CN115304551A - 2- ((4-morpholinyl phenyl) amino) pyrimidine amino acid derivative and preparation method and application thereof - Google Patents
2- ((4-morpholinyl phenyl) amino) pyrimidine amino acid 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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- A61P35/02—Antineoplastic agents specific for leukemia
Abstract
The invention belongs to the field of organic compound synthesis and medical application, and discloses 2- ((4-morpholinyl phenyl) amino) pyrimidine amino acid derivatives, and a preparation method and application thereof. The structures of the 2- ((4-morpholinyl phenyl) amino) pyrimidine amino acid derivatives provided by the invention are shown as general formulas I and II
Description
Technical Field
The invention belongs to the field of organic compound synthesis and medical application, and particularly relates to a 2- ((4-morpholinylphenyl) amino) pyrimidine amino acid derivative, and a preparation method and application thereof.
Background
Histone Deacetylase (HDAC) is involved in the regulation of histone deacetylation in the body, acetylation is inhibited or HDAC expression dissimilatory leads to extremely tight binding of DNA to histone, resulting in no longer binding of DNA to regulatory factors in transcription, resulting in transcriptional repression and ultimately uncontrolled gene expression, resulting in excessive cell proliferation into cancer cells, and small molecule HDAC inhibitors have been shown to be promising novel anticancer agents for the treatment of hematologic malignancies and solid tumors (Blood 2016;127 (18): 2168-2170 and Blood 2013 (21): 5142. FMS-like tyrosine kinase 3 (FLT 3) is a member of the type iii receptor tyrosine kinase family, acts on the hematopoietic stem/group cell domain, and is an important target for the treatment of autoimmune diseases and hematological malignancies (see Blood 2018 (4): 426-438 and cancer riscov 2017 (5): 478-493. Preclinical and clinical studies show that the HDAC inhibitor and the FLT3 inhibitor have the effect of coordinately treating acute leukemia (see Blood2008;112 (11): 1618), so that the novel HDAC/FLT3 double-target-point medicine with a novel synthetic structure and druggability is designed and synthesized, and has very important significance for treating hematological malignancies.
Disclosure of Invention
The invention aims to provide 2- ((4-morpholinyl phenyl) amino) pyrimidine amino acid derivatives, and a preparation method and application thereof, wherein the compounds have HDAC/FLT3 dual inhibitory activity, have an anti-tumor effect, have excellent biological activity, and simultaneously improve the molecular diversity and novelty of the compounds.
In order to achieve the purpose, the technical scheme of the disclosure is as follows:
in a first aspect of the present invention, the present invention provides a 2- ((4-morpholinophenyl) amino) pyrimidine amino acid derivative or a pharmaceutically acceptable salt thereof, wherein the 2- ((4-morpholinophenyl) amino) pyrimidine amino acid derivative is a 2- ((4-morpholinophenyl) amino) pyrimidine amino acid derivative I having a structure shown in general formula I or a 2- ((4-morpholinophenyl) amino) pyrimidine amino acid derivative II having a structure shown in general formula II:
in the general formula I and the general formula II, n is 1,2,3,4,5,6,7; preferably, n is 2,3,4,5,6.
Preferably, the 2- ((4-morpholinylphenyl) amino) pyrimidine amino acid derivative I is selected from any one of the following compounds:
3- (5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) propionic acid methyl ester (I-1)
4- (5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) butyric acid methyl ester (I-2)
5- (5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) pentanoic acid methyl ester (I-3)
6- (5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) methyl hexanoate (I-4)
7- (5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) heptanoic acid methyl ester (I-5)
The 2- ((4-morpholinyl phenyl) amino) pyrimidine amino acid derivative II is selected from any one of the following compounds:
3- ((5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxypropanamide (II-1)
4- ((5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybutyramide (II-2)
5- ((5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxypentanamide (II-3)
6- ((5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxyhexanamide (II-4)
7- ((5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxyheptanamide (II-5)
The corresponding reference numbers are shown in parentheses after the names of the above 10 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 2- ((4-morpholinylphenyl) amino) pyrimidine amino acid derivative can exist in a free form or further in a salt form, and aims 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 present invention, the present invention also provides a process for the preparation of 2- ((4-morpholinophenyl) amino) pyrimidine amino acid derivatives I and 2- ((4-morpholinophenyl) amino) pyrimidine amino acid derivatives II, said process comprising carrying out the following reaction scheme:
wherein n is 1,2,3,4,5,6,7;
the method comprises the following specific steps:
(1) The compound 1 and amino acid methyl ester are condensed to obtain an intermediate 2.
