50.11,49.06.HRMS(ESI)m/z calcd for C₂₂H₂₄F₂N₇O₂[M+H]⁺456.1954,found 456.1949.

I-6: 2- ((5-fluoro-2- ((2-methoxy-4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide

¹H NMR(400MHz,DMSO-d₆)δ11.03(s,1H),9.40(s,1H),8.89(s,1H),7.98(d,J＝3.7Hz,1H),7.80(s,1H),7.76(d,J＝8.7Hz,2H),7.57(d,J＝8.8Hz,2H),7.42(d,J＝8.7Hz,1H),6.57(d,J＝2.5Hz,1H),6.40(dd,J＝8.8,2.5Hz,1H),3.71(s,3H),2.42–2.40(m,4H),2.41(m,4H),2.17(s,3H).¹³C NMR(101MHz,DMSO-d₆)δ164.48,157.06(d,J＝2.8Hz),152.44,149.59(d,J＝10.6Hz),149.04,142.41,141.67(d,J＝19Hz),140.77(d,J＝245Hz),127.76,126.75,124.64,121.23,119.94,107.20,100.59,55.89,55.19,49.29,46.25.HRMS(ESI)m/z calcd for C₂₃H₂₇FN₇O₃[M+H]⁺468.2154,found 468.2149.

I-7: 3- ((5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide

¹H NMR(400MHz,DMSO-d₆)δ11.21(s,1H),9.45(s,1H),9.08(s,1H),9.00(s,1H),8.07(d,J＝3.6Hz,1H),7.99(s,1H),7.97(s,1H),7.47(d,J＝9.0Hz,2H),7.44-7.36(m,2H),6.82(d,J＝9.1Hz,2H),3.73(t,J＝4.8Hz,4H),3.01(t,J＝4.8Hz,4H).¹³C NMR(101MHz,DMSO-d₆)δ164.70,156.27(d,J＝2.6Hz),150.03(d,J＝10.7Hz),146.38,141.39(d,J＝16.9Hz),140.70(d,J＝244Hz),139.48,133.89,133.70,128.90,124.39,121.79,120.86,120.63,116.04,66.66,49.81.HRMS(ESI)m/z calcd for C₂₁H₂₂FN₆O₃[M+H]⁺425.1732,found425.1728.

I-8: 3- ((5-fluoro-2- ((4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide

¹H NMR(400MHz,DMSO-d₆)δ11.18(s,1H),9.43(s,1H),9.07(s,1H),8.95(s,1H),8.07(d,J＝3.6Hz,1H),7.98(s,2H),7.45(d,J＝8.9Hz,2H),7.42–7.34(m,2H),6.81(d,J＝9.0Hz,2H),3.03(t,J＝4.4Hz,4H),2.44(t,J＝4.4Hz,4H),2.22(s,3H).¹³C NMR(101MHz,DMSO-d₆)δ164.60,156.30(d,J＝2.6Hz),150.03(d,J＝11Hz),146.43,141.37(d,J＝20.0Hz),140.70(d,J＝244Hz),139.48,134.11,133.37,128.87,124.22,121.69,120.77,120.66,116.28,55.22,49.39,46.28.HRMS(ESI)m/z calcd for C₂₂H₂₅FN₇O₂[M+H]⁺438.2048,found 438.2045.

I-9: 3- ((5-fluoro-2- ((4- (piperidin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide

1H NMR(400MHz,DMSO-d6)δ11.17(s,1H),9.43(s,1H),9.06(s,1H),8.94(s,1H),8.07(d,J＝3.6Hz,1H),8.05–7.95(m,2H),7.44-7.36(m,4H),6.80(d,J＝9.1Hz,2H),3.09–2.93(m,4H),1.69–1.54(m,4H),1.54–1.42(m,2H).13C NMR(101MHz,DMSO-d6)δ164.75,156.32(d,J＝2.7Hz),150.01(d,J＝10.7Hz),147.35,141.40(d,J＝18.9Hz),140.68(d,J＝244Hz)139.54,133.88,133.19,128.91,124.37,121.69,120.79,120.62,116.96,51.07,25.96,24.36.HRMS(ESI)m/z calcd for C22H24FN6O2[M+H]+423.1939,found423.1936.

