CN110437223B

CN110437223B - Thiazolone derivative of lenetinic acid and application thereof

Info

Publication number: CN110437223B
Application number: CN201910720100.4A
Authority: CN
Inventors: 李飞; 周新基; 张毅
Original assignee: Jiangsu Qianzhikang Biological Medicine Science And Technology Co ltd
Current assignee: Jiangsu Qianzhikang Biological Medicine Science And Technology Co ltd
Priority date: 2019-08-06
Filing date: 2019-08-06
Publication date: 2022-11-25
Anticipated expiration: 2039-08-06
Also published as: CN110437223A; WO2021023016A1

Abstract

4- [ 3-chloro-4- (cyclopropylaminocarbonyl) aminophenoxy]-7-methoxy-6-quinolinecarboxylthiazolone and application thereof, wherein the structure of the (E) corresponds to the general formula (I)

The medicine has weak efficacy under normal conditions, is unstable in a high-free-radical environment, and can generate molecules with stronger efficacy. The medicine has good drug effect and safety, and can be used for preparing medicines for treating tumors.

Description

Thiazolone derivative of lenetinic acid and application thereof

Technical Field

The invention belongs to the field of pharmacy, and provides a thiazolone derivative of lenvatinib acid 4- [ 3-chloro-4- (cyclopropylaminocarbonyl) aminophenoxy ] -7-methoxy-6-quinolinecarboxylthiazolone and an application thereof.

Background

Lenvatinib (Lenvatinib) is a Receptor Tyrosine Kinase (RTK) inhibitor, the receptors VEGFR1 (FLT 1), VEGFR2 (KDR), and VEGFR3 (FLT 4) that inhibit the kinase activity of Vascular Endothelial Growth Factor (VEGF). Lenvatinib also inhibits Fibroblast Growth Factor (FGF) receptors FGFR1,2,3 and 4, platelet-derived growth factor receptor alpha (PDGFR α), KIT, and RET9 (Int J cancer.2008,122, 664-671). Is suitable for treating patients with local recurrence or metastasis, progressive, radioactive iodine-refractory differentiated thyroid cancer and hepatocarcinoma. Lenvatinib can cause serious side effects including heart failure, thrombosis (arterial thromboembolic events), liver damage (hepatotoxicity), kidney damage (renal failure and injury), perforation of the gastrointestinal tract or abnormal connections between the stomach or intestines, changes in electrocardiographic activity (prolonged QT interval), hypocalcemia, concomitant headaches, seizures and visual changes (reversible leukoencephalopathy syndromes), severe bleeding (massive haemorrhage), risk of unborn babies if treated during pregnancy and impaired suppression of thyroid-stimulating hormone production (oncotarget.2016, 7: 44545-44557).

The applicant found that: the derivative of the lenvatinib, lenvatinib acid, has similar anti-tumor proliferation activity with lenvatinib, but the half-life period is short, and the drug effect is difficult to last.

Disclosure of Invention

The technical problem to be solved is as follows: aiming at the technical problems, the invention provides a thiazolone derivative of lenvatinib acid and application thereof. The medicine has similar anti-tumor cell proliferation activity with lenvatinib, and the activity is related to high free radical of tumor microenvironment. Because the concentration of free radicals in normal cells is lower, the medicine has lower toxicity to the normal cells. Has excellent anticancer effect and good safety, and can be used for preparing medicine for treating tumor.

The technical scheme is as follows: the thiazolone derivative of the lenetinic acid is named 4- [ 3-chloro-4- (cyclopropylaminocarbonyl) aminophenoxy ] -7-methoxy-6-quinolinecarboxylthiazolone, and the structure of the thiazolone derivative conforms to the general formula (I)

The application of the compound or the pharmaceutically acceptable salt thereof in preparing the medicine for treating the tumor.

The medicine for treating tumor contains the compound or its pharmaceutically acceptable salt as effective component.

When tumor cells are pretreated with N-acetylcysteine (NAC) as a free radical scavenger for 1 hour, the anti-tumor proliferation activity of the target compound of the present invention is significantly decreased, indicating that the anti-tumor proliferation activity of the target compound of the present invention is related to free radicals in the tumor cells. Because the concentration of free radicals in normal cells is lower, the medicine has lower toxicity to the normal cells.