(2) Condensing the intermediate 2 with 4-morpholine aniline under trifluoroacetic acid condition to obtain 2- ((4-morpholine phenyl) amino) pyrimidine amino acid derivative I.
(3) And reacting the 2- ((4-morpholinyl phenyl) amino) pyrimidine amino acid derivative I with a hydroxylamine potassium solution to obtain a 2- ((4-morpholinyl phenyl) amino) pyrimidine amino acid derivative II.
The preparation method of the 2- ((4-morpholinylphenyl) amino) pyrimidine amino acid derivative I comprises the following steps:
(i) The starting compound 1 and the amino acid methyl ester were dissolved in isopropanol, DIPEA was added, and the reaction was carried out at 85 ℃ for 4 hours. And (4) detecting by TLC, completely reacting, cooling to room temperature, separating out a large amount of solid, filtering, and recrystallizing a filter cake by using ethyl acetate to obtain an intermediate 2.
(ii) Dissolving the intermediate 2 in n-butanol, adding 4-morpholine aniline, dropwise adding trifluoroacetic acid into the solution, and reacting for 12h at 110 ℃. And (3) detecting by TLC (thin layer chromatography), completely reacting, cooling to room temperature, evaporating to remove the solvent under reduced pressure, and performing silica gel column chromatography to obtain a target compound I, namely the 2- ((4-morpholinyl phenyl) amino) pyrimidine amino acid derivative I.
The preparation method of the 2- ((4-morpholinyl phenyl) amino) pyrimidine amino acid derivative II comprises the following steps:
preparation of NH from potassium hydroxide, hydroxylamine hydrochloride and anhydrous methanol 2 OK solution. Dissolving 2- ((4-morpholinylphenyl) amino) pyrimidine amino acid derivative I in NH 2 In OK solution, react for 2h at room temperature. TLC detection, reaction is completed, the solvent is evaporated under reduced pressure, water is added, the pH value is adjusted to 6-7 by dilute hydrochloric acid, solid is separated out, filtration is carried out, and a filter cake is recrystallized by methanol or ethyl acetate to obtain the 2- ((4-morpholinylphenyl) amino) pyrimidine amino acid derivative II.
In a third aspect of the present invention, the present invention also provides a pharmaceutical composition containing the above-mentioned 2- ((4-morpholinophenyl) amino) pyrimidine amino acid derivative or a pharmaceutically acceptable salt thereof.
The pharmaceutical composition of the 2- ((4-morpholinylphenyl) amino) pyrimidine amino acid derivative 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 a fourth aspect of the present invention, the present invention also provides a pharmaceutical preparation, which comprises the above-mentioned 2- ((4-morpholinylphenyl) amino) pyrimidine amino acid derivative or a pharmaceutically acceptable salt thereof or a composition containing the above-mentioned compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable adjuvant and/or carrier.
The 2- ((4-morpholinophenyl) amino) pyrimidine amino acid derivatives of the invention or pharmaceutical compositions containing them can 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 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.
In a fifth aspect of the present invention, the present invention further provides an application of the above amino acid derivative containing 2- ((4-morpholinyl phenyl) amino) pyrimidine or a pharmaceutically acceptable salt thereof, or a composition containing the above amino acid derivative containing 2- ((4-morpholinyl phenyl) amino) pyrimidine or a pharmaceutically acceptable salt thereof in preparation of a FLT3 and/or HDAC inhibitor drug.
