Use of chromenone compound

Abstract

The present invention relates to use of a chromenone compound, and specifically, to use of a small molecule substance X in preparing a medicament for treating cancer. Substance X is a compound of formula I or a pharmaceutically acceptable salt thereof. The chromenone compound is used for treating cancer such as lung cancer, liver cancer, colon cancer, breast cancer, bone marrow cancer, or melanoma. Compared with macromolecules, the chromenone compound features good penetrability, good absorption and penetrability after injection or intragastric administration, and targeted actions in the tumor microenvironment.

Description

Applications of Chonenones

[0001] This application claims priority to Chinese patent application 2024109077498, filed on July 8, 2024. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention relates to the application of a chromone compound. Background Technology

[0003] Prior art US20180344862A1 discloses a method for using cytosine-linked isoflavone antitumor drugs to treat cancer and the following general formula.

[0004] Cytosine-linked isoflavones can be used to treat conditions that lead to cell-dependent peroxisome HSD17B4 degradation of ultra-long-chain fatty acids and provide the necessary energy for cell proliferation, such as in the treatment of colorectal and prostate cancer.

[0005] Existing technology (Development of Targeted Antitumor Drugs: Peroxisome Enzymes: Cytosine-Linked Flavonoids as Inhibitors of Hydroxysteroids 17-β-Dehydrogenase-4 (HSD17B4), Org. Biomol. Chem., 2017, 15, 7623) reported that cytosine-linked isoflavones are selective inhibitors of the fatty acid MAP protease activity in the bifunctional peroxisome enzyme hydroxysteroid 17β-dehydrogenase-4 (HSD17B4). The results suggest that peroxisome enzymes may be novel targets for cancer therapy. Summary of the Invention

[0006] The technical problem this invention aims to solve is that existing clinically marketed PD-1 / PD-L1 inhibitors are all large molecular weight compounds. Due to their large molecular weight, they suffer from poor penetration ability, strong antibody immunogenicity leading to significant side effects, and the need for injection administration. This invention provides an application of chromone compounds for the treatment of cancers such as lung cancer, liver cancer, colon cancer, breast cancer, bone marrow cancer, or melanoma. Compared with large molecules, they have strong penetration ability, exhibiting good absorption and penetration after injection or oral administration, reaching the tumor microenvironment to exert their effects.

[0007] This invention provides the use of substance X in the preparation of a cancer treatment drug, wherein substance X is a compound as shown in Formula I or a pharmaceutically acceptable salt thereof.

[0008] Among them, R 1 It is chlorine or methoxy;

[0009] X is CHR X-1 NR X-2 Or oxygen;

[0010] R X-1 It is hydrogen, C1-C4 alkyl, 6-10 aryl, C(O)N(R) X-1-1 )2 or a C1-C4 alkyl group substituted with one or more hydroxyl groups;

[0011] R X-1-1 Independently hydrogen or C1-C4 alkyl;

[0012] R X-2 It is hydrogen, C1-C4 alkyl, 6-10 aryl, or C1-C4 alkyl substituted with one or more hydroxyl groups;

[0013] m can be 1, 2, 3 or 4.

[0014] In a preferred embodiment, certain groups in the compound of formula (I) or its pharmaceutically acceptable salt are defined as follows, and the definitions of groups not mentioned are as described in any embodiment of the invention (hereinafter referred to as "in some embodiments").

[0015] In some implementations, the cancer is lung cancer, liver cancer, colon cancer, breast cancer, or bone marrow cancer.

[0016] In some implementations, the cancer is melanoma.

[0017] In some embodiments, the substance X inhibits the binding of PD-1 to PD-L1 by a rate of >10%, preferably >30%, and more preferably >40%.

[0018] In some embodiments, the molecular weight of substance X is less than 500 Daltons.

[0019] In some implementation schemes, R X-1 and R X-2 In the C1-C4 alkyl group and the C1-C4 alkyl group substituted with one or more hydroxyl groups, the C1-C4 alkyl group is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl, for example methyl or ethyl.

[0020] In some implementation schemes, R X-1 and R X-2 In this context, the 6-10 aryl groups are independently phenyl or naphthyl, such as phenyl.

[0021] In some implementation schemes, R X-1 and R X-2 In this context, the plurality of C1-C4 alkyl groups substituted with one or more hydroxyl groups is one or two, for example, one.

[0022] In some implementation schemes, R X-1-1 In this context, the C1-C4 alkyl groups are independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl.

[0023] In some implementation schemes, R X-1 C(O)N(R) X-1-1 )2.

[0024] In some implementation schemes, R X-1-1 It is hydrogen.

[0025] In some implementation schemes, R X-2 It is a C1-C4 alkyl, phenyl, or a C1-C4 alkyl substituted with one or more hydroxyl groups; preferably, R X-2 It is a C1-C4 alkyl, phenyl, or a C1-C4 alkyl substituted with one hydroxyl group.

[0026] In some implementations, m is 2.

[0027] In some implementation schemes, R X-1 It is C(O)NH2.

[0028] In some implementation schemes, R X-2 Methyl, ethyl, Or phenyl.

[0029] In some embodiments, the pharmaceutically acceptable salt of the compound represented by Formula I is prepared by salting the compound represented by Formula I with an acid, wherein the acid may be an inorganic acid; preferably hydrochloric acid, hydrobromic acid, or hydroiodic acid; the pharmaceutically acceptable salt of the compound represented by Formula I is the hydrochloride, hydrobromide, or hydroiodic acid salt of the compound represented by Formula I.

[0030] In some embodiments, in the pharmaceutically acceptable salt of the compound as shown in Formula I, the molar ratio of the compound as shown in Formula I to the acid is 1:(1-4), for example 1:2.

[0031] In some implementation schemes, R 1 It is chlorine;

[0032] X is CHR X-1 or NR X-2 ;

[0033] R X-1 It is C(O)NH2;

[0034] R X-2 It is a C1-C4 alkyl, a 6-10 aryl, or a C1-C4 alkyl substituted with one or more hydroxyl groups;

[0035] m can be 1, 2, 3 or 4.

[0036] In some embodiments, the compound shown in Formula I is a compound shown in Formula I'.

[0037] R X-2 It is a C1-C4 alkyl group substituted with one or more hydroxyl groups;

[0038] m can be 1, 2, 3 or 4.

[0039] In some embodiments, the substance X is any of the following compounds:

[0040] In some embodiments, substance X is compound 1 as shown below.

[0041] In some embodiments, the substance X comprises 0.01% to 30% by weight in the drug, preferably 0.01% to 15%, more preferably 0.01% to 10%, and even more preferably 0.1% to 5%.

[0042] In some embodiments, the dosage of substance X may be administered according to the subject's weight, with a non-limiting range of 0.1 mg / kg to 300 mg / kg, referring to a single dose, calculated based on the active ingredient, such as 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg. , 30mg / kg, 35mg / kg, 40mg / kg, 45mg / kg, 50mg / kg, 55mg / kg, 60mg / kg, 65mg / kg, 70mg / kg, 75mg / kg, 80mg / kg, 85mg / kg, 90mg / kg, 95mg / kg, 100mg / kg, 105mg / kg, 110mg / kg, 115mg / kg, 120mg / kg, 125mg / kg, 130mg / kg, 135 mg / kg, 140mg / kg, 145mg / kg, 150mg / kg, 155mg / kg, 160mg / kg, 165mg / kg, 170mg / kg, 175mg / kg, 180mg / kg, 18 5mg / kg, 190mg / kg, 195mg / kg, 200mg / kg, 205mg / kg, 210mg / kg, 215mg / kg, 220mg / kg, 225mg / kg, 230mg / kg, 23 5 mg / kg, 240 mg / kg, 245 mg / kg, 250 mg / kg, 255 mg / kg, 260 mg / kg, 265 mg / kg, 270 mg / kg, 275 mg / kg, 280 mg / kg, 285 mg / kg, 290 mg / kg, 295 mg / kg or 300 mg / kg, preferably 1 mg / kg to 100 mg / kg, more preferably 1 mg / kg to 30 mg / kg, for example 5 mg / kg or 10 mg / kg.