Has the beneficial effects that: the 4- [ 3-chloro-4- (cyclopropylaminocarbonyl) aminophenoxy ] -7-methoxy-6-quinolinecarboxylthiazolone obtained by the invention has anti-tumor cell proliferation activity similar to that of lenvatinib, and has lower toxicity to normal cells. Has excellent anticancer effect and good safety, and can be used for preparing medicine for treating tumor.

Drawings

FIG. 1 is a scheme of the synthesis of Compound 1 of interest;

FIG. 2 is a schematic diagram of the in vitro inhibition effect of a target compound 1 on human liver cancer HepG2 tumor cell proliferation;

FIG. 3 is a schematic diagram showing the growth inhibition effect of a target compound 1 on human liver cancer HepG2 cell subcutaneous transplantation tumor;

FIG. 4 is a schematic diagram of the growth inhibition effect of a target compound 1 on human liver cancer HepG2 cell subcutaneous transplantation tumor-terminal tumor mass;

FIG. 5 is a schematic diagram showing the growth inhibition effect-body weight change of a target compound 1 on human liver cancer HepG2 cell subcutaneous transplantation tumor;

Detailed Description

The following examples are given to enable a person skilled in the art to fully understand the invention, but do not limit it in any way.

Example 1: synthesis of 4- [ 3-chloro-4- (cyclopropylaminocarbonyl) aminophenoxy ] -7-methoxy-6-quinolinecarboxylthiazolone (target compound), see FIG. 1;

1.1 Synthesis of methyl 4-chloro-7-methoxyquinoline-6-carboxylate (3)

2.56g of methyl 7-methoxy-4-oxo-1, 4-dihydroquinoline-6-carboxylate (2, synthesis according to the reference: J. Chem. China. J. Pharmacology, 2015, 285-288) was placed in a 50mL dry eggplant-shaped bottle, to which 20mL of thionyl chloride and 3 drops of DMF were added, and stirred at 125 ℃ under reflux for 3 hours. After the reaction, thionyl chloride was dried by spinning, 100mL of dichloromethane was added thereto until complete dissolution, poured into 200mL of saturated sodium bicarbonate solution, and stirred for 1h until no bubbles emerged. The extraction was washed once with 100mL of saturated brine, and the organic phase was collected and purified by flash column chromatography (DCM: meOH =300:1 to 100).

1.2 Synthesis of methyl 4- [ 3-chloro-4- (cyclopropylaminocarbonyl) aminophenoxy ] -7-methoxyquinoline-6-carboxylate (5)

Intermediate 4 (0.326g, 1.44mmol, synthesized by the reference method: journal of Chinese medicinal chemistry, 2015, 285-288) was weighed into a 25mL dry eggplant-shaped bottle, 10mL of DMF and anhydrous potassium carbonate (0.398g, 2.88mmol) were added thereto, and stirred at room temperature for 10min, and intermediate 3 (0.300g, 0.959mmol) was added thereto, and stirred at 75 ℃ for 30h under nitrogen. Cooled to room temperature, extracted twice with 20mL of dichloromethane, washed once with saturated brine and chromatographed by flash column (DCM: meOH = 80) to give 0.217g of a white solid.

1.3 Synthesis of 4- [ 3-chloro-4- (cyclopropylaminocarbonyl) aminophenoxy ] -7-methoxyquinoline-6-carboxylic acid (6)

Intermediate 5 (2.0 g, 4.53mmol) was weighed into a 50mL dry eggplant-shaped bottle, 10mL of 2N sodium hydroxide solution and 10mL of methanol solution were added thereto, and the mixture was stirred at 40 ℃ for 24 hours until the solid was completely dissolved. After the reaction was completed, the pH was adjusted to 6-7 with 2M hydrochloric acid solution, a large amount of pale yellow solid was precipitated, suction filtered, and the filter cake was washed with 10mL of methanol, and subjected to flash column chromatography (DCM: meOH = 20). ¹ H NMR(DMSO-d6,400MHz) δ(ppm):8.55(s,1H),8.18(s,1H),8.15(d,J＝2.6Hz,1H),7.53(s,1H),7.34(d,J＝3.5Hz,2H), 7.13(s,1H),6.46(s,1H),5.72(s,1H),3.86(s,3H),2.51(s,1H),0.61(s,2H),0.37(s,2H).

