CN115141150A - 2,4,5-trisubstituted pyrimidine hydroxylamine acyl derivative and preparation method and application thereof - Google Patents
2,4,5-trisubstituted pyrimidine hydroxylamine acyl derivative and preparation method and application thereof Download PDFInfo
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- 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
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Abstract
The invention belongs to the field of organic compound synthesis and medical application, and relates to a2,4,5-trisubstituted pyrimidine hydroxylamine acyl derivative, and a preparation method and application thereof. The 2,4,5-trisubstituted pyrimidine hydroxylamine acyl derivative provided by the invention has the structure shown as the general formula I
Wherein n is 1,2,3,4,5,6,7; r 1 One selected from hydrogen, halogen, trifluoromethyl and methyl. The 2,4,5-trisubstituted pyrimidine hydroxylamine acyl derivative has certain HDAC and/or FLT3 dual-inhibition activity, and can be applied to preparation of anti-cancer drugs.
Description
Technical Field
The invention belongs to the field of organic compound synthesis and medical application, and relates to a2,4,5-trisubstituted pyrimidine hydroxylamine acyl derivative, a preparation method and application thereof, in particular to a (2- ((4-phenoxyphenyl) amino) pyrimidine-4-yl) amino acid derivative, a preparation method and application thereof.
Background
In the body, the enzyme responsible for regulating histone deacetylation is Histone Deacetylase (HDAC). Under normal conditions, this enzyme is in a state of dynamic equilibrium, maintaining normal expression and transcription of genes, and HDAC is abnormally expressed in various malignancies, and thus, HDAC is currently considered to be an effective target for the treatment of hematologic malignancies (Blood 2016 (18): 2168-2170 and Blood 201122 (21): 5142. FMS-like tyrosine kinase 3 (FMS-like receptor tyrosine kinase, FLT 3) is a transmembrane protein, is uniformly expressed on leukemia primitive cells of Acute Myelogenous Leukemia (AML), acts on hematopoietic stem/histiocyte regions (see Blood 2018 132 ((Supplement _ 1): 903 and Blood 2020 (136 (21): 2442-2456), and is an important target for treating hematological malignancies and autoimmune diseases). Preclinical and clinical studies show that double inhibition of HDAC and FLT3 has a coordinating effect (see Blood 2019 (Supplement _ 1): 1268), so that a novel HDAC/FLT3 double-target drug with a novel structure and drug-forming property is designed and synthesized, and has a very important significance for treating hematological malignancies.
Disclosure of Invention
The invention aims to provide 2,4,5-trisubstituted pyrimidine hydroxylamine acyl derivatives, a preparation method and application thereof, the compounds have HDAC/FLT3 dual inhibition activity and 2,4,5-trisubstituted pyrimidine hydroxylamine acyl derivatives with anti-tumor effect, and the compounds have excellent biological activity.
In order to achieve the purpose, the technical scheme of the disclosure is as follows:
in a first aspect of the invention, the invention provides a2,4,5-trisubstituted pyrimidine hydroxylamine derivative or a pharmaceutically acceptable salt thereof, having a structure represented by formula I:
wherein n is 1,2,3,4,5,6,7; r is 1 One selected from hydrogen, halogen, trifluoromethyl and methyl.
Preferably, n is 2,3,4,5,6; r is 1 Selected from fluorine.
3- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxypropionamide (A14) (I-1)
4- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybutyramide (A18) (I-2)
5- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxypentanamide (A29) (I-3)
6- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxyhexanamide (A22) (I-4)
7- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxyheptanamide (A33) (I-5)
The corresponding reference numbers are shown in parentheses after the names of the above 5 compounds, and for the convenience of description and the simplicity of expression, the reference numbers in the 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.
In a second aspect of the invention, there is also provided a process for the preparation of the 2,4,5-trisubstituted pyrimidine hydroxyamide derivative described above comprising carrying out the reaction of the following reaction scheme:
the method comprises the following specific steps:
(1) Raw material 1 and amino acid methyl ester are condensed to obtain intermediate 2.