[0043] In some implementations, the application frequency of substance X is once a day, twice a day, three times a day, once every other day, once a week, twice a week, three times a week, once every other week, once every two weeks, once every three weeks, or once every four weeks, etc.

[0044] In some embodiments, the dosage of substance X is 5 mg / kg, and the frequency of administration is once a day.

[0045] In some embodiments, the dosage of substance X is 10 mg / kg, and the frequency of administration is once a day.

[0046] In some embodiments, the substance X can be administered by any suitable route of administration in the art, such as oral administration, injection administration, sublingual administration, rectal administration, local administration, etc., and the injection administration can be intravenous injection, intramuscular injection or subcutaneous injection.

[0047] In some embodiments, the drug further comprises substance Y, which is a compound as shown in Formula II or Formula III.

[0048] In some embodiments, the active ingredient of the drug is composed of substance X and substance Y.

[0049] In some embodiments, the mass ratio of substance Y to substance X is (0.1-5):1, preferably (0.5-2):1, for example 1:1.

[0050] In some embodiments, the active ingredient of the drug comprises substance X and substance Y, wherein substance X can be administered according to the subject's weight, and the dosage, in non-limiting examples, can range from 0.05 mg / kg to 150 mg / kg (single dose), for example 0.1 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, etc. mg / kg, 50 mg / kg, 55 mg / kg, 60 mg / kg, 65 mg / kg, 70 mg / kg, 75 mg / kg, 80 mg / kg, 85 mg / kg, 90 mg / kg, 95 mg / kg, 100 mg / kg, 105 mg / kg, 110 mg / kg, 115 mg / kg, 120 mg / kg, 125 mg / kg, 130 mg / kg, 135 mg / kg, 140 mg / kg, 145 mg / kg, 150 mg / kg, preferably 1 mg / kg to 50 mg / kg, more preferably 1 mg / kg to 15 mg / kg, for example 5 mg / kg.

[0051] In some embodiments, the active ingredient of the drug comprises substance X and substance Y, wherein substance Y can be administered according to the subject's weight, and the dosage, in non-limiting examples, can range from 0.005 mg / kg to 750 mg / kg (single dose), for example 0.01 mg / kg, 0.1 mg / kg, 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg. The concentrations are 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 250 mg / kg, 500 mg / kg, and 750 mg / kg, preferably 0.1 mg / kg to 250 mg / kg, more preferably 0.1 mg / kg to 75 mg / kg, for example, 5 mg / kg.

[0052] In some embodiments, substances X and Y can be administered independently using any suitable route of administration in the art, such as oral administration, injection administration, sublingual administration, rectal administration, local administration, etc., wherein the injection administration can be intravenous injection, intramuscular injection, or subcutaneous injection.

[0053] In some embodiments, both substance X and substance Y are administered via injection, such as intramuscular injection or intravenous injection.

[0054] In some implementations, substance X and substance Y can be administered simultaneously; alternatively, substance X and substance Y can be administered separately at intervals, for example, substance X can be administered first, followed by substance Y, or substance Y can be administered first, followed by substance X.

[0055] In some implementations, when the drug also contains substance Y, the frequency of administration of substance X and substance Y is independently once a day, twice a day, three times a day, every other day, once a week, twice a week, three times a week, every other week, once every two weeks, once every three weeks, or once every four weeks.

[0056] In some embodiments, when the drug also contains substance Y, the dosage of substance X and substance Y is 5 mg / kg, and the frequency of administration is once a day.

[0057] In some embodiments, the substance X is Substance Y is

[0058] In some embodiments, the pharmaceutical composition comprises a first pharmaceutical composition and a second pharmaceutical composition;

[0059] The first pharmaceutical composition comprises an active ingredient and pharmaceutical excipients; the active ingredient comprises substance X as described above;

[0060] The second pharmaceutical composition comprises an active ingredient and pharmaceutical excipients; the active ingredient comprises substance Y as described above.

[0061] In some embodiments, the mass ratio of substance Y to substance X is (0.1-5):1, preferably (0.5-2):1, for example 1:1.

[0062] In some embodiments, the sole active ingredient of the first pharmaceutical composition is substance X.

[0063] In some embodiments, the sole active ingredient of the second pharmaceutical composition is said substance Y.

[0064] In some embodiments, the substance X accounts for 0.01% to 30% by weight in the first pharmaceutical composition, preferably 0.01% to 15%, more preferably 0.01% to 10%, and even more preferably 0.1% to 5%.

[0065] In some embodiments, the substance Y accounts for 0.01% to 30% by weight in the second pharmaceutical composition, preferably 0.01% to 15%, more preferably 0.01% to 10%, and even more preferably 0.1% to 5%.

[0066] In some embodiments, the first pharmaceutical composition and the second pharmaceutical composition may be the same dosage form or different dosage forms.

[0067] In some embodiments, the first and second pharmaceutical compositions may be administered independently using any suitable route of administration in the art, such as oral administration, injection administration, sublingual administration, rectal administration, local administration, etc., wherein the injection administration may be intravenous administration, intramuscular administration, or subcutaneous administration.

[0068] In some embodiments, the first and second drug compositions may be administered simultaneously; or they may be administered at intervals, for example, the first drug composition may be administered first, followed by the second drug composition, or the second drug composition may be administered first, followed by the first drug composition.

[0069] This invention provides a combination medicine box, which includes,

[0070] A first container containing the first pharmaceutical composition as described above; and,

[0071] The second container contains the second pharmaceutical composition as described above.

[0072] The combination medicine box preferably also includes an instruction manual.

[0073] The present invention provides a method for treating cancer, comprising administering a therapeutically effective amount of the substance X to a subject in need of such treatment.

[0074] The present invention provides a method for treating cancer, comprising administering therapeutically effective amounts of said substance X and said substance Y to a subject in need of such treatment.

[0075] In some implementations, the cancer in the method is lung cancer, liver cancer, colon cancer, breast cancer, or bone marrow cancer.

[0076] In some implementations, the cancer in the method is melanoma.

[0077] The application methods (including application method, dosage, and application interval) of the substance X and the substance Y can be the same or different, and can be adjusted by those skilled in the art as needed to provide the optimal therapeutic effect.

[0078] The administration methods for substances X and Y can be as described above.

[0079] Terminology Explanation

[0080] As used herein, the term “treatment” refers to the process of intervening in or altering a particular health condition. When a specific ailment is involved, treatment means: (1) alleviating one or more biological manifestations of the disease or ailment; (2) interfering with (a) one or more points in a biological cascade that causes or induces the ailment or (b) one or more biological manifestations of the ailment; (3) improving one or more symptoms, effects or side effects associated with the ailment, or one or more symptoms, effects or side effects associated with the ailment or its treatment; or (4) slowing the development of the ailment or one or more biological manifestations of the ailment.

