CN114181161B - (2- ((substituted oxy) phenyl) amino) pyrimidin-4-yl) aminobenzoyl derivative and preparation method and application thereof - Google Patents

(2- ((substituted oxy) phenyl) amino) pyrimidin-4-yl) aminobenzoyl derivative and preparation method and application thereof Download PDF

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CN114181161B
CN114181161B CN202111628318.0A CN202111628318A CN114181161B CN 114181161 B CN114181161 B CN 114181161B CN 202111628318 A CN202111628318 A CN 202111628318A CN 114181161 B CN114181161 B CN 114181161B
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amino
pyrimidin
phenyl
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fluoro
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CN114181161A (en
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冉凡胜
凌勇
刘云
谢旭东
陶维志
何遥
陈莹
郭李婷
胡义荣
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Nantong University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/48—Two nitrogen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia

Abstract

The invention belongs to the technical field of synthesis and medical application of organic compounds, and discloses a (2- ((substituted oxygen) phenyl) amino) pyrimidine-4-yl) aminobenzoyl derivative, and a preparation method and application thereof. The structure of the (2- ((substituted oxy) phenyl) amino) pyrimidine-4-yl) aminobenzoyl derivative is shown as a general formula I, IIWherein R is 1 Selected from C 1‑6 Linear or branched alkyl ester groups; r is R 2 Selected from C containing or not containing halogen/nitrogen/oxygen/sulfur atoms 1‑6 Linear or branched alkyl, aryl; r is R 3 Selected from hydrogen, cyano, halogen, trifluoromethyl, methyl, methoxy; r is R 4 Is derived from hydroxylamine acyl. The compound has certain BTK/FLT3 double inhibition activity and antitumor activity.

Description

(2- ((substituted oxy) phenyl) amino) pyrimidin-4-yl) aminobenzoyl derivative and preparation method and application thereof
Technical Field
The invention belongs to the technical field of synthesis and medical application of organic compounds, and particularly relates to a (2- ((substituted oxygen) phenyl) amino) pyrimidine-4-yl) aminobenzoyl derivative, and a preparation method and application thereof.
Background
Bruton's tyrosine kinase, BTK) is a key node of the B Cell Receptor (BCR) and Fc receptor (FcR) signaling pathways. BCR and FcR are expressed on the surface of B cells and myeloid cells (macrophages, monocytes, mast cells, etc.), respectively. BTK regulates B cell and myeloid cell survival and biological function through BCR and FcR signaling pathways, and is an important target for the treatment of various diseases involving B cell and/or macrophage abnormalities (see: drug discovery today 2014;19 (8): 1200-1204 and Mol cancer.201)8, 8;17 (1):57). FMS-like tyrosine kinase 3 (FMS-like tyrosine kinase, FLT 3) is a III receptor tyrosine kinase, expressed predominantly in CD34 + The surface of hematopoietic stem cells and immature hematopoietic progenitor cells plays an important role in the development of hematopoietic stem cells and DC progenitor cells (see: physiological reviews 2019;99 (3): 1433-1466). FLT3 is an important target for the treatment of hematological malignancies and autoimmune diseases. Preclinical and clinical studies show that the dual inhibition of BTK and FLT3 has a synergistic effect (see: blood 2019;134 (supplement_1): 5477-5477), so the design and synthesis of novel BTK/FLT3 double-target drugs with novel structure and drug-forming property have important significance for treating malignant tumors of Blood systems and autoimmune diseases.
Disclosure of Invention
In view of the above, the present invention aims to provide a (2- ((substituted oxy) phenyl) amino) pyrimidin-4-yl) aminobenzoyl derivative, a preparation method and application thereof, which have dual BTK/FLT3 inhibitory activity and have anti-tumor effect. The compound has excellent biological activity, and simultaneously improves the molecular diversity and novelty of the compound.
In order to achieve the above object, the present disclosure discloses the following technical solutions:
in a first aspect of the invention, the invention provides a compound which is a (2- ((substituted oxy) phenyl) amino) pyrimidin-4-yl) aminobenzoyl derivative having the structure shown in formulas I and II:
wherein R is 1 Selected from C 1-6 Linear or branched alkyl ester groups; r is R 2 Selected from C containing or not containing halogen/nitrogen/oxygen/sulfur atoms 1-6 Linear or branched alkyl, aryl; r is R 3 Selected from hydrogen, cyano, halogen, trifluoromethyl, methyl, methoxy; r is R 4 Is derived from hydroxylamine acyl.
