CN118684691A - A ROS-responsive compound, a preparation method and application thereof, and a drug - Google Patents

Abstract

The invention relates to the field of medicines, in particular to a ROS response compound, a preparation method and application thereof, and a medicine. The invention provides an ROS response compound, a preparation method and application thereof, and a medicament. The invention provides a small molecular compound which responds to ROS and releases LDK378, has high targeting property, high anti-tumor activity and low toxic and side effects, can inhibit proliferation of various cancer cells, and obviously reduces the toxic and side effects on a normal cell line compared with LDK 378. The invention aims to endow drug molecules with the capability of distinguishing cancer cells from normal cells through oxidative response modification, and enhance the cell targeting capability of the drug, thereby reducing the toxic and side effects of LDK 378. Meanwhile, the drug modification at the piperidine terminal can improve the pharmacokinetics property and the anti-tumor activity of the parent drug, and is hopeful to keep or even improve the toxicity of the drug to tumor cells.

Description

A ROS-responsive compound, a preparation method and application thereof, and a drug

Technical Field

The present invention relates to the field of medicines, in particular to a ROS responsive compound, a preparation method and application thereof, and a medicine.

Background Art

In recent years, anti-tumor drugs have developed rapidly, but most anti-tumor drugs lack targeting, and damage to normal cells and tissues leads to serious toxic side effects during chemotherapy. The abnormality of the tumor environment, especially the oxidative stress caused by the excessive production of reactive oxygen species (ROS) in tumor cells, is the physiological basis for many fluorescent probes and drug-targeted tumor controlled release [Eur. J. Med. Chem., 2020, 207, 112670.]. In countries around the world, cancer is the leading cause of death and an important obstacle to improving life expectancy. The increasing incidence and mortality of cancer under the influence of many factors needs to be solved urgently [CA Cancer J Clin., 2021, 71, 209.]. There are many clinical drugs and treatments for cancer. With the progress of drug research and development, more and more efficient new generation anti-tumor drugs are entering the clinic.

The clinical anticancer drug Ceritinib (LDK378) is an ALK tyrosine kinase inhibitor developed by Novartis. It was approved for marketing by the FDA in 2014 and is approved for the treatment of ALK-positive non-small cell lung cancer. At the same time, LDK378 also has a highly effective inhibitory effect on targets such as IGF-1R, IR and ACK1. LDK378 has shown excellent anti-tumor activity against a variety of cancer cell lines. For example, the IC ₅₀ of LDK378 measured by us for breast cancer cells MCF-7 is 4.72μmol/L, and the IC ₅₀ for human non-small cell lung cancer cells H2228 is 0.26μmol/L. LDK378 is a clinical chemotherapy drug with broad application prospects. However, along with its excellent anti-tumor activity, its use is often accompanied by obvious toxic side effects and is highly toxic to normal cell lines. We measured that the IC50 of LDK378 for human liver cells LO2 was 3.71 μmol/L, and the _IC50 for human breast cells MCF-10A was 1.47 μmol/L. At the same time, it is reported that LDK378 also has side effects such as hyperglycemia, metabolic and nutritional disorders in clinical practice, and these negative factors affect its application in clinical medication [Internal Med., 2019, 58, 817.].

LDK378, a multi-target protein kinase small molecule inhibitor, can effectively inhibit the proliferation of various tumor cell lines and is a clinical anti-tumor drug with wide application potential. However, its use is also accompanied by obvious toxic and side effects, and gastrointestinal reactions of varying degrees are common. The drug also often exhibits hepatotoxicity, and cases of interstitial lung disease (ILD), hyperglycemia, and bradycardia during medication have also been reported [Lung Cancer, 2017, 111, 51.]. The above toxic and side effects during the use of LDK378 are largely due to the lack of cell targeting of small molecule inhibitors. LDK378 not only inhibits the proliferation of tumor cells, but also has strong toxic and side effects on normal human cell lines. This inhibitory effect that lacks distinction between normal cells and tumor cells leads to strong toxic and side effects of LDK378, limiting its clinical application.

On the other hand, some drug modification efforts have attempted to modify the terminal piperidine structure of LDK378 to achieve modifications in the activity and pharmacokinetics of LDK378. However, most of the structures reported so far not only fail to impart new functions to the drug, but also significantly reduce the activity of the drug. [Eur.J.Med.Chem., 2015, 93, 1.][Eur.J.Med.Chem., 2017, 126, 536.]

The lack of differentiation between tumor cells and normal cells by LDK378 is one of the fundamental reasons for its toxic side effects. ROS, as one of the markers of abnormal tumor expression, is often used to target tumor cells and tissues. Reactive oxygen species (ROS) molecules include hydroxyl radicals (·OH), superoxide (O ₂ ·-), and hydrogen peroxide (H ₂ O ₂ ). Among them, H ₂ O ₂ is considered to be the main active reactant of ROS because its diffusion capacity is stronger than that of free radicals. High levels of ROS are one of the important characteristics of cancer tissues. The concentration of H ₂ O ₂ in normal cells is 1 to 10 nmol/L, while the concentration of H ₂ O ₂ overexpressed in malignant tumors can reach 10 to 100 μmol/L. Compared with normal cells, cancer cells have a high level of H ₂ O ₂ , and have a rich and efficient response structure tool library for H ₂ O ₂ , which makes H ₂ O ₂ an ideal target for ROS response.

The aryl boronic acid/ester structure is one of the classic reactive oxygen species responsive structures and has a wide range of applications. It was first used in reactive oxygen species activated fluorescent probe structures, and later gradually applied to tumor-targeted, reactive oxygen species activated small molecule prodrug structures. The mechanism of the ROS response mechanism H ₂ O ₂ of borate compounds is shown in Figure 1, which is a schematic diagram of the mechanism of the ROS response mechanism H ₂ O ₂ of borate compounds. As shown in Figure 1, H ₂ O ₂ oxidizes aryl boronic acid esters to form borate salts. The borate salts are hydrolyzed to release non-toxic boric acid, and the generated phenolic ions trigger the cleavage of the modified structure, thereby releasing the parent drug. Drugs modified with aryl boronic acid/ester structures will not only enhance the targeting of the drug, but also have uncertain effects on the toxicity and pharmacokinetic properties of the drug. Modification and combination of different drugs often yield drugs with new properties. Therefore, research on aryl boronic acid/ester modified drugs is still developing. At present, there is no similar ROS response modification work reported for LDK378.