[0081] As used herein, the term "therapeutic effective amount" refers to an amount of compound sufficient to effectively treat the disease or condition described herein when administered to a subject. The amount of compound constituting a "therapeutic effective amount" will vary depending on the compound, the condition and its severity, and the age of the subject to be treated, but may be adjusted as needed by those skilled in the art.

[0082] As used in this article, the term "container" refers to any container and cap suitable for storing, transporting, dispensing and / or handling pharmaceuticals.

[0083] As used herein, the term "subject" refers to any animal, preferably a mammal, that is about to be or has already been administered the compound or composition according to embodiments of the invention. As used herein, the term "mammal" includes any mammal. Examples of mammals include, but are not limited to, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, and humans, with humans being the most preferred.

[0084] The term "pharmaceutical excipients" as used in this article refers to excipients and additives used in the production of pharmaceuticals and the dispensing of prescriptions. It includes all substances contained in pharmaceutical preparations, excluding the active ingredient. See the Pharmacopoeia of the People's Republic of China (2015 Edition), Volume IV, or the Handbook of Pharmaceutical Excipients (Raymond C. Rowe, 2009 Sixth Edition).

[0085] The term "pharmaceutically acceptable" as used in this article means that the acids or bases, solvents, excipients, etc. (used in the preparation of salts) are generally non-toxic, safe, and suitable for patient use.

[0086] As used herein, the term "pharmaceutically acceptable salt" refers to a salt prepared by reacting a compound with a relatively non-toxic, pharmaceutically acceptable acid or base. When a compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of a pharmaceutically acceptable base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include, but are not limited to, lithium, sodium, potassium, calcium, aluminum, magnesium, zinc, bismuth, ammonium, and diethanolamine salts. When a compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of a pharmaceutically acceptable acid in a pure solution or a suitable inert solvent. The pharmaceutically acceptable acid includes inorganic acids, including but not limited to, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, phosphorous acid, sulfuric acid, and hydrogen sulfate. The pharmaceutically acceptable acids include organic acids, including but not limited to: acetic acid, propionic acid, oxalic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, octanoic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, salicylic acid, tartaric acid, methanesulfonic acid, isonicotinic acid, acidic citric acid, oleic acid, tannic acid, pantothenic acid, hydrogen tartrate, ascorbic acid, gentic acid, fumaric acid, gluconic acid, succinic acid, formic acid, ethanesulfonic acid, dihydroxynaphthyl acid (i.e., 4,4'-methylene-bis(3-hydroxy-2-naphthylcarboxylic acid)), amino acids (e.g., glutamic acid, arginine), etc. When a compound contains functional groups with relatively acidic and relatively basic properties, it can be converted into a base addition salt or an acid addition salt. For details, see Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science 66: 1-19 (1977), or Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl and Camille G. Wermuth, ed., Wiley-VCH, 2002).

[0087] The "pharmaceutically acceptable salts" described in this article can exist in amorphous or crystalline forms. The term "amorphous" refers to a disordered distribution of ions or molecules, meaning there is no periodic arrangement between them. The term "crystalline" refers to a strictly periodic arrangement of ions or molecules in three-dimensional space, with a regular repetition at certain intervals; due to different periodsic arrangements, multiple crystalline forms can exist, a phenomenon known as polymorphism.

[0088] The term "alkyl" refers to an alkyl group having a specified number of carbon atoms (e.g., C40, C50, C6 ... 1-6 Or C1-C6, C 1-4 Alkyl groups (or C1-C4), straight-chain or branched, saturated monovalent hydrocarbon groups. Alkyl groups include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, etc.

[0089] The term "aryl" refers to an aryl group having a specified number of carbon atoms in its ring (e.g., C10, C20, C30, C40, C50, C60, C7 ... 6-10 Or C6-C 10 Aryl groups are aromatic groups. Examples of aryl groups include, but are not limited to, phenyl or naphthyl groups.

[0090] The pharmaceutical excipients described in this invention refer to substances other than the active ingredient that are contained in the dosage form.

[0091] The pharmaceutical excipients described in this invention may possess certain physiological activities, but their addition will not alter the dominant role of the aforementioned drugs in the disease treatment process; rather, they will only exert auxiliary effects, which are merely the utilization of the known activities of the excipient. If the aforementioned auxiliary components are used in combination with the drugs of this invention, they should still fall within the scope of protection of this invention.

[0092] In this invention, depending on the therapeutic purpose, the pharmaceutical composition can be formulated into various types of dosage forms, such as tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, suppositories, and injections (solutions and suspensions).

[0093] In this invention, any excipient known and widely used in the art can be used to form the pharmaceutical composition in tablet form. Examples of carriers include lactose, white sugar, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, and silica; binders include water, ethanol, propanol, common syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, and polyvinylpyrrolidone; disintegrants include dry starch, sodium alginate, agar powder, and kelp powder, sodium bicarbonate, calcium carbonate, fatty acid esters of polyvinyl sorbitol, sodium lauryl sulfate, monoglyceride stearate, starch, and lactose; disintegration inhibitors include white sugar, glyceryl tristearate, coconut oil, and hydrogenated oil; adsorption promoters include quaternary ammonium base and sodium lauryl sulfate; wetting agents include glycerin and starch; adsorbents include starch, lactose, kaolin, bentonite, and colloidal silica; and lubricants include pure talc, stearates, boric acid powder, and polyethylene glycol. It can also be made into sugar-coated tablets, gelatin-coated tablets, sausage-coated tablets, coated tablets, double-layered tablets and multilayered tablets by selecting common coating materials as needed.

[0094] In this invention, any known and widely used excipients in the art can be used to form the pharmaceutical composition in pill form, such as carriers, like lactose, starch, coconut oil, hardened vegetable oil, kaolin, and talc; binders, like gum arabic, tragacanth, gelatin, and ethanol; and disintegrants, like agar and kelp powder.

[0095] In this invention, any excipient known and widely used in the art can be used to form the pharmaceutical composition in suppository form, such as polyethylene glycol, coconut oil, higher alcohols, esters of higher alcohols, gelatin, and semi-synthetic glycerides.

[0096] In this invention, to prepare the pharmaceutical composition in injectable form, the solution or suspension can be sterilized (preferably with the addition of appropriate amounts of sodium chloride, glucose, or glycerol, etc.) to prepare an injectable form with isotonicity similar to blood. Any commonly used carrier in the art can also be used when preparing the injectable form. Examples include water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and fatty acid esters of polyvinyl sorbitol. Furthermore, common solvents, buffers, and analgesics can also be added.

[0097] In this invention, the method of administration of the pharmaceutical composition can be selected from various dosage forms according to the patient's age, gender, other conditions and symptoms. For example, tablets, pills, solutions, suspensions, emulsions, granules or capsules can be administered orally; injections can be administered alone or mixed with injection delivery solutions (such as glucose solutions and amino acid solutions) for intravenous injection; suppositories are administered rectally.

[0098] In this invention, the dosage mentioned refers to the dosage administered to mice. In practical applications, the dosage for different test subjects can be adjusted based on the conversion factor given by the FDA in "Estimating the Safe Starting Dose in Clinical Trials for Therapeutics for in Adult Healthy Volunteers" or according to the actual application.