Preferably, said R 1 、R 4 The substitution position of (C-2),C-3 and C-4;
preferably, R 1 Selected from-COOMe; r is R 2 Selected from methoxyethyl and phenyl; r is R 3 Selected from fluorine; r is R 4 Is taken from-CONHOH;
preferably, among the above-mentioned compounds,
the compound having the structure shown in the general formula I is selected from any one of the following compounds:
methyl 2- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) benzoate (I-1)
Methyl 2- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) benzoate (I-2)
3- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) benzoic acid methyl ester (I-3)
3- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) benzoic acid methyl ester (I-4)
Methyl 4- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) benzoate (I-5)
Methyl 4- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) benzoate (I-6)
The compound having the structure shown in the general formula II is selected from any one of the following compounds:
2- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzoamide (II-1)
2- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzoamide (II-2)
3- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzoamide (II-3)
3- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzoamide (II-4)
4- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzoamide (II-5)
4- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzoamide (II-6)
The brackets after the names of the above compounds are given their corresponding codes, and the codes in the brackets will be used directly in the following of the present specification for convenience of description and simplicity of expression.
The compounds of the present invention may be present in free form or further in salt form in order to increase water solubility and bioavailability.
The term "pharmaceutically acceptable salt" as used herein refers to conventional non-toxic salts, and includes salts formed with basic amino groups of the compounds of the present application. These salts are well known to those skilled in the art and any pharmaceutically acceptable salt provided by the skilled artisan can be prepared. In addition, the skilled artisan may discard one salt from another based on factors such as solubility, stability, ease of formulation, etc. Determination and optimization of these salts is within the experience of the skilled artisan.
The invention also provides a preparation method of the structural compound shown in the general formula I, II, which comprises the following reaction route:
wherein R is 1 Selected from C 1-6 Linear or branched alkyl ester groups; r is R 2 Selected from C containing or not containing halogen/nitrogen/oxygen/sulfur atoms 1-6 Linear or branched alkyl, aryl; r is R 3 Selected from hydrogen, cyano, halogen, trifluoromethyl, methyl, methoxy; r is R 4 Is derived from hydroxylamine acyl.
Reagents and conditions: (a) Different methyl aminobenzoate, N, N-Diisopropylethylamine (DIPEA), isopropanol, 85℃for 4h; (b) Substituted aniline, trifluoroacetic acid, n-butanol, 110 ℃ for 12h; (c) Potassium hydroxide, hydroxylamine hydrochloride, anhydrous methanol, 0-r.t., 2h.
The method comprises the following specific steps:
(i) Starting compound 1 and methyl aminobenzoate were dissolved in isopropanol, DIPEA was added and reacted at 85 ℃ for 4 hours. TLC detection, complete reaction, cooling to room temperature, precipitation of a large amount of solid, filtration, and recrystallization of the filter cake with ethyl acetate, to obtain intermediate 2.
(ii) Dissolving the intermediate 2 in n-butanol, adding substituted aniline, and then dropwise adding trifluoroacetic acid into the solution for reaction for 12 hours at 110 ℃. TLC detection, complete reaction, cooling to room temperature, decompression evaporating solvent, silica gel column chromatography, and obtaining compound I, namely (2- ((substituted oxygen) phenyl) amino) pyrimidine-4-yl) aminobenzoyl derivative with the structure shown in the general formula I.
(iii) Preparation of NH by potassium hydroxide, hydroxylamine hydrochloride and anhydrous methanol 2 OK solution. Dissolving Compound I in NH 2 In OK solution, the reaction is carried out for 2h at room temperature. TLC detection, complete reaction, decompression evaporation of solvent, water addition, pH value adjustment to 6-7 with dilute hydrochloric acid, solid precipitation, filtering, re-crystallization of filter cake with methanol or ethyl acetate to obtain (2- ((substituted oxy) phenyl) amino) pyrimidine-4-yl) amino benzoyl derivative with structure shown in the general formula II.
The room temperature of the invention is 20-30 ℃.