(2) Condensing the intermediate 2 and 4-phenoxyaniline under the condition of trifluoroacetic acid to obtain an intermediate 3.
(3) The intermediate 3 reacts with hydroxylamine potassium solution to obtain 2,4,5-trisubstituted pyrimidine hydroxylamine acyl derivative I.
The preparation methods of the intermediate 3 and 2,4,5-trisubstituted pyrimidine hydroxylamine acyl derivatives I are concretely as follows.
The preparation method of the intermediate 3 comprises the following steps:
(i) Dissolving the compound 1 and the amino acid methyl ester 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 4-phenoxyaniline, dropwise adding trifluoroacetic acid into the solution, and reacting for 12h at 110 ℃. TLC detection, complete reaction, cooling to room temperature, reduced pressure evaporation to remove solvent, silica gel column chromatography to obtain intermediate 3.
The preparation method of the 2,4,5-trisubstituted pyrimidine hydroxylamine acyl derivative I comprises the following steps:
preparation of NH from potassium hydroxide, hydroxylamine hydrochloride and anhydrous methanol 2 OK solution. Dissolving intermediate 3 in NH 2 In OK solution, react for 2h at room temperature. TLC detection, complete reaction, decompression evaporation to remove solvent, water addition, pH value adjustment to 6-7 with dilute hydrochloric acid, solid precipitation, filtration, and recrystallization of filter cake with methanol or ethyl acetate to obtain 2,4,5-trisubstituted pyrimidine hydroxylamine acyl derivative I.
In a third aspect of the invention, the invention also provides a composition comprising the 2,4,5-trisubstituted pyrimidine hydroxylamine derivative or a pharmaceutically acceptable salt thereof as described above.
The pharmaceutical composition provided by the invention can be applied in any mode selected from the following modes: 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 the fourth aspect of the invention, the invention also provides a pharmaceutical preparation, which comprises the 2,4,5-trisubstituted pyrimidine hydroxylamine derivative or its pharmaceutically acceptable salt or a composition containing the 2,4,5-trisubstituted pyrimidine hydroxylamine derivative or its pharmaceutically acceptable salt and pharmaceutically acceptable auxiliary materials and/or carriers.
The 2,4,5-trisubstituted pyrimidine hydroxylamine derivatives 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 different carrier and drug concentration, when making injection solution, the compound is dissolved in water, filtered and sterilized, and then filled into sealed bottle or ampoule.
It will be appreciated that the optimum dosage and spacing of administration of 2,4,5-trisubstituted pyrimidine hydroxylamine derivatives provided by the present invention is determined by the nature of the compound and the conditions of the outer 2,4,5-trisubstituted pyrimidine hydroxylamine derivative 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 must also be recognized that the optimal course of treatment, i.e., the daily dosage of 2,4,5-trisubstituted pyrimidine hydroxyamide derivative over a nominal period of time, can be determined by methods known in the art.
In a fifth aspect of the invention, the invention also provides an application of the 2,4,5-tri-substituted pyrimidine hydroxylamine derivative or a pharmaceutically acceptable salt thereof or a composition containing the compound or the pharmaceutically acceptable salt thereof in preparing FLT3 and/or HDAC inhibitor drugs.
The invention also provides the application of the 2,4,5-trisubstituted pyrimidine hydroxylamine acyl derivative or the pharmaceutically acceptable salt thereof or the composition containing the compound or the pharmaceutically acceptable salt thereof in preparing a medicament for treating tumors.
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 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 to which this invention belongs. 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 raw materials 2,4-dichloro-5-substituent-pyrimidine (12mmol, 1.2eq), amino acid methyl ester (10mmol, 1.0eq) and DIPEA (10mmol, 1.0eq) were dissolved in 20mL of isopropanol and reacted 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
The intermediate 2 (1mmol, 1eq) was dissolved in 30mL of n-butanol, 4-phenoxyaniline (1.1mmol, 1.1eq) was added, 3 drops of trifluoroacetic acid were added dropwise to the solution, and the reaction was heated at 110 ℃ for 12 hours. 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 = 100.