Prodrug strategy is a classic new drug research strategy. Prodrugs are structurally modified on the basis of existing drugs. The resulting new drugs have low activity when the structure is unchanged, and can only produce drug effects after being decomposed in the body to release the parent drug. Therefore, prodrugs can effectively reduce the toxic side effects of drugs, achieve selective killing of tumor cells, and minimize damage to normal tissues.

Summary of the invention

In view of this, the technical problem to be solved by the present invention is to provide a ROS responsive compound and a preparation method and application thereof, and a drug. The compound provided by the present invention has high targeting and low toxic side effects.

The present invention provides a ROS-responsive compound having a structure shown in Formula 1:

The R is selected from the group of the structure shown in Formula 2 or Formula 3;

Wherein, said R ₁ ' and R ₂ ' are independently selected from substituted or unsubstituted C _n H _2n+1 ; wherein n is an integer from 0 to 10; or, at least one of said R ₁ ' and R ₂ ' is connected with its O and B to form a heterocyclic ring; the heteroatoms in said heterocyclic ring include B, O and optional N.

The compound of the above formula 1 structure has a parent molecule of LDK378, and its molecular ends all contain oxidation responsive groups such as phenylboronic acid and phenylboronic acid ester. R in the formula 1 of the present invention is selected from the group of the structure shown in formula 2 or formula 3; in formula 2 or formula 3, the R ₁ 'and R ₂ 'are independently selected from substituted or unsubstituted C _n H _2n+1 ; wherein n is an integer from 0 to 10. When n is 0, the R ₁ 'and R ₂ 'are independently selected from H; when n is not 0, the R ₁ 'and R ₂ 'are independently selected from substituted or unsubstituted C _n H _2n+1 . In certain embodiments of the present invention, in Formula 1, R is 4-boric acid (ester)-benzyloxycarbonyl, 4-boric acid (ester)-benzyl, 2-boric acid (ester)-benzyloxycarbonyl, 2-boric acid (ester)-benzyl, R ₁ 'or R ₂ 'is a hydrogen atom and a straight or branched alkyl group with a carbon chain length of 1 to 10.

At least one of R ₁ 'and R ₂ 'in Formula 2 or Formula 3 of the present invention is connected with O where it is located and B where O is located to form a heterocycle; the heteroatoms in the heterocycle include B, O and optional N, that is, the heteroatoms in the heterocycle of the present invention include B and O, and may or may not include N. In certain embodiments of the present invention, R ₁ 'and R ₂ 'in the compound of the above Formula 1 structure form a cyclic ester group with boronic acid, and its structure includes diol analogs of the structures shown in Formula a1, Formula b1, Formula c1, Formula d1, Formula e1, Formula f1, Formula g1 and Formula h1;

In some embodiments, the R is selected from any one of the structures shown in formula a to formula t;

In certain embodiments of the present invention, R is any one of the groups of structures shown in Formulae 4 to 7;

In some embodiments, R is selected from any one of the structures shown in Formula 8 to Formula 47;

In one embodiment, the compound provided by the present invention is:

The present invention aims at the disadvantage that LDK378 has toxic and side effects in clinical practice, and provides an LDK378 derivative modified by a ROS-sensitive borate. The compound utilizes the difference in ROS levels between tumor cells and normal cells to achieve the selective release of LDK378 by drug molecules between different cells. Compared with the prior art, the present invention has the following significant advantages: (1) The compound has good ROS responsiveness and can effectively release drugs in an in vitro oxidative environment and tumor cells, thereby achieving ROS-targeted drug release, which helps to enrich the drug in tumor tissues. (2) The compound has good anti-tumor activity and can show activity comparable to that of the parent drug LDK378 in most tumor cell lines, especially in the human breast cancer cell line MCF-7, showing anti-tumor activity superior to that of the parent drug. (3) The compound has excellent targeting, and compared with the parent drug LDK378, the toxic and side effects of the drug on normal human tissues are reduced.

The present invention provides an application of the above-mentioned compound in the preparation of an anti-tumor drug. Specifically, the present invention provides an application of the above-mentioned compound in the preparation of an anti-tumor drug having at least one of the effects of reducing toxicity to normal cells or enhancing toxicity of the drug to cancer cells. The anti-tumor drug of the present invention has no obvious toxic side effects on normal tissues; wherein the tumor is at least one of breast cancer or lung cancer; the lung cancer is non-small cell lung cancer; and the cell is MCF-10A cell, LO2 cell, H2228 cell or MCF-7 cell.

The present invention provides a drug, comprising at least one of the above-mentioned compounds and pharmaceutically acceptable salts thereof and a pharmaceutically acceptable excipient. The drug provided by the present invention includes a pharmaceutically acceptable excipient, and the present invention has no special restrictions on the excipient, and the excipient is a pharmaceutically acceptable excipient well known to those skilled in the art, such as at least one of a diluent, a lubricant, a binder, a disintegrant, a surfactant, a film-forming material, a coating material, and a capsule material.

The medicine provided by the present invention includes the above-mentioned compound, and may also include a pharmaceutically acceptable salt of the above-mentioned compound, wherein the salt is a salt formed by the above-mentioned compound with an acid or a salt formed with a base; wherein the acid is an inorganic acid or an organic acid, and the base is an inorganic base or an organic base; wherein the organic acid includes at least one of common organic acids such as formic acid, acetic acid, oxalic acid, benzoic acid, phenylacetic acid, and p-toluenesulfonic acid; the inorganic acid includes at least one of common inorganic acids such as hydrochloric acid, sulfuric acid, and nitric acid. The organic base includes at least one of common organic bases such as diethylamine, triethylamine, diisopropylethylamine, pyridine, or sodium methoxide; the inorganic base includes at least one of common inorganic bases such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, or cesium carbonate.