The invention also provides a pharmaceutical composition containing the compound or pharmaceutically acceptable salt thereof.
The above pharmaceutical composition may be administered in any manner selected from the group consisting of: oral, spray 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 modes of administration being preferred.
The invention also provides a pharmaceutical preparation which comprises the compound or pharmaceutically acceptable salt thereof or a composition containing the compound or the pharmaceutically acceptable salt thereof and pharmaceutically acceptable auxiliary materials and/or carriers.
The compounds of the present invention or pharmaceutical compositions containing them may be administered in unit dosage form. The administration dosage form may be liquid dosage form or solid dosage form. The liquid dosage form can be true solution, colloid, microparticle, emulsion, and gyratory. 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 compositions or formulations of the present invention may also contain conventional carriers, including but not limited to: ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum proteins, buffer substances such as phosphates, glycerol, sorbitol, 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, polyvinylpyrrolidone, 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 from 1% to 98% by weight, typically about 80% by weight. For convenience, local anesthetics, preservatives, buffers, and the like may be directly dissolved in the carrier.
Oral tablets and capsules may contain excipients such as binding agents, for example syrup, acacia, sorbitol, tragacanth, or polyvinylpyrrolidone, fillers, for example lactose, sucrose, corn starch, calcium phosphate, sorbitol, glycine, lubricants, for example magnesium stearate, talc, polyethylene glycol, silica, disintegrants, for example potato starch, or acceptable wetting agents, for example sodium lauryl sulfate. The tablets may be coated by methods known in the pharmaceutical arts.
The oral liquid can be made into water and oil suspension, solution, emulsion, syrup, or dry product, and can be 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 (possibly containing edible oils) such as almond oil, fats and oils such as glycerin, ethylene glycol, or ethyl alcohol; preservatives, such as methyl or propyl parahydroxybenzoate, sorbic acid. Flavoring or coloring agents may be added as desired.
Suppositories may contain conventional suppository bases such as cocoa butter or other glycerides.
For parenteral administration, liquid dosage forms are typically made of the compound and a sterile carrier. The carrier is water. Depending on the carrier and drug concentration selected, the compound may be dissolved in either the carrier or in suspension, and when preparing an injectable solution, the compound is first dissolved in water, filtered and sterilized, and filled into sealed bottles or ampoules.
It must be appreciated that the optimal dosage and spacing of administration of the compounds of formula I, II is 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 optimal dosage may be determined by conventional techniques. It must also be appreciated that the optimal course of treatment, i.e., the simultaneous daily doses of I, II compound over the nominal time period, can be determined by methods well known in the art.
The invention also provides a pharmaceutical composition comprising the compound or pharmaceutically acceptable salt thereof.
The invention also provides a pharmaceutical agent which comprises an active ingredient and pharmaceutically acceptable auxiliary materials and/or carriers, wherein the active ingredient comprises the compound or pharmaceutically acceptable salts thereof.
The invention also provides an inhibitor which is one of a BTK inhibitor, a FLT3 inhibitor and a BTK/FLT3 dual inhibitor, and the inhibitor comprises the compound or pharmaceutically acceptable salt thereof as an active ingredient.
Preferably, the inhibitor is a dual BTK/FLT3 inhibitor comprising 2- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzoamide or a pharmaceutically acceptable salt thereof as an active ingredient.
The invention also provides application of the compound in preparing antitumor drugs, wherein the compound is the compound or pharmaceutically acceptable salt thereof
Further, the tumors are lymphomas and leukemias.
Compared with the prior art, the inventionThe invention provides a BTK/FLT3 double-target inhibitor with a brand new structure framework, and a representative compound has strong double-inhibition activity IC on BTK and FLT3 50 The values are at low nanomolar levels (IC 50 <200 nM) which has antiproliferative activity on hematological malignant cells comparable to the marketed drugs i Lu Dini and sorafenib. The invention provides a new scheme for the research and development of new antitumor drugs.
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.
Detailed Description
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions or under conditions recommended by the manufacturer. 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 methods and materials described herein are presented for illustrative purposes only.
