CN115232076A - Phenylamino substituted pyrimidine amino acid derivative and preparation method and application thereof - Google Patents

Abstract

其中，n选自1，2，3，4，5，6，7，8，9，10。该类苯氨基取代的嘧啶氨基酸衍生物具有一定的FLT3/HDAC双重抑制活性和抗肿瘤活性，可应用于抗肿瘤药物的制备。The invention belongs to the field of organic compound synthesis and medical application, and discloses an phenylamino-substituted pyrimidine amino acid derivative and a preparation method and application. The structures of the phenylamino-substituted pyrimidine amino acid aminobenzoyl derivatives provided by the invention are shown in general formulas I and II:

wherein, n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The phenylamino-substituted pyrimidine amino acid derivatives have certain FLT3/HDAC dual inhibitory activity and anti-tumor activity, and can be used in the preparation of anti-tumor drugs.

Description

Pyrimidine amino acid derivatives substituted with phenylamino groups and preparation method and application thereof

technical field

The invention belongs to the field of organic compound synthesis and medical application, and relates to an phenylamino-substituted pyrimidine amino acid derivative and a preparation method and application thereof.

Background technique

The abnormal expression and mutation of histone deacetylase (HDAC) are closely related to the occurrence and development of tumors. The high expression of HDAC in hematological malignancies leads to histone hypoacetylation. Therefore, inhibiting HDAC can block the cell cycle. thereby inducing apoptosis. (Best Practice & Research Clinical Haematology 2012, 25(2): 191-200.) Vorinostat (SAHA) is the first HDAC inhibitor approved by the FDA for the treatment of cutaneous T-cell lymphoma. HDAC has become an important target for the treatment of hematological malignancies. FMS-likereceptortyrosine kinase 3 (FLT3) is a member of the type III receptor tyrosine kinase family, which is homologous to the stem cell growth factor c-Kit and acts on the hematopoietic stem/histomic region (see Blood 2017 ; 130(6):732-741 and CancerDiscov 2017;7(5):478-493), an important target for the treatment of hematological malignancies and autoimmune diseases. Preclinical and clinical studies have shown that dual inhibition of HDAC and FLT3 has a coordinated effect (see: Blood 2019; 134(Supplement_1): 5477-5477), so a novel HDAC/FLT3 dual-target drug with novel structure and druggability is designed and synthesized. It is of great significance for the treatment of hematological malignancies.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an phenylamino-substituted pyrimidine amino acid derivative, a preparation method and application thereof. The phenylamino-substituted pyrimidine amino acid derivative has HDAC/FLT3 dual inhibitory activity and antitumor activity.

In order to achieve the above purpose, the technical solution of the present disclosure is:

In the first aspect of the present invention, the present invention provides an phenylamino-substituted pyrimidine amino acid derivative or a pharmaceutically acceptable salt thereof, wherein the phenylamino-substituted pyrimidine amino acid derivative is a benzene having a structure shown in general formula I Amino-substituted pyrimidine amino acid derivative I or phenylamino-substituted pyrimidine amino acid derivative II of the structure represented by general formula II:

Among them, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

Preferably, n is 2,3,4,5,6.

Methyl 3-(5-fluoro-2-(4-(2-methoxyethoxy)phenyl)amino)pyrimidin-4-yl)amino)propanoate (I-1)

4-(5-Fluoro-2-(4-(2-methoxyethoxy)phenyl)amino)pyrimidin-4-yl)amino)butyric acid methyl ester (I-2)

Methyl 5-(5-fluoro-2-(4-(2-methoxyethoxy)phenyl)amino)pyrimidin-4-yl)amino)pentanoate (I-3)

Methyl 6-(5-fluoro-2-(4-(2-methoxyethoxy)phenyl)amino)pyrimidin-4-yl)amino)hexanoate (I-4)

Methyl 7-(5-fluoro-2-(4-(2-methoxyethoxy)phenyl)amino)pyrimidin-4-yl)amino)heptanoate (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-hydroxybutanamide (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-hydroxycaproamide (II-4)

7-((5-Fluoro-2-((4-(2-methoxyethoxy)phenyl)amino)pyrimidin-4-yl)amino)-N-hydroxyheptamide (II-5)

The parentheses after the names of the above 10 compounds are their corresponding codes. For the convenience of description and the brevity of expression, the codes in the above parentheses will be directly used in the following contents of this specification.

