CN114591336A - 5-hydroxy-3-methylpyrido [2,3-d ] pyrimidine compound and preparation and application thereof - Google Patents

Abstract

5-hydroxy-3-methylpyrido [2,3-d ] of the invention]A pyrimidine compound, a preparation method and an application thereof, which belong to the technical field of medicine, in particular to 3-methylpyrido [2,3-d]The pyrimidine-2, 4,7(1H,3H,8H) -trione compound has a structural general formula shown in the formula (I): r₁Is hydrogen, C1-C4 alkyl, halogen substituted C1-C4 alkyl, C1-C4 alkoxy, halogen substituted C1-C4 alkoxy, halogen, nitro, amino or C1-C4 alkylamino; r₂Is hydrogen, C1-C6 alkyl, C1-C4 alkyl substituted aryl, C1-C4 alkoxy, halogen substituted C1-C4 alkoxy or C1-C6 alkylamino; r₃Is hydrogen, C1-C6 alkyl, C1-C6 alkylamino, C1-C4 alkyl substituted or unsubstituted phenyl or benzyl; the compound of the invention has simple and convenient synthesis method, is suitable for industrial production and biological activity measurementExperiments show that the compounds have antitumor activity and can be applied to antitumor drugs.

Description

5-hydroxy-3-methylpyrido [2,3-d ] pyrimidine compound and preparation and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a 5-hydroxy-3-methylpyrido [2,3-d ] pyrimidine compound, and a preparation method and an application thereof.

Background

Mutations affecting codon 600 of the serine/threonine kinase BRAF are among the highest genetic variations in melanoma that recur most frequently. These mutations activate the downstream kinases MEK and ERK in the mitogen-activated protein kinase (MAPK) pathway, thereby enhancing cell proliferation and survival. BRAF^V600The discovery that mutations predict sensitivity to MAPK pathway inhibitors revolutionized the approach to melanoma treatment. MAPK pathway inhibitors at 80-90% BRAF^V600The clinical effect is achieved in patients with the mutant melanoma. However, resistance is commonly acquired in patients who respond to MAPK pathway inhibitors. In addition, 10-20% of patients do not respond due to innate or intrinsic resistance. (Garcia JI, Zaliba G, et al. Mamm Genome,1996,7(11):810-

Mitogen-activated protein kinase (MAPK) is a tandem of three highly conserved protein kinases, proline-directed serine/threonine kinase, activated by dual phosphorylation. MAPK affects many cellular processes, such as proliferation, tumorigenesis, development and differentiation, cell cycle and cell death. Three major MAPK cascades are found in mammalian cells: NK, p38 and ERK cascades, the basic structure of each cascade is very similar. MAPK is activated by phosphorylating its substrate, which in most cases is a transcription factor or another protein kinase. MAPK is activated by dual phosphorylation of MAPKK at the motif threonine-X-tyrosine, which itself is activated by MAPKKK at two conserved serine residues. The MAPK pathway inhibitor small molecule drugs which are on the market at present mainly comprise:

trametinib (Trametinib) is an oral small molecule ATP noncompetitive selective MEK1 and MEK2 inhibitor. In preclinical studies, phosphorylated extracellular signal-regulated kinase (Perk) -1/2 was potently inhibited. The same was true for V600E and V600K mutant patients, approved by the FDA for the treatment of BRAF mutant metastatic melanoma 5 months in 2013. (Grimaldi. AM, Simeone E, Ascieto P A. Current Opinion in Oncology,2014,26(2):196-

Cobicistinib (Cobimetinib) was approved by the FDA as a second MEK inhibitor on month 11 of 2015 as a single agent or in combination with vemurafenib for treatment of patients with BRAFV600E or V600K mutated unresectable or metastatic melanoma. A quick review for 6 months was received by the FDA's priority review program and orphan designation was obtained. (Signorelli J, Gandhi AS. Annals of Pharmacotherapy,2016:146-

Disclosure of Invention

The invention aims to provide a 5-hydroxy-3-methylpyrido [2,3-d ] pyrimidine compound and a preparation method and application thereof, in particular to a 5-hydroxy-3-methylpyrido [2,3-d ] pyrimidine-2, 4,7(1H,3H,8H) -trione compound, which is correspondingly applied to the preparation of drugs for treating or/and preventing tumor diseases related to MAPK pathways such as B-Raf kinase, Ras/Raf/MEK/ERK signal pathway, p38MAPK signal pathway, JNK-SAPK signal pathway and the like, and also comprises the application of the compound as a multi-target inhibitor in the aspect of tumor resistance.

5-hydroxy-3-methylpyrido [2,3-d ] pyrimidines, in particular to 5-hydroxy-3-methylpyrido [2,3-d ] pyrimidine-2, 4,7(1H,3H,8H) -triones shown in formula I, prodrugs and pharmaceutically acceptable salts thereof;

R₁is hydrogen, C1-C4 alkyl, halogen substituted C1-C4 alkyl, C1-C4 alkoxy, halogenSubstituted C1-C4 alkoxy, halogen, nitro, amino, or C1-C4 alkylamino;

R₂is hydrogen, C1-C6 alkyl, C1-C4 alkyl substituted aryl, C1-C4 alkoxy, halogen substituted C1-C4 alkoxy or C1-C6 alkylamino;

R₃is hydrogen, C1-C6 alkyl, C1-C6 alkylamino, C1-C4 alkyl substituted or unsubstituted phenyl or benzyl;

R₁、R₂and R₃May be one or more.