Example 1: preparation of intermediate 2
The starting material 2, 4-dichloro-5-fluoropyrimidine (12 mmol,1.2 eq), methyl aminobenzoate (10 mmol,1.0 eq) and DIPEA (10 mmol,1.0 eq) were dissolved in 20mL isopropanol and reacted by heating at 85 ℃ for 4 hours. After the reaction, the reaction solution was cooled to room temperature, a large amount of solids were precipitated, filtered, and the cake was recrystallized from ethyl acetate to give intermediate 2.
Example 2: preparation of target Compound I
Intermediate 2 (1 mmol,1 eq) was dissolved in 30mL n-butanol, various substituted anilines (1.1 mmol,1.1 eq) were added, and 5 drops of trifluoroacetic acid were added dropwise to the solution and reacted for 12h with heating at 110 ℃. After the reaction, the reaction solution was cooled to room temperature, the solvent was distilled off under reduced pressure, and the target compound I was obtained by silica gel column chromatography (dichloromethane/methanol=200:1-20:1).
I-1:2- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) benzoic acid methyl ester
1 H NMR(400MHz,DMSO-d 6 )δ10.93(d,J＝2.3Hz,1H),9.22(s,1H),8.91(d,J＝8.5Hz,1H),8.20(d,J＝3.2Hz,1H),8.03(dd,J＝8.0,1.6Hz,1H),7.63(dt,J＝8.0,1.6Hz,1H),7.53(d,J＝9.0Hz,2H),7.16(dt,J＝8.0,1.2Hz,1H),6.92–6.86(m,2H),4.09–4.03(m,2H),3.90(s,3H),3.68–3.63(m,2H),3.32(s,3H). 13 C NMR(101MHz,DMSO-d 6 )δ168.76,156.23,154.01,149.33(d,J＝11.2Hz),142.43,141.70,141.56(d,J＝17Hz),141.21(d,J＝244Hz),134.84,134.21,131.33,122.30,121.64,120.97,115.59,114.72,70.98,67.51,58.64,53.08.HRMS(ESI)m/z calcd for C 21 H 22 FN 4 O 4 [M+H] + 413.1620,found 413.1621.
I-2:2- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) benzoic acid methyl ester
1 H NMR(400MHz,DMSO-d 6 )δ10.93(d,J＝2.2Hz,1H),9.43(s,1H),8.89(d,J＝8.5Hz,1H),8.24(d,J＝3.2Hz,1H),8.03(dd,J＝8.0,1.6Hz,1H),7.68(d,J＝9.0Hz,2H),7.65–7.58(m,1H),7.40–7.33(m,2H),7.21–7.13(m,1H),7.13–7.05(m,1H),7.03–6.94(m,4H),3.90(s,3H). 13 C NMR(101MHz,DMSO-d 6 )δ168.73,158.35,155.94(d,J＝2.7Hz),150.68,149.43(d,J＝9.7Hz),142.65,141.57,141.35,140.20,137.18,134.78,131.33,130.37,123.08,122.43,121.40,121.11,120.06,117.85,115.83,53.09.HRMS(ESI)m/z calcd for C 24 H 20 FN 4 O 3 [M+H] + 431.1514,found 431.1516.
I-3:3- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) benzoic acid methyl ester
1 H NMR(400MHz,DMSO-d 6 )δ9.54(s,1H),9.06(s,1H),8.21(t,J＝1.7Hz,1H),8.18(d,J＝8.3Hz,1H),8.11(d,J＝3.6Hz,1H),7.66(d,J＝7.8Hz,1H),7.54–7.42(m,3H),6.78(d,J＝9.0Hz,2H),4.02(dd,J＝5.4,3.8Hz,2H),3.83(s,3H),3.67–3.61(m,2H),3.31(s,3H). 13 C NMR(101MHz,DMSO-d 6 )δ166.65,156.18(d,J＝2.7Hz),153.62,149.90(d,J＝10.8Hz),141.61(d,J＝19.7Hz),140.74(d,J＝245Hz),139.85,134.41,130.41,129.39,126.25,123.97,122.39,121.09,114.54,70.96,67.41,58.62,52.59.HRMS(ESI)m/z calcd for C 21 H 22 FN 4 O 4 [M+H] + 413.1620,found 413.1620.