The invention also provides application of the amino acid derivative containing the 2- ((4-morpholinyl phenyl) amino) pyrimidine or pharmaceutically acceptable salt thereof or a composition containing the compound or the pharmaceutically acceptable salt thereof in preparing a medicament for treating tumors. Preferably, the tumor is a lymphoma or leukemia.
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. 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), 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 2- ((4-morpholinylphenyl) amino) pyrimidine amino acid derivative I
The intermediate 2 (1mmol, 1eq) was dissolved in 30mL of n-butanol, 4-morpholinoaniline (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 evaporated under reduced pressure, and silica gel column chromatography (dichloromethane/methanol =100: 1-20) was performed to obtain 2- ((4-morpholinophenyl) amino) pyrimidine amino acid derivative I.
I-1:3- (5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) propionic acid methyl ester
1 H NMR(400MHz,DMSO-d 6 )δ8.81(s,1H),7.82(d,J＝3.7Hz,1H),7.58(d,J＝9.0Hz,2H),7.35(t,J＝5.4Hz,1H),6.86(d,J＝9.0Hz,2H),3.76–3.70(m,4H),3.60-3.55(m,2H),3.41(s,3H),3.05–2.96(m,4H),2.33(t,J＝7.2Hz,2H). 13 C NMR(101MHz,DMSO-d 6 )δ167.85,156.52(d,J＝2.4Hz),152.37(d,J＝12.1Hz),145.92,141.16(d,J＝242Hz),139.02(d,J＝18.8Hz),134.48,119.81,116.19,66.67,51.60,49.96,37.04,32.48.HRMS(ESI)m/z calcd for C 18 H 23 FN 5 O 3 [M+H] + 376.17,found 376.19.
I-2:4- (5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) butyric acid methyl ester
1 H NMR(400MHz,DMSO-d 6 )δ8.79(s,1H),7.82(d,J＝3.7Hz,1H),7.58(d,J＝9.0Hz,2H),7.44(t,J＝5.4Hz,1H),6.86(d,J＝9.1Hz,2H),3.79–3.69(m,4H),3.42(s,3H),3.04–2.96(m,4H),2.06(t,J＝7.5Hz,2H),1.86–1.76(m,2H). 13 C NMR(101MHz,DMSO-d 6 )δ169.28,156.52(d,J＝2.5Hz),152.53(d,J＝12.2Hz),145.89,141.12(d,J＝242Hz),138.95(d,J＝18.7Hz),134.53,119.72,116.18,66.67,51.62,49.95,40.07,30.44,25.43.HRMS(ESI)m/z calcd for C 19 H 25 FN 5 O 3 [M+H] + 390.19,found 390.16.
I-3:5- (5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) pentanoic acid methyl ester
1 H NMR(400MHz,DMSO-d 6 )δ8.75(s,1H),7.77(d,J＝3.7Hz,1H),7.57(d,J＝8.9Hz,2H),7.40(t,J＝5.5Hz,1H),6.84(d,J＝9.0Hz,2H),3.77–3.68(m,4H),3.40(s,3H),3.04–2.96(m,4H),2.03–1.93(m,2H),1.62-1.50(m,4H). 13 C NMR(101MHz,DMSO-d 6 )δ169.40,156.56,152.49(d,J＝12.2Hz),145.92,141.12(d,J＝242Hz),138.88(d,J＝18.2Hz),134.53,119.77,116.11,66.68,51.61,49.93,32.60,29.07,23.28.HRMS(ESI)m/z calcd for C 20 H 27 FN 5 O 3 [M+H] + 404.21,found404.18.