[0099] For example, if the dosage for mice is 100 mg / kg, the equivalent human dosage is 100 / 12.3 = 8.1 mg / kg. If the dosage for mice is 10 mg / kg / day, then the dosage for humans could be ~1 mg / kg / day, meaning a 60 kg person would receive 60 mg per day.

[0100] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0101] The reagents and raw materials used in this invention are all commercially available.

[0102] The positive and progressive effects of this invention are as follows: This invention applies chromone small molecule compounds to the treatment of cancer, such as lung cancer, liver cancer, colon cancer, breast cancer or bone marrow cancer. Compared with macromolecules, it has the characteristics of strong penetrability. After injection or oral administration, it has good absorption and penetration, and reaches the tumor microenvironment to exert its effects. Attached Figure Description

[0103] Figure 1 shows that compound 1 significantly inhibited tumor growth in a mouse LLC lung cancer model, where * indicates P < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0104] Figure 2 shows the therapeutic effect of compound 1 on 4T1 (breast cancer) tumor-bearing mice.

[0105] Figure 3 shows the therapeutic effect of compound 1 on SP20 (myeloma) tumor-bearing mice.

[0106] Figure 4 shows the therapeutic effect of compound 1 on H22 (hepatocellular carcinoma) tumor-bearing mice.

[0107] Figure 5 shows the therapeutic effect of compound 1 on MC38 (colon cancer) tumor-bearing mice.

[0108] Figure 6 shows the therapeutic effect of the combination of compound 1 and the compound shown in formula II on a mouse LLC lung cancer model.

[0109] Figure 7 is a line graph of the measured blood drug concentration. Detailed Implementation

[0110] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0111] Example 1: Verification test of the compound's effect in inhibiting PD-1 / PD-L1 binding

[0112] The inhibitory effect of the compound of this application on PD-1 / PD-L1 binding was detected using a PD-1 / PD-L1 in vitro detection kit (Perkin Elmer / cisbio, catalog number: 64PD1PEG) in accordance with the product instructions. The results are shown below.

[0113] Table a

[0114] *All compounds were tested for activity at a concentration of 20 μM. The effect of inhibiting the binding of PD-1 and PD-L1 is indicated by "+", where "+" indicates 10% < inhibition rate ≤ 20%, "++" indicates 20% < inhibition rate ≤ 30%, "+++" indicates 30% < inhibition rate ≤ 40%, and "++++" indicates 40% < inhibition rate.

[0115] The experimental results above show that the compounds of the present invention have a good effect on inhibiting PD-1 / PD-L1 binding, and their inhibition rates on PD-1 / PD-L1 binding are all greater than 10%.

[0116] Example 2: Validation of the antitumor efficacy of compound 1 in a mouse LLC lung cancer model

[0117] (1) Methods: 6-8 week old SPF grade C57BL / 6 male mice were subcutaneously injected with 200 μL of Lewis cell (LLC cell) suspension, at a ratio of 1 × 10⁻⁶ cells per mouse. 6 LLC cells (Nanjing Kebai Biotechnology Co., Ltd., catalog number CBP60188) were subcutaneously injected at a rate of / mL. After tumor formation, tumors with a volume of approximately 50-70mm were selected. 3 Mice bearing tumors were randomly divided into a model control group, a positive control group, and a PC group, with 5 mice per group. After grouping, PC group mice received intraperitoneal injections of the drug (compound 1) once daily (10 mg / kg) for 18 consecutive days. Positive control group mice received intravenous injections of PD-1 antibody (sintilimab injection, batch number: DP2111020) at a dose of 200 μg / mouse on days 1, 4, 7, 10, 13, 16, and 19 after grouping. The general clinical condition of the animals was observed daily, and body weight and tumor volume were measured every other day. Twenty-four hours after the last administration, the animals were sacrificed, and tumors were dissected for measurement of tumor weight and immunohistochemical analysis.

[0118] Tumor volume measurement: The long and short diameters of the tumor are measured every two days to plot the tumor growth curve.

[0119] Tumor volume is calculated using the formula: V = (ab) 2 ) / 2 is measured and calculated, where a and b are the longest and shortest diameters, respectively.

[0120] (2) Results: Apart from tumor growth, the animals showed no other abnormalities, and tissue samples were collected from their bodies. There was no statistically significant difference in body weight between the groups and the model control group during the drug administration period. In the positive control group, the tumor volume on days 3 and 5 of drug administration was significantly lower than that in the model control group (P<0.05), and the tumor volume on day 7 of drug administration was also significantly lower than that in the model control group (P<0.01) (as shown in Figure 1 and Table 1). In the PC group, the tumor volume on days 7, 9, 11, 13, 15, 17, and 19 of drug administration was significantly lower than that in the model control group (P<0.01) (as shown in Figure 1 and Table 1).

[0121] Table 1. Effect of the test drug PC on tumor volume in tumor-bearing mice ( mm 3 (n=5) Note: One-way ANOVA and rank-sum test were used. Compared with the model control group, "*" p < 0.05, "**" < 0.01.

[0122] Table 2: Effect of the test drug PC on tumor volume inhibition rate in tumor-bearing mice

[0123] The tumor volume inhibition rates of the positive control group and the PC group after 19 days of administration were 16.29% and 77.14%, respectively (as shown in Table 2).

[0124] Compared with the model control group, the tumor weight of the PC group mice was reduced and the difference was statistically significant (P<0.01), and the tumor weight inhibition rate on the day of dissection was 70.36%.

[0125] Compared with the model control group, the percentage of CD8 positive area in the tumors of PC group mice was significantly increased (P<0.01); the percentage of PD-L1 positive area in the tumors of PC group mice was significantly decreased (P<0.01) (as shown in Table 3).

[0126] Table 3: Effects of the test drug PC on the expression of CD4, CD8, and PD-L1 in tumors of tumor-bearing mice Note: Compared with the model control group, "*" p < 0.05, "**" < 0.01.

[0127] (3) Conclusion: Based on the above research results, the drugs in the PC group have the effect of increasing the expression of CD8 T cells in tumor tissue and reducing the expression level of PD-L1 in tumor tissue, and show a significant inhibitory effect on the growth of LLC lung cancer tumors. The anti-tumor effect of the small molecule drugs in the PC group is superior to that of sintilimab injection (positive control).

[0128] Example 3: Effects of Compound 1 in the treatment of breast cancer, myeloma, liver cancer, and colon cancer.

[0129] (1) Objective: To study the therapeutic effects of compound 1 on tumor-bearing mice with 4T1 (breast cancer), SP20 (myeloma), H22 (liver cancer) and MC38 (colon cancer) models.

[0130] (2) Methods: SPF-grade mice (n=8-10 per group, female mice were used for the 4T1 breast cancer model, and male mice were used for the others) were injected axillarily with 200 μL of 4T1 cell, SP20 cell, H22 cell, and MC38 cell suspensions (Nanjing Kebai Biotechnology Co., Ltd., catalog numbers: 4T1–CBP60352, SP20–CBP60881, H22–CBP60230, MC38–CBP60825, respectively). Each mouse was injected with 4.0 × 10⁶ 4T1 cell suspensions. 6 The SP20 cell suspension was 3.0 × 10⁶ cells / mL. 6 The H22 cell suspension was 3.0 × 10⁶ cells. 6 The MC38 cell suspension was 3.0 × 10⁶ cells / mL. 6 One. After inoculation of tumor cells, at a tumor volume of 40-80 mm. 3 Mice were randomly divided into a model control group and a drug-treated PC group, with 8-10 mice per group. After grouping, the PC group received an intraperitoneal injection of the drug (compound 1) once daily (10 mg / kg) for 15-21 consecutive days. The general clinical condition of the animals was observed daily, body weight was measured twice weekly, and tumor volume was measured every other day. Twenty-four hours after the last administration, the animals were sacrificed, and the tumors were dissected and their weight measured. The thymus and spleen were weighed, and organ coefficients were calculated.