I-4:3- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) benzoic acid methyl ester
1 H NMR(400MHz,DMSO-d 6 )δ9.60(s,1H),9.29(s,1H),8.22(t,J＝1.8Hz,1H),8.18-8.14(t,J＝6.8Hz,2H),7.69–7.61(m,3H),7.47(t,J＝7.9Hz,1H),7.38–7.31(m,2H),7.08(t,J＝7.4Hz,1H),6.97–6.91(m,2H),6.88(d,J＝9.0Hz,2H),3.79(s,3H). 13 C NMR(101MHz,DMSO-d 6 )δ166.59,158.32,155.94(d,J＝2.9Hz),150.34,150.00(d,J＝10.9Hz),141.58(d,J＝19.6Hz),140.80(d,J＝245Hz),139.74,137.35,130.37(d,J＝6.9Hz),129.42,126.48,124.12,123.04,122.60,120.84,119.91,117.84,52.58.HRMS(ESI)m/z calcd for C 24 H 20 FN 4 O 3 [M+H] + 431.1514,found 431.1516.
I-5:4- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) benzoic acid methyl ester
1 H NMR(400MHz,DMSO-d 6 )δ9.67(s,1H),9.13(s,1H),8.15(d,J＝3.6Hz,1H),8.01(d,J＝8.8Hz,2H),7.88(d,J＝8.9Hz,2H),7.52(d,J＝9.0Hz,2H),6.87(d,J＝9.0Hz,2H),4.08–4.03(m,2H),3.84(s,3H),3.68–3.63(m,2H),3.32(s,3H). 13 C NMR(101MHz,DMSO-d 6 )δ166.39,155.99(d,J＝2.6Hz),154.07,149.66(d,J＝10.7Hz),144.22,141.29(d,J＝20.3Hz),140.82(d,J＝246Hz),134.06,130.30,123.63,121.88,120.26,114.72,70.94,67.49,58.62,52.36.HRMS(ESI)m/z calcd for C 21 H 22 FN 4 O 4 [M+H] + 413.1620,found 413.1621.
I-6:4- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) benzoic acid methyl ester
1 H NMR(400MHz,DMSO-d 6 )δ9.99(s,1H),9.59(s,1H),8.24(d,J＝4.0Hz,1H),7.98(d,J＝8.8Hz,2H),7.89(d,J＝8.8Hz,2H),7.61(d,J＝8.9Hz,2H),7.43–7.34(m,2H),7.11(t,J＝7.4Hz,1H),7.04–6.95(m,4H),3.83(s,3H). 13 C NMR(101MHz,DMSO-d 6 )δ166.25,157.88,154.14,152.12,150.84(d,J＝11.0Hz),143.24,140.48(d,J＝247Hz),137.37(d,J＝15.5Hz),135.43,130.46,130.25,124.69,123.43,123.14,121.19,119.85,118.22,52.45.HRMS(ESI)m/z calcd for C 24 H 20 FN 4 O 3 [M+H] + 431.1514,found 431.1516.
Example 3: preparation of target Compound II
KOH (28.55 g,509 mmol) and NH 2 OH HCI (23.84 g, 323 mmol) was dissolved in 70mL and 120mL dry methanol, respectively, to give solution A and solution B. Under ice bath condition, the solution A is dripped into the solution B, white solid is separated out, the reaction is continued for 1 hour, and the precipitate is filtered to obtain NH 2 OK solution. Compound I (0.50 mmol) was dissolved in 10mL NH 2 In the OK solution, stir for 2h at room temperature. After the reaction, the solvent was distilled off under reduced pressure, 20mL of water was added, the pH was adjusted to 6-7 with 1M HCl, solids were precipitated, filtered, and the cake was recrystallized from methanol/ethyl acetate to give the objective compound II.
II-1:2- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzoamide
1 H NMR(400MHz,DMSO-d 6 )δ11.56(s,1H),11.22(s,1H),9.37(s,1H),9.16(s,1H),8.77(d,J＝8.4Hz,1H),8.14(d,J＝3.3Hz,1H),7.62(d,J＝7.9Hz,1H),7.57–7.48(m,3H),7.11(t,J＝7.5Hz,1H),6.91–6.85(m,2H),4.07–4.04(m,2H),3.67–3.63(m,2H),3.31(s,3H). 13 C NMR(101MHz,DMSO-d 6 )δ166.43,156.24(d,J＝2.9Hz),153.89,149.34(d,J＝9.9Hz),141.20(d,J＝20.6Hz),141.17(d,J＝244Hz),139.75,134.33,132.24,128.03,122.24,121.63,121.48,121.27,118.87,114.69,70.98,67.49,58.64.HRMS(ESI)m/z calcd for C 20 H 21 FN 5 O 4 [M+H] + 414.1572,found 414.1568.