1.4 Synthesis of 4- [ 3-chloro-4- (cyclopropylaminocarbonyl) aminophenoxy ] -7-methoxyquinoline-6-carboxythiazolone (1)

To a 100mL eggplant-shaped flask were added 1.00g (2.3 mmol) of 4- [ 3-chloro-4- (cyclopropylaminocarbonyl) aminophenoxy ] -7-methoxyquinoline-6-carboxylic acid, 20mL of dichloromethane, 0.531g (2.5 mmol) of dicyclohexylcarbodiimide, and 0.315g (2.5 mmol) of 4-dimethylaminopyridine, and after stirring at room temperature for 20min, 0.242g (2.3 mmol) of 1, 3-thiazolidin-2-one was further added, and the mixture was further stirred at room temperature for 4 hours and filtered under suction. The filtrate was collected, the appropriate amount of water was added, extracted three times with dichloromethane, the organic phases were combined and dried with anhydrous sodium sulfate and purified by flash column chromatography (dichloromethane: methanol = 85) to give 0.625g of beige solid in 53% yield. Beating with ethyl acetate and petroleum ether gave 0.512g of white crystals in 43% yield.

¹ H NMR(400MHz,DMSO-D6)δ：8.61(d,J＝5.2Hz，1H),8.23(d,J＝9.1Hz,1H),8.14(s,1H), 7.94(s,1H),7.45(d,J＝2.7Hz,1H),7.40(s,1H),7.21(dd,J＝9.1,2.7Hz,1H),7.16(d,J＝2.6 Hz,1H),6.48(d,J＝5.2Hz,1H),4.22(s,2H),3.89(s,3H),3.45(s,2H),2.54(dt,J＝10.1,3.3Hz,1H), 0.63(d,J＝5.2Hz,2H),0.39(s,2H).

Example 2: screening of inhibitory Activity of target Compound on tyrosine kinase in vitro

Incubating a series of tested compounds with gradient concentration with an enzyme solution with specific concentration for 5min at room temperature, adding a proper amount of enzyme reaction substrate and ATP, starting an enzyme reaction process, adding a proper amount of reaction stop solution and detection solution into an enzyme reaction system after 30min, incubating for 1h, measuring the enzyme activity of the compounds with specific concentration on a multifunctional enzyme-labeling instrument, calculating the inhibition activity of the compounds with different concentrations on the enzyme activity, fitting the inhibition activity of the compounds with different concentrations according to a four-parameter equation, and calculating the IC50 value.

TABLE 1 inhibitory Activity of the target Compounds on tyrosine kinase in vitro (IC 50, nM)

Compound (I)	VEGFR2(KDR)
		1	26
Levatinib	4
		Levatinib acid	2

The results show that the target compound 1 has significantly lower in vitro tyrosine kinase inhibitory activity than lenvatinib or lenvatinib acid. Suggesting that the target compound 1 has relatively small cell activity under normal conditions.

Example 3: stability study of target compound in normal environment

1.0mL of acetonitrile solution of 100 mu moL L/L of target compound 1, adding 4.0mL of PBS buffer solution with pH of 7.4, uniformly mixing, and measuring the peak area of the target compound 1 by adopting high performance liquid chromatography; after standing at room temperature for 6 and 12 hours, the peak area of the target compound 1 was measured by high performance liquid chromatography and compared with the peak area at 0 hour. A relative value is obtained.

TABLE 2 stability examination of the target Compounds in PBS buffer at pH 7.4 for 6 and 12 hours

Compound numbering	6 hours	13 hours
			1	92％	85％

The above experimental results show that: the target compound 1 was stable in PBS buffer at pH 7.4 and was not easily hydrolyzed.

Example 4: target compound in H ₂ O ₂ Stability survey in an environment

1.0mL of acetonitrile solution of 100 mu moL L/L of lenvatinib acid, 4.0mL of PBS buffer solution with pH of 7.4 are added, the mixture is uniformly mixed, and the peak area of the lenvatinib acid is measured by adopting high performance liquid chromatography.

100. Mu. MoL/L acetonitrile solution of the objective Compound 1 (1.0 mL) to which 250. Mu. MoL/L H was added ₂ O ₂ 4.0mL of PBS buffer solution with the pH value of 7.4, uniformly mixing, and measuring the peak area of the target compound 1 by adopting high performance liquid chromatography; standing at room temperature for 0.5 and 1 hr, and measuring by high performance liquid chromatographyPeak area of target compound 1, and comparison with 0 hour peak area. A relative value is obtained.

TABLE 3 target compounds in H ₂ O ₂ Stability survey in an environment

Compound number	0.5 hour	1 hour (h)
			1	51％	25％
Levatinib acid	45％	65％

The results show that the target compound 1 is in H ₂ O ₂ Can be rapidly converted into the lenvatinib acid under the environmental condition.