[0131] (3) Results: Apart from tumor growth, the animals showed no other abnormalities and were dissected for tissue collection. There was no statistically significant difference in body weight between the groups and the model control group during the drug administration period.

[0132] 4T1 tumor-bearing mice: Compared with the control group, the tumor volume reduction in the PC group (n=10) was statistically significant at days 5, 9, and 17-21 after drug administration (P<0.05); the tumor volume reduction was also statistically significant at days 11-13 after drug administration (P<0.01) (as shown in Figure 2). The tumor volume inhibition rate in the PC group after 21 days of drug administration was 42.86%; on the day of dissection, the tumor weight of the mice in the PC group was significantly reduced (P<0.01), with a tumor weight inhibition rate of 48.24%. Two mice (2 / 10) in the PC group showed no tumor growth after 21 days of drug administration followed by a 30-day observation period after drug withdrawal, suggesting tumor cure (as shown in Figure 2).

[0133] SP20 tumor-bearing mice: Compared with the control group, the tumor volume reduction in the PC-treated group (n=8) was statistically significant at days 7, 13, 17 to 21 after administration (P<0.05); the tumor volume reduction at day 3 after administration was significantly significant (P<0.01) (as shown in Figure 3); the tumor volume inhibition rate in the PC-treated group at day 21 after administration was 52.6%. On the day of dissection, the tumor weight reduction in the PC-treated group was significantly significant (P<0.01), and the tumor weight inhibition rate was 84.0% (as shown in Table 4).

[0134] Table 4. Effect of the test drug PC on tumor inhibition rate in SP20 tumor-bearing mice ( n=8)

[0135] Note: One-way ANOVA and rank-sum test were used. Compared with the model control group, "*" p < 0.05, "**" < 0.01.

[0136] H22 tumor-bearing mice: Compared with the model control group, the tumor volume of mice in the PC drug group (n=8) decreased significantly at days 3, 5, and 7 after drug administration (P<0.05) (as shown in Figure 4); the tumor volume inhibition rate of the PC drug group after day 15 was 23.98%. On the day of dissection, compared with the model control group, the tumor weight of mice in the PC drug group decreased significantly (P<0.05), and the tumor weight inhibition rate was 54.02%.

[0137] MC38 tumor-bearing mice: Compared with the control group, the tumor volume reduction in the PC drug group (n=8) was statistically significant at days 4, 6, 8, and 14 after drug administration (P<0.05); the tumor volume reduction at days 10-12 after drug administration was also statistically significant (P<0.01) (as shown in Figure 5). The tumor volume inhibition rate in the PC drug group after 16 days of administration was 20.32%.

[0138] (4) Conclusions and Discussion: The drugs in the PC group had a significant tumor-suppressing effect on breast cancer (4T1) tumor-bearing mice throughout the observation process. Among them, the tumor volume of two tumor-bearing mice (20%) did not increase after one month of observation after drug withdrawal, indicating that the tumor was cured. The drugs in the PC group had a significant inhibitory effect on tumor growth in SP20 myeloma tumor-bearing mice. Under the conditions of this experiment, the drugs in the PC group had an early tumor-suppressing effect on liver cancer (H22) and colon cancer (MC38) tumor-bearing mouse models.

[0139] Example 4: Enhanced efficacy of compound 1 and the compound shown in Formula II in a mouse LLC lung cancer model.

[0140] (1) Objective: To verify the efficacy of antitumor drugs PC (i.e., compound 1) and compound shown in Formula II (i.e., ZP-1) using the Lewis lung cancer subcutaneous xenograft model and to discuss the mechanism of combined drug use.

[0141] (2) Methods: SPF grade 6-8 week old, male C57BL / 6 mice were subcutaneously injected with 200 μL Lewis cell suspension at a dose of 1 × 10⁻⁶ cells per mouse. 6 LLC cells were subcutaneously injected at a dose of / mL. Tumor-bearing mice selected based on tumor volume after tumor cell inoculation were randomly divided into four groups: a model control group, a positive control group (PD-1 antibody), a PC group (5mg / kg), a ZP-1 group (5mg / kg), and a PC+ZP-1 combination therapy group (5mg / kg PC + 5mg / kg ZP-1), with 8 mice per group. After grouping, the PC group (5mg / kg), ZP-1 group (5mg / kg), and PC+ZP-1 combination therapy group (5mg / kg PC + 5mg / kg ZP-1) received intraperitoneal injections of the drug once daily for 15 consecutive days. The positive control group mice received intravenous injections of PD-1 antibody (sintilimab injection, batch number: DP2111020) at a dose of 200μg / mouse on days 1, 4, 7, 10, and 13 after grouping. The general clinical condition of the animals was observed daily, animal body weight was measured twice weekly, and tumor volume was measured every other day. Twenty-four hours after the last administration, animals were sacrificed, and tumors were dissected to measure tumor weight and for immunohistochemical analysis. The thymus and spleen were weighed, and organ coefficients were calculated.

[0142] (3) Results: Apart from tumor growth, the animals showed no other abnormalities. There was no statistically significant difference in body weight between the groups and the model control group during the treatment period. As shown in Figure 6 and Table 5, the tumor volume of the PC group mice was lower than that of the model control group on days 2, 4, and 6 after administration, with statistically significant differences (P<0.05). The tumor volume of the ZP-1 group mice was lower than that of the model control group on days 2, 4, 6, 8, 10, 12, and 16 after administration, with statistically significant differences (P<0.05). The tumor volume of the PC+ZP-1 group mice was lower than that of the model control group from days 2 to 16 after administration, with statistically significant differences (P<0.05). The tumor volume inhibition rates of the positive control group, PC group, ZP-1 group, and PC+ZP-1 group after 16 days of administration were 14.05%, 17.53%, 38.94%, and 51.88%, respectively (as shown in Table 6), indicating that the combined administration was significantly more effective than the single drug. Compared with the model control group, the tumor weight of mice in the PC+ZP-1 group was reduced and statistically significant (P<0.01), with a tumor weight inhibition rate of 55% (as shown in Table 7). Compared with the model control group, the percentage of CD4-positive area in the tumor area of ​​mice in the PC+ZP-1 group was significantly increased (P<0.01); the percentage of PD-L1-positive area in the tumor area of ​​mice in the PC group was significantly decreased (P<0.05); and the percentage of PD-L1-positive area in the tumor area of ​​mice in the ZP-1 group and the PC+ZP-1 group was significantly decreased (P<0.01) (as shown in Table 8).

[0143] Table 5: Effect of combined administration of PC and ZP-1 on tumor volume in LLC tumor-bearing mice mm3, n=8)

[0144] Table 6: Effect of combined administration of PC and ZP-1 on tumor volume inhibition rate in LLC tumor-bearing mice

[0145] Table 7 Effects of combined PC and ZP-1 administration on organ coefficients and tumor weight inhibition rates in LLC tumor-bearing mice

[0146] Table 8. Effects of combined administration of PC and ZP-1 on the expression of CD4, CD8, and PD-L1 in LLC mouse tumors. Note: Compared with the model control group, "*" P<0.05 and "***" P<0.01.