The present invention also provides a method for preparing the above compound, comprising the following steps:

The above-mentioned compound is obtained by subjecting an aryl boronic acid compound or an aryl boronic ester compound to a carbamate reaction with LDK378. Specifically, an aryl boronic acid compound or an aryl boronic ester compound is modified to obtain an intermediate product, and the intermediate product is reacted with LDK378 to obtain the above-mentioned compound. In certain embodiments of the present invention, an aryl boronic acid compound or an aryl boronic ester compound is subjected to a carbonation reaction, a sulfonation reaction or a halogenation reaction to obtain an intermediate product, and the intermediate product is subjected to a carbamate reaction or a nucleophilic substitution reaction with LDK378 to obtain the above-mentioned compound.

In one embodiment of the present invention, the present invention first performs a carbonation reaction on an aryl boronic acid compound or an aryl boronic ester compound to obtain an intermediate product. Specifically, the present invention performs a carbonation reaction on any one of an aryl boronic acid compound or an aryl boronic ester compound, di(p-nitrobenzene) carbonate and a base in an organic solvent to obtain an intermediate product. In one embodiment, the present invention performs a carbonation reaction on any one of an aryl boronic acid compound or an aryl boronic ester compound, di(p-nitrobenzene) carbonate and a base in an organic solvent to obtain an intermediate product. In one embodiment, the aryl boronic ester compound is The intermediate product is obtained by reacting it with di(p-nitrobenzene) carbonate and a base in an organic solvent to obtain a carbonate ester. The intermediate product is 4-nitrophenyl (4-(4,4,5,5-tetramethyl-1,3,2-dioxolane-2-yl) benzyl) carbonate. The structural formula of the intermediate product is

In certain embodiments of the present invention, the base includes an organic base and an inorganic base; wherein the organic base includes at least one of common organic bases such as diethylamine, triethylamine, diisopropylethylamine, pyridine or sodium methoxide; the inorganic base includes at least one of common inorganic bases such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate or cesium carbonate, preferably diisopropylethylamine. In certain embodiments of the present invention, the organic solvent is at least one of common organic solvents such as acetone, ethanol, dichloromethane, chloroform, tetrahydrofuran, dioxane, acetonitrile, N,N-dimethylformamide or dimethyl sulfoxide, preferably dichloromethane.

The carbonation reaction of the present invention is carried out at room temperature. In one embodiment, the carbonation reaction is carried out at 20 to 30°C. In one embodiment, the carbonation reaction time is 2h to 72h, preferably 2h to 12h, and more preferably 6h to 8h. In certain embodiments of the present invention, the molar ratio of any one of the aryl boronic acid compound or aryl boronic ester compound and di(p-nitrobenzene) carbonate is 1:0.5 to 5, preferably 1:1 to 2, and more preferably 1:1.5. In certain embodiments of the present invention, the amount ratio of any one of the aryl boronic acid compound or aryl boronic ester compound, di(p-nitrobenzene) carbonate, base and organic solvent is 1 mole part: 0.5 to 5 mole parts: 0.5 to 5 mole parts: 1 to 20 parts by volume, preferably 1 mole part: 1 to 2 mole parts: 1 to 2 mole parts: 5 to 10 parts by volume, more preferably 1 mole part: 1.5 mole parts: 1.5 mole parts: 5 parts by volume. In one embodiment, the usage ratio of any one of the aryl boronic acid compound or the aryl boronic ester compound, di(p-nitrobenzene) carbonate, base and organic solvent is 2 mmol:1.5 mmol:1.5 mmol:15 mL.

After the intermediate product is obtained in the present invention, the intermediate product is subjected to a carbamate reaction with LDK378 to obtain the above-mentioned compound. Specifically, the intermediate product, LDK378 and a base are subjected to a carbamate reaction in an organic solvent to obtain the above-mentioned compound. In certain embodiments of the present invention, the intermediate product, LDK378 and a base are added to an organic solvent to carry out a carbamate reaction to obtain the above-mentioned compound. In one embodiment, the intermediate product is 4-nitrophenyl (4-(4,4,5,5-tetramethyl-1,3,2-dioxolan-2-yl) benzyl) carbonate, and its structural formula is After being dissolved in an organic solvent, it is reacted with LDK378 to form carbamate to obtain the structural formula of the above compounds.

In the carbamate reaction of the present invention, the molar ratio of the LDK378 to the intermediate product is 1:1-10, preferably 1:2-5, and more preferably 1:2. In certain embodiments of the present invention, the usage ratio of the LDK378, the intermediate product, the base and the organic solvent is 1 mol part: 1-10 mol parts: 1-10 mol parts: 1-20 volume parts, preferably 1 mol part: 2-5 mol parts: 2-5 mol parts: 5-10 volume parts, and more preferably 1 mol part: 2 mol parts: 2 mol parts: 5 volume parts. In one embodiment, the usage ratio of the LDK378, the intermediate product, the base and the organic solvent is 0.1 mmol: 0.15 mmol: 0.3 mmol: 5 mL.

The carbamate reaction of the present invention is carried out at room temperature. In one embodiment, the carbamate reaction is carried out at 20 to 30°C. In one embodiment, the time of the carbamate reaction is 2h to 72h, preferably 6h to 24h, and more preferably 12h to 16h. The base and organic solvent of the present invention are the same as those described above and will not be described in detail. The structural formula of LDK378 of the present invention is

In another embodiment of the present invention, the present invention first performs a sulfonation reaction on an aryl boronic acid compound or an aryl boronic ester compound to obtain an intermediate product. Specifically, the present invention performs a sulfonation reaction on any one of an aryl boronic acid compound or an aryl boronic ester compound, a sulfonyl halide compound and a base in an organic solvent to obtain an intermediate product. In one embodiment, the present invention adds any one of an aryl boronic acid compound or an aryl boronic ester compound, a sulfonyl halide compound and a base to an organic solvent for a sulfonation reaction to obtain an intermediate product. The sulfonyl halide compound of the present invention is selected from at least one of an alkyl sulfonyl chloride or an aryl sulfonyl chloride; the alkyl sulfonyl chloride is preferably selected from methyl sulfonyl chloride. The base and the organic solvent of the present invention are the same as those described above and will not be repeated.

