CN115806523A

CN115806523A - Substituted quinoline compound and application thereof in preparation of antitumor drugs

Info

Publication number: CN115806523A
Application number: CN202211485572.4A
Authority: CN
Inventors: 张吉泉; 张娜娜; 王丽丽; 班玉娟; 吴春风; 董永喜
Original assignee: Guizhou Medical University
Current assignee: Guizhou Medical University
Priority date: 2022-11-24
Filing date: 2022-11-24
Publication date: 2023-03-17

Abstract

The invention belongs to the technical field of medicines, and relates to a substituted quinoline compound and application thereof in preparing antitumor medicines, wherein the inhibitor is a compound shown as a formula (I) obtained by virtually screening an mTOR inhibitor PI-103 serving as a shape questioning molecule from a ChemDiv library, or an isomer, a tautomer or a pharmaceutically acceptable salt thereof, and the structure of the formula (I) is as follows:

Description

Substituted quinoline compound and application thereof in preparation of antitumor drugs

Technical Field

The invention belongs to the technical field of medicines, and relates to a substituted quinoline compound and application thereof in preparing antitumor medicines.

Background

mTOR, as an important serine-threonine protein kinase downstream of PI3K/Akt, is involved in regulating cell growth, proliferation, survival and autophagy. Activation of the mTOR signaling pathway is closely associated with cancer, diabetes, alzheimer's disease, and autoimmune diseases. mTOR includes mTOR complex 1 (mTORC 1) and complex 2 (mTORC 2). mTORC1 fully activates AKT, regulating cell growth and energy metabolism, and is sensitive to rapamycin, mainly by phosphorylating Ser 473. And mTORC2 is mainly involved in cytoskeleton reconstruction and cell survival and is not sensitive to rapamycin. Eukaryotic translation initiation factor 4E-binding protein 1 (4 EBP 1) and ribosomal protein S6 kinase (S6 Ks) belong to two signaling pathways downstream of mTOR that are involved in mRNA translation and protein synthesis. Highly phosphorylated 4EBP1 can release elF4E, promote binding of elF4G and eIF4E, and initiate translation of the relevant mRNA. The S6Ks protein is another downstream target protein of the mTOR pathway, encoded by two cellular genes, S6K1 and S6K 2. Many studies have shown that S6K1 activates ribosomes involved in protein synthesis by promoting translation of mRNA and affects cell growth, differentiation, and autophagy. Since mTOR signaling pathways control cellular metabolism, growth, proliferation and survival, excessive mTOR activation leads to increased cellular metabolism, increased cell life, and can directly or indirectly induce metabolic diseases, cancer and aging diseases, and inhibition of this state is effective in delaying or treating cancer, cardiovascular damage, and other diseases caused by excessive mTOR activation. Therefore, the mTOR inhibitor is a hot field of the current targeted antitumor drug research, and a plurality of mTOR inhibitors (with a specific structural formula shown in figure 1) are currently used in clinical trials or on the market.

mTOR inhibitors can be divided into allosteric inhibitors and ATP competitive inhibitors by protein pocket. Allosteric mTOR inhibitors are mainly rapamycin and its derivatives (Rapalogs). However, the clinical efficacy of Rapalogs in some cancers is not expected, and the negative feedback of mTORC1 to AKT will be activated to greatly reduce the efficacy and easily generate drug resistance. In addition, rapalogs have the defects of unstable structure, difficult synthesis and the like. ATP-competitive mTOR inhibitors include PI-103, WAY-001, AZD8055, PQR620, and the like. The ATP competitive mTOR inhibitor regulates an mTOR downstream signal pathway by competing with an upstream molecule of the mTOR kinase for an ATP binding site on the mTOR kinase, and has the advantages of stable chemical structure, high kinase inhibition activity, good drug-like property and the like. Although ATP-competitive mTOR inhibitors overcome some of the disadvantages of rapamycin, no inhibitors are currently available on the market, most are in clinical trials, and there are structural or mechanism-related side effects. Therefore, a novel ATP competitive inhibitor with novel structure type, high kinase inhibition activity and selectivity and good drug-like property needs to be further developed, and a candidate drug is provided for research of an anti-tumor innovative drug targeting mTOR. Meanwhile, theoretical reference is provided for the research of mTOR-based innovative drugs.