[0147] (4) Conclusion and discussion: Based on the above research results, both PC and ZP-1 can reduce the expression level of PD-L1 in Lewis tumor tissue and inhibit tumor growth. After combined administration of PC and ZP-1, the expression level of PD-L1 in tumor tissue can be more significantly inhibited, and the expression of CD4 and CD8 of T cells in the tissue can be increased, resulting in better tumor treatment effect.

[0148] Example 5: Enhanced efficacy of compound 1 and the compound shown in Formula III in a mouse LLC lung cancer model.

[0149] The preparation of the tumor-bearing mouse model and the efficacy experiment were conducted in accordance with the specific steps of Example 4, except that the compound shown in Formula II was replaced with the compound shown in Formula III for administration.

[0150] Example 6 Acute oral toxicity test of compound 1

[0151] (1) Objective: To study the toxicity of different doses of the test substance administered orally for a short period of time using the up-down method.

[0152] (2) Methods: SPF-grade ICR mice (18-22g, male) were first administered the drug at a dose of 2000 mg / kg body weight. If the animal died within 48 hours, the main experiment was conducted. The limit experiment used a dose sequence of 1.75 mg / kg, 5.5 mg / kg, 17.5 mg / kg, 55 mg / kg, 175 mg / kg, 550 mg / kg, and 2000 mg / kg, with an initial dose of 175 mg / kg and a dose order factor of 3.2. One animal was used for each main experiment, and observation continued for 48 hours. If the animal survived, the next animal was given a higher dose; if the animal died or showed signs of death, the next animal was given a lower dose. The experiment was terminated based on animal survival and the results of AOT 425 (version 1.0) statistical software analysis. Each animal was observed at least once within the first 30 minutes after administration, and regularly for the first 24 hours, followed by daily observation for 14 days. Observe and record the poisoning process and the poisoning and death of animals during the observation period. Perform gross autopsies on dead animals and surviving animals at the end of the observation period, and conduct histopathological examinations on the organs with observed lesions.

[0153] (3) Results: Five animals were treated with PC drug (i.e., compound 1) at a dose of 2000 mg / kg body weight. No abnormalities were observed in any of the surviving mice during the experiment. Five animals survived at the end of the observation period, and the experiment was terminated. No gross lesions were found in any of the animals that were dissected at the end of the observation period. The LD50 of PC drug is >2000 mg / kg (as shown in Table 9).

[0154] Table 9 Results of acute oral toxicity tests of PC drugs

[0155] (4) Conclusion and discussion: The acute oral toxicity LD50 of PC drug in mice is >2000 mg / kg, indicating a low risk of acute toxicity.

[0156] Example 7 Repeated-dose toxicity test of compound 1

[0157] (1) Objective: To study the toxic effects of compound 1 by repeatedly administering single doses of the corresponding test substance.

[0158] (2) Methods: SPF-grade male C57BL / 6J mice, aged 8-10 weeks, were randomly divided into a solvent control group and a PC group (n=3 per group) based on body weight. The solvent control group received intraperitoneal injection of 1.3% DMSO at 10 mL / kg body weight once daily for 28 consecutive days; the PC group received intraperitoneal injection of PC drug (compound 1, 5 mg / kg, dissolved in 1.3% DMSO) at 10 mL / kg body weight once daily for 28 consecutive days. During the experiment, the general clinical condition of the animals was observed daily, body weight was measured every 2 days, and food intake was measured weekly. After the last administration, the animals were fasted for 12 hours but allowed free access to water. Blood was collected after anesthesia for routine blood tests and blood biochemistry tests. Blood biochemistry tests included alanine aminotransferase (ALT), alanine aminotransferase (AST), urea (UREA), creatinine (CR), total protein (TP), albumin (ALB), total cholesterol (TC), and triglycerides (TG); blood glucose (GLU) was measured using serum.

[0159] (3) Results: During the experiment, no abnormalities were observed in the body size, coat, feces, muscle tone, gait, mental state, or respiration of the mice in each group. All mice were necropsy at the end of the experiment. Compared with the solvent control group, there was no statistically significant difference in body weight at any time point in the PC group (P>0.05); the Bas% of the PC group mice was decreased in blood routine tests, which was statistically significant (P<0.05), while there were no statistically significant differences in other blood routine test indicators (P>0.05) (as shown in Table 10). Blood biochemistry tests showed that ALT, AST, UREA, CR, TP, ALB, TC, TG, and GLU were all at normal levels (as shown in Table 11).

[0160] (4) Conclusion: The toxicity results of repeated administration over 28 days showed that PC drug administered intraperitoneally at a daily dose of 5 mg / kg had no significant effect on the general clinical manifestations, body weight, hematology and blood biochemistry of C57BL / 6 mice.

[0161] Table 10 Effects of repeated 28-day administration of PC group on routine blood tests in mice Note: "*" indicates P < 0.05, "**" indicates P < 0.01.

[0162] Table 11 Effects of repeated 28-day administration of PC group on blood biochemical assays in mice Note: "#" indicates P < 0.05, "##" indicates P < 0.01.

[0163] Example 8: Pharmacokinetic Analysis of Compound 1

[0164] (1) Experimental Methods: Male SD rats, weighing 280–300 g, were administered the corresponding test drug solution (1% DMSO solution of compound 1) via intravenous injection or gavage at a dose of 10 mL / kg body weight. Rats in the gavage group were fasted for 12 h before administration but allowed free access to water, and were fasted for another 2 h after administration. Blood samples (0.5–0.6 mL) of EDTA·Na2 were collected from the jugular vein before injection (0 h) and at 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h after administration. Plasma was collected by centrifugation and stored at -80℃ for later use. The concentration of the test drug in the rat whole blood supernatant was determined using a validated LC-MS / MS method (results are shown in Figure 7 and Tables 13-15).

[0165] (2) Results:

[0166] Table 12 Dosage Table

[0167] Table 13 Measured values ​​of blood drug concentration

[0168] Table 14 Pharmacokinetic parameters of the intravenous injection group

[0169] Table 15 Pharmacokinetic parameters of the gavage group

[0170] Example 9: Therapeutic effect of the test drug on B16F10 melanoma C57BL / 6J tumor-bearing mice:

[0171] 9.1 Cell Culture: Cells were cultured in 10cm Hyclone cell culture dishes. B16F10 cells were cultured at 10... 6 Cells were seeded at 10 mL of RPMI-1640 cell culture medium containing 10% fetal bovine serum. After 3 days, when the cells reached 80% confluence, they were passaged. Logarithmic growth phase cells were harvested, digested with 0.25% trypsin, centrifuged to remove the supernatant, and then washed twice with serum-free culture medium. The cell count was calculated, and the cell concentration was adjusted. The cells were then resuspended in PBS to a concentration of 1.0 × 10⁶ cells / mL.7 per mL.

[0172] 9.2 B16F10 cells (1.0×10⁻⁶) 6 (Catalog No.: CL-0319, Wuhan Pronosei Life Sciences Co., Ltd.) and 0.1 ml of frozen PBS were mixed, and then mixed with an equal volume of frozen Matrigel Matrix, and inoculated into the right abdomen of mice. Tumor diameter was measured every 2 days after tumor formation, and the tumor volume was approximately 60–90 mm. 3 Treatment should begin at that time.

[0173] 9.3 Tumor-bearing mice were randomly divided into a model control group, a positive control group (sintilimab), and a drug PC (i.e., compound 1, 10 mg / kg), with 5 mice per group.