3-1:3- (5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) propionic acid methyl ester
1 H NMR(400MHz,DMSO-d 6 )δ9.10(s,1H),7.87(d,J＝3.7Hz,1H),7.79–7.74(m,2H),7.46(t,J＝5.4Hz,1H),7.37–7.32(m,2H),7.08(t,J＝7.4Hz,1H),6.95–6.90(m,4H),3.57(q,J＝6.9Hz,2H),3.40(s,3H),2.31(t,J＝7.2Hz,2H). 13 C NMR(101MHz,DMSO-d 6 )δ167.79,158.52,156.27(d,J＝2.5Hz),152.45(d,J＝12.3Hz),149.76,141.36(d,J＝243Hz),139.00(d,J＝19.2Hz),138.09,130.31,122.87,120.18,120.06,117.66,37.13,32.45.MS(ESI)m/z calcd for C 20 H 20 FN 4 O 3 [M+H] + 383.15,found 383.11.
3-2:4- (5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) butyric acid methyl ester
1 H NMR(400MHz,DMSO-d 6 )δ9.04(s,1H),7.87(d,J＝3.7Hz,1H),7.74(d,J＝9.0Hz,2H),7.52(t,J＝5.3Hz,1H),7.38–7.30(m,2H),7.07(t,J＝7.4Hz,1H),7.00–6.89(m,4H),3.42(s,3H),3.36-3.32(m,2H),2.02(t,J＝7.4Hz,2H),1.88–1.75(m,2H). 13 C NMR(101MHz,DMSO-d 6 )δ169.25,158.58,156.30(d,J＝2.5Hz),152.58(d,J＝12.4Hz),149.64,141.36(d,J＝242Hz),138.88(d,J＝18.7Hz),138.17,130.34,122.82,120.27,119.99,117.56,30.42,25.39.MS(ESI)m/z calcd for C 21 H 22 FN 4 O 3 [M+H] + 397.16,found 397.14.
3-3:5- (5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) pentanoic acid methyl ester
1 H NMR(400MHz,DMSO-d 6 )δ9.08(s,1H),7.84(d,J＝3.7Hz,1H),7.74(d,J＝9.0Hz,2H),7.45(t,J＝5.4Hz,1H),7.39–7.30(m,2H),7.06(t,J＝7.4Hz,1H),6.99–6.88(m,4H),3.43(s,3H),2.06–1.92(m,2H),1.63–1.47(m,4H). 13 C NMR(101MHz,DMSO-d 6 )δ169.42,158.51,156.30(d,J＝2.6Hz),152.57(d,J＝12.4Hz),149.71,141.38(d,J＝243Hz),138.78(d,J＝18.3Hz),138.17,130.34,122.87,120.14,120.03,117.63,32.54,28.99,23.25.MS(ESI)m/z calcd for C 22 H 24 FN 4 O 3 [M+H] + 411.18,found 411.15.
3-4:6- (5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) hexanoic acid methyl ester
1 H NMR(400MHz,DMSO-d 6 )δ9.05(s,1H),7.84(d,J＝3.7Hz,1H),7.79–7.71(m,2H),7.46(t,J＝5.5Hz,1H),7.38–7.31(m,2H),7.06(t,J＝7.4Hz,1H),6.96-6.92(m,4H),3.40(s,3H),1.94(t,J＝7.3Hz,2H),1.62–1.46(m,4H),1.32-1.25(m,2H). 13 C NMR(101MHz,DMSO-d 6 )δ169.43,158.52,156.32(d,J＝2.5Hz),152.60(d,J＝12.4Hz),149.77,141.38(d,J＝243Hz),138.73(d,J＝19.3Hz),138.15,130.31,122.89,120.07,120.04,117.69,40.32,32.69,29.03,26.59,25.42.MS(ESI)m/z calcd for C 23 H 26 FN 4 O 3 [M+H] + 425.19,found 425.16.