Disclosure of Invention

The invention provides a substituted quinoline compound and application thereof in preparing antitumor drugs aiming at the defects of the prior art.

The method is realized by the following technical scheme:

in one aspect of the present invention, a substituted quinoline compound is a compound represented by formula (I) or an isomer, a tautomer, or a pharmaceutically acceptable salt thereof, wherein the structure of formula (I) is as follows:

in another aspect of the present invention, there is provided a pharmaceutical composition comprising at least one pharmaceutically acceptable adjuvant, adjuvant or carrier, and a therapeutically effective amount of at least one of the above substituted quinoline compounds.

In another aspect of the present invention, an application of the above substituted quinoline compound or the above pharmaceutical composition in the preparation of an antitumor drug is provided.

In particular to an application of the substituted quinoline compound or the pharmaceutical composition in preparing a medicament for preventing and/or treating proliferative diseases with mTOR kinase action.

The value range of the IC50 value of the effective therapeutic dose is 8.90 +/-0.09 mu M.

In still another aspect of the present invention, a method for obtaining the above substituted quinoline compound is provided. PI-103 is an ATP-competitive mTOR kinase inhibitor obtained by high throughput screening from Astella. The structure is simple, the molecular weight is small, the modification is easy, and the crystal structure of the protein 4JT6 (https:// www.rcsb.org /) is analyzed, so that the accuracy of the combination mode comparison and analysis of the protein is improved. The compound is obtained by taking an ATP competitive mTOR inhibitor PI-103 as a shape questioning molecule and virtually screening from a ChemDiv library.

Specifically, the method for obtaining the substituted quinoline compound comprises the steps of comparing the shapes of molecules in a ChemDiv library with an mTOR inhibitor PI-103, selecting molecules with similarity larger than 80%, carrying out high-precision (XP) docking on the molecules and an mTOR kinase protein (protein code: 4JT 6), selecting 10 promising compounds according to docking scoring and combination mode analysis, carrying out in-vitro kinase inhibition experiments and cell proliferation inhibition experiments, and screening the compounds according to experimental results.

The substituted quinoline compounds are targeted PI3K/mTOR signaling pathway inhibitors.

In the shape screening, the lowest similarity of 0.8 is set to reduce the screening range and reduce the workload of next butt joint, and if the lowest similarity of 0.9 or 0.85 is set, the range is too small, so that the result is inaccurate. The high precision docking is adopted instead of the common Standard (SP) docking because the high precision docking can dock the compound into the mTOR kinase protein binding pocket more accurately, and the docking result is more accurate and reliable.

Has the advantages that:

the compound with the structure of the formula (I) or the isomer thereof or the pharmaceutical composition containing the compound with the structure of the formula (I) has broad-spectrum anticancer effect, has higher inhibitory activity on various cancer cell lines such as HCT116, TE-1, 5637, hepG2 and the like, has excellent stability, is particularly stable in artificial gastrointestinal fluids, rat plasma and rat liver microsomes, is not easy to degrade, and has micromolar inhibitory activity on mTOR kinase. The compound has the characteristics of excellent activity, high selectivity, excellent stability and high safety.

The method is a virtual screening method and has the advantages of low cost and high calculation speed.

Drawings

FIG. 1: 9 reported compound structural formulas of mTOR kinase inhibitors;

FIG. 2: preliminarily screening the structural formulas of the 10 obtained potential compounds;

FIG. 3: histogram of inhibition of 20 cancer cell lines by compound L971-0652;

FIG. 4: dose-response curves for compound L971-0652 against HCT116 cell line;

FIG. 5: dose-response curves for compound L971-0652 against TE-1 cell line;

FIG. 6: dose-response curves for compound L971-0652 versus 5637 cell line;

FIG. 7 is a schematic view of: dose-response curves for compounds L971-0652 against HepG2 cell line;

FIG. 8: the average percent remaining in artificial gastrointestinal fluids-time curve for compound L971-0652 (n = 3);

FIG. 9: mean percent remaining in rat plasma-time bar or line graph for compound L971-0652 (n = 3);

FIG. 10: average percent remaining versus time curve for compound L971-0652 in rat liver microsome incubation system (n = 3).