I-4:6- (5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) methyl hexanoate
1 H NMR(400MHz,DMSO-d 6 )δ8.79(s,1H),7.79(d,J＝3.8Hz,1H),7.57(d,J＝9.0Hz,2H),7.36(t,J＝5.5Hz,1H),6.82(d,J＝8.9Hz,2H),3.79–3.69(m,4H),3.42(s,3H),3.08–2.95(m,4H),1.95(t,J＝7.3Hz,2H),1.61-1.49(m,4H),1.35–1.25(m,2H). 13 C NMR(101MHz,DMSO-d 6 )δ156.57(d,J＝2.5Hz),152.51(d,J＝12.3Hz),145.89,141.11(d,J＝242Hz),138.87(d,J＝19.0Hz),134.54,119.75,116.07,66.68,51.60,49.92,32.72,29.13,26.67,25.50.HRMS(ESI)m/z calcdfor C 21 H 29 FN 5 O 3 [M+H] + 418.21,found 418.22.
I-5:7- (5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) heptanoic acid methyl ester
1 H NMR(400MHz,DMSO-d 6 )δ8.77(s,1H),7.78(d,J＝3.8Hz,1H),7.55(d,J＝9.1Hz,2H),7.35(t,J＝5.5Hz,1H),6.83(d,J＝9.1Hz,2H),3.76–3.65(m,4H),3.40(s,3H),3.04–2.95(m,4H),1.94(t,J＝7.3Hz,2H),1.60–1.46(m,4H),1.35–1.23(m,4H). 13 C NMR(101MHz,DMSO-d 6 )δ169.52,156.55(d,J＝2.5Hz),152.51(d,J＝12.2Hz),145.86,141.11(d,J＝242Hz),138.87(d,J＝18.6Hz),134.56,119.75,116.07,66.67,51.60,49.94,32.72,29.26,28.95,26.79,25.68.MS(ESI)m/z calcd for C 22 H 31 FN 5 O 3 [M+H] + 432.24,found 432.26.
Example 3: preparation of 2- ((4-morpholinylphenyl) amino) pyrimidine amino acid derivative II
KOH (2.85g, 50.9mmol) and NH 2 OH & HCI (2.38g, 34.3 mmol) was dissolved in 7mL and 12mL of anhydrous methanol, respectively, to give solution A and solution B. Under the 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. Target Compound I (0.50 mmol) was dissolved in 10mL NH 2 OK solution was stirred 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 1M HCl, a solid is separated out, the filtration is carried out, and a filter cake is recrystallized by methanol/ethyl acetate to obtain the 2- ((4-morpholine phenyl) amino) pyrimidine amino acid derivative II.
II-1:3- ((5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxypropanamide
1 H NMR(400MHz,DMSO-d 6 )δ10.45(s,1H),8.82(s,1H),8.80(s,1H),7.82(d,J＝3.7Hz,1H),7.58(d,J＝9.0Hz,2H),7.35(t,J＝5.4Hz,1H),6.84(d,J＝9.0Hz,2H),3.76–3.70(m,4H),3.60-3.55(m,2H),3.05–2.96(m,4H),2.33(t,J＝7.2Hz,2H). 13 C NMR(101MHz,DMSO-d 6 )δ167.87,156.52(d,J＝2.4Hz),152.39(d,J＝12.1Hz),145.92,141.16(d,J＝242Hz),139.04(d,J＝18.8Hz),134.48,119.82,116.19,66.67,49.96,37.05,32.48.HRMS(ESI)m/z calcd for C 17 H 22 FN 6 O 3 [M+H] + 377.1737,found 377.1729.
II-2:4- ((5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybutyramide
1 H NMR(400MHz,DMSO-d 6 )δ10.42(s,1H),8.78(s,1H),8.74(s,1H),7.80(d,J＝3.7Hz,1H),7.57(d,J＝9.0Hz,2H),7.42(t,J＝5.4Hz,1H),6.85(d,J＝9.1Hz,2H),3.79–3.69(m,4H),3.04–2.96(m,4H),2.04(t,J＝7.5Hz,2H),1.86–1.76(m,2H). 13 C NMR(101MHz,DMSO-d 6 )δ169.29,156.52(d,J＝2.5Hz),152.51(d,J＝12.2Hz),145.88,141.12(d,J＝242Hz),138.95(d,J＝18.7Hz),134.51,119.72,116.17,66.67,49.94,40.07,30.44,25.43.HRMS(ESI)m/z calcd for C 18 H 24 FN 6 O 3 [M+H] + 391.1894,found 391.1883.
