CN115232076A - Phenylamino substituted pyrimidine amino acid derivative and preparation method and application thereof - Google Patents
Phenylamino substituted pyrimidine amino acid derivative and preparation method and application thereof Download PDFInfo
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- C07—ORGANIC CHEMISTRY
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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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- 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 field of organic compound synthesis and medical application, and discloses a phenylamino substituted pyrimidine amino acid derivative, a preparation method and application thereof. The structure of the phenylamino substituted pyrimidine amino acid aminobenzoyl derivative provided by the invention is shown as general formulas I and II:
Description
Technical Field
The invention belongs to the field of organic compound synthesis and medical application, and relates to a phenylamino substituted pyrimidine amino acid derivative, and a preparation method and application thereof.
Background
Abnormal expression and mutation of Histone Deacetylase (HDAC) are closely related to the occurrence and development of tumors, and high expression of HDAC in malignant tumors of the blood system leads to low acetylation of histone, so that inhibition of HDAC can block cell cycle, thereby inducing apoptosis. (Best Practice & Research Clinical Haematology 2012,25 (2): 191-200.) Vorinostat (SAHA) is the first HDAC inhibitor to be marketed, FDA approved for the treatment of cutaneous T-cell lymphoma. HDACs have become important targets for the treatment of hematological malignancies. FMS-like tyrosine kinase 3 (FLT 3) is a member of the type III receptor tyrosine kinase family, is homologous with stem cell growth factor c-Kit, acts on hematopoietic stem/group cell regions (see Blood 2017 (6): 732-741 and Cancer Discov2017;7 (5): 478-493), and is an important target for the treatment of hematological malignancies and autoimmune diseases. Preclinical and clinical studies show that the double inhibition of HDAC and FLT3 has a coordination effect (see: blood2019;134 (Supplement _ 1): 5477-5477), so that the design and synthesis of a novel HDAC/FLT3 double-target-point drug with a novel structure and a drug property have a very important significance for treating hematological malignancies.
Disclosure of Invention
The invention aims to provide a phenylamino substituted pyrimidine amino acid derivative, and a preparation method and application thereof, wherein the phenylamino substituted pyrimidine amino acid derivative has HDAC/FLT3 dual inhibitory activity and anti-tumor activity.
In order to achieve the purpose, the technical scheme of the disclosure is as follows:
in a first aspect of the present invention, the present invention provides a phenylamino substituted pyrimidine amino acid derivative or a pharmaceutically acceptable salt thereof, wherein the phenylamino substituted pyrimidine amino acid derivative is a phenylamino substituted pyrimidine amino acid derivative I having a structure represented by general formula I or a phenylamino substituted pyrimidine amino acid derivative II having a structure represented by general formula II:
wherein n is 1,2,3,4,5,6,7,8,9,10;
preferably, n is 2,3,4,5,6.
3- (5-fluoro-2- (4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) propionic acid methyl ester (I-1)
4- (5-fluoro-2- (4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) butanoic acid methyl ester (I-2)
5- (5-fluoro-2- (4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) pentanoic acid methyl ester (I-3)
6- (5-fluoro-2- (4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) hexanoic acid methyl ester (I-4)
7- (5-fluoro-2- (4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) heptanoic acid methyl ester (I-5)
3- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxypropionamide (II-1)
4- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybutyramide (II-2)
5- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxypentanamide (II-3)
6- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxyhexanamide (II-4)
7- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxyheptanamide (II-5)
The parenthesis following the names of the above 10 compounds are the corresponding reference numbers, and for the convenience of description and brevity of expression, the above parenthesis reference numbers will be used directly in the following description of the present specification.
The compounds of the invention may be present in free form or further in salt form for the purpose of improving water solubility and increasing 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 worker.
In a second aspect of the present invention, the present invention also provides a process for preparing phenylamino substituted pyrimidine amino acid derivatives I or phenylamino substituted pyrimidine amino acid derivatives II, the reaction scheme being as follows:
wherein n is selected from 1,2,3,4,5,6,7,8,9, 10.
Reagents and conditions: (a) N, N-Diisopropylethylamine (DIPEA), isopropanol, 85 ℃,4h; (b) 4- (2-methoxyethoxy) aniline, trifluoroacetic acid, n-butanol, 110 ℃ and 12h; (c) Potassium hydroxide, hydroxylamine hydrochloride, anhydrous methanol, 0-r.t., 2h.