Further, the compounds represented by formula I, prodrugs and pharmaceutically active metabolites thereof, and pharmaceutically acceptable salts thereof, according to the present invention:

R₁is hydrogen, methyl or ethyl;

R₂is hydrogen, methyl or cyclopropyl;

R₃is hydrogen, methyl, benzyl or 2-methylbenzyl.

Further, the following compounds are preferred in the present invention:

6-benzyl-8-cyclopropyl-5-hydroxy-1, 3-dimethylpyrido [2,3-d ] pyrimidine-2, 4,7(1H,3H,8H) -trione (XQB01), having the formula I-1:

5-hydroxy-1, 3, 8-trimethyl-6- (2-methylbenzyl) pyrido [2,3-d ] pyrimidine-2, 4,7(1H,3H,8H) -trione (XQB02), having the formula I-2 below:

8-cyclopropyl-5-hydroxy-1, 3-dimethyl-6- (2-methylbenzyl) pyrido [2,3-d ] pyrimidine-2, 4,7(1H,3H,8H) -trione (XQB03), having the formula I-3 below:

5-hydroxy-1, 3-dimethyl-6- (2-methylbenzyl) pyrido [2,3-d ] pyrimidine-2, 4,7(1H,3H,8H) -trione (XQB04), having the formula I-4 below:

6-benzyl-5-hydroxy-3, 8-dimethylpyrido [2,3-d ] pyrimidine-2, 4,7(1H,3H,8H) -trione (XQB05), which has the following structural formula I-5:

6-benzyl-1-ethyl-5-hydroxy-3, 8-dimethylpyrido [2,3-d ] pyrimidine-2, 4,7(1H,3H,8H) -trione (XQB06), having the formula I-6:

6-benzyl-8-cyclopropyl-1-ethyl-5-hydroxy-3-methylpyrido [2,3-d ] pyrimidine-2, 4,7(1H,3H,8H) -trione (XQB07), having the formula I-7:

a pharmaceutical composition comprising as active ingredient a compound of any one of the compounds of the present invention, prodrugs and pharmaceutically active metabolites thereof, and pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier or diluent.

"pharmaceutically acceptable salt" refers to conventional acid addition salts or base addition salts that retain the biological potency and properties of the compounds of formula I and are formed with non-toxic organic or inorganic acids or organic or inorganic bases. Acid addition salts include hydrochloride, hydrobromide, hydroiodide, nitrate, phosphate, sulfate, perchlorate, thiocyanate, bisulfate, persulfate, borate, formate, acetate, propionate, valerate, pivalate, hexanoate, heptanoate, octanoate, isooctanoate, undecanoate, laurate, palmitate, stearate, oleate, cyclopropionate, oxalate, malonate, succinate, maleate, fumarate, adipate, azelate, acrylate, strawberry, crotonate, glatironate, itaconate, sorbate, cinnamate, glycolate, lactate, malate, tartrate, citrate, tartrate, mandelate, diphenoxylate, troponate, ascorbate, gluconate, glucoheptonate, mandelate, dibenzolate, trogoplate, ascorbate, gluconate, glucoheptonate, and the like, Glucarate, mannonate, lactobionate, benzoate, phthalate, paraththalate, furoate, nicotinate, isonicotinate, salicylate, acetylsalicylate, caseinate, gallate, caffeate, ferulate, picrate, camphorate, camphorsulfonate, methanesulfonate, ethanesulfonate, propanesulfonate, benzenesulfonate, p-toluenesulfonate, sulfanilate, sulfamate, taurate, 2-hydroxyethanesulfonate, glycinate, alaninate, valine, leucine, isoleucine, phenylalanine, tryptophan, caseinate, aspartate, asparagine, glutamate, lysine, glutamine, methionine, serine, threonine, cysteine, proline, histidine, arginine, and salts thereof, Edetate, pyruvate, alpha-ketoglutarate, alginate, cyclopentanepropionate, 3-phenylpropionate, 3-cyclohexylpropionic acid, 2-naphthoate, 2-naphthalenesulfonate, pamoate, lauryl sulfate, glycerophosphate, lauryl sulfate, pectin oleate. Preferred acids for the formation of acid addition salts include hydrochloric acid, p-toluenesulfonic acid, methanesulfonic acid, maleic acid, malic acid, picric acid, citric acid, sulfanilic acid. Base addition salts include ammonium salts, alkali metal salts, such as sodium and potassium salts, alkaline earth metal salts, such as calcium and magnesium salts, salts with organic bases, such as dicyclohexylamine salts, N-methyl-D-glucamine salts, and basic nitrogen-containing groups may be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkyl sulfates such as dimethyl sulfate, diethyl sulfate, dibutyl sulfate and diamyl sulfate; long chain halides, such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; aralkyl halides such as benzyl and phenethyl bromides.