3-5:7- (5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) heptanoic acid methyl ester
1 H NMR(400MHz,DMSO-d 6 )δ9.06(s,1H),7.85(d,J＝3.7Hz,1H),7.77(d,J＝9.0Hz,2H),7.43(t,J＝5.3Hz,1H),7.37–7.29(m,2H),7.07(t,J＝7.4Hz,1H),6.98–6.88(m,4H),3.40(s,3H),1.91(t,J＝7.3Hz,2H),1.60-1.53(m,2H),1.51–1.42(m,2H),1.35–1.20(m,4H). 13 C NMR(101MHz,DMSO-d 6 )δ169.48,158.49,156.33,152.58(d,J＝12.3Hz),149.81,142.62,140.17,138.75(d,J＝19.3Hz),138.13,130.31,122.92,120.01(d,J＝3.1Hz),117.73,32.67,29.18,28.86,26.74,25.62.MS(ESI)m/z calcd for C 24 H 28 FN 4 O 3 [M+H] + 439.21,found 439.23.
Example 3: preparation of target compound 2,4,5-trisubstituted pyrimidine hydroxylamine acyl derivative I
KOH (2.85g, 50.9 mmol) and NH were added 2 OH. HCI (2.38g, 34.3mmol) was dissolved in 7mL and 12mL of anhydrous methanol, respectively, to obtain 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 NH 2 OK solution. Will be intermediateBody 3 (0.50 mmol) was dissolved in 10mL NH 2 In OK solution, stir at room temperature for 2h. 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 1MHCl, solid is separated out, the filtration is carried out, and a filter cake is recrystallized by methanol/ethyl acetate to obtain the hydroxylamine acyl target compound I.
I-1:3- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxypropanamide
1 H NMR(400MHz,DMSO-d 6 )δ10.45(s,1H),9.12(s,1H),8.78(s,1H),7.86(d,J＝3.7Hz,1H),7.79–7.74(m,2H),7.45(t,J＝5.4Hz,1H),7.37–7.32(m,2H),7.06(t,J＝7.4Hz,1H),6.97–6.91(m,4H),3.59(q,J＝6.9Hz,2H),2.33(t,J＝7.2Hz,2H). 13 C NMR(101MHz,DMSO-d 6 )δ167.81,158.54,156.29(d,J＝2.5Hz),152.45(d,J＝12.3Hz),149.76,141.38(d,J＝243Hz),139.01(d,J＝19.2Hz),138.09,130.31,122.87,120.18,120.06,117.66,37.11,32.47.HRMS(ESI)m/z calcd for C 19 H 19 FN 5 O 3 [M+H] + 384.1472,found 384.1461.
I-2:4- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybutyramide
1 H NMR(400MHz,DMSO-d 6 )δ10.41(s,1H),9.07(s,1H),8.73(s,1H),7.85(d,J＝3.7Hz,1H),7.75(d,J＝9.0Hz,2H),7.50(t,J＝5.3Hz,1H),7.38–7.30(m,2H),7.06(t,J＝7.4Hz,1H),7.00–6.89(m,4H),3.36-3.32(m,2H),2.04(t,J＝7.4Hz,2H),1.88–1.75(m,2H). 13 C NMR(101MHz,DMSO-d 6 )δ169.26,158.59,156.30(d,J＝2.5Hz),152.58(d,J＝12.4Hz),149.64,141.36(d,J＝242Hz),138.88(d,J＝18.7Hz),138.17,130.31,122.82,120.27,119.98,117.56,30.41,25.39.HRMS(ESI)m/z calcd for C 20 H 21 FN 5 O 3 [M+H] + 398.1628,found 398.1617.