Detailed Description

The following is a detailed description of the embodiments of the present invention, but the present invention is not limited to these embodiments, and any modifications or substitutions in the basic spirit of the embodiments are included in the scope of the present invention as claimed in the claims.

Example 1

A substituted quinoline compound is a compound shown as a formula (I) and is numbered as L971-0652:

the method for obtaining the substituted quinoline compound shown in the formula (I) specifically comprises the following steps: utilizing Schrodinger software to compare the shapes of molecules in a ChemDiv library with an mTOR inhibitor PI-103, selecting molecules with similarity larger than 80%, then carrying out high-precision (XP) docking with an mTOR kinase protein (protein code: 4JT 6), carrying out in-vitro kinase inhibition experiment and cell proliferation inhibition experiment after preliminarily screening 10 promising compounds according to docking scoring and combination mode analysis, and screening the compounds according to experiment results; the structural formula of the 10 potential compounds is shown in figure 2;

1. in vitro mTOR kinase inhibition assay

The inhibitory effect of such compounds on mTOR kinase was evaluated by the lancet Ultra fluorescence assay method described below.

The detection principle is as follows: lance Ultra fluorescence assay is a homogeneous, non-radioactive assay that quantitatively determines the activity of purified kinases by measuring the ATP content in the system after the kinase reaction. ATP content was determined by measuring the amount of ATP contained in the sample from Mg ²⁺ ATP and oxygen catalyze the light intensity generated by the oxidation of firefly luciferin (beette luciferase). A certain amount of ATP is added into a reaction system, the Kinase reaction needs to consume the ATP, and the residual ATP can react with the firefly luciferase in the Kinase Glo reagent to emit light, so that the amount of the residual ATP can be quantitatively detected, and the activity of the reaction Kinase can be indirectly measured.

The detection method comprises the following steps: first, 1 Xkinase buffer containing 50mM HEPES, pH 7.5,1mM EGTA,0.01% Tween-20 was prepared; the compounds were dissolved in 100% DMSO and diluted in a gradient, and 10nL of the diluted compounds were transferred to an assay plate to prepare a Control group containing no compound and a blank Control group containing no kinase. Adding mTOR into 1 × kinase buffer to prepare kinase solution, adding 5 μ L of the kinase solution into a detection plate, and uniformly mixing by vortex. Separately preparing 1 Xkinase reactionbuffer containing 4E-BP1 (Thr 37/46, PE) polypeptide and ATP substrate, adding 5 μ L into the well plate to start reaction, reacting at room temperature for 1h, adding EDTA and Eu-anti-P-4E-BP1 (Thr 37/46, P)E) And adding 10 mu L of antibody PBS buffer solution into the well plate, incubating for 60min at room temperature, reading the well plate, and counting data to calculate the inhibition rate of the compound on mTOR kinase. Substituting the inhibition rate and the corresponding concentration into GraphPadprism software for curve fitting, and calculating IC ₅₀ The value is obtained. The experimental results are shown in table 1;

TABLE 1 kinase inhibitory Activity of PI-103 and 10 compounds purchased

Compounds	IC ₅₀ (uM)
		PI-103	0.005±0.00
4229-0124	38.19±0.18
		D337-1379	>100
E130-0745	>100
		F043-0262	>100
L971-0652	8.90±0.09
		8018-9599	>100
6197-5126	29.87±0.23
		E130-0641	46.98±0.96
F167-0048	>100
		Y503-0661	>100

From the kinase activity data in Table 1, it is clear that the kinase inhibitory activity of the compound L971-0652 is the best. Therefore, the compound shown in the formula (I) has positive and foreseeable anti-proliferative diseases, especially anti-tumor clinical application value, and has good development prospect; the compounds of the invention inhibit mTOR kinase activity, thereby inhibiting transduction of cellular signaling pathways, which affect cell cycle and cell proliferation.