The compounds of the present invention can exist in free form or further in salt form, in order to improve water solubility and increase bioavailability.

The "pharmaceutically acceptable salts" in the present invention refer to conventional non-toxic salts, mainly including the salts formed by the basic amino groups of the compounds of the present application. These salts are well known to those skilled in the art, who can prepare any pharmaceutically acceptable salt provided by the knowledge in the art. In addition, the skilled artisan can choose one salt and leave out another based on factors such as solubility, stability, ease of formulation, and the like. Determination and optimization of these salts is within the experience of the skilled artisan.

In the second aspect of the present invention, the present invention also provides the preparation method of the phenylamino-substituted pyrimidine amino acid derivative I or the phenylamino-substituted pyrimidine amino acid derivative II, and the reaction scheme is as follows:

wherein, n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

Reagents and conditions: (a) N,N-diisopropylethylamine (DIPEA), isopropanol, 85°C, 4h; (b) 4-(2-methoxyethoxy)aniline, trifluoroacetic acid , n-butanol, 110℃, 12h; (c) potassium hydroxide, hydroxylamine hydrochloride, anhydrous methanol, 0℃～r.t., 2h.

Specific steps are as follows:

(1) Condensation of raw material 1 and amino acid methyl ester 2 to obtain intermediate 3.

(2) Intermediate 3 is condensed with 4-(2-methoxyethoxy)aniline under the condition of trifluoroacetic acid to obtain phenylamino-substituted pyrimidine amino acid derivative I.

(3) Anilino-substituted pyrimidine amino acid derivative I reacts with potassium hydroxylamine solution to obtain anilino-substituted pyrimidine amino acid derivative II.

(4) the preparation method of the pyrimidine amino acid derivative I substituted by phenylamino is as follows:

(i) Compound 1 and amino acid methyl ester 2 were dissolved in isopropanol, DIPEA was added, and the reaction was carried out at 85° C. for 4 hours. TLC detected that the reaction was complete, cooled to room temperature, a large amount of solid was precipitated, filtered, and the filter cake was recrystallized with ethyl acetate to obtain intermediate 3.

(ii) Intermediate 3 was dissolved in n-butanol, 4-(2-methoxyethoxy)aniline was added, trifluoroacetic acid was added dropwise to the solution, and the reaction was carried out at 110° C. for 12 h. TLC detected that the reaction was complete, cooled to room temperature, evaporated the solvent under reduced pressure, and chromatographed on silica gel column to obtain the phenylamino-substituted pyrimidine amino acid derivative I.

(5) the preparation method of the pyrimidine amino acid derivative II substituted by phenylamino is as follows:

An _NH2OK solution was prepared using potassium hydroxide and hydroxylamine hydrochloride and anhydrous methanol. The phenylamino-substituted pyrimidine amino acid derivative I was dissolved in NH ₂ OK solution and reacted at room temperature for 2 h. TLC detection, the reaction was complete, the solvent was evaporated under reduced pressure, water was added, the pH value was adjusted to 6-7 with dilute hydrochloric acid, a solid was precipitated, filtered, and the filter cake was recrystallized with methanol or ethyl acetate to obtain phenylamino-substituted pyrimidine amino acid derivatives Object II.

In the third aspect of the present invention, the present invention also provides a pharmaceutical composition comprising the above-mentioned phenylamino-substituted pyrimidine amino acid derivative or a pharmaceutically acceptable salt thereof.