The method comprises the following specific steps:
(1) The raw material 1 and amino acid methyl ester 2 are condensed to obtain an intermediate 3.
(2) Condensing the intermediate 3 with 4- (2-methoxyethoxy) aniline under the condition of trifluoroacetic acid to obtain the phenylamino substituted pyrimidine amino acid derivative I.
(3) And reacting the phenylamino substituted pyrimidine amino acid derivative I with a hydroxylamine potassium solution to obtain the phenylamino substituted pyrimidine amino acid derivative II.
(4) The preparation method of the phenylamino substituted pyrimidine amino acid derivative I comprises the following steps:
(i) The compound 1 and the amino acid methyl ester 2 are dissolved in isopropanol, DIPEA is added, and the reaction is carried out for 4 hours at 85 ℃. 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 3.
(ii) Dissolving the intermediate 3 in n-butanol, adding 4- (2-methoxyethoxy) aniline, then dropwise adding trifluoroacetic acid into the solution, and reacting for 12h at 110 ℃. And (4) detecting by TLC, completely reacting, cooling to room temperature, distilling under reduced pressure to remove the solvent, and performing silica gel column chromatography to obtain the phenylamino substituted pyrimidine amino acid derivative I.
(5) The preparation method of the phenylamino substituted pyrimidine amino acid derivative II comprises the following steps:
preparation of NH from potassium hydroxide, hydroxylamine hydrochloride and anhydrous methanol 2 OK solution. Dissolving phenylamino substituted pyrimidine amino acid derivative I 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 filter cake recrystallization with methanol or ethyl acetate to obtain phenylamino substituted pyrimidine amino acid derivative II.
In a third aspect of the present invention, the present invention also provides a pharmaceutical composition comprising the above phenylamino substituted pyrimidine amino acid derivative or a pharmaceutically acceptable salt thereof.
The pharmaceutical composition of the phenylamino substituted 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 the fourth aspect of the present invention, the present invention also provides a pharmaceutical preparation, which comprises the phenylamino substituted pyrimidine amino acid derivative or its pharmaceutically acceptable salt or a composition containing the phenylamino substituted pyrimidine amino acid derivative or its pharmaceutically acceptable salt and a pharmaceutically acceptable adjuvant and/or carrier.
The phenylamino substituted pyrimidine amino acid 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 dry product, and is supplemented with water or other suitable medium before use. Such liquid preparations may contain conventional additives such as suspending agents, sorbitol, cellulose methyl ether, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gelatin, hydrogenated edible fats and oils, emulsifying agents such as lecithin, sorbitan monooleate, gum arabic; or a non-aqueous carrier (which may comprise an edible oil), such as almond oil, an oil such as glycerol, ethylene glycol, or ethanol; preservatives, e.g. methyl or propyl p-hydroxybenzoates, sorbic acid. Flavoring or coloring agents may be added if desired.
Suppositories may contain conventional suppository bases such as cocoa butter or other glycerides.
For parenteral administration, liquid dosage forms are generally prepared from the compound and a sterile carrier. The carrier is preferably water. The compound can be dissolved in the carrier or made into suspension solution according to the concentration of the carrier and the drug, and the compound is firstly dissolved in water when made into the solution for injection, filtered and sterilized and then filled into a sealed bottle or ampoule.
It will be appreciated that the optimum dosage and interval for administration of the phenylamino substituted pyrimidine amino acid derivatives provided herein 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 optimum dosage can be determined by conventional techniques. It should also be recognized that the optimal course of treatment, i.e., the daily dosage of the phenylamino-substituted pyrimidine amino acid derivatives provided herein 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 phenylamino substituted pyrimidine amino acid derivative or its pharmaceutically acceptable salt or a composition containing the phenylamino substituted pyrimidine amino acid derivative or its pharmaceutically acceptable salt in the preparation of a medicament for inhibiting HDAC and/or FLT 3.
The invention also provides the application of the phenylamino substituted pyrimidine amino acid derivative or the pharmaceutically acceptable salt thereof or the composition containing the phenylamino substituted pyrimidine amino acid derivative or the pharmaceutically acceptable salt thereof in preparing the medicine for treating tumors. The tumor is preferably 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 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 exemplary only.
Example 1: preparation of intermediate 3
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 3.