[0174] 9.4 Mice in each treatment group were injected intraperitoneally once a day for 13 consecutive days after grouping; the positive control group (PD-1 sintilimab, batch number DP2212001) was treated with PD-1 antibody intravenously at a dose of 200 μg / mouse (20g) on ​​days 1, 4, 7, 10 and 13 after grouping.

[0175] Table 16 Dosage Table for Each Group

[0176] 9.5 Preparation of test solution

[0177] Drug administration group solution: Weigh 1 mg of drug PC group, add 20 uL DMSO to dissolve, then add 980 μl of physiological saline containing 1% DMSO to prepare 1 ml of each drug administration group solution, and vortex with sonication for later use;

[0178] PD1 monoclonal antibody positive control group 200μg / animal (20g): Take 0.1ml of PD1 sintilimab (10mg / ml), add 900μl of physiological saline to prepare 1ml of drug administration group solution, and vortex with sonication for later use;

[0179] 9.6 Detection indicators:

[0180] Tumor volume measurement: The long and short diameters of the tumor are measured every two days, and a tumor growth curve is plotted. Tumor volume is calculated using the formula: V = 1 / 2ab 2 Measure and calculate that a and b are the longest and shortest diameters, respectively.

[0181] Tumor weight and organ coefficients: 24 hours after the last administration, animals were euthanized, tumors were dissected, fat and connective tissue were removed, tumor weight was measured, photographed, and weighed to calculate the tumor inhibition rate. The thymus and spleen were harvested, weighed, and organ coefficients were calculated.

[0182] Calculate the tumor growth inhibition rate according to the formula.

[0183] Tumor weight: At the end of the experiment, the tumors were completely dissected, weighed, grouped, and photographed to calculate the tumor weight inhibition rate.

[0184] 9.7 Analysis and Statistics

[0185] All data are adopted This indicates that statistical analysis is performed using application software; if the variances of the measurement data are homogeneous, or if the variances are homogeneous after data transformation, then one-way ANOVA with pairwise comparisons between groups is used; if the variances are still heterogeneous after data transformation, then the rank-sum test is used for statistical analysis. P ≤ 0.05 is considered statistically significant.

[0186] 9.8 Results

[0187] (1) Apart from tumor growth, the animals showed no other abnormalities and were dissected for tissue collection. There were no statistically significant differences in body weight between the groups and the model control group during the drug administration period.

[0188] (2) Tumor volume and tumor volume inhibition rate of B16F10 (see Tables 17 and 18)

[0189] Table 17 Effects of the test drugs on tumor volume in B16F10 model tumor-bearing mice mm 3 (n=5) Note: One-way ANOVA and rank-sum test were used. Compared with the B16F10 model control group, "*" p<0.05, "**" p<0.01.

[0190] Table 18 Effects of the test drugs on tumor volume inhibition rate in B16F10 model tumor-bearing mice ( mm 3 (n=5)

[0191] Compared with the model control group, the tumor volume of mice in the positive control group and the drug PC group was significantly reduced on days 11 and 13 after drug administration, with statistically significant differences (P<0.01); the tumor volume inhibition rates of the positive control group and the drug PC group at the last measurement were 34.8% and 42.0%, respectively.

[0192] Table 19 Effects of the test drugs on organ coefficients and tumor weight inhibition rates in B16F10 tumor-bearing mice. Note: One-way ANOVA and rank-sum test were used. Compared with the B16F10 model control group, "*" p<0.05, "**" p<0.01.

[0193] At the experimental endpoint (day 14), compared with the model control group, the tumor weight inhibition rates of the positive control group and the drug PC group were 45.1% and 56.5%, respectively; compared with the model control group, the reduction in tumor weight in the positive control group and the drug PC group was statistically significant (P<0.01).

[0194] 9.9 Conclusion:

[0195] Under the conditions of this experiment, the drug PC inhibited tumor growth in B16F10 tumor-bearing mice.

[0196] Example 10: Pharmacokinetics and tissue distribution of drug PC (compound 1) in SD rats.

[0197] 10.1 Preparation of test solution

[0198] PC solution (1 mg / mL): Take 25 uL of the stock solution and add 2475 uL of 1% DMSO physiological saline to prepare a 1 mg / mL solution (100 times dilution). Shake well and store at 2-8℃. This solution can be used for intravenous injection and gavage.

[0199] 10.2 Procedure: After 3 days of acclimatization to the SPF-grade barrier system and passing quarantine, 14 SD rats were randomly selected and divided into three groups: a blank control group (n=2, 1 male and 1 female), an intravenous injection group (n=6, males), and a gavage group (n=6, females). Any excess animals were euthanized. All rats were fasted for 12 hours before administration of the test substance, but allowed free access to water, and were fasted for another 2 hours after administration. Rats in the intravenous injection and gavage groups were administered 10 mg / kg of the test drug solution once via intravenous injection or gavage at a dose of 10 mL / kg body weight. The blank control group received no intravenous injection or gavage treatment.

[0200] 10.3 Detection methods and indicators

[0201] (1) Immediately after the quarantine of rats in the blank control group, whole blood was collected from the abdominal aorta after isoflurane anesthesia (K2-EDTA anticoagulation). Male rats were rapidly collected on ice for brain, heart, lung, liver, stomach, spleen, kidney, skeletal muscle, abdominal fat, testes, epididymis, bladder, small intestine, large intestine, skin and other tissues. Female rats were rapidly collected on ice for brain, heart, lung, liver, stomach, spleen, kidney, skeletal muscle, abdominal fat, uterus, ovary, mammary gland, bladder, small intestine, large intestine, skin and other tissues. They were rinsed with pre-cooled 0.9% NaCl to remove blood clots, removed with filter paper to remove moisture, weighed and stored at -80℃.

[0202] (2) Whole blood (K2-EDTA anticoagulated) was collected from the abdominal aorta of one rat in each of the intravenous injection and gavage groups at six time points after injection (before administration, 1 h, 4 h, 8 h, 24 h, 48 h, and 120 h after administration). For male rats, tissues including brain, heart, lungs, liver, stomach, spleen, kidneys, skeletal muscle, abdominal fat, testes, epididymis, bladder, small intestine, large intestine, and skin were rapidly collected on ice. For female rats, tissues including brain, heart, lungs, liver, stomach, spleen, kidneys, skeletal muscle, abdominal fat, uterus, ovaries, mammary glands, bladder, small intestine, large intestine, and skin were rapidly collected on ice. The tissues were rinsed with pre-cooled 0.9% NaCl to remove blood clots, removed with filter paper, weighed, and stored at -80℃.

[0203] (4) Accurately weigh each tissue, cut it into small pieces and place it in a homogenization tube. Homogenize the tissue and physiological saline at a ratio of 1:3 (g:mL). Store the homogenate at -80℃ for testing.

[0204] (5) The concentration of the test drug in rat whole blood supernatant was detected using a validated LC-MS / MS method.

[0205] 10.4 Results

[0206] The pharmacokinetic results and key parameters of drug PC (compound 1) in SD rats are shown in Tables 20 and 21.

[0207] Table 20. Measured plasma CT results in rats after a single administration of PC (unit: ug / L)

[0208] Table 21 Main pharmacokinetic parameters of plasma in rats after a single administration of PC.