2. Cell proliferation inhibition assay

Cell Counting Kit (CCK-8) method for evaluating Cell proliferation inhibitory activity of compounds, and determining half inhibitory concentration IC by single-concentration activity preliminary screening and multiple concentrations ₅₀ The value is obtained.

The detection principle is as follows: the CCK-8 reagent contains WST-8, which is reduced into a yellow Formazan product (Formazan) with high water solubility by dehydrogenase in cell mitochondria under the action of an electron carrier 1-Methoxy-5-methylphenazinium dimethyl sulfate (1-Methoxy PMS). The amount of formazan product generated is directly proportional to the number of viable cells.

The detection method comprises the following steps: (1) seeding of cells: the cells were made into single cell suspensions in culture medium containing 10% fetal bovine serum, and 90. Mu.L of 5X 10 cells were inoculated into each well of a 96-well plate ⁴ Adherent cells/mL and 9X 10 ⁴ Suspension cells/mL, in 5% CO ₂ Preculture was carried out at 37 ℃ for 24h. (2) adding a sample solution to be tested: add 10. Mu.L of sample solution to each well and perform a preliminary Activity Screen on each wellSetting 1 concentration of a sample, and setting 3 multiple holes; IC (integrated circuit) ₅₀ Measuring 8 concentrations (including 0 concentration), wherein each concentration is provided with 3 multiple holes; placing in an incubator for 48h. The experiment was set up for Blank (Blank), control (Control) and Drug (Drug). (3) color development: adherent cells were aspirated from old medium and drug solution (10. Mu.L of CCK-8 stock solution was added directly to suspension cells), 100. Mu.L of CCK-8 solution was added ten times diluted per well, and the CO was 5% at 37% ₂ And continuously culturing for 1-4h (operation in dark place and real-time observation). And (4) detecting: and (5) measuring the absorbance at 450nm by using a microplate reader, and recording the result of the original data. (5) The raw data were normalized using Excel software, and the inhibition rate of cell proliferation was calculated by primary screening from the OD value per well (formula = (ODControl-ODDrug)/(ODControl-ODBlank) × 100%), and the inhibition rate was counted. IC (integrated circuit) ₅₀ The results are expressed as ± SD calculated by GraphPadPrism 8 (version 8.0.2, graphpad Software Inc).

(6) Positive control: doxorubicin hcl Doxorubicin (Dox).

Selecting a compound L971-0652 with the best in vitro kinase inhibition activity to carry out a cell proliferation inhibition experiment, wherein the experiment results are shown in a table 2 and a figure 3;

TABLE 2 inhibition of 20 cancer cell lines by L971-0652

Celllines	Dox	L971-0652
			A549	70.03％±0.94％	32.50％±0.60％
MKN-45	75.65％±0.08％	68.99％±0.54％
			HCT116	84.45％±0.17％	94.28％±0.17％
HeLa	7.98％±0.39％	58.83％±0.98％
			K-562	77.64％±0.23％	56.11％±1.04％
786-O	92.98％±0.43％	83.15％±0.94％
			TE-1	73.98％±1.30％	93.48％±0.88％
5637	98.81％±0.45％	90.95％±1.25％
			GBC-SD	77.12％±0.64％	64.99％±0.32％
LO2	98.98％±0.44％	62.97％±1.30％
			MCF7	41.88％±1.46％	70.49％±1.27％
HepG2	86.56％±2.02％	89.80％±0.92％
			SF126	87.56％±1.71％	60.86％±1.31％
DU145	72.86％±0.54％	32.74％±0.78％
			CAL-62	92.63％±0.69％	80.01％±1.75％
PATU8988T	98.54％±1.19％	85.59％±1.19％
			HOS	94.52％±1.47％	14.32％±0.62％
A-375	95.22％±1.35％	85.24％±1.15％
			A-673	99.33％±0.46％	92.74％±2.12％
293T	88.02％±0.19％	94.24％±1.18％

As shown in Table 2 and FIG. 3, the compound L971-0652 has strong inhibitory effects on various cancer cell lines such as HCT116, TE-1, 5637, hepG2, A-673 and 293T.