In one embodiment, the aryl borate compound is The intermediate product is subjected to a sulfonate reaction with methanesulfonyl chloride and a base in an organic solvent, wherein the intermediate product is 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)methanesulfonate, and the structural formula of the intermediate product is

The sulfonation reaction of the present invention is carried out at room temperature. In one embodiment, the sulfonation reaction is carried out at 20 to 30°C. In one embodiment, the sulfonation reaction time is 2h to 72h, preferably 2h to 24h, and more preferably 8h to 10h. In certain embodiments of the present invention, the molar ratio of any one of the aryl boronic acid compound or aryl boronic ester compound to the sulfonyl halide compound is 1:0.5 to 5, preferably 1:1 to 2, and more preferably 1:1.2. In certain embodiments of the present invention, the amount ratio of any one of the aryl boronic acid compound or aryl boronic ester compound, sulfonyl halide compound, base and organic solvent is 1 mole part: 0.5 to 5 mole parts: 0.5 to 5 mole parts: 1 to 20 parts by volume, preferably 1 mole part: 1 to 2 mole parts: 1 to 2 mole parts: 5 to 10 parts by volume, more preferably 1 mole part: 1.2 mole parts: 1.5 mole parts: 5 parts by volume. In one embodiment, the usage ratio of any one of the aryl boronic acid compound or the aryl boronic ester compound, the sulfonyl halide compound, the base and the organic solvent is 1 mmol: 1.2 mmol: 2 mmol: 15 mL.

After the intermediate product is obtained in the present invention, the intermediate product is subjected to a nucleophilic substitution reaction with LDK378 to obtain the above-mentioned compound. Specifically, the intermediate product, LDK378 and a base are subjected to a nucleophilic substitution reaction in an organic solvent to obtain the above-mentioned compound. In certain embodiments of the present invention, the intermediate product, LDK378 and a base are added to an organic solvent to carry out a carbamate reaction to obtain the above-mentioned compound. In one embodiment, the intermediate product is (4-(4,4,5,5-tetramethyl-1,3,2-dioxolan-2-yl)benzyl) methanesulfonate, and its structural formula is After being dissolved in an organic solvent, it reacts with LDK378 for nucleophilic substitution to obtain the structural formula: of the above compounds.

The nucleophilic substitution reaction of the present invention is carried out at room temperature. In one embodiment, the nucleophilic substitution reaction is carried out at 20 to 30° C. In one embodiment, the time of the nucleophilic substitution reaction is 2 h to 72 h, preferably 6 h to 24 h, and more preferably 8 h to 12 h. The base and organic solvent of the present invention are the same as those described above and will not be described in detail.

In the nucleophilic substitution reaction of the present invention, the molar ratio of the LDK378 to the intermediate product is 1:1-10, preferably 1:1-5, and more preferably 1:1.5. In certain embodiments of the present invention, the usage ratio of the LDK378, the intermediate product, the base and the organic solvent is 1 mol part: 1-10 mol parts: 1-10 mol parts: 1-20 volume parts, preferably 1 mol part: 1-5 mol parts: 2-5 mol parts: 5-10 volume parts, and more preferably 1 mol part: 1.5 mol parts: 2 mol parts: 5 volume parts. In one embodiment, the usage ratio of the LDK378, the intermediate product, the base and the organic solvent is 0.1 mmol: 0.15 mmol: 0.3 mmol: 5 mL.

After the intermediate product is subjected to a carbamate reaction or a nucleophilic substitution reaction with LDK378, the present invention further comprises: subjecting the product obtained after the carbamate reaction or the nucleophilic substitution reaction to a borate exchange reaction. Specifically, the present invention further comprises: subjecting the product obtained after the carbamate reaction or the nucleophilic substitution reaction to a borate exchange reaction with diethanolamine in an organic solvent. In certain embodiments of the present invention, the present invention further comprises: adding the product obtained after the carbamate reaction or the nucleophilic substitution reaction and diethanolamine to an organic solvent to conduct a borate exchange reaction. In one embodiment, the product obtained after the carbamate reaction or the nucleophilic substitution reaction is It and diethanolamine are added to an organic solvent for borate exchange reaction to obtain

In one embodiment, the organic solvent is selected from at least one of common organic solvents such as diethyl ether, acetone, ethanol, dichloromethane, chloroform, tetrahydrofuran, dioxane, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, etc., preferably a mixed solvent of dichloromethane and diethyl ether, and more preferably a mixed solvent of dichloromethane and diethyl ether in a volume ratio of 1:1.

In certain embodiments of the present invention, the molar ratio of the product obtained after the carbamate reaction or the nucleophilic substitution reaction to diethanolamine is 1:1-10, preferably 1:1-2, and more preferably 1:1.2. In certain embodiments of the present invention, the amount ratio of the product obtained after the carbamate reaction or the nucleophilic substitution reaction, diethanolamine and the organic solvent is 1 mol part: 1-10 mol parts: 1-20 volume parts, preferably 1 mol part: 1-2 mol parts: 5-10 volume parts, and more preferably 1 mol part: 1.2 mol parts: 5 volume parts. In one embodiment, the amount ratio of the product obtained after the carbamate reaction or the nucleophilic substitution reaction, diethanolamine and the organic solvent is 0.24mmol: 0.27mmol: 5mL.

The temperature of the borate exchange reaction of the present invention is room temperature. In one embodiment, the temperature of the borate exchange reaction is 20°C to 30°C; the borate exchange reaction is 2h to 72h, preferably 2h to 12h, and more preferably 3h to 6h.

After the borate exchange reaction, the present invention further includes: hydrolyzing the product obtained after the borate exchange reaction. Specifically, it also includes: hydrolyzing the product obtained after the borate exchange reaction and an acid in an organic solvent. In certain embodiments of the present invention, it also includes: adding the product obtained after the borate exchange reaction and an acid into an organic solvent for hydrolysis reaction. In one embodiment, the product obtained after the borate exchange reaction is The product obtained after the borate exchange reaction and the acid are added to an organic solvent for hydrolysis reaction to obtain

In one embodiment, the acid includes an organic acid and an inorganic acid; wherein the organic acid includes at least one of common organic acids such as formic acid, acetic acid, oxalic acid, benzoic acid, phenylacetic acid, and p-toluenesulfonic acid; the inorganic acid includes at least one of common inorganic acids such as hydrochloric acid, sulfuric acid, and nitric acid, preferably dilute hydrochloric acid. In one embodiment, the organic solvent is at least one of common organic solvents such as acetone, ethanol, dichloromethane, chloroform, tetrahydrofuran, dioxane, acetonitrile, N,N-dimethylformamide, and dimethyl sulfoxide, preferably dichloromethane.