Example 5: research on in-vitro inhibition effect of target compound on tumor cell proliferation under normal state and state of removing free radicals in advance

Collecting human liver cancer HepG2 tumor cells in logarithmic growth phase, adding 0.25% pancreatin, digesting for 3min, suspending the cells with 10% calf serum RPMI-1640, counting, and adjusting cell concentration to 1 × 10 ⁵ cell/mL, 100. Mu.L/well in Top-count specific 96 well cell culture plate, 37 ℃,5% ₂ And (5) incubating for 24h. The cells were then divided into experimental and control groups, and the experimental group was added with the target compound solution, each concentration was four replicates of wells, and the volume of each well was made up to 200 μ L. After each group of samples is added, respectivelyContinuing to culture for 72h, adding into each well before the culture is finished ³ H-TdR 3×10 ⁵ Bq, the CPM (count per minute) value of each well was determined using a Top-count. Calculating the half Inhibition Concentration (IC) of each experimental group drug to cell proliferation ₅₀ )。

Removing free radical groups in advance: collecting human liver cancer HepG2 tumor cells in logarithmic growth phase, adding 0.25% pancreatin, digesting for 3min, suspending the cells with 10% calf serum RPMI-1640, counting, and adjusting cell concentration to 1 × 10 ⁵ cell/mL, 100. Mu.L/well in Top-count specific 96 well cell culture plate, 37 ℃,5% ₂ And (5) incubating for 24h. Then, the cells are pretreated for 1 hour by using a free radical scavenger N-acetylcysteine (NAC, 20 mmoL/L) and divided into an experimental group and a control group, the target compound solution is added into the experimental group, each concentration is four-time-of-use, and each pore volume is complemented to 200 mu L. Continuously culturing for 72h, respectively, adding into each well before culturing ³ H-TdR 3×10 ⁵ Bq, the CPM (count per minute) value of each well was determined using a Top-count.

The results of the above experiments show (fig. 2): levatinib acid has stronger in-vitro inhibition effect on human liver cancer HepG2 tumor cell proliferation than Levatinib. The compound 1 of the embodiment of the invention has stronger in-vitro inhibition effect on the proliferation of human liver cancer HepG2 tumor cells in a normal state than lenvatinib, but has smaller in-vitro inhibition effect on the proliferation of human liver cancer HepG2 tumor cells under the condition of removing free radicals in advance.

Example 6: growth inhibition effect of target compound on human liver cancer HepG2 cell subcutaneous transplantation tumor

Selecting 6 weeks old BALB/c-nu female and male 15 cells respectively, collecting human liver cancer HepG2 cells at logarithmic growth phase, and culturing at 5 × 10 ⁶ Individual cell 0.2mL ^-1 Only a ^-1 The concentration of (4) is inoculated subcutaneously in the right underarm of a nude mouse.

The administration scheme is that the nude mice of the tumor model are divided into a negative control group, a positive control group (Levatinib, 30 mg/kg), a low dose group (compound 1, 15 mg/kg), a medium dose group (compound 1, 30 mg/kg) and a high dose group (compound 1, 60 mg/kg), the nude mice are respectively administrated by stomach irrigation (1 time/day) for 2 weeks, the nude mice body mass is measured once every 3 days, the tumor major diameter and the tumor minor diameter are measured by a vernier caliper, and a subcutaneous tumor growth curve is drawn. And (4) killing the nude mice by cervical dislocation after the last administration for 24 hours, stripping subcutaneous transplanted tumors, and weighing the mass of a terminal tumor body.

The inhibition effect is shown in figure 3, the terminal tumor mass is shown in figure 4, and the weight change is shown in figure 5: after administration, each group showed significant tumor growth inhibition, and the high dose group showed better therapeutic effect. All the test groups had no significant difference in body weight of nude mice, but all were smaller than the negative control group.

The above examples are only for illustrating the technical idea and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the content of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims

1. The thiazolone derivative of the lenetinic acid is characterized by being specifically named as 4- [ 3-chloro-4- (cyclopropylaminocarbonyl) aminophenoxy ] -7-methoxy-6-quinolinecarboxylthiazolinone, and the structure of the thiazolone derivative is shown as the formula (I):

2. use of a compound of claim 1 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of a tumour.

3. A drug for treating tumor, characterized in that the active ingredient is the compound of claim 1 or a pharmaceutically acceptable salt thereof.