CN114031559A

CN114031559A - Aryl nitrogen-containing heterocycle modified 5-fluoro-pyrimidinediamine benzoate and preparation method and application thereof

Info

Publication number: CN114031559A
Application number: CN202111628291.5A
Authority: CN
Inventors: 冉凡胜; 刘云; 许中原; 孟迟; 陶维志; 姚俊辰; 刘星星; 胡义荣
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2021-12-28
Filing date: 2021-12-28
Publication date: 2022-02-11

Abstract

The invention belongs to the technical field of organic compound synthesis and medical application, and discloses aryl nitrogen-containing heterocycle modified 5-fluoro-pyrimidinediamine benzoate as well as a preparation method and application thereof. The structure of the aryl nitrogen-containing heterocycle modified 5-fluoro-pyrimidinediamine benzoate is shown as a general formula I

Wherein R is₁Is selected from C_1‑6Straight chain alkyl ester group or C_1‑6A branched alkyl ester group; r₂Selected from morpholinyl, piperidinyl, substituted piperazinyl, C_1～6A linear or branched alkyl group; r₃Selected from hydrogen, halogen, methoxy, trifluoromethyl, cyano, C_1～6Straight or branched chain alkyl. The compound has certain BTK/FLT3 dual inhibitory activity and also has anti-tumor activity.

Description

Aryl nitrogen-containing heterocycle modified 5-fluoro-pyrimidinediamine benzoate and preparation method and application thereof

Technical Field

The invention relates to the field of organic compound synthesis and medical application, in particular to aryl nitrogen heterocyclic ring modified 5-fluoro-pyrimidinediamine benzoate 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 aryl nitrogen heterocycle modified 5-fluoro-pyrimidinediamine benzoate as well as a preparation method and application thereof, and the compound has the double inhibition effect of BTK/FLT3 and has antitumor activity.

In order to achieve the purpose, the invention discloses the following technical scheme:

the invention provides a compound or pharmaceutically acceptable salt thereof, wherein the compound is aryl nitrogen heterocyclic ring modified 5-fluoro-pyrimidinediamine benzoate, and has a structure shown in a general formula I:

wherein R is₁Is represented by C_1-6Straight chain alkyl ester group or C_1-6A branched alkyl ester group; r₂Represents morpholinyl, piperidinyl, substituted piperazinyl, C_1～6A linear or branched alkyl group; r₃Represents hydrogen, halogen, methoxy, trifluoromethyl, cyano, C_1～6A linear or branched alkyl group;

preferably, R₁represents-COOMe; r₂Represents morpholinyl, piperidinyl, N-methylpiperazinyl or N-ethylpiperazinyl; r₃Represents hydrogen, fluorine, methoxy;

preferably, the compound is selected from any one of the following compounds:

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

Methyl 2- ((5-fluoro-2- ((4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) benzoate (I-2)

Methyl 2- ((5-fluoro-2- ((4- (piperidin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) benzoate (I-3)

Methyl 2- ((2- ((4- (4-ethylpiperazin-1-yl) phenyl) amino) -5-fluoropyrimidin-4-yl) amino) benzoate (I-4)

Methyl 2- ((5-fluoro-2- ((3-fluoro-4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) benzoate (I-5)

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

Methyl 3- ((5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) benzoate (I-7)

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

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

Methyl 3- ((2- ((4- (4-ethylpiperazin-1-yl) phenyl) amino) -5-fluoropyrimidin-4-yl) amino) benzoate (I-10)

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

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

Methyl 4- ((5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) benzoate (I-13)

Methyl 4- ((5-fluoro-2- ((4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) benzoate (I-14)

Methyl 4- ((5-fluoro-2- ((4- (piperidin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) benzoate (I-15)

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

Methyl 4- ((5-fluoro-2- ((3-fluoro-4- (4-methylpiperazin-1-yl) phenyl) amino) pyrimidin-4-yl) amino) benzoate (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 preparation method of the compound, which comprises the following reaction routes:

wherein R is₁Is represented by C_1-6Straight chain alkyl ester group or C_1-6A branched alkyl ester group; r₂Represents morpholinyl, piperidinyl, substituted piperazinyl, C_1～6A linear or branched alkyl group; r₃Represents hydrogen, halogen, methoxy, trifluoromethyl, cyano, C_1～6Straight or branched chain alkyl.

Reagents and conditions: (a) methyl aminobenzoate compound, N, N-Diisopropylethylamine (DIPEA), isopropanol, 85 ℃ and 4 h; (b) aniline compound, trifluoroacetic acid, n-butanol, 110 ℃ and 12 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, precipitation of a large amount of solid, filtration, and recrystallization of 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. And (3) TLC detection, completely reacting, cooling to room temperature, evaporating under reduced pressure to remove the solvent, and performing silica gel column chromatography to obtain a compound I, namely the 5-fluoro-pyrimidinediamine benzoate of which the structure shown in the general formula I is based on aryl nitrogen-containing heterocyclic ring modification.

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

The pharmaceutical composition can be administered in any mode selected from the group consisting of: 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.