The invention also relates to a pharmaceutical composition for inhibiting MEK kinase and B-RAF kinase, which comprises the compound shown in the formula I or the derivative or the pharmaceutically acceptable acid addition salt thereof and a pharmaceutically acceptable carrier.

"pharmaceutically acceptable" such as pharmaceutically acceptable carriers, excipients, prodrugs, means pharmacologically acceptable and substantially non-toxic to a patient to whom a particular compound is administered.

"pharmaceutically active metabolite" refers to a pharmaceutically acceptable and effective metabolite of a compound of formula I.

The term "halogen" as used in the present invention includes fluorine, chlorine, bromine and iodine.

The compounds of the present invention may be administered to a patient by various methods, including orally in capsules or tablets, as sterile solutions or suspensions, and in some cases, intravenously in the form of solutions. The free base compounds of the present invention may be formulated and administered in the form of their pharmaceutically acceptable acid addition salts.

The compound is used as a small molecule inhibitor related to MAPK signal pathways such as B-Raf kinase, Ras/Raf/MEK/ERK signal pathways, p38MAPK signal pathways, JNK-SAPK signal pathways and the like in a brand new structure type, has the characteristics of novel structure type, capability of acting on multiple targets and the like, can be used for preparing medicaments for treating or/and preventing tumor diseases related to MAPK pathways such as lung cancer, liver cancer, melanoma, colon cancer, rectal cancer, breast cancer, ovarian cancer, cervical cancer and renal cancer, such as B-Raf kinase, Ras/Raf/MEK/ERK signal pathways, p38MAPK signal pathways, JNK-SAPK signal pathways and the like, and has good application value and development and application prospect.

The preparation method of the 5-hydroxy-3-methylpyrido [2,3-d ] pyrimidine compound comprises the following steps:

the compound XQB01-XQB07, pharmaceutically acceptable salts and prodrugs thereof can act on a Ras/Raf/MEK/ERK signal channel and inhibit phosphorylation of ERK1/2, thereby achieving the purpose of inhibiting tumor cell proliferation.

The invention has the beneficial effects that:

the invention provides a structure of a novel antitumor compound, which has potential patent medicine value, and the corresponding compound has simple preparation route, easy synthesis and low cost. Compared with the marketed drugs, the compound has more remarkable anti-tumor cell proliferation activity.

Detailed Description

The present invention is described in detail by the following examples. It should be understood, however, that the present invention is not limited to the following examples which are specifically set forth.

Example 1: 6-benzyl-8-cyclopropyl-5-hydroxy-1, 3-dimethylpyrido [2,3-d ] pyrimidine-2, 4,7(1H,3H,8H) -trione (XQB 01):

step I: 6-chloro-3-methyluracil (4.80g,30mmol), potassium carbonate (4.98g,36mmol), TBAB (0.2g,0.6mmol), 3mL of water and 50mL of acetone were weighed into a 250mL round-bottomed flask, stirred at room temperature until the solids dissolved first and then a large amount of solids precipitated, and then a solution of dimethyl sulfate (4.16g,33mmol) dissolved in 10mL of acetone was slowly dropped into the flask, stirred and refluxed for 2 hours. After TLC monitoring reaction is completed, solid is filtered out, mother liquor is subjected to reduced pressure distillation to obtain white solid, ether is used for pulping, and solid is filtered out again to obtain the product 6-chloro-1, 3-dimethyl uracil, wherein the yield is 5.10g, and the yield is 91.70%.

Step II: 6-chloro-1, 3-dimethyluracil (2.61g,15.00mmol), cyclopropylmethylamine (1.39g,19.50mmol), TEA (3.04g,30.00mmol) and 15mL of ethanol were weighed into a 100mL round-bottomed flask and stirred at reflux overnight for reaction. TLC monitored the reaction was complete, after ethanol was distilled off under reduced pressure, 10mL of diethyl ether was added and slurried, the solid was filtered off and dried to give 6- (cyclopropylamino) -1, 3-dimethylpyrimidine-2, 4(1H,3H) -dione as a pale yellow solid in 1.93g yield 89.12%.

Step III: weighing 6- (cyclopropylamino) -1, 3-dimethylpyrimidine-2, 4(1H,3H) -dione (1.95g,10mmol), diethyl benzylmalonate (3.25g,13mmol) and 10.00g diphenyl ether in a 50mL three-neck round-bottom flask, heating to 256 ℃ on a heating jacket, refluxing for 40min, monitoring by TLC that the raw materials are completely reacted, pouring the reaction liquid into 20mL petroleum ether, separating out a solid, performing suction filtration, drying, and purifying by column chromatography to obtain a pure product of 6-benzyl-8-cyclopropyl-5-hydroxy-1, 3-dimethylpyrido [2,3-d ]]Pyrimidine-2, 4,7(1H,3H,8H) -trione, white solid, yield 1.02g, 41.13% yield; m.p, 206.2 to 207.3 ℃; MS 354.14319[ M + H ]]⁺；¹H NMR(400MHz,DMSO-d₆)δ12.49(s,1H),7.24–7.18(m,4H),7.08–7.14(m,1H),3.67(s,2H),3.58(s,3H),3.34–3.39(m,1H),3.22(s,3H),1.19–1.11(m,2H),0.65–0.75(m,2H)。

Example 2: 5-hydroxy-1, 3, 8-trimethyl-6- (2-methylbenzyl) pyrido [2,3-d ] pyrimidine-2, 4,7(1H,3H,8H) -trione (XQB 02):

the compound 6-chloro-1, 3-dimethyluracil was obtained by the method of example 1.