Example 2: preparation of phenylamino substituted pyrimidine amino acid derivatives I
Intermediate 3 (1mmol, 1eq) was dissolved in 30mL of n-butanol, 4- (2-methoxyethoxy) aniline (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, and the solvent was evaporated under reduced pressure, followed by silica gel column chromatography (dichloromethane/methanol =100: 1-20).
I-1:3- (5-fluoro-2- (4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) propionic acid methyl ester
1 H NMR(400MHz,DMSO-d 6 )δ8.85(s,1H),7.80(d,J＝3.7Hz,1H),7.66(d,J＝9.1Hz,2H),7.38(t,J＝5.4Hz,1H),6.85(d,J＝9.1Hz,2H),4.05–4.00(m,2H),3.64–3.60(m,2H),3.57(dd,J＝13.1,6.8Hz,2H),3.40(s,3H),3.31(s,3H),2.32(t,J＝7.1Hz,2H). 13 C NMR(101MHz,DMSO-d 6 )δ167.78,156.43(d,J＝2.5Hz),153.16,152.40(d,J＝12.5Hz),141.25(d,J＝241Hz),139.01(d,J＝18.8Hz),135.17,120.11,114.69,70.99,67.44,58.62,51.61,37.01,32.42.MS(ESI)m/z calcdfor C 17 H 22 FN 4 O 4 [M+H] + 365.14,found 365.16.
I-2:4- (5-fluoro-2- (4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) butanoic acid methyl ester
1 H NMR(400MHz,DMSO-d 6 )δ8.82(s,1H),7.82(d,J＝3.7Hz,1H),7.59(d,J＝9.1Hz,2H),7.43(t,J＝5.3Hz,1H),6.85(d,J＝9.1Hz,2H),4.08–3.97(m,2H),3.66–3.60(m,2H),3.37–3.32(m,2H),3.40(s,3H),3.30(s,3H),2.04(t,J＝7.4Hz,2H),1.89-1.75(m,2H). 13 C NMR(101MHz,DMSO-d 6 )δ169.28,156.49(d,J＝2.4Hz),153.15,152.53(d,J＝12.3Hz),141.19(d,J＝242Hz),138.93(d,J＝18.6Hz),135.19,120.09,114.69,71.00,67.44,58.62,51.63,40.09,30.42,25.42.MS(ESI)m/z calcd for C 18 H 24 FN 4 O 4 [M+H] + 379.17,found 379.15.
I-3:5- (5-fluoro-2- (4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) pentanoic acid methyl ester
1 H NMR(400MHz,DMSO-d 6 )δ8.81(s,1H),7.81(d,J＝3.7Hz,1H),7.58(d,J＝9.0Hz,2H),7.43(t,J＝6.0Hz,1H),6.82(d,J＝9.0Hz,2H),4.05–3.98(m,2H),3.68–3.59(m,2H),3.40(s,3H),3.30(s,3H),2.05-1.95(m,2H),1.59-1.51(m,4H). 13 C NMR(101MHz,DMSO-d 6 )δ169.46,156.49(d,J＝2.4Hz),153.11,152.49(d,J＝12.2Hz),141.18(d,J＝242Hz),138.86(d,J＝18.4Hz),135.19,120.09,114.62,70.99,67.43,58.61,51.60,32.59,30.46,29.06,23.27.MS(ESI)m/z calcdfor C 19 H 26 FN 4 O 4 [M+H] + 393.19,found 393.18.
I-4:6- (5-fluoro-2- (4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) hexanoic acid methyl ester
1 H NMR(400MHz,DMSO-d 6 )δ8.80(s,1H),7.81(d,J＝3.8Hz,1H),7.61(d,J＝9.1Hz,2H),7.38(t,J＝5.3Hz,1H),6.83(d,J＝9.1Hz,2H),4.04-4.02(m,2H),3.66–3.62(m,2H),3.40(s,3H),3.30(s,3H),1.96(t,J＝7.3Hz,2H),1.62–1.49(m,4H),1.36–1.25(m,2H). 13 C NMR(101MHz,DMSO-d 6 )δ169.51,156.51(d,J＝2.4Hz),153.14,152.53(d,J＝12.2Hz),141.18(d,J＝242Hz),138.84(d,J＝18.1Hz),135.22,120.17,114.62,71.00,67.48,58.61,51.61,32.75,29.19,26.62,25.44.MS(ESI)m/z calcd for C 20 H 28 FN 4 O 4 [M+H] + 407.21,found407.20.
