CN111393374A

CN111393374A - Oxo-dihydropyridazine derivative and application thereof in antitumor drugs

Info

Publication number: CN111393374A
Application number: CN202010380730.4A
Authority: CN
Inventors: 张建蒙
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-05-08
Filing date: 2020-05-08
Publication date: 2020-07-10

Abstract

The invention belongs to the technical field of medicines, and particularly relates to oxo-dihydropyridazine and application thereof in antitumor medicines. MTT results show that the oxo-dihydropyridazine derivative has the advantage of strong anti-tumor activity and can be used for preparing anti-tumor drugs.

Description

Oxo-dihydropyridazine derivative and application thereof in antitumor drugs

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to oxo-dihydropyridazine and application thereof in antitumor medicines.

Background

Malignant tumors arise due to the abnormal changes and accumulation of the genome that result in changes in the biological behavior of the cells. Under the combined action of various carcinogenic factors, certain cells of human tissues undergo gene mutation and accumulate continuously, and the normal regulation and control on the growth cycle is lost, so that the cells are abnormally proliferated, the normal tissue structure is damaged, and the normal functions of various human organs are influenced. The data of the national statistical administration in 2015 show that the incidence rate of malignant tumors in China accounts for about 21.8 percent of the incidence rate of malignant tumors in the world, and the total incidence rate is on the middle upper level. The mortality rate of malignant tumor is the first of the mortality rates of various diseases of Chinese residents, mainly comprises lung cancer, liver cancer, gastric cancer, esophagus cancer, colorectal cancer and breast cancer, and is a serious disease seriously threatening the public health of residents.

The cell cycle is a fundamental process in a living body, and the cell cycle controls the transition of cells from a resting phase or a cytokinesis phase to cell proliferation, and is strictly controlled at various check points to ensure stable inheritance of a genome. Cyclin-dependent kinases (CDKs) and cyclins (cyclins) are key molecules involved in the regulation of the cell cycle, CDKs are at the core of the overall regulatory network, and there is an overexpression of CDKs in most tumor cells. The research shows that the inhibition of the expression of CDK2-cyclin A/E can block the circulation of a tumor cell cycle, inhibit the proliferation of tumor cells and induce apoptosis.

In recent years, studies on CDK2 inhibitors have been reported, and small molecule compounds such as miciclib, Seliciclib, Dinaciclib and the like have entered clinical phase II or phase III studies and show good antitumor activity. Selective small molecule CDK2 inhibitors have become one of the hot spots in the development of antitumor targeted therapeutic drugs.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides the oxo-dihydro-pyridazine derivative and the application thereof in antitumor drugs.

In order to achieve the above object, the present invention provides oxodihydropyridazines represented by the general formula (I):

r in the general formula₁Is hydrogen, halogen, amino, cyano, (C)₁-C₆) Alkyl, (C)₂-C₆) Alkenyl, (C)₂-C₆) Alkynyl or (C)₁-C₆) An alkoxy group.

Said R₂Is hydrogen radical, halogen or (C)₁-C₆) An alkyl group.

The compounds of the general formula I according to the invention are preferably the following compounds, but these are not intended to limit the invention in any way:

the compound and the pharmaceutically acceptable salt, hydrate and solvate thereof can be applied to antitumor drugs.

The anti-tumor comprises anti-breast cancer, thyroid cancer, lung cancer or osteosarcoma and the like.