The IC50 values of compound L971-0652 on HCT116, TE-1, 5637 and HepG2 cell lines are shown in Table 3, and the dose-response graphs are shown in FIGS. 4-7.

TABLE 3 IC50 values of Compound L971-0652 on HCT116, TE-1, 5637 and HepG2 cell lines

Note: doxorubicin hydrochloride (Dox) was measured at a concentration of 10 μ M, L971-0652 at a concentration of 20 μ M;

as can be seen from table 3 and fig. 4-7: selective mTOR inhibitors are useful in antitumor studies.

3. L971-0652 in vitro stability test

Using an Agilent 1290Infinity type II ultra performance liquid chromatograph (USA), an Agilent Eclipse Plus C18 column (2.1 mm. Times.50mm, 1.8 μm, USA); the column temperature is 40 ℃; the mobile phase is 0.1 percent of formic acid water-0.1 percent of formic acid acetonitrile; the sample volume is 2 mu L; sample introduction time is 5.0min; the gradient elution procedure is shown in table 4 below:

TABLE 4 gradient of mobile phase

Triple quadrupole mass spectrometer (USA) model Agilent 6470 was used; ESI ion source; scanning in a positive ion mode; capillary voltage: 4.0kV; temperature of the drying gas: 300 ℃; flow rate of drying gas: 5.0L/min; temperature of sheath gas: 250 ℃; flow rate of sheath gas: 11.0L/min; the monitoring mode was Multiple Reaction Monitoring (MRM), fragment 210V, precursor 469.23, product 346.1 (m/z), and Collision Energy 41V.

1 in vitro artificial gastrointestinal fluid stability

1.1 preparation of the solution

1.1.1 preparation of Artificial gastrointestinal fluids

Preparing artificial gastric juice: according to Chinese pharmacopoeia (2020 edition), taking 16.4mL of dilute hydrochloric acid, adding 800mL of water and 10g of pepsin, shaking up to dissolve, adjusting the pH value to 1.3, adding ultrapure water to fix the volume to 1000mL, and obtaining the artificial gastric juice. Preparing blank gastric juice: the artificial gastric juice is blank gastric juice without adding pepsin. Artificial intestinal juice: taking 6.8g of potassium dihydrogen phosphate, adding 500mL of ultrapure water, mixing and dissolving by vortex, and adjusting the pH value to 6.8 by using 0.1mol/L sodium hydroxide solution: dissolving 10g of trypsin in a proper amount of water, mixing the two solutions, adding ultrapure water to a constant volume of 1000mL, and obtaining the artificial intestinal juice. Blank intestinal juice: the artificial intestinal juice is blank intestinal juice without adding trypsin.

1.1.2 preparation of Standard solution

A proper amount of the compound L971-0652 is precisely weighed, dissolved by methanol and subjected to constant volume to prepare a stock solution with the concentration of 500 mu g/mL, and the stock solution is stored at 4 ℃ for later use.

1.2 in vitro Artificial gastrointestinal fluid stability test

Taking a proper amount of L971-0652 stock solution under the item of '1.1.2', diluting to 100 mu g/mL with methanol, precisely measuring 50 mu L, adding into 4 10mL centrifuge tubes, and respectively adding blank gastric juice, artificial gastric juice, blank intestinal juice and 4950 mu L of artificial intestinal juice into each tube. Immediately after mixing, the mixture was dispensed into 1.5mL EP tubes (4 sets, 6 tubes/set), 200. Mu.L each. The reaction was stopped by adding 400. Mu.L of ice methanol at 0, 1, 2, 4, 6, 8h, respectively, and each system was run in parallel 3 times. Vortex mixing for 10min, centrifuging at 13000rpm for 10min, collecting 200 μ L supernatant, detecting, recording peak area, and calculating the residual percentage of L971-0652 incubated in artificial gastrointestinal fluid for different times with the drug content of 0h as 100%.