I-5:7- (5-fluoro-2- (4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) heptanoic acid methyl ester
1 H NMR(400MHz,DMSO-d 6 )δ8.85(s,1H),7.80(d,J＝3.7Hz,1H),7.61(d,J＝9.1Hz,2H),7.39(t,J＝5.2Hz,1H),6.82(d,J＝9.1Hz,2H),4.05–3.95(m,2H),3.68–3.60(m,2H),3.40(s,3H),3.30(s,3H),1.94(t,J＝7.3Hz,2H),1.59-1.48(m,4H),1.35–1.22(m,4H). 13 C NMR(101MHz,DMSO-d 6 )δ169.48,156.50(d,J＝2.4Hz),153.15,152.53(d,J＝12.3Hz),141.20(d,J＝242Hz),138.80(d,J＝17.8Hz),135.26,120.12,114.60,70.99,67.46,58.61,51.61,32.69,29.20,28.89,26.75,25.65.MS(ESI)m/z calcd for C 21 H 30 FN 4 O 4 [M+H] + 421.22,found421.23.
Example 3: preparation of phenylamino-substituted pyrimidine amino acid derivatives II
KOH (28.55g, 509mmol) and NH 2 OH. HCI (23.84g, 343mmol) was dissolved in 70mL and 120mL of anhydrous methanol, respectively, to give solution A and solution B. Dropwise adding the solution A into the solution B under the ice bath condition, separating out white solid, continuing to react for 1 hour, filtering the precipitate to obtain NH 2 OK solution. Compound I (0.50 mmol) was dissolved in 30mL NH 2 OK solution was stirred at room temperature for 2h. After the reaction is finished, the solvent is evaporated under reduced pressure, 10mL of water is added, the pH value is adjusted to 6-7 by 1M HCl, solid is precipitated, the mixture is filtered, and a filter cake is recrystallized by methanol/ethyl acetate to obtain the phenylamino substituted pyrimidine amino acid derivative II.
II-1:3- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxypropionamide
1 H NMR(400MHz,DMSO-d 6 )δ10.45(s,1H),8.87(s,1H),8.79(s,1H),7.82(d,J＝3.7Hz,1H),7.61(d,J＝9.1Hz,2H),7.38(t,J＝5.4Hz,1H),6.82(d,J＝9.1Hz,2H),4.04–4.00(m,2H),3.65–3.61(m,2H),3.58(dd,J＝13.1,6.8Hz,2H),3.31(s,3H),2.33(t,J＝7.2Hz,2H). 13 C NMR(101MHz,DMSO-d 6 )δ167.84,156.47(d,J＝2.5Hz),153.16,152.40(d,J＝12.3Hz),141.22(d,J＝242Hz),139.02(d,J＝18.6Hz),135.14,120.16,114.69,70.99,67.44,58.62,37.05,32.47.HRMS(ESI)m/z calcd for C 16 H 21 FN 5 O 4 [M+H] + 366.1578,found 366.1567.
II-2:4- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybutyramide
1 H NMR(400MHz,DMSO-d 6 )δ10.43(s,1H),8.83(s,1H),8.75(s,1H),7.81(d,J＝3.7Hz,1H),7.59(d,J＝9.0Hz,2H),7.43(t,J＝5.3Hz,1H),6.84(d,J＝9.0Hz,2H),4.08–3.97(m,2H),3.66–3.60(m,2H),3.37–3.32(m,2H),3.30(s,3H),2.04(t,J＝7.4Hz,2H),1.89-1.75(m,2H). 13 C NMR(101MHz,DMSO-d 6 )δ169.28,156.49(d,J＝2.4Hz),153.15,152.53(d,J＝12.3Hz),141.19(d,J＝242Hz),138.93(d,J＝18.6Hz),135.19,120.09,114.69,71.00,67.44,58.62,40.09,30.42,25.42.HRMS(ESI)m/z calcd for C 17 H 23 FN 5 O 4 [M+H] + 380.1734,found 380.1724.
II-3:5- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxypentanamide
1 H NMR(400MHz,DMSO-d 6 )δ10.36(s,1H),8.81(s,1H),8.69(s,1H),7.80(d,J＝3.7Hz,1H),7.59(d,J＝9.0Hz,2H),7.42(t,J＝6.0Hz,1H),6.82(d,J＝9.0Hz,2H),4.05–3.98(m,2H),3.66–3.59(m,2H),3.30(s,3H),2.02-1.95(m,2H),1.59-1.51(m,4H). 13 C NMR(101MHz,DMSO-d 6 )δ169.48,156.49(d,J＝2.4Hz),153.14,152.49(d,J＝12.2Hz),141.18(d,J＝242Hz),138.86(d,J＝18.4Hz),135.19,120.09,114.62,70.99,67.43,58.62,32.59,30.47,29.06,23.27.HRMS(ESI)m/z calcd for C 18 H 25 FN 5 O 4 [M+H] + 394.1891,found 394.1881.