The derivatives of the invention comprising formula I may be synthesized by methods well known in the chemical arts, including, inter alia, methods according to the routes disclosed herein, in particular: diazotizing different substituted anilines, reacting with ethyl acetoacetate to obtain an intermediate 2, condensing with N, N-dimethyl phthalein amine dimethyl acetal (DMF-DMA) to obtain an intermediate 3, performing cyclization reaction on the intermediate 3 under the condition of weak base, performing hydrolysis reaction with strong base to obtain an intermediate 5, and performing amide reaction with substituted 2-amino-2-phenyl ethanol to obtain a target product. The specific synthetic route is as follows:

synthetic scheme 1 reagents and conditions: (a) ethyl acetate, NaNO₂,EtOH,0℃；(b)DMF-DMA,80℃；(c)EtOH,5％NaOH,80℃；(d)2N NaOH,MeOH/H₂O；(e)EDCI,HOBt,DIEA,rt。

The positive progress effects of the invention are as follows: the invention provides an oxo-dihydropyridazine derivative, a preparation method, a pharmaceutical composition and application thereof. MTT results show that the oxo-dihydropyridazine derivative has the advantage of strong anti-tumor activity and can be used for preparing anti-tumor drugs.

Detailed Description

The examples provided below are intended to illustrate but not limit the scope of the invention. The starting materials may generally be obtained from commercial sources or prepared using methods well known to those skilled in the art, or prepared according to the methods described herein. The reagents used are, without particular reference, analytically or chemically pure.

Example 1N- (2-hydroxy-1-phenylethyl) -4-oxo-1-phenyl-1, 4-dihydropyridazine-3-carboxamide.

Step 1 synthesis of intermediate 2.

Aniline (3.00g,32.21mmol) was added to the mixed solution of water and hydrochloric acid (1:1), stirred for 30 minutes, and then an aqueous solution of sodium nitrite (2.33g,33.82mmol) was slowly added dropwise to the reaction mixture, stirred at room temperature for 20 minutes to form a diazonium salt solution, which was used in the next reaction without purification.

Adding ethyl acetoacetate (4.19g,32.21mmol) and potassium acetate (31.61g,322.13mmol) into 100m L ethanol, stirring at room temperature for 10 minutes, slowly dropwise adding the prepared diazonium salt solution into the reaction solution, continuing to react at room temperature for 2 hours after dropwise adding, detecting that the reaction is finished by T L C, pouring the reaction solution into water, separating out a large amount of solid, and performing suction filtration to obtain 5.20g of yellow solid with the yield of 68.91%.

Step 2 synthesis of intermediate 3.

Adding the intermediate 2(5g,21.3mmol) into 60m L DMF-DMA, heating to 80 ℃ for reaction, detecting that the reaction is finished by T L C after 6h of reaction, cooling the reaction liquid to room temperature, adding 200m L petroleum ether, separating out a large amount of solid, and filtering to obtain 5.80g of yellow solid with the yield of 93.92%.

Step 3 synthesis of intermediate 4.

Dissolving the intermediate 3(5.0g,17.28mmol) in 100m L ethanol, adding 10m L5% NaOH aqueous solution, heating to reflux reaction, detecting the reaction progress by T L C, evaporating the solvent under reduced pressure after the reaction is finished, adding water to precipitate a large amount of solid, and performing suction filtration to obtain 3.6g of white solid with the yield of 85.3%.

Step 4 synthesis of intermediate 5.

Dissolving the intermediate 4(3g,12.28mmol) in ethanol, adding 10m L2N sodium hydroxide solution, stirring at room temperature for 2h, detecting reaction completion by T L C, evaporating the solvent under reduced pressure, adding water, adjusting pH to 4-5 with 3N hydrochloric acid, precipitating a large amount of solid, and filtering to obtain 2.4g of white solid with yield of 90.38%.

Step 5 Synthesis of N- (2-hydroxy-1-phenylethyl) -4-oxo-1-phenyl-1, 4-dihydropyridazine-3-carboxamide.

Dissolving the intermediate 5(1.0g,4.63mmol) in dried DMF, adding EDCI (0.98g,5.09mmol) and HOBt (0.69g,4.4mmol), reacting at room temperature for 1h, adding phenylglycinol (0.63g,4.63mmol) and DIEA (1.20g,9.25mmol), heating to 70 deg.C, reacting at 8 h.T L C, detecting reaction completion, cooling to room temperature, pouring the reaction liquid into 100m L water, precipitating solid, filtering, drying, purifying the crude product by silica gel column chromatography to obtain 0.96g white solid with yield of 61.9%.