I-3:5- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxypentanamide
1 H NMR(400MHz,DMSO-d 6 )δ10.35(s,1H),9.06(s,1H),8.69(s,1H),7.84(d,J＝3.7Hz,1H),7.74(d,J＝9.0Hz,2H),7.48(t,J＝5.4Hz,1H),7.39–7.30(m,2H),7.06(t,J＝7.4Hz,1H),6.99–6.88(m,4H),2.06–1.92(m,2H),1.63–1.47(m,4H). 13 C NMR(101MHz,DMSO-d 6 )δ169.40,158.54,156.30(d,J＝2.6Hz),152.57(d,J＝12.4Hz),149.71,141.38(d,J＝243Hz),138.78(d,J＝18.3Hz),138.15,130.31,122.86,120.14,120.03,117.63,32.54,28.99,23.25.HRMS(ESI)m/z calcd for C 21 H 23 FN 5 O 3 [M+H] + 412.1785,found 412.1775.
I-4:6- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxyhexanamide
1 H NMR(400MHz,DMSO-d 6 )δ10.34(s,1H),9.06(s,1H),8.68(s,1H),7.84(d,J＝3.7Hz,1H),7.79–7.71(m,2H),7.46(t,J＝5.5Hz,1H),7.38–7.31(m,2H),7.06(t,J＝7.4Hz,1H),6.96-6.92(m,4H),1.94(t,J＝7.3Hz,2H),1.62–1.46(m,4H),1.32-1.25(m,2H). 13 C NMR(101MHz,DMSO-d 6 )δ169.43,158.52,156.32(d,J＝2.5Hz),152.60(d,J＝12.4Hz),149.77,141.38(d,J＝243Hz),138.73(d,J＝19.3Hz),138.15,130.31,122.89,120.07,120.04,117.69,40.32,32.69,29.03,26.59,25.42.HRMS(ESI)m/z calcd for C 22 H 25 FN 5 O 3 [M+H] + 426.1941,found 426.1931.
I-5:7- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxyheptanamide
1 H NMR(400MHz,DMSO-d 6 )δ10.33(s,1H),9.06(s,1H),8.68(s,1H),7.83(d,J＝3.7Hz,1H),7.75(d,J＝9.0Hz,2H),7.45(t,J＝5.3Hz,1H),7.37–7.29(m,2H),7.06(t,J＝7.4Hz,1H),6.98–6.88(m,4H),1.92(t,J＝7.3Hz,2H),1.60-1.53(m,2H),1.51–1.42(m,2H),1.35–1.20(m,4H). 13 C NMR(101MHz,DMSO-d 6 )δ169.46,158.48,156.31,152.58(d,J＝12.3Hz),149.80,142.60,140.16,138.75(d,J＝19.3Hz),138.13,130.31,122.92,120.01(d,J＝3.1Hz),117.73,32.68,29.18,28.87,26.73,25.61.HRMS(ESI)m/z calcd for C 23 H 27 FN 5 O 3 [M+H] + 440.2098,found 440.2088.
Experimental example: 2,4,5-trisubstituted pyrimidine hydroxylamine derivative is used for testing FLT3 and HDAC (histone deacetylase) inhibitory activity and anti-proliferative activity of tumor cells
1) Experiment on FLT3 inhibitory activity:
experimental materials and instruments: the FLT3 kinase inhibitory activity assay was performed with the assistance of eurofinshoma, uk.
The experimental method comprises the following steps: the compounds of the invention were formulated using DMSO in water to give working solutions at 50-fold final assay concentrations. Compound working solution was first added as a first component to the test wells, followed by kinase buffer diluted 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%).
Half maximal Inhibitory Concentration (IC) was calculated by fitting a curve using Gragphadprism6.0 software with the logarithm of concentration as the abscissa and the inhibition ratio as the center 50 ) The 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 FLT3 at a concentration of 1. Mu.M
IC 50 : half maximal inhibitory concentration
A：IC 50 <1μM；B:1μM<IC 50 <10μM；C:10μM<IC 50
Table 1 experimental data show that most compounds show FLT3 inhibitory activity comparable to that of the positive control drug Tandutinib, especially the IC of compounds I-3, II-1, II-3 and II-5 on FLT3 50 The value is less than 1 mu M, and is obviously superior to the positive control drug Tandatinib.