Step II: 6-chloro-1, 3-dimethyluracil (2.61g,15.00mmol), methylamine (40%) in water (1.51g,19.50mmol), TEA (3.04g,30.00mmol) and 15mL of ethanol were weighed into a 100mL round-bottomed flask and stirred at reflux overnight for reaction. TLC monitored the reaction was complete, after ethanol was distilled off under reduced pressure, approximately 10mL of diethyl ether was added and slurried, the solid was filtered off and dried to give 1, 3-dimethyl-6- (methylamino) pyrimidine-2, 4(1H,3H) -dione as a pale yellow solid in 2.33g yield 91.67%.

Step III: weighing 1, 3-dimethyl-6- (methylamino) pyrimidine-2, 4(1H,3H) -diketone (0.85g,5mmol), diethyl 2- (2-methylbenzyl) malonate (1.72g,6.5mmol) and 10.00g diphenyl ether in a 50mL three-neck round-bottom flask, heating to 256 ℃ on a heating jacket, refluxing for 40min, monitoring the reaction of the raw materials by TLC, pouring the reaction liquid into 10mL petroleum ether, precipitating a solid, performing suction filtration, drying, and purifying by column chromatography to obtain a pure product of 5-hydroxy-1, 3, 8-trimethyl-6- (2-methylbenzyl) pyrido [2,3-d ] of a pure product]Pyrimidine-2, 4,7(1H,3H,8H) -trione, white solid, yield 0.47g, yield 38.09%; m.p, 298.2-304.8 ℃; MS 314.11292[ M + H ]]⁺；¹H NMR(400MHz,DMSO-d₆)δ12.31(s,1H),11.99(s,1H),7.27–7.15(m,4H),7.15–7.06(m,1H),3.67(s,2H),3.43(s,3H),3.21(s,3H)。

Example 3: 8-cyclopropyl-5-hydroxy-1, 3-dimethyl-6- (2-methylbenzyl) pyrido [2,3-d ] pyrimidine-2, 4,7(1H,3H,8H) -trione (XQB 03):

the compound 6- (cyclopropylamino) -1, 3-dimethylpyrimidine-2, 4(1H,3H) -dione was obtained by the method of example 1.

Step III: weighing 6- (cyclopropylamino) -1, 3-dimethylpyrimidine-2, 4(1H,3H) -dione (0.98g,5mmol), diethyl 2- (2-methylbenzyl) malonate (1.72g,6.5mmol) and 10.00g diphenyl ether in a 50mL three-neck round-bottom flask, heating to 256 ℃ on a heating jacket, carrying out reflux reaction for 40min, monitoring the reaction of raw materials by TLC, pouring the reaction liquid into 10mL petroleum ether, separating out a solid, carrying out suction filtration, drying, and purifying by column chromatography to obtain a pure product of 8-cyclopropyl-5-hydroxy-1, 3-dimethyl-6- (2-methylbenzyl) pyrido [2,3-d ] of a pure product]Pyrimidine-2, 4,7(1H,3H,8H) -trione, white solid, yield 0.49g, 37.76% yield; m.p, 167.7 to 170.8 ℃; MS 342.14429[ M + H ]]⁺；¹H NMR(400MHz,DMSO-d₆)δ12.52(s,1H),7.31–7.08(m,5H),3.97(q,J＝6.9Hz,2H),3.70(s,2H),3.48(s,3H),3.22(s,3H),1.36(t,J＝6.9Hz,3H)。

Example 4: 5-hydroxy-1, 3-dimethyl-6- (2-methylbenzyl) pyrido [2,3-d ] pyrimidine-2, 4,7(1H,3H,8H) -trione (XQB 04):

the compound 6-chloro-1, 3-dimethyluracil was obtained by the method of example 1.

Step II: 6-chloro-1, 3-dimethyluracil (2.61g,15.00mmol), aqueous ammonia (25%) solution (1.33g,19.50mmol), TEA (3.04g,30.00mmol) and 15mL of ethanol were weighed into a 100mL round-bottomed flask and stirred at reflux overnight for reaction. TLC monitored the reaction was complete, after distilling off the majority of ethanol under reduced pressure, approx.10 mL of diethyl ether was added and slurried, the solid was filtered off and dried to give 6-amino-1, 3-dimethylpyrimidine-2, 4(1H,3H) -dione as a pale yellow solid in 1.80g yield 86.89%.