II-2:2- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzoamide
1 H NMR(400MHz,DMSO-d 6 )δ11.57(s,1H),11.25(s,1H),9.38(s,1H),9.37(s,1H),8.77(d,J＝8.3Hz,1H),8.18(d,J＝3.2Hz,1H),7.69(d,J＝9.0Hz,2H),7.63(d,J＝7.8Hz,1H),7.49(t,J＝7.9Hz,1H),7.39–7.34(m,2H),7.13–7.06(m,2H),7.01–6.94(m,4H). 13 C NMR(101MHz,DMSO-d 6 )δ166.37,158.38,155.99(d,J＝3.2Hz),150.58,149.43(d,J＝10.0Hz),141.40(d,J＝245Hz),141.10(d,J＝19.9Hz),139.64,137.31,132.18,130.36,128.06,123.05,122.37,121.38,121.30,120.07,119.06,117.82.HRMS(ESI)m/z calcd for C 23 H 19 FN 5 O 3 [M+H] + 432.1466,found 432.1464.
II-3:3- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzoamide
1 H NMR(400MHz,DMSO-d 6 )δ11.19(s,1H),9.45(s,1H),9.07(s,1H),9.03(s,1H),8.09(d,J＝3.6Hz,1H),7.98(s,1H),7.96(d,1H),7.50(d,J＝9.0Hz,2H),7.45–7.37(m,2H),6.80(d,J＝9.0Hz,2H),4.03(t,J＝4.4Hz,2H),3.62(t,J＝4.4Hz,2H),3.31(s,3H). 13 C NMR(101MHz,DMSO-d 6 )δ164.79,156.18(d,J＝2.7Hz),153.63,150.10(d,J＝10.8Hz),141.98,141.33(d,J＝19.2Hz),139.50(d,J＝8.2Hz),134.40,133.84,128.97,124.60,121.86,121.00,114.64,70.98,67.40,58.62.HRMS(ESI)m/z calcd for C 20 H 21 FN 5 O 4 [M+H] + 414.1572,found 414.1567.
II-4:3- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzoamide
1 H NMR(400MHz,DMSO-d 6 )δ11.19(s,1H),9.51(s,1H),9.26(s,1H),9.06(s,1H),8.13(d,J＝3.6Hz,1H),8.01(d,J＝7.5Hz,1H),7.97(s,1H),7.65(d,J＝8.8Hz,2H),7.46–7.29(m,4H),7.06(t,J＝7.4Hz,1H),6.94-6.89(m,4H). 13 C NMR(101MHz,DMSO-d 6 )δ164.67,158.44,155.98(d,J＝2.9Hz),150.19,150.18(d,J＝10Hz),141.33(d,J＝18.8Hz),141.01(d,J＝246Hz),139.44,137.47,133.85,130.33,128.97,124.74,122.94,121.91,121.01,120.79,120.15,117.69.HRMS(ESI)m/z calcd for C 23 H 19 FN 5 O 3 [M+H] + 432.1466,found 432.1465.
II-5:4- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzoamide
1 H NMR(400MHz,DMSO-d 6 )δ11.16(s,1H),9.52(s,1H),9.11(s,1H),8.98(s,1H),8.12(d,J＝3.7Hz,1H),7.92(d,J＝8.7Hz,2H),7.71(d,J＝8.8Hz,2H),7.53(d,J＝9.0Hz,2H),6.87(d,J＝9.1Hz,2H),4.08–4.02(m,2H),3.68–3.63(m,2H),3.31(s,3H). 13 C NMR(101MHz,DMSO-d 6 )δ164.39,156.22(d,J＝2.8Hz),153.80,149.69(d,J＝10.5Hz),142.35,141.56(d,J＝19.7Hz),140.85(d,J＝245Hz),134.41,127.80,127.00,121.32,120.31,114.69,70.97,67.48,58.63.HRMS(ESI)m/z calcd for C 20 H 21 FN 5 O 4 [M+H] + 414.1572,found 414.1569.