¹H-NMR(400MHz,DMSO-d₆)9.00(d,J＝7.8Hz,1H),8.62(d,J＝7.8Hz,1H),7.85(d,J＝8.5Hz,2H),7.61(t,J＝7.9Hz,2H),7.34-7.25(m,6H),6.90(d,J＝7.8Hz,1H),4.92-4.88(m,1H),3.77(dd,J＝14.1,4.8Hz,1H),3.68(dd,J＝14.0,9.8Hz,1H).ESI-MS m/z:336.2[M+H]⁺.

Examples 2-9 were prepared according to the procedure for example 1, using differently substituted anilines as starting materials, respectively, and following 5 steps of diazotization, condensation, ring closure, hydrolysis, condensation, and the like.

Example 2N- (2-hydroxy-1-phenylethyl) -4-oxo-1- (4-fluorophenyl) -1, 4-dihydropyridazine-3-carboxamide.

¹H-NMR(400MHz,DMSO-d₆)8.98(d,J＝8.0Hz,1H),8.61(d,J＝8.2Hz,1H),7.51(d,J＝8.5Hz,2H),7.42(d,J＝8.0Hz,2H),7.28-7.24(m,5H),6.90(d,J＝7.8Hz,1H),4.92-4.87(m,1H),3.78(dd,J＝14.2,4.6Hz,1H),3.69(dd,J＝14.0,9.8Hz,1H).ESI-MS m/z:354.3[M+H]⁺.

Example 3N- (2-hydroxy-1-phenylethyl) -4-oxo-1- (4-chlorophenyl) -1, 4-dihydropyridazine-3-carboxamide.

¹H-NMR(400MHz,DMSO-d₆)9.01(d,J＝7.6Hz,1H),8.62(d,J＝7.8Hz,1H),7.61(d,J＝8.0Hz,2H),7.44(d,J＝7.6Hz,2H),7.31-7.25(m,5H),6.91(d,J＝7.8Hz,1H),4.92-4.87(m,1H),3.77(dd,J＝14.1,4.8Hz,1H),3.66(dd,J＝14.1,9.6Hz,1H).ESI-MS m/z:370.1[M+H]⁺.

Example 4N- (2-hydroxy-1-phenylethyl) -4-oxo-1- (4-bromophenyl) -1, 4-dihydropyridazine-3-carboxamide.

¹H-NMR(400MHz,DMSO-d₆)9.02(d,J＝7.6Hz,1H),8.61(d,J＝7.8Hz,1H),7.73(d,J＝7.5Hz,2H),7.39(d,J＝7.8Hz,2H),7.30-7.25(m,5H),6.90(d,J＝7.8Hz,1H),4.92-4.88(m,1H),3.74-3.69(m,2H).ESI-MS m/z:414.2[M+H]⁺.

Example 5N- (2-hydroxy-1-phenylethyl) -4-oxo-1- (4-methoxyphenyl) -1, 4-dihydropyridazine-3-carboxamide.

¹H-NMR(400MHz,DMSO-d₆)9.00(d,J＝7.8Hz,1H),8.62(d,J＝7.8Hz,1H),7.51(d,J＝7.7Hz,2H),7.30-7.24(m,5H),7.12(d,J＝8.1Hz,2H),6.91(d,J＝7.9Hz,1H),4.90-4.86(m,1H),3.81(s,3H),3.76(dd,J＝14.1,4.8Hz,1H),3.69(dd,J＝14.0,9.8Hz,1H).ESI-MS m/z:366.2[M+H]⁺.

Example 6N- (2-hydroxy-1-phenylethyl) -4-oxo-1- (3-chloro-4-fluorophenyl) -1, 4-dihydropyridazine-3-carboxamide.