I-10: 3- ((2- ((4- (4-ethylpiperazin-1-yl) phenyl) amino) -5-fluoropyrimidin-4-yl) amino) -N-hydroxybenzamide

¹H NMR(400MHz,DMSO-d₆)δ11.20(s,1H),9.45(s,1H),9.08(s,1H),8.99(s,1H),8.08(d,J＝3.6Hz,1H),7.99(s,1H),7.97(s,1H),7.46(d,J＝8.9Hz,2H),7.43–7.36(m,2H),6.83(d,J＝9.1Hz,2H),3.11(s,4H),2.70(s,4H),2.60-2.55(m,2H),1.09(t,J＝7.2Hz,3H).¹³C NMR(101MHz,DMSO-d₆)δ164.77,156.27(d,J＝2.7Hz),150.02(d,J＝10.7Hz),145.92,141.42(d,J＝19Hz),140.72(d,J＝242Hz),139.51,133.86,133.71,128.95,124.41,121.75,120.87,120.64,116.50,52.23,51.79,48.72,11.53.HRMS(ESI)m/z calcd for C₂₃H₂₇FN₇O₂[M+H]⁺452.2205,found 452.2200.

I-11: 3- ((5-fluoro-2- ((3-fluoro-4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide

¹H NMR(400MHz,DMSO-d₆)δ11.20(s,1H),9.54(s,1H),9.29(s,1H),9.07(s,1H),8.14(d,J＝3.6Hz,1H),7.99(d,J＝7.6Hz,1H),7.96(s,1H),7.60(dd,J＝15.4,1.8Hz,1H),7.46-7.39(m,2H),7.29(dd,J＝8.8,1.6Hz,1H),6.91(t,J＝9.4Hz,1H),3.02(s,4H),2.76(s,4H),2.43(s,3H).¹³C NMR(101MHz,DMSO-d₆)δ164.60,155.69(d,J＝2.8Hz),155.17(d,J＝240Hz),150.15(d,J＝10.9Hz),141.29(d,J＝19.3Hz),141.01(d,J＝245Hz),139.35,136.93(d,J＝10.9Hz),133.89,133.22(d,J＝9.6Hz),129.01,124.71,122.01,121.05,119.72(d,J＝4.0Hz),114.81(d,J＝2.4Hz),107.06(d,J＝25.8Hz),54.45,49.86,44.97.HRMS(ESI)m/z calcd for C₂₂H₂₄F₂N₇O₂[M+H]⁺456.1960,found 456.1954.

I-12: 3- ((5-fluoro-2- ((2-methoxy-4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide

¹H NMR(400MHz,DMSO-d₆)δ11.03(s,1H),9.40(s,1H),8.89(s,1H),7.98(d,J＝3.7Hz,1H),7.80(s,1H),7.76(d,J＝8.7Hz,2H),7.57(d,J＝8.8Hz,2H),7.42(d,J＝8.7Hz,1H),6.57(d,J＝2.5Hz,1H),6.40(dd,J＝8.8,2.5Hz,1H),3.71(s,3H),2.42–2.40(m,4H),2.41(m,4H),2.17(s,3H).¹³C NMR(101MHz,DMSO-d₆)δ164.76,156.64,151.33,149.99(d,J＝10.6Hz),148.03,142.00,141.42(d,J＝18.7Hz),139.58,133.80,128.87,124.06,123.23,121.57(d,J＝4.9Hz),120.43,107.39,100.63,56.01,54.72,48.79,45.53.HRMS(ESI)m/z calcd for C₂₃H₂₇FN₇O₃[M+H]⁺468.2154,found 468.2151.

I-13: 4- ((5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide

¹H NMR(400MHz,DMSO-d₆)δ11.12(s,1H),9.50(s,1H),9.06(s,1H),8.97(s,1H),8.11(d,J＝3.6Hz,1H),7.93(d,J＝8.7Hz,2H),7.70(d,J＝8.8Hz,2H),7.50(d,J＝8.9Hz,2H),6.88(d,J＝9.1Hz,2H),3.77–3.71(m,4H),3.07–3.01(m,4H).¹³C NMR(101MHz,DMSO-d₆)δ164.45,156.28(d,J＝2.6Hz),149.66(d,J＝10.4Hz),146.62,142.42,141.52(d,J＝19Hz),140.80(d,J＝246Hz),133.65,127.83,126.89,121.03,120.19,116.08,66.65,49.84.HRMS(ESI)m/z calcd for C₂₁H₂₂FN₆O₃[M+H]⁺425.1732,found 425.1727.