II-4:6- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxyhexanamide
1 H NMR(400MHz,DMSO-d 6 )δ10.36(s,1H),8.82(s,1H),8.69(s,1H),7.80(d,J＝3.8Hz,1H),7.60(d,J＝9.1Hz,2H),7.39(t,J＝5.3Hz,1H),6.82(d,J＝9.1Hz,2H),4.04-4.02(m,2H),3.66–3.62(m,2H),3.30(s,3H),1.96(t,J＝7.3Hz,2H),1.62–1.49(m,4H),1.35–1.25(m,2H). 13 C NMR(101MHz,DMSO-d 6 )δ169.51,156.51(d,J＝2.4Hz),153.14,152.53(d,J＝12.2Hz),141.18(d,J＝242Hz),138.82(d,J＝18.1Hz),135.22,120.07,114.62,71.00,67.46,58.61,32.75,29.11,26.62,25.44.HRMS(ESI)m/z calcd for C 19 H 27 FN 5 O 4 [M+H] + 408.2047,found408.2035.
II-5:7- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxyheptanamide
1 H NMR(400MHz,DMSO-d 6 )δ10.33(s,1H),8.82(s,1H),8.67(s,1H),7.79(d,J＝3.8Hz,1H),7.60(d,J＝9.0Hz,2H),7.39(t,J＝5.2Hz,1H),6.81(d,J＝9.0Hz,2H),4.07–3.97(m,2H),3.67–3.59(m,2H),3.30(s,3H),1.94(t,J＝7.3Hz,2H),1.59-1.46(m,4H),1.36–1.22(m,4H). 13 C NMR(101MHz,DMSO-d 6 )δ169.46,156.50(d,J＝2.4Hz),153.13,152.53(d,J＝12.3Hz),141.20(d,J＝242Hz),138.80(d,J＝17.8Hz),135.23,120.10,114.60,70.99,67.46,58.61,32.69,29.22,28.89,26.75,25.65.HRMS(ESI)m/z calcd for C 20 H 29 FN 5 O 4 [M+H] + 422.2204,found422.2193.
Experimental example 4: the phenylamino substituted pyrimidine amino acid derivative prepared in the embodiment of the invention has FLT3 and HDAC inhibitory activity test and anti-proliferation activity test experiment on tumor cells
1) Experiment of phenylamino substituted pyrimidine amino acid derivatives prepared in the embodiment of the invention on FLT3 inhibitory activity
Experimental materials and instruments: the FLT3 kinase inhibitory activity assay was performed with the assistance of Eurofins Pharma, UK.
The experimental method comprises the following steps: all phenylamino substituted pyrimidine amino acid derivatives used in the test were formulated into 50-fold final test concentrations of working solutions using DMSO in water. Compound working solution was first added as a first component to the test wells, followed by kinase buffer diluted FLT3 kinase solution. The kinase reaction is initiated by the addition of Mg/ATP. 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 the 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 using GragphadPrism6.0 software, fitting the curve with the logarithm of concentration as the abscissa and the inhibition as the middle ordinate 50 ) The value is obtained. The results of the assays for FLT3 kinase inhibitory activity are shown in Table 1.
Table 1 target compounds for FLT3 inhibitory activity
IC 50 : half maximal inhibitory concentration
A：IC 50 <1μM；B:1μM<IC 50 <10μM；C:10μM<IC 50
Table 1 shows that most compounds have stronger inhibitory activity (IC) on FLT3 50 <1μM)，In particular, compounds I-3, I-5, II-2, II-3, II-4 and II-5 showed FLT3 inhibitory activity superior to that of the positive control drug Tandutinib.
2) Experiment of the phenylamino substituted pyrimidine amino acid derivatives of the present invention on HDAC inhibitory activity:
experimental materials and methods: boc-Lys (acetyl) -AMC (HeLa cell nuclear extraction fluorescent substrate) was purchased from BachemAG, switzerland, tris-HCl, trypsin and EDTA were purchased from Sigma-Aldrich, TSA was purchased from Allantin technology Biochemical Co., ltd, and 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 spectral scanning multifunctional reading instrument.