¹H-NMR(400MHz,DMSO-d₆)9.01(d,J＝7.9Hz,1H),8.62(d,J＝7.8Hz,1H),7.75(d,J＝2.5Hz,1H),7.59(d,J＝7.9Hz,1H),7.34-7.25(m,6H),6.89(d,J＝7.8Hz,1H),4.92-4.89(m,1H),3.77-3.68(m,2H).ESI-MS m/z:388.3[M+H]⁺.

Example 7N- [1- (4-fluorophenyl) -2-hydroxyethyl) -4-oxo-1- (4-chlorophenyl) -1, 4-dihydropyridazine-3-carboxamide.

¹H-NMR(400MHz,DMSO-d₆)9.01(d,J＝7.6Hz,1H),8.62(d,J＝7.8Hz,1H),7.61(d,J＝8.0Hz,2H),7.44(d,J＝7.6Hz,2H),7.26(d,J＝7.6Hz,2H),7.16(d,J＝7.8Hz,2H),6.91(d,J＝7.8Hz,1H),4.93-4.87(m,1H),3.75(dd,J＝14.1,4.8Hz,1H),3.66(dd,J＝14.1,9.5Hz,1H).ESI-MS m/z:388.2[M+H]⁺.

Example 8N- [1- (4-chlorophenyl) -2-hydroxyethyl) -4-oxo-1- (4-chlorophenyl) -1, 4-dihydropyridazine-3-carboxamide.

¹H-NMR(400MHz,DMSO-d₆)9.01(d,J＝7.6Hz,1H),8.62(d,J＝7.8Hz,1H),7.61(d,J＝8.0Hz,2H),7.48-7.42(m,6H),6.91(d,J＝7.8Hz,1H),4.93-4.87(m,1H),3.74-3.68(m,2H).ESI-MS m/z:404.1[M+H]⁺.

Example 9N- [1- (4-fluorophenyl) -2-hydroxyethyl) -4-oxo-1- (4-methoxyphenyl) -1, 4-dihydropyridazine-3-carboxamide.

¹H-NMR(400MHz,DMSO-d₆)9.01(d,J＝7.8Hz,1H),8.60(d,J＝7.8Hz,1H),7.51(d,J＝7.7Hz,2H),7.26(d,J＝7.8Hz,2H),7.16-7.12(m,4H),6.91(d,J＝7.8Hz,1H),4.90-4.86(m,1H),3.81(s,3H),3.77(dd,J＝14.2,4.7Hz,1H),3.68(dd,J＝14.0,9.6Hz,1H).ESI-MS m/z:384.2[M+H]⁺.

Firstly, testing pharmacological activity:

CDK2 kinase inhibitory activity evaluation:

(1) compound preparation the compound powder was dissolved in 100% DMSO to prepare a 10mM stock solution.

(2) Preparation of Kinase 1 × Kinase buffer was prepared and used to prepare a CDK2 Kinase solution at 2.5 fold final concentration.

(3) CDK2 Kinase solution of 10 μ L at 2.5 times final concentration was added to the compound wells and the positive control wells, respectively, and 10 μ L of 1 × Kinase buffer.1000rpm was added to the negative control wells, followed by centrifugation for 30 seconds, shaking and mixing, followed by incubation at room temperature for 10 minutes.

(4) A mixed solution of 25/15 times final ATP concentration and Kinase substrate 18 was prepared using 1 × Kinase buffer, 10. mu. L was added to the wells to initiate the reaction, the 384 well plates were centrifuged at 1000rpm for 30 seconds, shaken well and incubated at room temperature for 30 minutes.

(5) Stopping the reaction, namely adding 30 mu L to stop the kinase reaction of the detection solution, centrifuging for 30 seconds at 1000rpm, shaking and uniformly mixing, and reading the conversion rate by using an enzyme-labeling instrument, wherein the calculation formula is as follows:

Inhibition％＝(Conversion％_max-Conversion％_sample)/(Conversion％_max-Conversion％_min)×100，

wherein Conversion%_maxIndicates positive control well Conversion reading, Conversion%_sampleIndicates the sample Conversion reading, Conversion%_minThe negative control well conversion readings are shown in table 1.