II-6:4- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzoamide
1 H NMR(400MHz,DMSO-d 6 )δ11.15(s,1H),9.57(s,1H),9.33(s,1H),8.98(s,1H),8.16(d,J＝3.6Hz,1H),7.93(d,J＝8.7Hz,2H),7.74–7.66(m,4H),7.39–7.34(m,2H),7.08(t,J＝7.4Hz,1H),7.00–6.93(m,4H). 13 C NMR(101MHz,DMSO-d 6 )δ164.35,158.36,155.99(d,J＝2.8Hz),150.46,149.77(d,J＝10.6Hz),142.23,141.48(d,J＝19.3Hz),141.07(d,J＝246Hz),137.37,130.38,127.80,127.15,123.03,121.16,120.42,120.08,117.77.HRMS(ESI)m/z calcd for C 23 H 19 FN 5 O 3 [M+H] + 432.1466,found 432.1465.
Experimental example: test for inhibitory Activity of Compounds against BTK and FLT3, test for antiproliferative Activity against tumor cells
1) Compound inhibition activity experiment on BTK and FLT3 kinase:
experimental materials and instruments: this experiment was carried out with the assistance of Eurofins Pharma, UK.
The experimental method comprises the following steps: all compounds tested were formulated using DMSO as working fluid at 50 times the final test concentration. Compound working fluid 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 stopped by incubating for 40 minutes at room temperature and adding a 0.5% phosphoric acid solution. mu.L of the reaction solution was spotted onto a P30 filter paper pad, washed 4 times with 0.425% phosphoric acid for 4 minutes each, then washed once with methanol, followed by drying and scintillation counting.
Compound test group (C), positive control group (P) and blank control group (B) were set in the experiment. No test compound was added to the positive control group, 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 the test compound in the placebo group to eliminate kinase activity and establish a baseline (residual kinase activity 0%).
Using Gragphadprism 6.0 software, using the logarithm of concentration as the abscissa, the inhibition rate as the middle coordinate, fitting a curve, calculating IC 50 Values. The test results of the target compounds on the inhibition activity of BTK and FLT3 kinase are shown in table 1.
TABLE 1 target compounds inhibit BTK and FLT3 kinase Activity
A: the inhibition rate is more than 60 percent; b60% > inhibition >40%; the inhibition rate is less than 40%
The experimental data in Table 1 show that most compounds have strong inhibitory activity against BTK and FLT3 (kinase inhibition >60% at 1. Mu.M concentration). Wherein, the compound II-1 shows strong dual inhibition.
2) Growth inhibitory Activity of Compounds on tumor cells experiments:
experimental materials and instruments: jeko-1 and RAW264.7 cell lines, RPMI-1640 medium, fetal bovine serum, PBS buffer, penicillin sodium (10000 units/mL) -streptomycin sulfate (10 mg/mL), CCK-8 kit, inverted light microscope, cell incubator, ultra clean bench, bench centrifuge, enzyme label machine, ultra low temperature refrigerator.
The experimental method comprises the following steps:
inoculating tumor cells in logarithmic growth phase into 96-well culture plate with cell number of 1×10 4 And (3) adding cell culture solutions of the compounds with different concentrations into the wells, setting a positive control group and a DMSO blank control group at the same time, and adjusting the DMSO concentration to be less than or equal to 1 per mill. 3 compound holes are arranged at each concentration, the mixture is placed at 37 ℃ and 5 percent of CO 2 Incubate in a constant temperature incubator for 72h. Subsequently, 20. Mu.L of CCK-8 solution was added to each well and the plates were placed at 37℃with 5% CO 2 Incubating in a constant temperature incubator for 1-4h, measuring absorbance value at 450nm with enzyme-labeled instrument, normalizing the obtained value with negative DMSO control group, and calculating IC with Prism6.0 software 50 Value of
TABLE 2 inhibition of Jeko-1 and RAW264.7 cell growth by target Compounds
IC 50 : half inhibition concentration
A：IC 50 <2μM；B:2μM<IC 50 <20μM；C:20μM<IC 50
Table 2 shows that most of the compounds have significant antiproliferative activity on Jeko-1 cells, IC 50 The values were comparable to or even lower than the positive control, ibrutinib, most compounds having moderate antiproliferative activity on RAW264.7 cells.