II-3:5- ((5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxypentanamide
1 H NMR(400MHz,DMSO-d 6 )δ10.37(s,1H),8.76(s,1H),8.71(s,1H),7.79(d,J＝3.7Hz,1H),7.56(d,J＝8.9Hz,2H),7.40(t,J＝5.5Hz,1H),6.84(d,J＝9.0Hz,2H),3.77–3.68(m,4H),3.04–2.96(m,4H),2.03–1.93(m,2H),1.62-1.50(m,4H). 13 C NMR(101MHz,DMSO-d 6 )δ169.39,156.55,152.49(d,J＝12.2Hz),145.91,141.12(d,J＝242Hz),138.88(d,J＝18.2Hz),134.52,119.77,116.11,66.67,49.93,32.60,29.07,23.28.HRMS(ESI)m/z calcd for C 19 H 26 FN 6 O 3 [M+H] + 405.2050,found 405.2040.
II-4:6- ((5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxyhexanamide
1 H NMR(400MHz,DMSO-d 6 )δ10.35(s,1H),8.78(s,1H),8.68(s,1H),7.79(d,J＝3.8Hz,1H),7.57(d,J＝9.0Hz,2H),7.38(t,J＝5.5Hz,1H),6.84(d,J＝8.9Hz,2H),3.79–3.69(m,4H),3.08–2.95(m,4H),1.96(t,J＝7.3Hz,2H),1.61-1.49(m,4H),1.35–1.25(m,2H). 13 C NMR(101MHz,DMSO-d 6 )δ156.55(d,J＝2.5Hz),152.50(d,J＝12.3Hz),145.89,141.11(d,J＝242Hz),138.86(d,J＝19.0Hz),134.54,119.73,116.07,66.68,49.92,32.72,29.13,26.67,25.50.HRMS(ESI)m/z calcd for C 20 H 28 FN 6 O 3 [M+H] + 419.2107,found 419.2198.
II-5:7- ((5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxyheptanamide
1 H NMR(400MHz,DMSO-d 6 )δ10.33(s,1H),8.76(s,1H),8.67(s,1H),7.79(d,J＝3.8Hz,1H),7.57(d,J＝9.1Hz,2H),7.37(t,J＝5.5Hz,1H),6.82(d,J＝9.1Hz,2H),3.76–3.65(m,4H),3.04–2.95(m,4H),1.94(t,J＝7.3Hz,2H),1.60–1.46(m,4H),1.35–1.23(m,4H). 13 C NMR(101MHz,DMSO-d 6 )δ169.50,156.55(d,J＝2.5Hz),152.51(d,J＝12.2Hz),145.88,141.11(d,J＝242Hz),138.86(d,J＝18.6Hz),134.56,119.75,116.07,66.67,49.94,32.72,29.26,28.93,26.78,25.68.HRMS(ESI)m/z calcd for C 21 H 30 FN 6 O 3 [M+H] + 433.2363,found 433.2353.
Experimental example: experiments for testing FLT3 and HDAC inhibitory activity and anti-proliferation activity of tumor cells by using 2- ((4-morpholinyl phenyl) amino) pyrimidine amino acid derivative
1) Experiment of FLT3 inhibitory activity of 2- ((4-morpholinyl phenyl) amino) pyrimidine amino acid derivative:
experimental materials and instruments: the FLT3 kinase inhibitory activity assay was performed with the aid of Eurofins Pharma, UK.
The experimental method comprises the following steps: the 2- ((4-morpholinylphenyl) amino) pyrimidine amino acid derivative obtained in the embodiment of the invention is prepared into working solution with the final test concentration 50 times by using DMSO (dimethylsulfoxide) aqueous solution. 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 was not added with test compound, DMSO was used instead (final concentration 2%), and the other components were the same as 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 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
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 have potent inhibitory activity (IC) against FLT3 50 <1 μ M), especially compounds of the II series and IC thereof 50 The value was significantly lower than the positive control drug Tandutinib.