2 in vitro plasma stability

Taking a proper amount of L971-0652 stock solution under the item of '1.1.2', diluting the stock solution to 50 mu g/mL with methanol, adding the stock solution into a 1.5mLEP tube by a2 mu L/tube, adding 198 mu L/tube of rat plasma in parallel by 3 parts, after shaking and mixing, incubating for 0, 0.5, 1, 2 and 3 hours in water bath at 37 ℃, adding 400 mu L of ice methanol to terminate the reaction, vortex mixing for 5min, centrifuging at 13000rpm at 4 ℃ for 10min, taking supernatant N2, drying, redissolving by 200 mu L of methanol, centrifuging at 13000rpm at 4 ℃ for 10min, taking the supernatant for detection, recording peak area, taking the drug content of incubating for 0 hour as 100%, and calculating the residual percentage of L971-0652 incubated in rat plasma for different times.

In vitro hepatic microsomal metabolic stability

3.1 preparation of the solution

3.1.1 preparation of reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) coenzyme solution

Solution A: respectively weighing 200mg of nicotinamide adenine dinucleotide disodium phosphate (NADP-Na 2), 200mg of glucose-6-phosphate-disodium (G-6-P-Na 2) and 133mg of magnesium chloride, adding water to dissolve and fixing the volume to 10mL; and B, liquid B: respectively weighing 44mg of sodium citrate and 1000U of glucose-6-phosphate dehydrogenase (G-6-P-DH), adding water to dissolve and fixing the volume to 25mL; all are stored at-20 ℃ for standby. Before use, mixing solution A and solution B according to the ratio of 5:1 volume ratio to obtain a coenzyme solution with a concentration of 1mmol/L (calculated as NADPH).

3.1.2 liver microsome dilution

The liver microsomes of 20mg/mL were diluted to 5mg/mL with a mixture of 80% PBS buffer (0.01M) and 20% glycerol, and were stored at-80 ℃ for further use after dispensing.

3.2 Metabolic stability experiments in rat liver microparticles

mu.L of rat liver microsomes (final concentration 0.5 mg/mL) was added to 150. Mu.L of the diluted L971-0652 solution. The above solution was preheated in a 37 ℃ water bath for 5min, and then 30. Mu.L of NADPH solution was added to start the reaction. The total volume of the incubation system is 200 μ L, wherein the mass concentration of L971-0652 is 500ng/mL, the final concentration of NADPH is 1mmol/L, and the organic solvent is not more than 1%. The incubation system is continuously placed in a water bath at 37 ℃, 400 mu L of ice methanol is added at 0min, 5min, 15 min, 30min, 45 min and 60min respectively to stop the reaction, and 3 parts of ice methanol are added at each time point. And (3) uniformly mixing the mixture by vortex for 5min, centrifuging at 13000rpm for 10min at 4 ℃, collecting supernatant, drying at 37 ℃ under N2, redissolving residues by 200 mu L of methanol, centrifuging at 13000rpm for 10min, taking the supernatant, detecting, and recording peak areas.

3.3 data processing

(1) Percent residual = Ct/C0X 100%

(2) Half-life: t1/2= -0.693/k

(3) Intrinsic clearance rate: CLint =0.693/T1/2 × V/0.1

In the formula: ct is the residual concentration of the drug at different incubation times; c0 is the drug concentration at time 0; k is the slope obtained by linear regression of the natural logarithm of the percent residual rate of the drug at each time point to the incubation time; v is total volume of incubation fluid (mL); 0.1 is the content of liver microsomes (mg) in the incubation system.