I-14: 4- ((5-fluoro-2- ((4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide

¹H NMR(400MHz,DMSO-d₆)δ11.15(s,1H),9.50(s,1H),9.04(s,2H),8.10(d,J＝3.7Hz,1H),7.93(d,J＝8.7Hz,2H),7.71(d,J＝8.8Hz,2H),7.48(d,J＝9.0Hz,2H),6.87(d,J＝9.1Hz,2H),3.10–3.02(m,4H),2.48–2.42(m,4H),2.22(s,3H).¹³C NMR(101MHz,DMSO-d₆)δ164.42,156.29(d,J＝2.7Hz),149.65(d,J＝10.8Hz),146.62,142.41,141.51(d,J＝18.0Hz),140.77(d,J＝245Hz),133.33,127.86,126.87,121.01,120.15,116.30,55.19,49.43,46.27.HRMS(ESI)m/z calcd for C₂₂H₂₅FN₇O₂[M+H]⁺438.2048,found 438.2044.

I-15: 4- ((5-fluoro-2- ((4- (piperidin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide

¹H NMR(400MHz,DMSO-d₆)δ11.14(s,1H),9.49(s,1H),9.02(s,1H),8.96(s,1H),8.10(d,J＝3.7Hz,1H),7.93(d,J＝8.7Hz,2H),7.70(d,J＝8.8Hz,2H),7.46(d,J＝9.0Hz,2H),6.86(d,J＝9.1Hz,2H),3.10–2.97(m,4H),1.66-1.61(m,4H),1.55–1.47(m,2H).¹³C NMR(101MHz,DMSO-d₆)δ164.51,156.34(d,J＝2.5Hz),149.64(d,J＝10.7Hz),147.55,142.43,141.54(d,J＝19.5Hz),140.76(d,J＝245Hz),133.13,127.82,126.87,121.07,120.15,116.96,51.10,25.93,24.35.HRMS(ESI)m/z calcd for C₂₂H₂₄FN₆O₂[M+H]⁺423.1939,found 423.1934.

I-16: 4- ((2- ((4- (4-ethylpiperazin-1-yl) phenyl) amino) -5-fluoropyrimidin-4-yl) amino) -N-hydroxybenzamide

¹H NMR(400MHz,DMSO-d₆)δ11.14(s,1H),9.49(s,1H),9.03(s,1H),8.96(s,1H),8.10(d,J＝3.7Hz,1H),7.93(d,J＝8.7Hz,2H),7.70(d,J＝8.8Hz,2H),7.48(d,J＝9.0Hz,2H),6.87(d,J＝9.1Hz,2H),3.13–2.99(m,4H),2.40-2.34(m,2H),1.04(t,J＝7.2Hz,3H).¹³C NMR(101MHz,DMSO-d₆)δ164.47,156.30(d,J＝2.8Hz),149.65(d,J＝10.5Hz),146.66,142.42,141.52(d,J＝20.1Hz),140.78(d,J＝245Hz),133.33,127.82,126.88,121.05,120.17,116.29,52.87,52.11,49.50,12.42.HRMS(ESI)m/z calcd for C₂₃H₂₇FN₇O₂[M+H]⁺452.2205,found 452.2199.

I-17: 4- ((5-fluoro-2- ((3-fluoro-4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide

¹H NMR(400MHz,DMSO-d₆)δ11.16(s,1H),9.59(s,1H),9.32(s,1H),8.98(s,1H),8.17(d,J＝3.6Hz,1H),7.91(d,J＝8.7Hz,2H),7.73(d,J＝8.8Hz,2H),7.67(dd,J＝15.5,2.3Hz,1H),7.26(dd,J＝8.8,1.8Hz,1H),6.94(t,J＝9.6Hz,1H),2.94(s,4H),2.42(s,4H),2.23(s,3H).¹³C NMR(101MHz,DMSO-d₆)δ164.42,155.69(d,J＝2.9Hz),155.20(d,J＝241Hz),149.78(d,J＝10.8Hz),142.19,141.41(d,J＝19.6Hz),141.09(d,J＝246Hz),136.57(d,J＝11.0Hz),134.01(d,J＝9.3Hz),127.88,127.18,120.46,119.57(d,J＝4.3Hz),115.13(d,J＝1.6Hz),107.36(d,J＝26Hz),55.21,50.86(d,J＝2.1Hz,2C),46.21.HRMS(ESI)m/z calcd for C₂₂H₂₄F₂N₇O₂[M+H]⁺456.1954,found 456.1948.

Experimental example: test of BTK and FLT3 inhibitory activity of compound and test experiment of antiproliferative activity of tumor cell line

1) Compound activity assay for BTK, FLT3 kinase inhibition:

experimental materials and instruments: this experiment was performed with the aid of Eurofins Pharma, british.