Step III: weighing 6-amino-1, 3-dimethylpyrimidine-2, 4(1H,3H) -diketone (0.78g,5mmol), diethyl 2- (2-methylbenzyl) malonate (1.72g,6.5mmol) and 10.00g diphenyl ether in a 50mL three-neck round-bottom flask, heating to 256 ℃ on a heating sleeve, carrying out reflux reaction for 40min, monitoring by TLC that the raw materials are completely reacted, pouring the reaction liquid into 10mL petroleum ether, separating out a solid, carrying out suction filtration, drying, and purifying by column chromatography to obtain a pure product of 5-hydroxy-1, 3-dimethyl-6- (2-methylbenzyl) pyrido [2,3-d ] of a pure product]Pyrimidine-2, 4,7(1H,3H,8H) -trione, white solid, yield 0.48g, yield 40.05%; m.p, at 218.2-221.4 deg.C; MS 368.15973[ M + H ]]⁺；¹H NMR(400MHz,DMSO-d₆)δ12.48(s,1H),7.29–7.08(m,5H),4.41(q,J＝7.0Hz,2H),3.68(s,2H),3.34–3.39(m,1H),3.23(s,3H),1.13(q,J＝6.8Hz,5H),0.70(s,2H)。

Example 5: 6-benzyl-5-hydroxy-3, 8-dimethylpyrido [2,3-d ] pyrimidine-2, 4,7(1H,3H,8H) -trione (XQB 05):

the compound R₁Step I is therefore omitted and step II is carried out directly from the starting 6-chloro-3-methyluracil.

Step II: 6-chloro-3-methyluracil (4.00g,25mmol), methylamine (40%) in water (2.52g,32.50mmol), TEA (5.06g,50.00mmol) and 40mL ethanol were weighed into a 250mL round bottom flask and stirred at reflux overnight for reaction. TLC monitored the reaction was complete, after distilling off most of the ethanol under reduced pressure, approx.20 mL of diethyl ether was added and slurried, the solid was filtered off and dried to give 3-methyl-6- (methylamino) pyrimidine-2, 4(1H,3H) -dione as a pale yellow solid in 3.69g yield 89.21%.

Step III: weighing 3-methyl-6- (methylamino) pyrimidine-2, 4(1H,3H) -diketone (1.55g,10mmol), diethyl benzylmalonate (3.25g,13mmol) and 15.00g diphenyl ether in a 50mL three-neck round-bottom flask, heating to 256 ℃ on a heating jacket, refluxing for 40min, after TLC monitors that the raw materials are completely reacted, pouring the reaction liquid into 15mL petroleum ether, separating out a solid, performing suction filtration, drying, and purifying by column chromatography to obtain a pure product of 6-benzyl-5-hydroxy-3, 8-dimethylpyrido [2,3-d ]]Pyrimidine-2, 4,7(1H,3H,8H) -trione, white solid, yield 0.91g, 36.98% yield; m.p, 218.7-222.1 ℃ C; MS 342.14423[ M + H ]]⁺；¹H NMR(400MHz,DMSO-d₆)δ12.48(s,1H),7.15–6.93(m,4H),3.65(s,2H),3.49(s,3H),3.47(s,3H),3.24(s,3H),2.37(s,3H)。

Example 6: 6-benzyl-1-ethyl-5-hydroxy-3, 8-dimethylpyrido [2,3-d ] pyrimidine-2, 4,7(1H,3H,8H) -trione (XQB 06):

step I: 6-chloro-3-methyluracil (4.80g,30mmol), potassium carbonate (4.98g,36mmol), TBAB (0.2g,0.6mmol), 3mL of water and 50mL of acetone were weighed into a 250mL round-bottomed flask, stirred at room temperature until the solid dissolved first and then a large amount of solid precipitated, and then a solution of diethyl sulfate (5.09g,33mmol) dissolved in 10mL of acetone was slowly dropped into the flask, stirred and refluxed for 2 hours. After TLC monitoring reaction was complete, the solid was filtered off, the mother liquor was distilled under reduced pressure to give a white solid, which was slurried with diethyl ether and filtered off again to give 6-chloro-1-ethyl-3-methylpyrimidine-2, 4(1H,3H) -dione as a white solid with a yield of 4.69g and a yield of 87.73%.

Step II: 6-chloro-1-ethyl-3-methylpyrimidine-2, 4(1H,3H) -dione (4.70g,25.00mmol), methylamine (40%) in water (2.52g,32.50mmol), TEA (5.06g,50.00mmol) and 40mL of ethanol were weighed into a 250mL round-bottomed flask and stirred at reflux overnight. TLC monitored the reaction was complete, after ethanol was distilled off under reduced pressure, 20mL of diethyl ether was added and slurried, the solid was filtered off and dried to give 1-ethyl-3-methyl-6- (methylamino) pyrimidine-2, 4(1H,3H) -dione as a pale yellow solid in 3.66g yield 88.79%.