L971-0652 in vitro stability test results

1 in vitro artificial gastrointestinal fluid stability

The remaining percentage of L971-0652 at different times was calculated at 100% concentration at 0h of incubation, and the results are shown in Table 5 and FIG. 8. After 8h of incubation, the residual rates of L971-0652 in the blank gastric juice, the artificial gastric juice, the blank intestinal juice and the artificial intestinal juice are 100.36%, 88.36%, 90.37% and 98.13%, respectively, which indicates that L971-0652 is stable in the artificial gastrointestinal juice and is not easy to degrade.

( Note: greater than 100% due to operation and detection errors, within the allowable range )

TABLE 5 remaining percentage of L971-0652 in artificial gastrointestinal fluids (Mean. + -. SD, n = 3)

2 in vitro plasma stability

The remaining percentage of L971-0652 at different times was calculated at 100% concentration at 0h of incubation, and the results are shown in Table 6 and FIG. 9. After 3h incubation in rat plasma, L971-0652 had a residual rate of 98.78%, indicating that L971-0652 is stable in rat plasma and not easily degraded.

Table 6 remaining percentage of L971-0652 in plasma (Mean ± SD, n = 3)

Time/h	Percentage remaining (%)
		0	100.00±0.00
0.5	99.82±10.83
		1	93.58±1.70
2	95.63±9.78
		3	98.78±13.27

3 hepatic microsomal metabolic stability

L971-0652 is shown in Table 7 below and in FIG. 10 for the residual rate of 60min incubation in rat liver microsomes, as well as the enzyme kinetic parameters half-life (T1/2), and intrinsic clearance in vitro (CLint). The metabolic stability is judged according to the T1/2, and the metabolic instability of the tested substance is shown when the T1/2 is less than 30 min; t1/2 is more than 30min and less than 90min, which indicates that the metabolic stability of the test substance is medium; t1/2 > 90min indicates that the metabolic stability of the test substance is good. The half-life T1/2 of L971-0652 in rat liver microsome is 277.20min, which shows that the L971-0652 has good stability in rat liver microsome and is not easy to be metabolized.

TABLE 7 metabolic stability of L971-0652 in rat liver microsomes (Mean + -SD, n = 3)

Compound (I)	Species of species	Residual ratio (%)	T _1/2 (min)	CL _int (mL/min/mg)
					L971-0652	Rat	83.01±5.55	277.20	0.005

The compound L971-0652 obtained by the screening method of the invention has target mTOR kinase inhibition activity, micromolar antiproliferative activity on various tumor cells, and good in vitro stability. The compound L971-0652 is a lead compound with novel structure and target inhibition effect on mTOR kinase, and lays a solid foundation for obtaining a novel high-activity mTOR kinase inhibitor through subsequent modification.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A substituted quinoline compound, which is a compound represented by formula (I) or an isomer, a tautomer, or a pharmaceutically acceptable salt thereof, wherein the structure of formula (I) is as follows:

2. a pharmaceutical composition comprising at least one pharmaceutically acceptable adjuvant, adjuvant or carrier, and a therapeutically effective amount of at least one substituted quinoline compound of claim 1.

3. Use of the substituted quinoline compound of claim 1 or the pharmaceutical composition of claim 2 for the preparation of an anti-tumor medicament.

4. Use of a substituted quinoline compound as claimed in claim 1 or a pharmaceutical composition as claimed in claim 2 in the manufacture of a medicament for the prevention and/or treatment of a proliferative disease in which an mTOR kinase acts.

5. A method for obtaining a substituted quinoline compound according to claim 1, wherein the mTOR inhibitor PI-103 is used as a shape questioning molecule, and is obtained by virtual screening from ChemDiv library.

6. The method for obtaining the substituted quinoline compound according to claim 5, wherein the molecules in the ChemDiv library are compared with the mTOR inhibitor PI-103 in shape, the molecules with similarity greater than 80% are selected, then high precision (XP) docking is carried out, 10 potential compounds are screened according to docking scoring and binding pattern analysis, then in vitro kinase inhibition experiments and cell proliferation inhibition experiments are carried out, and the screening is carried out according to experimental results.

7. A substituted quinoline compound according to claim 1 which is a targeted PI3K/mTOR signaling pathway inhibitor.