In certain embodiments of the present invention, the ratio of the product obtained after the borate exchange reaction, the acid and the organic solvent is 1 mole part: 10 to 50 mole parts: 10 to 50 parts by volume, preferably 1 mole part: 20 mole parts: 20 parts by volume. In one embodiment, the ratio of the product obtained after the borate exchange reaction, the acid and the organic solvent is 0.12mmol: 5mmol: 5mL. The temperature of the hydrolysis reaction of the present invention is room temperature. In one embodiment, the temperature of the hydrolysis reaction is 20°C to 30°C; the hydrolysis reaction is 2h to 72h, preferably 2h to 12h, and more preferably 4h to 8h.

After the hydrolysis reaction, the present invention further comprises: performing a borate esterification reaction on the product obtained after the hydrolysis reaction. Specifically, the present invention further comprises: performing a borate esterification reaction on the product obtained after the hydrolysis reaction and a diol compound in an organic solvent. In certain embodiments of the present invention, the present invention further comprises: adding the product obtained after the hydrolysis reaction and the diol compound into an organic solvent for a borate esterification reaction.

The structural formula of the diol compound of the present invention is The R ₁ ' and R ₂ ' are the same as above and will not be described in detail. In one embodiment, the product obtained after the hydrolysis reaction is The product obtained after the hydrolysis reaction, neopentyl glycol and anhydrous magnesium sulfate are added to an organic solvent for borate esterification reaction to obtain

In one embodiment, the organic solvent is at least one of common organic solvents such as acetone, ethanol, dichloromethane, chloroform, tetrahydrofuran, dioxane, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, etc., preferably dichloromethane.

In certain embodiments of the present invention, the molar ratio of the product obtained after the hydrolysis reaction to neopentyl glycol is 1:1-10, preferably 1:2-5, and more preferably 1:2. In certain embodiments of the present invention, the amount ratio of the product obtained after the hydrolysis reaction, neopentyl glycol and organic solvent is 1 mole part: 1-10 mole parts: 1-20 volume parts, preferably 1 mole part: 2-5 mole parts: 5-10 volume parts, more preferably 1 mole part: 2 mole parts: 5 volume parts. In one embodiment, the amount ratio of the product obtained after the hydrolysis reaction, neopentyl glycol, anhydrous magnesium sulfate and organic solvent is 0.041mmol: 0.048mmol: 0.2mmol: 5mL.

The temperature of the borate esterification reaction of the present invention is room temperature. In one embodiment, the temperature of the borate esterification reaction is 20°C to 30°C; the borate esterification reaction is 2h to 72h, preferably 4h to 12h, and more preferably 6h to 8h.

The present invention provides a ROS-responsive compound, a preparation method and application thereof, and a drug. The present invention provides a small molecule compound that responds to ROS and releases LDK378, which has high targeting and low toxic side effects, can inhibit the proliferation of multiple cancer cells, and has significantly reduced toxic side effects on normal cell lines compared with LDK378. The present invention aims to endow drug molecules with the ability to distinguish cancer cells from normal cells through oxidation-responsive modification, enhance the cell targeting ability of the drug, and thus reduce the toxic side effects of LDK378. At the same time, drug modification at the piperidine end can also improve the pharmacokinetic properties and anti-tumor activity of the parent drug, and is expected to maintain or even improve the toxicity of the drug to tumor cells.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of the mechanism of ROS response to H2O2 of borate compounds;

FIG2 is a full NMR image of the in vitro oxidation of compound I;

FIG3 is a partial enlarged view of FIG2;

FIG4 is a graph showing the cell viability of human breast cancer cells MCF-7 treated with compound I for 48 hours;

FIG5 is a graph showing the cell viability of human non-small cell lung cancer cells H2228 treated with compound I for 48 hours;

FIG6 is a graph showing the cell viability of human normal breast epithelial cell line MCF-10A treated with compound I for 48 hours;

FIG7 is a graph showing the cell viability of human normal liver cell line LO2 treated with compound I for 48 hours;

FIG8 is a graph showing changes in tumor volume in the low-dose group of Compound I and LDK378;

Figure 9 shows the changes in tumor volume in the high-dose groups of Compound I and LDK378;

Figure 10 is a hematoxylin-eosin stained section of mouse tissue after 30 days of administration of Compound I;

FIG11 is a graph showing the in vitro inhibition of tumor cell viability by compounds I, VI, VII, VIII, and IX.

DETAILED DESCRIPTION

The present invention discloses a ROS responsive compound, a preparation method and application thereof, and a drug. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

The compounds of the present invention, namely compounds I, VI, VII, VIII and IX, are prepared according to the following reaction schemes:

The present invention will be further described below in conjunction with embodiments:

Example 1

468 mg (2 mmol) of 4-(hydroxymethyl)phenylboronic acid pinacol ester, 463 mg (1.5 mmol) of di(p-nitrobenzene) carbonate, 194 mg (1.5 mmol) of N,N-diisopropylethylamine and 15 mL of dichloromethane were added to the reactor and reacted at room temperature for 6 hours. After the reaction was completed, the solvent was drained. Column chromatography was used for separation and purification, and DCM was used as the elution. The mixture was collected and concentrated to obtain 426 mg of compound IV as a white solid with a yield of 71.15%. 1H NMR (500 MHz, CDCl ₃ ) δ8.29-8.26 (m, 2H), 7.85 (d, J＝8.1 Hz, 2H), 7.44 (d, J＝8.0 Hz, 2H), 7.39-7.36 (m, 2H), 5.31 (s, 2H), 1.35 (s, 12H).