The pharmaceutical composition of the phenylamino-substituted pyrimidine amino acid derivatives of the present invention can be administered in any of the following modes: oral administration, spray inhalation, rectal administration, nasal administration, vaginal administration, topical administration, parenteral administration Such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal or intracranial injection or infusion, or via an explanted reservoir, wherein oral, intramuscular, intraperitoneal or intravenous administration is preferred Way.

In the fourth aspect of the present invention, the present invention also provides a pharmaceutical preparation comprising the above-mentioned anilino-substituted pyrimidine amino acid derivative or a pharmaceutically acceptable salt thereof or the above-mentioned anilino-substituted pyrimidine amino acid derivative or its Compositions of pharmaceutically acceptable salts and pharmaceutically acceptable excipients and/or carriers.

The phenylamino-substituted pyrimidine amino acid derivative of the present invention or the pharmaceutical composition containing it can be administered in unit dosage form. The dosage form for administration can be a liquid dosage form, a solid dosage form. Liquid dosage forms can be true solutions, colloids, particulate dosage forms, emulsion dosage forms, and swirl dosage forms. Other dosage forms such as tablets, capsules, dropping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, suppositories, lyophilized powders, inclusions, embeddings, patches, wipes agent, etc.

The pharmaceutical combination or pharmaceutical preparation of the present invention may also contain commonly used carriers, and the pharmaceutically acceptable carriers herein include but are not limited to: ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin , buffer substances such as phosphates, glycerol, sorbate, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, Zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, beeswax, lanolin, etc. The content of the carrier in the pharmaceutical composition can be 1% by weight to 98% by weight, and usually accounts for about 80% by weight. For convenience, local anesthetics, preservatives, buffers, etc. can be dissolved directly in the vehicle.

Oral tablets and capsules may contain excipients such as binders, such as syrup, acacia, sorbitol, tragacanth, or polyvinylpyrrolidone, fillers such as lactose, sucrose, cornstarch, calcium phosphate, sorbitol, amino acids Acetic acid, lubricants such as magnesium stearate, talc, polyethylene glycols, silica, disintegrants such as potato starch, or acceptable emollients such as sodium lauryl sulfate. Tablets can be coated by methods known in pharmacy.

Oral liquids can be made into water and oil suspensions, solutions, emulsions, syrups, and can also be made into dry products, which can be supplemented with water or other suitable vehicles 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 gel, hydrogenated edible Fats, emulsifiers such as lecithin, sorbitan monooleate, acacia; or non-aqueous carriers (may contain edible oils) such as almond oil, oils such as glycerol, glycol, or ethanol; preservatives, Such as methyl or propyl paraben, 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 usually prepared from the compound and a sterile carrier. The preferred carrier is water. According to the selected carrier and drug concentration, the compound can be dissolved in the carrier or can be made into a suspension solution. When preparing an injection solution, the compound is first dissolved in water, filtered and sterilized, and then put into a sealed bottle or ampule.

It must be recognized that the optimal dosage and interval for administration of the Anilino-Substituted Pyrimidine Amino Acid Derivatives provided by the present invention are 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 this optimal dosage can be determined by conventional techniques. It must also be recognized that the optimal course of treatment, that is, the daily dose of the anilino-substituted pyrimidine amino acid derivatives provided by the present invention for a specified period of time can be determined by methods known in the art.

In the fifth aspect of the present invention, the present invention also provides the above-mentioned phenylamino-substituted pyrimidine amino acid derivatives or pharmaceutically acceptable salts thereof or the above-mentioned phenylamino-substituted pyrimidine amino acid derivatives or pharmaceutically acceptable salts thereof Application of the composition in the preparation of HDAC and/or FLT3 inhibitor medicines.

And, the present invention also provides the above-mentioned anilino-substituted pyrimidine amino acid derivative or a pharmaceutically acceptable salt thereof or a composition containing the above-mentioned anilino-substituted pyrimidine amino acid derivative or a pharmaceutically acceptable salt thereof in the preparation of the treatment of tumors application in medicines. The tumor is preferably lymphoma or leukemia.