The experimental method comprises the following steps: all compounds tested were formulated in DMSO as 50-fold final assay concentration working solution. Compound working solution was first added as a first component to the test wells, followed by addition of kinase buffer diluted BTK or FLT3 kinase solution. The addition of Mg/ATP initiates the kinase reaction. Subsequently, the reaction was incubated at room temperature for 40 minutes, and a 0.5% phosphoric acid solution was added to terminate the reaction. 10 μ L of the reaction was spotted onto a pad of P30 filter paper, washed 4 times with 0.425% phosphoric acid for 4 minutes each, then washed once with methanol, followed by drying and scintillation counting.

The test was set up with a compound test group (C), a positive control group (P) and a blank control group (B). The positive control group contained no test compound, DMSO was used instead (final concentration 2%), and the other components were identical to the test group (residual kinase activity 100%); staurosporine (staurosporine) was used in place of test compound in the blank control group to eliminate kinase activity and establish a baseline (residual kinase activity 0%).

IC was calculated by fitting a curve using Gragopd prism6.0 software with the logarithm of concentration as the abscissa and the inhibition ratio as the center₅₀The value is obtained. The test results of the target compound on the BTK and FLT3 kinase inhibition activity are shown in the table 1.

TABLE 1 inhibitory Activity of the Compounds of interest on BTK and FLT3 kinase

A: the inhibition rate is more than 60%; b, 60% > inhibition rate is more than 40%; c, the inhibition rate is less than 40 percent

Table 1 shows that most compounds have stronger inhibitory activity to BTK and FLT3 (the inhibition rate of kinase at 1 mu M concentration)>60%). Compounds I-1, I-2, I-4, I-5, I-13, I-16, I-17 showed potent dual inhibitory effects on BTK and FLT3, representative compounds were IC for BTK and FLT3₅₀At low nanomolar level (IC)₅₀<20nM)。

2) Growth inhibitory activity of compounds on tumor cells:

experimental materials and instruments: jeko-1, MV4-11 and MCF-7 cell strains, RPMI-1640 culture medium, fetal bovine serum, PBS buffer solution, penicillin sodium (10000units/mL) -streptomycin sulfate (10mg/mL), CCK-8 kit, an inverted optical microscope, a cell culture box, a super clean bench, a bench centrifuge, a microplate reader and an ultra-low temperature refrigerator.

The experimental method comprises the following steps:

inoculating the tumor cells in logarithmic growth phase in 96-well culture plate with the number of cells being 1 × 10⁴Adding cell culture solutions of the compounds to be detected with different concentrations into each well, simultaneously establishing a positive control group and a DMSO blank control group, and adjusting the DMSO concentration to be less than or equal to 1 per thousand. Each concentration is provided with 3 compound holes, after the addition is finished, the mixture is placed at 37 ℃ and 5 percent CO₂Incubate in the incubator for 72 h. Then 20. mu.L of CCK-8 solution was added to each well and the plates were placed at 37 ℃ in 5% CO₂Continuously incubating for 1-4h in a constant temperature incubator, measuring absorbance value of the sample at 450nm wavelength by using an enzyme-labeling instrument, normalizing the obtained value and a negative DMSO control group, and calculating IC by using Prism6.0 software₅₀The value is obtained.

TABLE 2 inhibition of Jeko-1, MV4-11 and MCF-7 cell growth by compounds of interest

IC₅₀: half maximal inhibitory concentration

A：IC₅₀<2μM；B:2μM<IC₅₀<20μM；C:20μM<IC₅₀ND-not tested

The experimental data in Table 2 show that most of the compounds have antiproliferative activity on Jeko-1 and MV4-11 cells comparable to or even superior to that of the positive control drug Sorafenib. The half inhibition concentration of the compounds I-1 to I-5, I-7 to I-11 and I-13 to I-17 on two cell lines is in low micromolar level (less than 2 mu M), and the IC of a representative compound on a plurality of malignant tumor cells in a blood system₅₀At low nanomolar level (IC)₅₀<100nM) and the antiproliferative activity is 12-1000 times better than that of the marketed drug sorafenib. Compound I-2 showed potent growth inhibition on MCF-7 cells.

The above description is only a preferred embodiment of the present disclosure and is not intended to limit the present disclosure, and various modifications and changes may be made to the present disclosure by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present disclosure should be included in the protection scope of the present disclosure.