56 mg (0.1 mmol) of LDK378, 60 mg (0.15 mmol) of compound IV, 39 mg (0.3 mmol) of N,N-diisopropylethylamine and 5 mL of dichloromethane were added to the reactor and reacted at room temperature for 12 hours. After the reaction was completed, the solvent was drained. Column chromatography was used for separation and purification, and the eluent was DCM/MeOH (20/1) to obtain 30 mg of compound I as a white solid with a yield of 36.70%. 1HNMR (500 MHz, CDCl ₃ )δ9.57(s,1H),8.56(d,J＝8.0Hz,1H),8.14(s,1H),7.98(s,1H),7.94(dd,J＝8.0Hz,1H),7.82(d,J＝8.0Hz,2H),7.63-7.59(m,1H),7.57(m,1H),7.38(d ,J＝8.0Hz,2H),7 .27(m,1H),6.69(s,1H),5.18(s,2H),4.57-4.52(m,1H),4.35(s,2H),3.30-3.22(m,1H),2.90-2.80(m,3H),2.16(s,3H),1.76(m,2H),1.59(m,2 H),1.37-1.31(m,24H).

Example 2

200 mg (0.24 mmol) of compound I, 28 mg (0.27 mmol) of diethanolamine and 5 mL of a dichloromethane/ether mixed solvent were added to a reactor and reacted at room temperature for 3 hours. After the reaction was completed, the precipitate was centrifuged and washed three times with ether, dried, and the solvent was evaporated. 150 mg of compound VI was obtained as a white solid with a yield of 76.21%. 1H NMR (500 MHz, CDCl ₃ )δ9.50(s,1H),8.57(d,J＝8.0Hz,1H),8.15(s,1H),8.01(s,1H),7.92(dd,J＝8.0Hz,1H),7.62(m,1H),7.543H),7.30(d,J＝8.0Hz,2H),7.26(m,1H),6.6 9(s,1H),5.10(s,2H),4.72(m,4.54(m,1H),4.31(m,2H),4.08(m,2H),4.00(m,2H),3.67(m,1H),3.25(m,2.80(m,6H),2.16(s,3H),1.75(m,2H), 1.58(m,2H),1.35(m,12H).

Example 3

100 mg (0.12 mmol) of compound VI, 5 mL of 1 mol/L dilute hydrochloric acid and 5 mL of dichloromethane were added to the reactor and reacted at room temperature for 6 hours. After the reaction was completed, the liquids were separated, the aqueous phase was extracted with dichloromethane, the organic phases were combined, dried, and the solvent was evaporated to obtain 80 mg of compound VII as a white solid with a yield of 87.49%. 1H NMR (500MHz, CDCl ₃ ) δ9.71(m,1H),8.51(d,J＝8.0Hz,1H),8.28(m,2H),8.06(d,J＝7.8Hz,2H),7.92(dd,J＝8.0Hz,1H),7.85(s,1H),7.53(m,1H),7.43(d,J =8.0Hz,2H),7.28(m,1H),6.72(m,1H),5.20(s,2H),4.51(m,1H),4.35(s,2H),3.24(m,1H),2.91(m,3H),2.15(s,3H),1.76(m,2H),1.61(m,2H),1. 29(m,12H).

Example 4

30 mg (0.041 mmol) of compound VII, 5 mg (0.048 mmol) of neopentyl glycol, 24 mg (0.2 mmol) of anhydrous magnesium sulfate and 5 mL of dichloromethane were added to the reactor and reacted at room temperature for 6 hours. After the reaction was completed, the mixture was filtered and the filtrate was evaporated to dryness. Column chromatography was used for separation and purification, and the eluent was DCM/MeOH (20/1) to obtain 21.6 mg of compound VIII as a white solid with a yield of 65.90%. 1H NMR (500 MHz, CDCl ₃ )δ9.44(s,1H),8.58(d,J＝8.0Hz,1H),8.15(s,1H),8.01(s,1H),7.94(dd,J＝8.0Hz,1H),7.81(d,J＝8.0Hz,2H),7.62(m,1H),7.56(m,1H),7.38(d,J＝8. 0Hz,2H),7.27(m,1H),6.70(s,1H),5.18(s,2H),4.55(m,1H),4.35(s,2H),3.77(s,4H),3.26(m,1H),2.90(m,3H),2.16(s,3H),1.74(m,2H),1.60 (m,2H),1.36(m,12H) implementation. Example 5

59mg (0.25mmol) of 4-(hydroxymethyl)phenylboronic acid pinacol ester, 34mg (0.3mmol) of methylsulfonyl chloride, 38mg (0.375mmol) of triethylamine and 15mL of dichloromethane were added to the reactor, and the reaction was carried out in an ice-water bath for 1 hour, and then gradually warmed to room temperature. After the reaction was monitored by thin layer chromatography, the solvent was spin-dried. It was redissolved in 15mL of dichloromethane, and Ceritinib and triethylamine were added, and the reaction was carried out for 12h. After the reaction was monitored by thin layer chromatography, column chromatography was used for separation and purification, and the eluent was DCM/MeOH (20/1). Concentration gave 53mg of compound IX as a white solid with a yield of 61.0%. ¹ H NMR (500MHz, CDCl3) δ9.48(s,1H),8.57(d,J＝8.2Hz,1H),8.14(s,1H),7.98(s,1H),7.92(dd,J＝8.01H),7.79(d,J＝8.0Hz,2H),7.61(m,1H),7.54(s,1H) ,7.38(d,J＝8.0Hz,2H),7.25(m,1H),6,81(s,1H),4.55(m,1H),3.62(s,2H),3.48(s,2H),3.26(m,1H),3.04(m,2H),2.65(m,1H),2.13(s,3H),1.7 4(m,4H),1.35(m,24H).

Example 6

A small amount of compound I and different equivalents of H ₂ O ₂ were dissolved in MeOH, and the final concentrations of compound I were set to 50 μmol/L and 100 μmol/L, respectively. Mix well and incubate at 37°C for 24 hours. The reaction solution was then spun dry and redissolved in CDCl ₃ , and the changes in the marker peaks of compound I were detected by nuclear magnetic resonance. The results are shown in Figures 2 and 3. Figure 2 is a full nuclear magnetic resonance image of compound I in vitro oxidation; from top to bottom in Figure 2, the groups are 100 μmol/L compound I and 150 μmol/LH ₂ O ₂ incubated for 24 hours, 50 μmol/L compound I and 100 μmol/LH ₂ O ₂ incubated for 24 hours, 50 μmol/L compound I and 50 μmol/LH ₂ O ₂ incubated for 24 hours, and compound I control group; wherein, the treatment method of the compound I control group is to incubate only with compound I solution for 24 hours. Figure 3 is a partial enlarged view of Figure 2, which is used to monitor the process of compound I in vitro oxidation.