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 invention also provides a pharmaceutical preparation, which comprises an effective component and a pharmaceutically acceptable auxiliary material and/or a carrier, wherein the effective component comprises the compound or the pharmaceutically acceptable salt thereof, or comprises the pharmaceutical composition.

The pharmaceutical composition 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 an inhibitor which is a BTK and/or FLT3 inhibitor and contains the compound or the pharmaceutically acceptable salt thereof as an active ingredient.

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.

Compared with the prior art, the invention provides the aryl nitrogen heterocyclic ring modified 5-fluoro-pyrimidinediamine benzoate and the preparation method thereof, different from the prior art that the compounds have double inhibitory activities of BTK and FLT3, and most of the compounds have strong antiproliferative activity IC on malignant tumor cells (Jeko-1 and MV-4-11)₅₀The value is in a low micromolar level, and the antitumor activity of part of compounds is obviously superior to that of medicaments Ibrutinib and Sorafenib on the market. The compound provided by the invention can be used for developing a novel anti-tumor drug.

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.

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 compound (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 Compound I

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 200:1-20:1) was performed to obtain compound I. The method comprises the following specific steps:

i-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,found424.1781.

I-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 calcdfor C₂₃H₂₆FN₆O₂[M+H]⁺437.2096,found437.2095.

I-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,found422.1986.

I-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.

I-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/zcalcd for C₂₃H₂₅F₂N₆O₂[M+H]⁺455.2002,found455.2001.

I-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.

I-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.

I-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.

I-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.

I-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.

I-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.

I-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.

I-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.

I-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.

I-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.

I-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.

I-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/zcalcd for C₂₃H₂₅F₂N₆O₂[M+H]⁺455.2002,found455.2000.

Experimental example: test of BTK and FLT3 inhibitory Activity and anti-proliferative Activity of Compound I against tumor cell lines

1) Compound I 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 strong inhibitory activity to BTK and FLT3 (the inhibition rate of kinase is more than 60% at 1 muM concentration). Compounds I-2, I-5, I-14, I-16 and I-17 showed potent dual inhibitory effects on BTK and FLT 3.

2) Growth inhibitory activity of compound I on tumor cells assay:

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.

Cell survival% (% OD administration-OD blank)/(OD positive control-OD blank) × 100%.

TABLE 2 inhibitory Effect of the Compounds on Jeko-1 and MV4-11 cell growth

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 compounds have antiproliferative activity on Jeko-1 and MV4-11 cells comparable to or even superior to that of the positive control ibrutinib. The half inhibitory concentrations of the compounds I-2, I-4, I-5, I-7, I-8, I-11, I-16 and I-17 on both cell lines were all at low micromolar (less than 2. mu.M). Compound I-8 showed potent growth inhibition of 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 5-fluoro-pyrimidinediamine benzoate modified on the basis of an aryl nitrogen-containing heterocycle,

wherein R is₁Is represented by C_1-6Straight chain alkyl ester group or C_1-6A branched alkyl ester group; r₂Represents morpholinyl, piperidinyl, substituted piperazinyl, C_1-6Straight chain alkyl or C_1-6A branched alkyl group; r₃Selected from hydrogen, halogen, methoxy, trifluoromethyl, cyano, C_1-6Straight chain alkyl or C_1-6A branched alkyl group.

2. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R is₁represents-COOMe; r₂Represents morpholinyl, piperidinyl, N-methylpiperazinyl or N-ethylpiperazinyl; r₃Selected from hydrogen, fluorine or methoxy.

3. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is selected from any of the following compounds:

methyl 2- ((5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) benzoate;

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

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

methyl 2- ((2- ((4- (4-ethylpiperazin-1-yl) phenyl) amino) -5-fluoropyrimidin-4-yl) amino) benzoate;

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

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

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

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

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

methyl 3- ((2- ((4- (4-ethylpiperazin-1-yl) phenyl) amino) -5-fluoropyrimidin-4-yl) amino) benzoate;

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

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

methyl 4- ((5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) benzoate;

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

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

methyl 4- ((2- ((4- (4-ethylpiperazin-1-yl) phenyl) amino) -5-fluoropyrimidin-4-yl) amino) benzoate;

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

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 is₁Is selected from C_1-6Straight chain alkyl ester group or C_1-6A branched alkyl ester group; r₂Selected from morpholinyl, piperidinyl, substituted piperazinyl, C_1～6A linear or branched alkyl group; r₃Selected from hydrogen, halogen, methoxy, trifluoromethyl, cyano, C_1～6A linear or branched alkyl group;

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-butyl alcohol, adding an aniline compound, 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 a compound I, wherein the compound I is the aryl nitrogen-containing heterocycle modified 5-fluoro-pyrimidine diamine benzoate.

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. A pharmaceutical composition comprising a compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof.

8. An inhibitor, wherein the inhibitor is one of a BTK inhibitor, a FLT3 inhibitor and a BTK/FLT3 dual inhibitor, and the inhibitor comprises the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof as an active ingredient.

9. Use of a compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a malignant tumour which is mantle cell lymphoma or acute myeloid leukaemia.

10. Use of a compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a malignant tumour, which is breast cancer.