Step III: weighing 1-ethyl-3-methyl-6- (methylamino) pyrimidine-2, 4(1H,3H) -diketone (1.83g,10mmol), diethyl benzylmalonate (3.25g,13mmol) and 15.00g diphenyl ether in a 50mL three-neck round-bottom flask, heating to 256 ℃ on a heating jacket, refluxing for 40min, monitoring the reaction of the raw materials by TLC, pouring the reaction liquid into 15mL petroleum ether, separating out a solid, performing suction filtration, drying, and purifying by column chromatography to obtain a pure product of 6-benzyl-1-ethyl-5-hydroxy-3, 8-dimethylpyrido [2,3-d ]]Pyrimidine-2, 4,7(1H,3H,8H) -trione, white solid, yield 1.00g, 39.37% yield; m.p, 253.1 to 258.5 ℃; MS 368.15970[ M + H ]]⁺；¹H NMR(400MHz,DMSO-d₆)δ12.48(s,1H),7.17–6.88(m,4H),3.62(s,2H),3.60(s,3H),3.43–3.34(m,1H),3.24(s,3H),2.36(s,3H),1.16(d,J＝6.9Hz,2H),0.73(s,2H)。

Example 7: 6-benzyl-8-cyclopropyl-1-ethyl-5-hydroxy-3-methylpyrido [2,3-d ] pyrimidine-2, 4,7(1H,3H,8H) -trione (XQB 07):

the compound 6-chloro-1-ethyl-3-methylpyrimidine-2, 4(1H,3H) -dione was obtained by the method of example 6.

Step II: 6-chloro-1-ethyl-3-methylpyrimidine-2, 4(1H,3H) -dione (4.70g,25.00mmol), cyclopropylmethylamine (2.31g,32.50mmol), TEA (5.06g,50.00mmol) and 40mL ethanol were weighed into a 250mL round-bottomed flask and stirred at reflux overnight. TLC monitored the reaction was complete, after ethanol was distilled off under reduced pressure, 20mL of diethyl ether was added and slurried, the solid was filtered off and dried to give 6- (cyclopropylamino) -1-ethyl-3-methylpyrimidine-2, 4(1H,3H) -dione as a pale yellow solid in 3.78g yield 84.95%.

Step III: weighing 6- (cyclopropylamino) -1-ethyl-3-methylpyrimidine-2, 4(1H,3H) -diketone (2.09g,10mmol), diethyl benzylmalonate (3.25g,13mmol) and 15.00g diphenyl ether in a 50mL three-neck round-bottom flask, heating to 256 ℃ on a heating jacket, refluxing for 40min, monitoring by TLC that the raw materials are completely reacted, pouring the reaction liquid into 15mL petroleum ether, separating out a solid, performing suction filtration, drying, and purifying by column chromatography to obtain a pure product of 6-benzyl-8-cyclopropyl-1-ethyl-5-hydroxy-3-methylpyrido [2,3-d ]]Pyrimidine-2, 4,7(1H,3H,8H) -trione, white solid, yield 0.87g, 37.43% yield; m.p, 244.6 to 248.2 ℃; MS 328.12860[ M + H ]]⁺；¹H NMR(400MHz,DMSO-d₆)δ12.40(s,1H),7.15–6.81(m,4H),3.70(s,2H),3.48(s,3H),3.26(s,3H),2.36(s,3H)。

Pharmacological examples

Example 8: inhibitory Activity of test Compounds on MCF7, A375, SK-Mel-2 cell proliferation

(1) Experimental Material

Cell line: MCF7, A375 and SK-Mel-2 cells were plated in 96-well plates at densities of 4000, 5000 and 4000/well, 200 ul/well, and used after 24 h.

Number XQB01-XQB07 target compound: dissolved in DMSO, diluted with culture medium to obtain six different concentrations of 100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M and 3.125. mu.M, and stored at-20 deg.C for use, with the final concentration of DMSO in the culture medium being less than 0.1%.

Positive control drug: 5-Fluorouracil (5-Fu, Fluorouracil).

MTT: dissolved in PBS to 2mg/mL and stored at-20 ℃.

(2) Experimental methods

MCF7, A375, SK-Mel-2 cells were selected to evaluate the anti-tumor proliferation activity of test samples using the MTT method. MCF7, A375, SK-Mel-2 cell lines were cultured in DMEM medium containing 10% bovine serum (FBS). When the cells proliferated to 80-90% they were pooled and subsequently subcultured for no more than 20 passages, and then they were acclimatized for 24h before the next disposal. These cells were plated in 96-well plates and then incubated in a medium containing 5% CO₂Culturing in an incubator at constant temperature of 37 ℃ until the cells are completely attached to the wall. After 24h, various concentrations of representative compounds of the invention were added. After an additional 24h of incubation, MTT (2mg/mL) was added and incubation continued for 4 h. The culture medium was removed, the crystals were dissolved in DMSO, and the absorbance was measured at a wavelength of 570nm using a microplate reader (Thermo Multiskan GO, Thermo Fisher, USA). According to the formula: the cell growth inhibition rate is (1-drug group OD value/control group OD value) × 100%, the cell growth inhibition rate under the corresponding concentration is calculated, and the IC corresponding to the tested compound is calculated according to the logarithmic curve of the inhibition rate of the tested compound to the cell and the different concentrations of the tested compound₅₀The value is obtained. Representative compounds of the invention were tested according to the methods described above.