The foregoing description of the preferred embodiments of the present disclosure is provided only and not intended to limit the disclosure so that various modifications and changes may be made to the present disclosure by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present disclosure should be included in the protection scope of the present disclosure.

Claims (9)

1. A compound which is a (2- ((substituted oxy) phenyl) amino) pyrimidin-4-yl) aminobenzoyl derivative of the structure shown in formula I or formula II:
，
wherein R is 1 is-COOMe; r is R 2 Selected from C containing or not containing halogen/nitrogen/oxygen/sulfur atoms 1-6 Linear or branched alkyl; or R is 2 Is phenyl; r is R 3 Selected from hydrogen, cyano, halogen, trifluoromethyl, methyl, methoxy; r is R 4 Is derived from hydroxylamine acyl.
2. The compound of claim 1, wherein R 1 、R 4 The substitution positions of (C) are C-2, C-3 and C-4; r is R 2 Selected from methoxyethyl and phenyl; r is R 3 Selected from fluorine; r is R 4 Is taken from-CONHOH.
3. A compound according to claim 2, wherein the compound of the structure of formula I is selected from any one of the following compounds:
methyl 2- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) benzoate;
methyl 2- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) benzoate;
3- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) benzoic acid methyl ester;
methyl 3- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) benzoate;
methyl 4- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) benzoate;
methyl 4- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) benzoate;
the compound of the structure shown in the general formula II is selected from any one of the following compounds:
2- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzoamide;
2- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzoamide;
3- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzoamide;
3- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzoamide;
4- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzoamide;
4- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzoamide.
4. The method for preparing the compound according to claim 1, wherein the compound 1 and the aminobenzoate are used as starting materials to prepare the (2- ((substituted oxy) phenyl) amino) pyrimidin-4-yl) aminobenzoyl derivatives of the structures shown in the general formula I and the general formula II through the following reaction schemes:
，
wherein R is 1 Selected from C 1-6 Linear or branched alkyl ester groups; r is R 2 Selected from C containing or not containing halogen/nitrogen/oxygen/sulfur atoms 1-6 Linear or branched alkyl; or R is 2 Is phenyl; r is R 3 Selected from hydrogen, cyano, halogen, trifluoromethyl, methyl, methoxy; r is R 4 Is taken from hydroxylamine acyl;
the preparation method comprises the following steps:
s1, dissolving a compound 1 and methyl aminobenzoate in isopropanol, adding DIPEA, reacting at 85 ℃, cooling to room temperature after TLC detection reaction is complete, precipitating a large amount of solids, 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 ℃, cooling to room temperature after TLC detection reaction is completed, evaporating the solvent under reduced pressure, and performing silica gel column chromatography to obtain the (2- ((substituted oxy) phenyl) amino) pyrimidine-4-yl) aminobenzoyl derivative with the structure shown in the general formula I;
s3, dissolving the 2,4, 5-trisubstituted pyrimidine derivative with the structure shown in the general formula I in NH2OK solution, reacting at room temperature, completely detecting by TLC, decompressing and evaporating the solvent, adding water, adjusting the pH value to 6-7 by using dilute hydrochloric acid, precipitating solid, filtering, and recrystallizing the filter cake by using methanol or ethyl acetate to obtain the (2- ((substituted oxy) phenyl) amino) pyrimidine-4-yl) aminobenzoyl derivative with the structure shown in the general formula II.
5. A pharmaceutical composition comprising a compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof.
6. A pharmaceutical formulation comprising an active ingredient and pharmaceutically acceptable excipients and/or carriers, the active ingredient comprising a compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof.
7. An inhibitor, characterized in that the inhibitor is one of a BTK inhibitor, a FLT3 inhibitor and a BTK/FLT3 dual inhibitor, which comprises the compound of any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof as an active ingredient.
8. The inhibitor according to claim 7, wherein the inhibitor is a BTK/FLT3 dual inhibitor comprising 2- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybenzoamide or a pharmaceutically acceptable salt thereof as an active ingredient.
9. An application of a compound in preparing an anti-tumor medicament, wherein the compound is a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-3, and the tumor is lymphoma or leukemia.
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