[0209] The tissue distribution of drug PC (compound 1) in SD rats is shown in Table 22 (tissue distribution of drug in male rats in the intravenous injection group) and Table 23 (tissue distribution of drug in female rats in the gavage group).

[0210] Table 22. Measured organ CT results in rats after a single intravenous injection of PC (unit: μg / g).

[0211] Table 23. Measured organ CT results in rats after a single oral administration of PC (unit: μg / g)

Claims

1. The use of a substance X in the preparation of a cancer treatment drug, said substance X being a compound as shown in Formula I or a pharmaceutically acceptable salt thereof. in, R 1 It is chlorine or methoxy; X is CHR X-1 NR X-2 Or oxygen; R X-1 It is hydrogen, C1-C4 alkyl, 6-10 aryl, C(O)N(R) X-1-1 )2 or a C1-C4 alkyl group substituted with one or more hydroxyl groups; R X-1-1 Independently hydrogen or C1-C4 alkyl; R X-2 It is hydrogen, C1-C4 alkyl, 6-10 aryl, or C1-C4 alkyl substituted with one or more hydroxyl groups; m can be 1, 2, 3 or 4.

2. The application as described in claim 1, characterized in that, It meets one or more of the following conditions: (1)R X-1 and R X-2 In this context, the C1-C4 alkyl group and the C1-C4 alkyl group substituted with one or more hydroxyl groups are independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl, for example, methyl or ethyl; (2)R X-1 and R X-2 In this context, the 6-10 aryl groups are independently phenyl or naphthyl, such as phenyl; (3)R X-1 and R X-2 In this context, the plurality of C1-C4 alkyl groups substituted with one or more hydroxyl groups is one or two, for example, one; (4)R X-1-1 In this context, the C1-C4 alkyl groups are independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl.

3. The application as described in claim 1, characterized in that, It meets one or more of the following conditions: (1)R X-1 C(O)N(R) X-1-1 )2; for example, R X-1 It is C(O)NH2; (2)R X-1-1 It is hydrogen; (3)R X-2 It is a C1-C4 alkyl, phenyl, or a C1-C4 alkyl substituted with one or more hydroxyl groups; preferably, R X-2 It is a C1-C4 alkyl, phenyl, or a C1-C4 alkyl group substituted with one hydroxyl group; for example, R X-2 Methyl, ethyl, Or phenyl; (4) m is 2; (5) The pharmaceutically acceptable salt of the compound represented by Formula I is a hydrochloride, hydrobromide or hydroiodide; for example, hydrochloride; preferably, in the pharmaceutically acceptable salt of the compound represented by Formula I, the molar ratio of the compound represented by Formula I to the acid is 1:(1-4), for example 1:

2.

4. The application as described in claim 1, characterized in that: The compound shown in Formula I satisfies any of the following schemes: Option 1: R 1 It is chlorine; X is CHR X-1 or NR X-2 ; R X-1 C(O)N(R) X-1-1 )2; R X-1-1 Independently hydrogen; R X-2 It is a C1-C4 alkyl group, a C1-C4 alkyl group substituted with one or more hydroxyl groups, or a 6-10 aryl group; m can be 1, 2, 3, or 4; Option 2: The compound shown in Formula I is the same as the compound shown in Formula I'. R X-2 It is a C1-C4 alkyl group or a C1-C4 alkyl group substituted with one or more hydroxyl groups; m can be 1, 2, 3 or 4.

5. The application as described in claim 1, characterized in that: The substance X is any of the following compounds.

6. The application as described in any one of claims 1-5, characterized in that, The drug meets one or more of the following conditions: (1) The substance X inhibits the binding of PD-1 and PD-L1 by a rate of >10%, preferably >30%, more preferably >40%; (2) The substance X is compound 1 as shown below. (3) The weight percentage of substance X in the drug is 0.01% to 30%, preferably 0.01% to 15%, more preferably 0.01% to 10%, and even more preferably 0.1% to 5%; (4) The dosage of substance X is 0.1 mg / kg to 300 mg / kg, preferably 1 mg / kg to 100 mg / kg, more preferably 1 mg / kg to 30 mg / kg, for example 5 mg / kg or 10 mg / kg; the dosage refers to a single dose; (5) The application frequency of the substance X is once a day, twice a day, three times a day, once every other day, once a week, twice a week, three times a week, once every other week, once every two weeks, once every three weeks, or once every four weeks; (6) The administration route of the substance X is oral administration, injection administration, sublingual administration, rectal administration or local administration, wherein the injection administration may be intravenous injection administration, intramuscular injection administration or subcutaneous injection administration.

7. The application as described in any one of claims 1-5, characterized in that, The drug further comprises substance Y, which is a compound as shown in Formula II or Formula III.

8. The application as described in claim 7, characterized in that, It meets one or more of the following conditions: (1) The active ingredient of the drug is composed of substance X and substance Y; (2) The mass ratio of substance Y to substance X is (0.1-5):1, preferably (0.5-2):1, for example 1:1; (3) The dosage of substance X is 0.05 mg / kg to 150 mg / kg, preferably 1 mg / kg to 50 mg / kg, more preferably 1 mg / kg to 15 mg / kg, for example 5 mg / kg, and the dosage refers to a single dose; (4) The dosage of substance Y is 0.005 mg / kg to 750 mg / kg, preferably 0.1 mg / kg to 250 mg / kg, more preferably 0.1 mg / kg to 75 mg / kg, for example 5 mg / kg, and the dosage refers to a single dose; (5) The administration routes of the substances X and Y are independently oral administration, injection administration, sublingual administration, rectal administration or local administration, wherein the injection administration may be intravenous injection administration, intramuscular injection administration or subcutaneous injection administration; preferably, the administration routes of the substances X and Y are both injection administration, such as intramuscular injection administration or intravenous injection administration. (6) The substance X and substance Y are administered simultaneously or separately at intervals, for example, substance X is administered first, followed by substance Y; or substance Y is administered first, followed by substance X. (7) The application frequency of the substance X and substance Y is independently once a day, twice a day, three times a day, once every other day, once a week, twice a week, three times a week, once every other week, once every two weeks, once every three weeks, or once every four weeks.

9. The application as described in claim 1, characterized in that, When substance X is the only active ingredient in the drug, the dosage of substance X is 5 mg / kg or 10 mg / kg, and the frequency of administration is once a day; when the drug also contains substance Y, the dosage of substance X and substance Y is 5 mg / kg, and the frequency of administration is once a day.

10. The application as described in any one of claims 1-5 and 8-9, characterized in that, The cancers mentioned are lung cancer, liver cancer, colon cancer, breast cancer, bone marrow cancer, or melanoma.

11. A method of treating cancer, comprising administering to a subject in need a therapeutically effective amount of substance X as described in any one of claims 1-5.

12. The method for treating cancer as described in claim 11, characterized in that, The cancer is lung cancer, liver cancer, colon cancer, breast cancer, bone marrow cancer, or melanoma; preferably, the method of administration of the substance X is as described in claim 6.

13. A method of treating cancer, comprising administering to a subject in need a therapeutically effective amount of substance X and substance Y as described in any one of claims 1-5, wherein substance Y is a compound as shown in Formula II or a compound as shown in Formula III.

14. The method for treating cancer as described in claim 13, characterized in that, The cancers mentioned are lung cancer, liver cancer, colon cancer, breast cancer, bone marrow cancer, or melanoma.

15. The method for treating cancer as described in claim 13, characterized in that, The manner of application of substances X and Y is as described in claim 8.

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