2) Experiment of 2- ((4-morpholinophenyl) amino) pyrimidine amino acid derivatives on HDAC inhibitory activity:
experimental materials and methods: boc-Lys (acetyl) -AMC (HeLa cell nucleus extraction fluorogenic substrate) was purchased from BachemAG, switzerland, tris-HCl, trypsin and EDTA were purchased from Sigma-Aldrich, TSA was purchased from Alantin technology 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, stored at-20 ℃ and diluted to 300. Mu.M with HDAC Buffer at the time of use.
Stopping liquid: 10mg/ml of 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 mu LHDACs enzyme solution with 50 mu LHDAC Buffer, incubating for 5min at 37 ℃, adding 40 mu L substrate solution, continuing to incubate for 30min at 37 ℃, adding 100 mu L stop solution, continuing to incubate for 20min at 37 ℃, and measuring the fluorescence intensity of the reaction solution at 390nm/460nm by using a microplate reader, wherein the value is 100% of group fluorescence intensity.
Blank control group: and mixing 40 mu L of substrate solution with 60 mu L of LHDAC Buffer, incubating 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 a microplate reader, wherein the value is the fluorescence intensity of a blank group.
Experimental groups: mixing 10 mu of LHDACs enzyme solution with 50 mu of 2- ((4-morpholinyl phenyl) amino) pyrimidine amino acid derivative to be detected diluted by HDAC Buffer, incubating for 5min at 37 ℃, adding 40 mu of substrate solution, continuing to incubate for 30min at 37 ℃, adding 100 mu of stop solution, continuing to incubate for 20min at 37 ℃, and measuring the fluorescence intensity of the reaction solution at 390nm/460nm by using a microplate reader, 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 2.2- ((4-Morpholinylphenyl) amino) pyrimidine amino acid derivatives have HDAC inhibitory Activity
A：IC 50 <1μM；B:1μM<IC 50 <10μM；C:10μM<IC 50
Table 2 experimental data show that compounds II-3, II-4 and II-5 show potent inhibition of HDAC1 and HDAC6 (IC) 50 <1 μ M), IC thereof 50 IC of value and Positive drug SAHA 50 The values are comparable. Notably, compounds II-3, II-4 and II-5 showed dual inhibitory effects on HDAC and FLT 3.
3) 2- ((4-morpholinylphenyl) amino) pyrimidine amino acid derivative growth inhibition activity experiment on tumor cells:
experimental materials and instruments: the kit comprises hematological malignant tumor cells Jeko-1, MV4-11, K562, Z138, hel and Molt4 cell strains, 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 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 × 10 4 Adding cell culture solutions of 2- ((4-morpholinyl phenyl) amino) pyrimidine amino acid derivatives measured at 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 duplicate wells per concentration, after addition, 37 ℃ C.,. 5% CO 2 Incubate in the incubator for 72h. Subsequently adding 20 μ LCCK-8 solution per well, incubating the plate at 37 ℃ 5% 2 Continuously 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 software 50 The value is obtained.
TABLE 3 antiproliferative effect of target compounds on hematological malignant cells
A：IC 50 <2μM；B:2μM<IC 50 <20μM；C:20μM<IC 50
We selected representative compounds with potent HDAC/FLT3 dual inhibitor activity and further studied their antiproliferative activity against hematological malignancies, and the results are shown in Table 3, compounds II-3, II-4 and II-5 showed potent antiproliferative activity against MV4-11 and Molt4 cells, especially compound II-5 showed the best IC for the antiproliferative activity against 6 cell lines tested 50 The values are all less than 2 mu M, and are equivalent to or even better than the positive drug 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. The 2- ((4-morpholinyl phenyl) amino) pyrimidine amino acid derivative is 2- ((4-morpholinyl phenyl) amino) pyrimidine amino acid derivative I or 2- ((4-morpholinyl phenyl) amino) pyrimidine amino acid derivative II, the structure of the 2- ((4-morpholinyl phenyl) amino) pyrimidine amino acid derivative I is shown in a general formula I, and the structure of the 2- ((4-morpholinyl phenyl) amino) pyrimidine amino acid derivative II is shown in a general formula II:
wherein n is 1,2,3,4,5,6,7.