The following experimental examples are only used to illustrate the technical effects of the present invention, but are not intended to limit the present invention.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In the following examples, the experimental methods without specific conditions are usually in accordance with conventional conditions or in accordance with the conditions suggested by the manufacturer.

Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be used in the methods of the present invention. Methods and materials for preferred embodiments described herein are provided for illustrative purposes only.

Example 1: Preparation of Intermediate 3

The starting material 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, heated at 85 ° C for reaction 4 Hour. After the reaction was completed, the reaction solution was cooled to room temperature, a large amount of solid was precipitated, filtered, and the filter cake was recrystallized from ethyl acetate to obtain intermediate 3.

Example 2: Preparation of phenylamino-substituted pyrimidine amino acid derivative I

Intermediate 3 (1 mmol, 1 eq) was dissolved in 30 mL of n-butanol, 4-(2-methoxyethoxy)aniline (1.1 mmol, 1.1 eq) was added, and 3 drops of trifluoroacetic acid were added dropwise to the solution , 110 ℃ heating reaction 12h. After the reaction is completed, the reaction solution is cooled to room temperature, the solvent is evaporated under reduced pressure, and silica gel column chromatography (dichloromethane/methanol=100:1-20:1) is used to obtain the phenylamino-substituted pyrimidine amino acid derivative I.

I-1: methyl 3-(5-fluoro-2-(4-(2-methoxyethoxy)phenyl)amino)pyrimidin-4-yl)amino)propanoate

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 calcd for C ₁₇ H ₂₂ FN ₄ O ₄ [M+H] ⁺ 365.14, found 365.16.

I-2: methyl 4-(5-fluoro-2-(4-(2-methoxyethoxy)phenyl)amino)pyrimidin-4-yl)amino)butyrate

¹ H NMR (400MHz, DMSO-d ₆ ) δ 8.82 (s, 1H), 7.82 (d, J=3.7 Hz, 1H), 7.59 (d, J=9.1 Hz, 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 CNMR(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: Methyl 5-(5-fluoro-2-(4-(2-methoxyethoxy)phenyl)amino)pyrimidin-4-yl)amino)pentanoate

¹ H NMR (400MHz, DMSO-d ₆ ) δ 8.81(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 ) δ 169.46, 156.49 (d, J=2.4Hz), 153.11, 152.49 (d, J=12.2 Hz),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 calcd for C ₁₉ H ₂₆ FN ₄ O ₄ [M+H] ⁺ 393.19, found 393.18.

1-4: Methyl 6-(5-fluoro-2-(4-(2-methoxyethoxy)phenyl)amino)pyrimidin-4-yl)amino)hexanoate

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). 13CNMR( ^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.

1-5: Methyl 7-(5-fluoro-2-(4-(2-methoxyethoxy)phenyl)amino)pyrimidin-4-yl)amino)heptanoate

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 CNMR(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 (28.55 g, 509 mmol) and NH ₂ OH·HCl (23.84 g, 343 mmol) were dissolved in 70 mL and 120 mL of anhydrous methanol, respectively, to give solution A and solution B. Under ice bath conditions, solution A was added dropwise to solution B, and a white solid was precipitated. The reaction was continued for 1 hour, and the precipitate was filtered to obtain NH ₂ OK solution. Compound I (0.50 mmol) was dissolved in 30 mL of NH ₂ OK solution and stirred at room temperature for 2 h. After the reaction was completed, the solvent was evaporated under reduced pressure, 10 mL of water was added, the pH value was adjusted to 6-7 with 1M HCl, a solid was precipitated, filtered, and the filter cake was recrystallized with 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 ) δ 10.45(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,found366.1567.

II-2: 4-((5-Fluoro-2-((4-(2-methoxyethoxy)phenyl)amino)pyrimidin-4-yl)amino)-N-hydroxybutyramide

¹ H NMR (400MHz, DMSO-d ₆ )δ 10.43(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 )δ 10.36(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, HRMS (ESI) m/z calcd for C ₁₈ H ₂₅ FN ₅ O ₄ [M+H] ⁺ 394.1891, found394.1881.