Figures 2 and 3 show that compound I can be oxidized in a relatively low concentration H2O2 environment, proving that compound I is ROS responsive.

Example 7

In vitro anti-tumor activity assay 1:

The in vitro anti-tumor drug screening experiment of the above-mentioned compound is carried out with a variety of cancer cells and normal cells as experimental objects, and the in vitro anti-tumor activity of the sample is detected by MTT colorimetry. The detection principle is that the amber dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to blue-purple needle-shaped formazan crystals that are insoluble in water and deposited in the cells. The crystals can be dissolved by dimethyl sulfoxide, and the light absorption value is measured at a wavelength of 490nm using an enzyme-linked immunosorbent assay, which can indirectly reflect the number of cells. The specific steps of the MTT colorimetric method described in the present invention are as follows:

Remove the culture medium from the cell culture dish, wash the cells once with PBS, and then digest the cells with 2mL of trypsin for 2min. Remove the trypsin, add 1mL of complete culture medium to resuspend the cells, take 10μL for cell counting, and adjust the cell concentration to 1×10 ⁵ /mL. Add 1×10 ⁴ cells (100μL/well) to each well of the 96-well plate, place in a cell culture incubator for 24h, aspirate the supernatant, add complete culture medium containing different concentrations of drugs, and incubate for 48h. Each group of samples has 3 replicate wells.

After the incubation, the complete medium containing the drug was aspirated, 100 μL of basal medium and 10 μL of MTT solution were added, and the cells were placed back in the incubator for incubation for 4 hours. The supernatant was gently aspirated, 150 μL of dimethyl sulfoxide was added to each well, and the OD value at a wavelength of 490 nm was detected after shaking and mixing. According to the test results, the IC ₅₀ value was calculated by drug concentration and cell viability using graphpad prism software. IC ₅₀ represents the concentration of the corresponding compound I or LDK378 when the cell proliferation rate is inhibited by 50%. The cell viability was calculated according to the following formula: Cell viability (%) = (average OD ₄₉₀ of the experimental group - OD ₄₉₀ of the blank group) / (average OD ₄₉₀ of the control group - OD ₄₉₀ of the blank group) × 100%.

The cell line experiment was carried out according to the above MTT colorimetric method, and the data are as follows:

Compound I and LDK378 were used to treat human tumor cells and human normal cells, respectively, wherein the human tumor cells include human breast cancer cells MCF-7 and human non-small cell lung cancer cells H2228; human normal cells include human mammary epithelial cells MCF-10A and human liver cells LO2. The in vitro anti-tumor activity of the samples was detected by MTT colorimetry. The experimental results are shown in Tables 1 to 6 and Figures 4 to 7 below, wherein Table 1 is a table of IC ₅₀ values of Compound I and LDK378 against human tumor cells and human normal cells.

Table 1

Figure 4 is a comparison of the cell viability of human breast cancer cells MCF-7 treated with different concentrations of compound I and LDK378 for 48 hours, Figure 5 is a comparison of the cell viability of human non-small cell lung cancer cells H2228 treated with different concentrations of compound I and LDK378 for 48 hours, Figure 6 is a comparison of the cell viability of human normal breast epithelial cell line MCF-10A treated with different concentrations of compound I and LDK378 for 48 hours, and Figure 7 is a comparison of the cell viability of human normal liver cell line LO2 treated with different concentrations of compound I and LDK378 for 48 hours.

The specific data of Figures 4 to 7 and the blank group are shown in Table 2:

Table 2

As shown in Tables 1-2 and Figures 4-7, LDK378 is more toxic to normal cells than to tumor cells. Oxidation-responsive compound I is less toxic to normal cells and more toxic to tumor cells. The experimental results show that compound I can selectively inhibit tumor cell proliferation. This result shows that the piperidine modification strategy of LDK378 using arylboronic acid or arylboronic ester is effective. Compared with the parent skeleton of LDK378, the activity of inhibiting tumor cell proliferation is not weakened but enhanced.

In vitro anti-tumor activity assay 2:

The inhibition rate experiment of compound IX and LDK378 on human cancer cells and normal human cells was conducted again, and the steps were as follows: Digest the cells to be tested and remove the trypsin. Collect the cells, centrifuge at 1200rps, discard the supernatant, resuspend the cells with complete culture medium, count the cells and adjust the cell concentration to 1×10 ⁵ cells/mL. Add 100μL of cell suspension (1×10 ⁴ cells/well) to each well of the 96-well plate and incubate at 37°C in a cell culture incubator for 24h. Aspirate the supernatant culture medium, add complete culture medium containing different concentrations of compound IX or LDK378, and set up 3 replicate wells for each group of samples. Return to the cell culture incubator at 37°C for a fixed time.

After the incubation, the culture medium containing compound IX or LDK378 was aspirated, 100 μL of basal medium and 10 μL of thiazolyl blue (MTT) solution were added, and the cells were returned to the incubator for incubation for 4 h. The supernatant was then aspirated, 150 μL of dimethyl sulfoxide was added to each well, and OD ₄₉₀ was detected after oscillation and mixing. According to the test results, the IC ₅₀ value was calculated by drug concentration and cell viability using graphpad prism software. The IC ₅₀ value represents the concentration of compound IX or LDK378 at which 50% of cell proliferation is inhibited. The cell viability was calculated according to the following formula: Cell viability (%) = (average OD ₄₉₀ of the experimental group - OD ₄₉₀ of the blank group) / (average OD ₄₉₀ of the control group - OD ₄₉₀ of the blank group) × 100%.

The experimental results are shown in Table 3, which is a table of IC ₅₀ values of compound IX and LDK378 against MCF-7 (tumor cells):

Table 3

Group MCF-7 (tumor cells) Compound IX 6.96μmol/L LDK378 4.72μmol/L

As shown in Table 3, the toxicity of LDK378 to breast cancer cells is comparable to the oxidative responsiveness and toxicity of compound IX. This result indicates that the piperidine modification strategy of LDK378 using this type of aryl boronic acid or aryl boronic ester structure is effective. Compared with the parent skeleton of LDK378, the activity of compound IX in inhibiting tumor cell proliferation has not been significantly reduced.