Table 1CDK2 kinase inhibitory activity.

Compound (I)	IC₅₀(μM)
		Example 1	2.6
Example 2	2.8
		Example 3	1.9
Example 4	10.5
		Example 5	0.9
Example 6	4.9
		Example 7	20.1
Example 8	0.8
		Example 9	2.9

Secondly, MTT method anticancer cell proliferation experiment:

cancer cells are inoculated to a 96-well plate, an RPMI1640 complete medium containing 5% CO2, 100U/m L penicillin and 100 mu g/m L streptomycin is applied to culture for 24h at 37 ℃, compounds with different concentrations are added, each concentration is set to be 5 multiple wells, after the medicine acts for 48h, the culture solution is discarded, and MTT reagent is used for measuring the cell activity.

The determination method comprises adding pre-prepared MTT reaction solution into 15 μ L/well, culturing for 4h, removing supernatant, adding DMSO into 100 μ L/well to dissolve the reduction product, reacting for 5min in dark, reading absorbance at 490nm, calculating cell activity, and determining absorbance of intervention/control wells as cell activity₅₀Refers to the concentration of inhibitor at which cell growth is inhibited by half, as shown in Table 2.

Table 2 MTT assay compounds were tested for their inhibitory activity against cancer cells.

While the invention has been described with reference to specific embodiments, modifications and equivalent arrangements will be apparent to those skilled in the art and are intended to be included within the scope of the invention.

Claims

1. Oxo-dihydropyridazine derivatives shown in a general formula I,

r in the general formula₁Is hydrogen radical, halogen, amino, cyano, (C)₁-C₆) Alkyl, (C)₂-C₆) Alkenyl, (C)₂-C₆) Alkynyl, (C)₁-C₆) Alkoxy group, (C)₁-C₆) Alkylthio, or substituted by halogen (C)₁-C₆) Alkyl or (C)₁-C₆) An alkoxy group;

said R₂Is hydrogen radical, halogen or (C)₁-C₆) An alkyl group.

2. Oxodihydropyridazine derivatives represented by the general formula I, selected from:

n- (2-hydroxy-1-phenylethyl) -4-oxo-1-phenyl-1, 4-dihydropyridazine-3-carboxamide,

N- (2-hydroxy-1-phenylethyl) -4-oxo-1- (4-fluorophenyl) -1, 4-dihydropyridazine-3-carboxamide,

N- (2-hydroxy-1-phenylethyl) -4-oxo-1- (4-chlorophenyl) -1, 4-dihydropyridazine-3-carboxamide,

N- (2-hydroxy-1-phenylethyl) -4-oxo-1- (4-bromophenyl) -1, 4-dihydropyridazine-3-carboxamide,

N- (2-hydroxy-1-phenylethyl) -4-oxo-1- (4-methoxyphenyl) -1, 4-dihydropyridazine-3-carboxamide,

N- (2-hydroxy-1-phenylethyl) -4-oxo-1- (3-chloro-4-fluorophenyl) -1, 4-dihydropyridazine-3-carboxamide,

N- [1- (4-fluorophenyl) -2-hydroxyethyl) -4-oxo-1- (4-chlorophenyl) -1, 4-dihydropyridazine-3-carboxamide,

N- [1- (4-chlorophenyl) -2-hydroxyethyl) -4-oxo-1- (4-chlorophenyl) -1, 4-dihydropyridazine-3-carboxamide,

N- [1- (4-fluorophenyl) -2-hydroxyethyl) -4-oxo-1- (4-methoxyphenyl) -1, 4-dihydropyridazine-3-carboxamide.

3. An application of the oxo-dihydro-pyridazine derivative shown in the general formula I as an anti-tumor drug.

4. The oxodihydropyridazine derivative according to claim 3, which is used as an antitumor drug, wherein the tumor is breast cancer, thyroid cancer, lung cancer or osteosarcoma.