II-4: 6-((5-Fluoro-2-((4-(2-methoxyethoxy)phenyl)amino)pyrimidin-4-yl)amino)-N-hydroxycaproamide

¹ H NMR (400MHz, DMSO-d ₆ ) δ 10.36(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-hydroxyheptamide

¹ H NMR (400MHz, DMSO-d ₆ ) δ 10.33(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 derivatives prepared in the example of the present invention were tested for their inhibitory activity against FLT3 and HDAC, and their anti-proliferative activity against tumor cells.

1) Experiment on the inhibitory activity of the phenylamino-substituted pyrimidine amino acid derivatives prepared in the embodiment of the present invention to FLT3

Experimental Materials and Instruments: The FLT3 kinase inhibitory activity assay was assisted by Eurofins Pharma, a British company.

Experimental method: All the phenylamino-substituted pyrimidine amino acid derivatives used for the test were prepared into a working solution with a final test concentration of 50 times using an aqueous solution of DMSO. The compound working solution was first added to the test wells as the first component, and then the FLT3 kinase solution diluted in kinase buffer was added. With the addition of Mg/ATP, the kinase reaction is initiated. Subsequently, after incubation at room temperature for 40 minutes, 0.5% phosphoric acid solution was added to stop the reaction. 10 μL of the reaction solution was spotted onto a P30 filter paper pad, washed 4 times with 0.425% phosphoric acid for 4 minutes each, then washed once with methanol, followed by drying and scintillation counting.

In the experiment, a compound test group (C), a positive control group (P) and a blank control group (B) were set. No test compound was added in the positive control group, and DMSO was used instead (final concentration 2%), and other components were the same as the test group (100% residual kinase activity); in the blank control group, staurosporine was used instead of the test compound , eliminating kinase activity and establishing a baseline (0% residual kinase activity).

Using Gragphad Prism 6.0 software, taking the logarithm of the concentration as the abscissa and the inhibition rate as the middle coordinate, the curve was fitted to calculate the median inhibitory concentration (IC ₅₀ ) value. Table 1 shows the results of the assay for FLT3 kinase inhibitory activity.

Table 1 Inhibitory activity of target compounds on FLT3

IC ₅₀ : half inhibitory concentration

A: _IC50 <1μM; B: 1μM< _IC50 <10μM; C: 10μM< _IC50

The experimental data in Table 1 show that most of the compounds have strong inhibitory activity against FLT3 (IC ₅₀ <1 μM), especially compounds I-3, I-5, II-2, II-3, II-4 and II- 5 showed that the FLT3 inhibitory activity was initially better than that of the positive control drug Tandutinib.

2) Experiment on HDAC inhibitory activity of phenylamino-substituted pyrimidine amino acid derivatives of the present invention:

Experimental materials and methods: Boc-Lys(acetyl)-AMC (a fluorescent substrate for nuclear extraction from HeLa cells) was purchased from Bachem AG, Switzerland, Tris-HCl, Trypsin and EDTA were purchased from Sigma-Aldrich, and TSA was purchased from Aladdin Technology Biochemical Co., Ltd. , Glycerol, NaCl, and 96-well flat-bottom fluorescent plates were purchased from Hongfei Reagent Company.

Buffer: 15mM Tris-HCl (PH8.0), 250mM NaCl, 250μM EDTA, 10% glycerol

HDAC enzyme solution: Dilute with Buffer according to the test requirements.

Fluorescent substrate solution: The substrate was dissolved in DMSO to prepare a 30mM stock solution and stored at -20°C, and diluted to 300μM with HDACBuffer when used.

Stop solution: 10 mg/mL Trypsin, 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 2 μM TSA.

Microplate reader: Varioskan Flash spectral scanning multifunction reader.

Experimental steps:

100% control group: Mix 10 μL HDACs enzyme solution with 50 μL HDAC Buffer, incubate at 37 °C for 5 min, add 40 μL substrate solution, continue to incubate at 37 °C for 30 min, add 100 μL stop solution, continue to incubate at 37 °C for 20 min, use The fluorescence intensity of the reaction solution at 390nm/460nm was measured by a microplate reader, and the value was the fluorescence intensity of the 100% group.