Animal Experimentation:

MCF-7 cells were injected into the subcutaneous tissue of the back of nude mice. On the second day of inoculation, mice were randomly assigned to the control group, the low-dose group treated with LDK378, the high-dose group treated with LDK378, the low-dose group treated with compound I, and the high-dose group treated with compound I, with n=5 in each group, and each group was kept separately in one cage. When the subcutaneous tumor grew to an appropriate size, the inhibitor treatment was started. The mice were administered once a day by intraperitoneal injection. The low-dose group of LDK378 was 20 mg/kg per day, and the high-dose group of LDK378 was 50 mg/kg per day; the low-dose group of compound I was 29 mg/kg per day, and the high-dose group of compound I was 73 mg/kg per day, ensuring that the daily molar concentrations of the two drugs in the low-dose group and the high-dose group were equal. After the start of treatment, the body weight was weighed every other day, the tumor diameter was measured once, and the tumor volume (width ² × length × 0.52) (mm ³ ) was calculated. The experiment ended when the drug was administered for 30 days, the mice were killed, the subcutaneous tumors were removed, and the photos and weights were taken. The experimental results are shown in Figures 8 to 10, where Figure 8 is a graph showing the changes in tumor volume in the low-dose group of Compound I and LDK378, Figure 9 is a graph showing the changes in tumor volume in the high-dose group of Compound I and LDK378, and Figure 10 is a hematoxylin-eosin stained section of mouse tissue 30 days after administration of Compound I.

The specific data of Figures 8 and 9 and their control groups are shown in Table 4:

Table 4

Note: SEM stands for Standard Error of Mean, which means the standard error of the mean.

Table 4 and Figures 8-9 show the inhibition of subcutaneous tumor volume growth in mice by LDK378 and oxidation-responsive compound I. The experimental results show that both LDK378 and compound I can inhibit the growth of subcutaneous tumors in mice, and the inhibitory effect increases with the dose. At the same time, the tumor inhibition activity of compound I compared to LDK378 has not weakened, but slightly enhanced. Figure 10 shows the changes in hematoxylin-eosin stained sections of mouse tissues after 30 days of administration of compound I. The experimental results show that compared with the control group, compound I has no obvious toxic side effects on normal tissues of mice.

In summary, the compound I provided by the present invention exhibits a better anti-tumor effect than the parent drug LDK378, especially in the treatment of breast cancer. The parent drug LDK378 has a good inhibitory effect on the proliferation of the human breast cancer cell line MCF-7, while the compound I has a stronger inhibitory effect on the proliferation of MCF-7. At the same time, LDK378 still exhibits a strong inhibitory effect on the human normal breast cell line MCF-10A, while the inhibitory effect of compound I on MCF-10A is greatly reduced.

Example 8

Boronate replacement experiment: The MTT method was used to determine the in vitro tumor cell growth inhibition activity of compounds I, VI, VII, and human breast cancer cells MCF-7 obtained in Examples 1 to 4. The experimental results are shown in Table 5 and Figure 11, where Table 5 is an IC ₅₀ table of compounds I, VI, VII, and VIII against MCF-7; Figure 11 is a graph of the cell viability of compounds I, VI, VII, and VIII in vitro tumor cell inhibition.

Table 5

The specific values of the cell survival rates of the compounds I, VI, VII, VIII shown in FIG11 and the control group LDK378 and the blank group in vitro inhibition of tumor cells are shown in Table 6:

Table 6

The experimental results show that the overall trend of drug effects is completely consistent when different diol structures are modified on boronic acid, and different substituents also slightly affect the anti-tumor activity of the drug. For example, the aryl boronic acid structure has the best effect, while the effect of aryl boronic acid neopentyl glycol ester is poor. Therefore, different boric acid ester structures have a certain effect on the drug effect, but the effect is limited and will not significantly affect the IC ₅₀ value of the drug, but will only produce slight differences.

The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims (11)

1. An ROS-responsive compound having the structure of formula 1:

r is selected from a group with a structure shown in a formula 2 or a formula 3;

wherein, the R ₁ 'and R ₂' are independently selected from substituted or unsubstituted C _nH_2n+1; wherein n is an integer from 0 to 10;

Or alternatively

At least one of R ₁ 'and R ₂' is connected with O where the at least one of R ₁ 'and R ₂' is connected with B where the at least one of O and O is located to form a heterocycle;

the heteroatoms in the heterocycle include B, O and optionally N.

2. The compound according to claim 1, wherein R is any one of groups of structures represented by formulae 4 to 7;

3. the compound according to claim 1 or 2, wherein R is selected from any one of the structures represented by formulae a to t;

4. a compound according to claim 3, characterized in that it is

5. Use of a compound according to any one of claims 1 to 4 for the preparation of an antitumor drug.

6. A medicament, which is characterized by comprising at least one of the compounds and pharmaceutically acceptable salts thereof according to any one of claims 1-4 and pharmaceutically acceptable excipients.

7. A process for the preparation of a compound according to any one of claims 1 to 4, comprising the steps of:

Reacting an arylboronic acid compound or arylboronic acid ester compound with LDK378 to obtain a compound according to any one of claims 1 to 4.

8. The method of claim 7, wherein an aryl borate compound or an aryl borate compound is subjected to a carbonation reaction, a sulfonation reaction or a halogenation reaction to obtain an intermediate product, and the intermediate product is subjected to a urethanization reaction or a nucleophilic substitution reaction with LDK378 to obtain the compound.

9. The method of claim 7, wherein after the intermediate product has been subjected to a urethanization reaction or a nucleophilic substitution reaction with LDK378, further comprising: and carrying out boric acid transesterification on the product obtained after the carbamate reaction or nucleophilic substitution reaction.

10. The method of claim 9, further comprising, after performing the boric acid transesterification reaction: and (3) carrying out hydrolysis reaction on the product obtained after the boric acid transesterification reaction.

11. The method of claim 10, further comprising, after the hydrolysis reaction: and carrying out boric acid esterification reaction on the product obtained after the hydrolysis reaction.