Formulation examples

The following formulation examples are merely illustrative of the scope of the invention and are not to be construed as limiting in any way. The active compounds described in the following examples refer to the compounds XQB01-XQB07 prepared in the above examples

Example 9: tablet formulation

25-1000mg of active compound, 45mg of starch, 35mg of microcrystalline cellulose, 4mL of polyvinylpyrrolidone (as a 10% aqueous solution), 4.5mg of sodium carboxymethylcellulose, 0.5mg of magnesium stearate, and 1mg of talc.

Example 10: suspending agent formulation

0.1-1000mg of active compound, 50mg of sodium carboxymethylcellulose, 1.25mg of syrup, 0.1mg of sodium benzoate, 25mg of flavoring agent and 5mg of coloring agent, and pure water is added to the volume of 5 mL.

Example 11: aerosol formulations

0.25mg of active compound, 25-75mL of ethanol and 70mg of propellant 22 (chlorodifluoromethane).

Example 12: suppository formula

250mg of active compound, 2000mL of saturated fatty acid glycerides.

Example 13: injectable formulation

50mg of active compound, 1000mL of isotonic saline solution.

Example 14: ointment formulation

0.025g of micronized active compound, 10g of liquid paraffin, and 100g of soft white wax.

Example 15: ointment formulation

0.025g of active compound, 5g of propylene glycol, 5g of sorbitan sesquioleate, 10g of liquid paraffin and 100g of soft white wax.

Example 16: oil-in-water cream formulation

0.025g of active compound, 5g of cetyl alcohol, 5g of glycerol monostearate, 10g of liquid paraffin, 2g of cetyl polyoxyethylene ether, 0.1g of citric acid, 0.2g of sodium citrate, 35g of propylene glycol and water to 100 g.

Example 17: oil-in-water cream formulation

0.025g of micronized active compound, 15g of soft white wax, 5g of liquid paraffin, 5g of cetyl alcohol, 2g of Sorbimcarol stearate (Tween 65 of the particular pharmaceutical excipient grade), 0.5g of sorbitan monostearate, 0.2g of sorbic acid, 0.1g of citric acid, 0.2g of sodium citrate, and water to 100 g.

Example 18: water-in-oil cream formulation

0.025g of active compound, 35g of soft white wax, 5g of liquid paraffin, 5g of sorbitan sesquioleate, 0.2g of sorbic acid, 0.1g of citric acid and 0.2g of sodium citrate, and water is added until the weight is 100 g.

Example 19: lotion formulation

0.25g of active compound, 0.5mL of isopropanol, 3mg of carboxyvinyl polymer, 2mg of NaOH and water to 1 g.

Example 20: injectable suspension formulations

10mg of the active compound, 7mg of sodium carboxymethylcellulose, 7mg of NaCl, 0.5mg of polyoxyethylene (20) sorbitan monooleate, 8mg of benzyl alcohol, and sterile water to 1 mL.

Example 21: aerosol formulation for oral and nasal inhalation

0.1% w/w active compound, 0.7% w/w sorbitan trioleate, 24.8% w/w trichlorofluoromethane, 24.8% w/w dichlorotetrafluoroethane and 49.6% w/w dichlorodifluoromethane.

Example 22: atomized solution formulation

7mg of active compound, 5mg of propylene glycol, water to 10 g.

Example 23: powder formulations for inhalation

Gelatine capsules were filled with a mixture of the following ingredients, micronised active compound 0.1mg, lactose 20mg and the powder was inhaled with the aid of an inhalation device.

Example 24: powder formulations for inhalation

The spheronized powder was loaded into a multi-dose powder inhaler containing 0.1mg of micronized active compound per dose.

Example 25: powder formulations for inhalation

The spheronized powder was loaded into a multi-dose powder inhaler containing 0.1mg of micronized active compound and 1mg of micronized lactose per dose.

Example 26: capsule formulation

1.0mg of active compound, 321mg of small sugar spheres, 306.6 mg of Aquacoat ECD in ethyl cellulose aqueous dispersion, 0.5mg of acetyl tributyl citrate, 800.1 mg of Tween-E, 100-5517.5 mg of acrylic resin aqueous dispersion Eudragit L, 1.8mg of triethyl citrate, 8.8mg of talcum powder and 0.lmg of defoaming agent MMS.

Example 27: capsule seedling formula

2.0mg of active compound, 305mg of small sugar spheres, 305.0 mg of aqueous ethyl cellulose dispersion Aquocoat ECD, 0.4mg of acetyl tributyl citrate, 800.14 mg of Tween-E, 12.6mg of aqueous acrylic resin dispersion Eudragit NE 30D, 10012.6 mg of aqueous acrylic resin dispersion Eudragit S, and 0.l6mg of talcum powder.

Example 28: enema formula

2mg of active compound, 25mg of sodium carboxymethylcellulose, 0.5mg of disodium ethylenediaminetetraacetate, 0.8mg of methylparaben, 0.2mg of propylparaben, 7mg of sodium chloride, 1.8mg of citric acid, 800.01 mg of tween-tween and 1mL of pure water.