2) Assay of the inhibitory Activity of Compounds on HDAC:
experimental materials and methods: boc-Lys (acetyl) -AMC (HeLa cell nuclear extraction fluorogenic substrate) was purchased from Bachem AG, switzerland, tris-HCl, trypsin and EDTA were purchased from Sigma-Aldrich, TSA was purchased from Allantin scientific and Biochemical Co., ltd., glycerol, naCl, 96-well flat-bottom fluorescent plate was purchased from Hongfei reagent.
Buffer solution: 15mM Tris-HCl (pH 8.0), 250mM NaCl, 250. Mu.M EDTA,10% glycerol
HDAC enzyme solution: diluting with Buffer according to the test requirement.
Fluorogenic substrate solution: the substrate was dissolved in DMSO to prepare a 30mM stock solution, which was stored at-20 ℃ and diluted to 300. Mu.M with HDAC Buffer at the time of use.
Stopping liquid: 10mg/mL Trypsin,50mM Tris-HCl (pH 8.0), 100mM NaCl, 2. Mu.M TSA.
An enzyme-labeling instrument: the Varioskan Flash spectrographic scanning multifunctional reader.
The experimental steps are as follows:
100% control group: mixing 10 μ L of HDACs enzyme solution with 50 μ L of HDAC Buffer, incubating at 37 deg.C for 5min, adding 40 μ L of substrate solution, further incubating at 37 deg.C for 30min, adding 100 μ L of stop solution, further incubating at 37 deg.C for 20min, and measuring the fluorescence intensity of reaction solution at 390nm/460nm with a microplate reader to obtain 100% group fluorescence.
Blank control group: mixing 40 μ L of the substrate solution with 60 μ L of HDAC Buffer, incubating at 37 deg.C for 30min, adding 100 μ L of stop solution, further incubating at 37 deg.C for 20min, and measuring the fluorescence intensity of the reaction solution at 390nm/460nm with microplate reader, wherein the value is the fluorescence intensity of the blank group.
Experimental groups: mixing 10 mu L of HDACs enzyme solution with 50 mu L of the compound of the invention to be detected diluted by HDAC Buffer, incubating for 5min at 37 ℃, adding 40 mu L of substrate solution, continuing to incubate for 30min at 37 ℃, adding 100 mu L of stop solution, continuing to incubate for 20min at 37 ℃, and measuring the fluorescence intensity of the reaction solution at 390nm/460nm by using an enzyme-labeling instrument, wherein the value is the fluorescence of the compound to be detected at the concentration.
Calculating the inhibition rate under different concentrations according to a formula, performing S-curve fitting by using origin software, and calculating the IC of the compound to be detected 50 Values, as shown in table 2.
TABLE 2 HDAC inhibitory Activity of target Compounds
A：IC 50 <1μM；B:1μM<IC 50 <10μM；C:10μM<IC 50
The experimental data in Table 2 show that the compound I shows better inhibition effect on HDAC, and especially the inhibition activity of the compounds I-3, I-4 and I-5 on HDAC1 and HDAC6 is equivalent to or even better than that of the positive control drug SAHA. Notably, compounds I-3, I-4, I-5 all showed potent inhibition of HDAC and FLT 3.
3) Growth inhibitory activity of compounds on tumor cells experiment:
experimental materials and instruments: jeko-1 cell strain, RPMI-1640 culture medium, fetal bovine serum, PBS buffer solution, penicillin sodium (10000 units/mL) -streptomycin sulfate (10 mg/mL), a CCK-8 kit, an inverted optical microscope, a cell culture box, an ultra-clean workbench, a desktop centrifuge, an enzyme labeling instrument 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 × 10 4 Adding 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. Setting 3 multiple wells per concentration, adding completely, placing at 37 deg.C, 5% 2 And (5) incubating in an incubator for 72h. Subsequently 20. Mu.L of CCK-8 solution per well was added and the plates were placed at 37 ℃ 5% CO 2 Continuously incubating for 1-4h in a constant temperature incubator, usingMeasuring absorbance value of the sample at 450nm wavelength by a microplate reader, normalizing the obtained value with a negative DMSO control group, and calculating IC by Prism6.0 software 50 The value is obtained.