Claims

1. A compound with a structure shown in a general formula I or a pharmaceutically acceptable salt thereof is characterized in that the compound is a (5-fluoro-2-anilinopyrimidin-4-yl) amino-N-hydroxybenzamide derivative,

2. A compound of claim 1, wherein R is₁Selected from morpholinyl, piperidinyl, N-methylpiperidylAn oxazinyl or N-ethylpiperazinyl group; r₂Selected from hydrogen, fluorine or methoxy.

3. The compound of claim 1, wherein the compound is selected from any one of the following compounds:

2- ((5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide;

2- ((5-fluoro-2- ((4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide;

2- ((5-fluoro-2- ((4- (piperidin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide;

2- ((2- ((4- (4-ethylpiperazin-1-yl) phenyl) amino) -5-fluoropyrimidin-4-yl) amino) -N-hydroxybenzamide;

2- ((5-fluoro-2- ((3-fluoro-4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide;

2- ((5-fluoro-2- ((2-methoxy-4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide;

3- ((5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide;

3- ((5-fluoro-2- ((4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide;

3- ((5-fluoro-2- ((4- (piperidin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide;

3- ((2- ((4- (4-ethylpiperazin-1-yl) phenyl) amino) -5-fluoropyrimidin-4-yl) amino) -N-hydroxybenzamide;

3- ((5-fluoro-2- ((3-fluoro-4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide;

3- ((5-fluoro-2- ((2-methoxy-4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide;

4- ((5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide;

4- ((5-fluoro-2- ((4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide;

4- ((5-fluoro-2- ((4- (piperidin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide;

4- ((2- ((4- (4-ethylpiperazin-1-yl) phenyl) amino) -5-fluoropyrimidin-4-yl) amino) -N-hydroxybenzamide;

4- ((5-fluoro-2- ((3-fluoro-4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzamide.

4. A preparation method of a compound with a structure shown in a general formula I, wherein the reaction route of the preparation method is shown as the following formula:

wherein R is₁Selected from morpholinyl, piperidinyl, C_1～6One of alkyl-substituted piperazinyl and C1-6 straight-chain or branched alkyl; r₂One selected from hydrogen, halogen, methoxy, trifluoromethyl, cyano and C1-6 straight chain or branched chain alkyl;

the preparation method comprises the following steps:

s1, dissolving a compound 1 and a methyl aminobenzoate compound in isopropanol, adding DIPEA, reacting at 85 ℃, cooling to room temperature after TLC detection reaction is completed, filtering, and recrystallizing a filter cake with ethyl acetate to obtain an intermediate 2;

s2, dissolving the intermediate 2 in n-butanol, adding substituted aniline, dropwise adding trifluoroacetic acid into the solution, reacting at 110 ℃, detecting by TLC (thin layer chromatography) to complete the reaction, cooling to room temperature, evaporating under reduced pressure to remove the solvent, and carrying out silica gel column chromatography to obtain an intermediate 3;

s3, dissolving the intermediate 3 in NH₂And (2) reacting in OK solution at 0-30 ℃, performing TLC detection, performing complete reaction, performing reduced pressure evaporation to remove the solvent, adding water, adjusting the pH value to 6-7 by using dilute hydrochloric acid, filtering, and recrystallizing a filter cake by using methanol or ethyl acetate to obtain a compound with a structure shown in a general formula I, namely the (5-fluoro-2-anilinopyrimidine-4-yl) amino-N-hydroxybenzamide derivative.

5. The method according to claim 4, wherein the reaction time in step S1 is 4 hours.

6. The method according to claim 4, wherein the reaction time in step S2 is 12 hours.

7. The method of claim 4, wherein the NH is₂The preparation method of the OK solution comprises the following steps: adding KOH and NH₂Respectively dissolving OH and HCI in anhydrous methanol to obtain potassium hydroxide methanol solution and hydroxylamine hydrochloride methanol solution, dropwise adding the potassium hydroxide methanol solution into the hydroxylamine hydrochloride methanol solution under ice bath condition, separating out white solid, continuing to react for 1h, filtering and precipitating to obtain NH₂OK solution.

8. Use of a compound according to any one of claims 1 to 3 or obtained by a process according to any one of claims 4 to 7 in the manufacture of a medicament for use as a BTK and/or FLT3 inhibitor.

9. Use of the compound according to any one of claims 1 to 3 or the compound obtained by the preparation method according to any one of claims 4 to 7 in preparation of an antitumor drug, wherein the tumor is one of mantle cell lymphoma, acute myeloid leukemia and breast cancer.

10. A pharmaceutical composition comprising a compound according to any one of claims 1 to 3 or a compound obtainable by a process according to any one of claims 4 to 7.