Example 29: formulations containing liposomes

A. Preparation of the instillation formulation

Dipalmitoyl lecithin (45mg), dimyristoyl lecithin (7mg), dipalmitoyl phosphatidyl glycerol (1mg) and active compound (5mg) were placed in a glass tube, all components were dissolved in chloroform and N was used₂Adding an aqueous solution (0.9% NaCl) to the lipids, forming liposomes at a temperature above the phase inversion temperature of the lipids, the resulting suspension containing liposomes ranging in size from very small vesicles to 2 μm.

B. Preparation of formulations for inhalation

Liposomes were prepared as in example A, with an aqueous solution containing 10% lactose at a 7:3 lactose to lipid ratio. The liposome suspension was frozen with dry ice and freeze-dried, and the dried product was micronized, the resulting particles had a mass-mean aerodynamic diameter (MMAD) of 2 μm.

The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. Any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention, without departing from the technical solution of the present invention, still belong to the protection scope of the technical solution of the present invention.

Claims (9)

1. A 5-hydroxy-3-methylpyrido [2,3-d ] pyrimidine compound represented by formula I, a prodrug and a pharmaceutically active metabolite thereof, and a pharmaceutically acceptable salt thereof, wherein the structure of formula I is:

R₁is hydrogen, C1-C4 alkyl, halogen substituted C1-C4 alkyl, C1-C4 alkoxy, halogen substituted C1-C4 alkoxy, halogen, nitro, amino or C1-C4 alkylamino;

R₂is hydrogen, C1-C6 alkyl, C1-C4 alkyl substituted aryl, C1-C4 alkoxy, halogen substituted C1-C4 alkoxy or C1-C6 alkylamino;

R₃is hydrogen, C1-C6 alkyl, C1-C6 alkylamino, C1-C4 alkyl substituted or unsubstituted phenyl or benzyl;

R₁、R₂and R₃Is one or more.

2. A 5-hydroxy-3-methylpyrido [2,3-d ] pyrimidine compound, its prodrugs and pharmaceutically active metabolites, and pharmaceutically acceptable salts thereof, according to claim 1, wherein:

R₁is hydrogen, methyl or ethyl;

R₂is hydrogen, methyl or cyclopropyl;

R₃is hydrogen, methyl, benzyl or 2-methylbenzyl.

3. A 5-hydroxy-3-methylpyrido [2,3-d ] pyrimidine compound, its prodrugs and pharmaceutically active metabolites, and pharmaceutically acceptable salts thereof according to claim 2, wherein the compound is any one of the following compounds:

6-benzyl-8-cyclopropyl-5-hydroxy-1, 3-dimethylpyrido [2,3-d ] pyrimidine-2, 4,7(1H,3H,8H) -trione;

5-hydroxy-1, 3, 8-trimethyl-6- (2-methylbenzyl) pyrido [2,3-d ] pyrimidine-2, 4,7(1H,3H,8H) -trione;

8-cyclopropyl-5-hydroxy-1, 3-dimethyl-6- (2-methylbenzyl) pyrido [2,3-d ] pyrimidine-2, 4,7(1H,3H,8H) -trione;

5-hydroxy-1, 3-dimethyl-6- (2-methylbenzyl) pyrido [2,3-d ] pyrimidine-2, 4,7(1H,3H,8H) -trione;

6-benzyl-5-hydroxy-3, 8-dimethylpyrido [2,3-d ] pyrimidine-2, 4,7(1H,3H,8H) -trione;

6-benzyl-1-ethyl-5-hydroxy-3, 8-dimethylpyrido [2,3-d ] pyrimidine-2, 4,7(1H,3H,8H) -trione;

6-benzyl-8-cyclopropyl-1-ethyl-5-hydroxy-3-methylpyrido [2,3-d ] pyrimidine-2, 4,7(1H,3H,8H) -trione.

4. A pharmaceutical composition comprising as an active ingredient a compound of any one of 5-hydroxy-3-methylpyrido [2,3-d ] pyrimidines, prodrugs and pharmaceutically active metabolites thereof, and pharmaceutically acceptable salts thereof according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier or diluent.

5. The pharmaceutical composition according to claim 4, which is a pharmaceutical composition for inhibiting mitogen-activated protein kinase.

6. A process for the preparation of 5-hydroxy-3-methylpyrido [2,3-d ] pyrimidines as claimed in claim 3, wherein the preparation is carried out by the following route:

7. use of a 5-hydroxy-3-methylpyrido [2,3-d ] pyrimidine compound according to any of claims 1-3, a prodrug and a pharmaceutically active metabolite thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to any of claims 4-5 for the preparation of a mitogen-activated protein kinase, a mitogen-activated protein kinase, an extracellular regulated protein kinase, a Raf protein kinase inhibitor.

8. Use of a 5-hydroxy-3-methylpyrido [2,3-d ] pyrimidine according to any of claims 1 to 3, a prodrug and a pharmaceutically active metabolite thereof, and a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to any of claims 4 to 5 for the manufacture of a medicament for the treatment of cancer.

9. The use of claim 8, wherein the tumor is lung cancer, liver cancer, melanoma, colon cancer, rectal cancer, breast cancer, ovarian cancer, cervical cancer, or renal cancer.