Blank control group: Mix 40μL of substrate solution with 60μL of HDAC Buffer, incubate at 37°C for 30min, add 100μL of stop solution, continue to incubate at 37°C for 20min, use a microplate reader to measure the fluorescence intensity of the reaction solution at 390nm/460nm, Values are the fluorescence of blank group.

Experimental group: Mix 10 μL of HDACs enzyme solution with 50 μL of the test compound diluted with HDAC Buffer, incubate at 37 °C for 5 min, add 40 μL of substrate solution, continue to incubate at 37 °C for 30 min, add 100 μL of stop solution, and continue to incubate at 37 °C Incubate at ℃ for 20min, use a microplate reader to measure the fluorescence intensity of the reaction solution at 390nm/460nm, and the value is the fluorescence intensity of the phenylamino-substituted pyrimidine amino acid derivative of the present invention at this concentration.

The inhibition rates at different concentrations were calculated according to the formula, and S-curve fitting was performed with the origin software to calculate the IC ₅₀ values of the compounds of the present invention to be tested, as shown in Table 2.

Table 2 HDAC inhibitory activity of phenylamino-substituted pyrimidine amino acid derivatives of the present invention

A: _IC50 <1μM; B: 1μM< _IC50 <10μM; C: 10μM< _IC50

The experimental data in Table 2 showed that compounds II-3, II-4 and II- ₅ exhibited potent inhibitory effects on HDAC1 and HDAC6 (IC ₅₀ <1 μM), and their IC ₅₀ values were comparable to that of the positive drug SAHA. Notably, compounds II-3, II-4 and II-5 exhibited dual inhibitory effects on HDAC and FLT3.

3) Experiment on the growth inhibitory activity of the phenylamino-substituted pyrimidine amino acid derivatives of the present invention on tumor cells:

Experimental materials and instruments: hematological malignant tumor cells Jeko-1, MV4-11, K562, Z138, Hel and Molt4 cell lines, RPMI-1640 medium, fetal bovine serum, PBS buffer, sodium penicillin (10000units/mL)- Streptomycin sulfate (10mg/mL), CCK-8 kit, inverted optical microscope, cell incubator, ultra-clean workbench, desktop centrifuge, microplate reader, ultra-low temperature refrigerator.

experimental method:

The tumor cells in the logarithmic growth phase were inoculated into a 96-well culture plate, the number of cells was 1×10 ⁴ /well, and the cell culture medium of the tested compounds with different concentrations was added. At the same time, a positive control group and a DMSO blank control group were set up, and the concentration of DMSO was adjusted. ≤1‰. Three replicate wells were set for each concentration, and after the addition was completed, the cells were incubated in a 37°C, 5% _CO2 constant temperature incubator for 72h. Then, 20 μL of CCK-8 solution was added to each well, and the culture plate was placed in a 37°C, 5% CO ₂ constant temperature incubator for 1-4 hours, and the absorbance value at 450 nm wavelength was measured with a microplate reader. The control group was normalized, and the IC ₅₀ value was calculated using Prism 6.0 software.

Table 3 Antiproliferative activity of representative compounds on hematological malignancy cells

A: _IC50 <2μM; B: 2μM< _IC50 <20μM; C: 20μM< _IC50

We selected representative compounds with potent HDAC/FLT3 dual inhibitor activity and further studied their anti-proliferative activity on tumor cells. The results are shown in Table 3. Compounds II-3, II-4 and II-5 It showed potent anti-proliferative activity on MV4-11, Hel and Molt4 cells, especially the anti-proliferative activity of compound II-5 on the tested 6 cell lines was comparable to the positive drug SAHA.

The above descriptions are only preferred embodiments of the present disclosure, and are not intended to limit the present disclosure. For those skilled in the art, the present disclosure may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present disclosure shall be included within 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.