2. The 2- ((4-morpholinophenyl) amino) pyrimidine amino acid derivative of claim 1, wherein the 2- ((4-morpholinophenyl) amino) pyrimidine amino acid derivative I is selected from any one of the following:
3- (5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) propionic acid methyl ester
4- (5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) butyric acid methyl ester
5- (5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) pentanoic acid methyl ester
6- (5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) hexanoic acid methyl ester
7- (5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) heptanoic acid methyl ester
The 2- ((4-morpholinyl phenyl) amino) pyrimidine amino acid derivative II is selected from any one of the following compounds:
3- ((5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxypropanamide
4- ((5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybutyramide
5- ((5-fluoro-2- ((4-morpholinophenyl) amino) pyrimidin-4-yl) amino) -N-hydroxypentanamide
6- ((5-fluoro-2- ((4-morpholinylphenyl) amino) pyrimidin-4-yl) amino) -N-hydroxyhexanamide
7- ((5-fluoro-2- ((4-morpholinophenyl) amino) pyrimidin-4-yl) amino) -N-hydroxyheptanamide.
3. The process for producing a 2- ((4-morpholinophenyl) amino) pyrimidine amino acid derivative according to claim 1 or 2, characterized by comprising producing the 2- ((4-morpholinophenyl) amino) pyrimidine amino acid derivative I and the 2- ((4-morpholinophenyl) amino) pyrimidine amino acid derivative II by the following reaction schemes starting with compound 1 and an amino acid methyl ester:
s1, dissolving a 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-butyl alcohol, adding 4-morpholine aniline, 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 a 2- ((4-morpholine phenyl) amino) pyrimidine amino acid derivative I;
s3, preparing NH by using potassium hydroxide, hydroxylamine hydrochloride and anhydrous methanol 2 OK solution, dissolving the 2- ((4-morpholinyl phenyl) amino) pyrimidine amino acid derivative obtained in the step S2 in NH 2 Reacting at room temperature for 2h in OK solution, detecting by TLC, completely reacting, distilling off the solvent under reduced pressure, adding water, adjusting pH to 6-7 with dilute hydrochloric acid, separating out solids, filtering, and recrystallizing the filter cake with methanol or ethyl acetate to obtain the 2- ((4-morpholinyl phenyl) amino) pyrimidine amino acid derivative II.
4. A pharmaceutical composition comprising the 2- ((4-morpholinophenyl) amino) pyrimidine amino acid derivative of claim 1 or 2, or a pharmaceutically acceptable salt thereof.
5. A pharmaceutical formulation comprising an active ingredient comprising the amino acid derivative of 2- ((4-morpholinophenyl) amino) pyrimidine according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, or comprising the pharmaceutical composition according to claim 4, and a pharmaceutically acceptable adjuvant and/or carrier.
6. An inhibitor, wherein the inhibitor is one of an HDAC inhibitor, an FLT3 inhibitor and a HDAC/FLT3 dual inhibitor, and the inhibitor comprises the 2- ((4-morpholinophenyl) amino) pyrimidine amino acid derivative or a pharmaceutically acceptable salt thereof according to claim 1 or 2 as an active ingredient.
7. The inhibitor according to claim 6, wherein the inhibitor is a HDAC/FLT3 dual inhibitor, and the active ingredient of the inhibitor comprises 1 or 2 of the amino acid derivative of 2- ((4-morpholinophenyl) amino) pyrimidine or a pharmaceutically acceptable salt thereof.
8. Use of a compound which is a 2- ((4-morpholinophenyl) amino) pyrimidine amino acid derivative as claimed in claim 1 or 2, or a pharmaceutically acceptable salt thereof, in the manufacture of an anti-neoplastic medicament.
9. The use according to claim 8, wherein the tumor is a lymphoma or leukemia.
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