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 the reaction solution at 390nm/460nm with a microplate reader to obtain 100% fluorescence intensity.
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 a compound 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 390nm/460nm of reaction solution by using an enzyme labeling instrument, wherein the value is the fluorescence of the aniline-substituted pyrimidine amino acid derivative 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 compound IC to be detected 50 Values, as shown in table 2.
TABLE 2 inhibitory Activity of phenylamino substituted pyrimidine amino acid derivatives of the invention on HDAC
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) The experiment of the growth inhibition activity of the phenylamino substituted pyrimidine amino acid derivative on tumor cells comprises the following steps:
experimental materials and instruments: blood system malignant tumor cells Jeko-1, MV4-11, K562, Z138, hel and Molt4 cell strains, RPMI-1640 culture medium, fetal calf 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 desk 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 adding 20. Mu.L of CCK-8 solution per well, placing 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 Value of
Anti-proliferative Activity of representative 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 tumor cells, and the results are shown in Table 3, and compounds II-3, II-4 and II-5 showed potent antiproliferative activity against MV4-11, hel and Molt4 cells, especially compound II-5 showed antiproliferative activity against 6 tested cells comparable to 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 (8)
1. A phenylamino substituted pyrimidine amino acid derivative or a pharmaceutically acceptable salt thereof, wherein the phenylamino substituted pyrimidine amino acid derivative is a phenylamino substituted pyrimidine amino acid derivative I with a structure shown in a general formula I or a phenylamino substituted pyrimidine amino acid derivative II with a structure shown in a general formula II:
wherein n is selected from 1,2,3,4,5,6,7,8,9, 10.
2. Phenylamino substituted pyrimidine amino acid derivatives according to claim 1, characterized in that the phenylamino substituted pyrimidine amino acid derivatives I are chosen from any of the following compounds:
3- (5-fluoro-2- (4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) propionic acid methyl ester,
4- (5-fluoro-2- (4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) butanoic acid methyl ester,
5- (5-fluoro-2- (4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) pentanoic acid methyl ester,
methyl 6- (5-fluoro-2- (4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) hexanoate,
methyl 7- (5-fluoro-2- (4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) heptanoate;
the phenylamino substituted pyrimidine amino acid derivative II is selected from any one of the following compounds:
3- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxypropionamide,
4- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxybutyramide,
5- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxypentanamide,
6- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxyhexanamide,
7- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) -N-hydroxyheptanamide.
3. A process for the preparation of phenylamino substituted pyrimidine amino acid derivatives according to claim 1 or 2, characterized in that phenylamino substituted pyrimidine amino acid derivatives I and phenylamino substituted pyrimidine amino acid derivatives II are prepared starting from compound 1 and amino acid methyl ester 2 by the following reaction schemes:
the preparation method comprises the following steps:
s1, dissolving a compound 1 and amino acid methyl ester 2 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 solids, filtering, and recrystallizing a filter cake with ethyl acetate to obtain an intermediate 3;
s2, dissolving the intermediate 3 in n-butyl alcohol, adding 4- (2-methoxyethoxy) 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 phenylamino substituted pyrimidine amino acid derivative I;
s3, preparing NH by using potassium hydroxide, hydroxylamine hydrochloride and anhydrous methanol 2 OK solution, dissolving phenylamino substituted pyrimidine amino acid derivative I 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 phenylamino substituted pyrimidine amino acid derivative II.
4. A pharmaceutical composition comprising the phenylamino substituted pyrimidine amino acid derivative of any one of claims 1 or 2, or a pharmaceutically acceptable salt thereof.
5. A pharmaceutical formulation comprising an active ingredient comprising the phenylamino substituted pyrimidine amino acid 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 an HDAC inhibitor, an FLT3 inhibitor and a HDAC/FLT3 dual inhibitor, and the active ingredient of the inhibitor is the phenylamino substituted pyrimidine amino acid derivative according to claim 1 or 2 or a pharmaceutically acceptable salt thereof.
7. Use of a compound which is the phenylamino substituted pyrimidine amino acid derivative of claim 1 or 2, or a pharmaceutically acceptable salt thereof, for the preparation of an anti-tumor medicament.
8. The use according to claim 7, wherein the tumor is a lymphoma or leukemia.
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