TABLE 3 inhibition of Jeko-1 cell growth by compounds of interest
A：IC 50 <2μM；B:2μM<IC 50 <20μM；C:20μM<IC 50
Table 3 experimental data show that most of the compounds have significant antiproliferative activity on Jeko-1 cells, IC of compounds I-1, I-2 and I-3 50 The values were comparable to or even lower than the positive control SAHA.
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 (9)
1. A2,4,5-trisubstituted pyrimidine hydroxylamine derivative is characterized in that the structure of the 2,4,5-trisubstituted pyrimidine hydroxylamine derivative is shown as a general formula I:
wherein n is 1,2,3,4,5,6,7; r 1 One selected from hydrogen, halogen, trifluoromethyl and methyl.
2. The 2,4,5-trisubstituted pyrimidine hydroxylamine derivative of claim 1, wherein the 2,4,5-trisubstituted pyrimidine hydroxylamine derivative is selected from any of the following compounds:
3- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxypropionamide,
4- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybutyramide,
5- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxypentanamide,
6- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxyhexanamide,
7- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxyheptanamide.
3. A process for preparing 2,4,5-trisubstituted pyrimidine hydroxylamine derivatives as claimed in claim 1 or 2, wherein compound 1 and amino acid methyl ester are used as starting materials to prepare 2,4,5-trisubstituted pyrimidine hydroxylamine derivatives I by the following reaction scheme:
the preparation method comprises the following steps:
s1, dissolving an initial compound 1 and amino acid methyl ester in isopropanol, adding DIPEA, reacting at 85 ℃ for 4 hours, detecting by TLC (thin layer chromatography), completely reacting, 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;
s2, dissolving the intermediate 2 in n-butanol, adding 4-phenoxyaniline, dropwise adding trifluoroacetic acid into the solution, reacting at 110 ℃ for 12h, detecting by TLC (thin layer chromatography), completely reacting, cooling to room temperature, evaporating the solvent under reduced pressure, and carrying out silica gel column chromatography to obtain an intermediate 3;
s3, preparing NH by using potassium hydroxide, hydroxylamine hydrochloride and anhydrous methanol 2 OK solution, dissolving intermediate 3 in NH 2 Reacting in OK solution at room temperature for 2h, detecting by TLC, reacting completely, distilling off solvent under reduced pressure, adding water, adjusting pH to 6-7 with dilute hydrochloric acid, precipitating solid, filtering,recrystallizing the filter cake by methanol or ethyl acetate to obtain 2,4,5-trisubstituted pyrimidine hydroxylamine derivative I.
4. A pharmaceutical composition comprising the 2,4,5-trisubstituted pyrimidine hydroxylamine derivative of claim 1 or 2 or a pharmaceutically acceptable salt thereof.
5. A pharmaceutical formulation comprising an active ingredient comprising the 2,4,5-trisubstituted pyrimidine hydroxyamidoyl derivative of claim 1 or 2 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition of claim 4 and a pharmaceutically acceptable adjuvant and/or carrier.
6. An inhibitor, wherein the inhibitor is one of HDAC inhibitor, FLT3 inhibitor and HDAC/FLT3 dual inhibitor, and the active ingredient of the inhibitor is 2,4,5-trisubstituted pyrimidine hydroxylamine derivative or pharmaceutically acceptable salt thereof according to claim 1 or 2.
7. The inhibitor according to claim 6, wherein the inhibitor is a HDAC/FLT3 dual inhibitor, and the active ingredient of the inhibitor is the 2,4,5-tri-substituted pyrimidine hydroxylamine derivative of claim 1 or 2 or a pharmaceutically acceptable salt thereof.
8. The use of a compound which is the 2,4,5-trisubstituted pyrimidine hydroxyamidoyl derivative of claim 1 or 2, or a pharmaceutically acceptable salt thereof, in the preparation of an anti-tumor medicament.
9. The use according to claim 8, wherein the tumor is a lymphoma or leukemia.
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