Anti-tumor drug and application thereof

Abstract

Phytol (PHY) is a metabolite of plant chlorophyll, is wide in source and is a chain-like diterpenoid substance consisting of four isoprene units, and the phytol ingested by animals is subjected to oxidative metabolism in vivo, so that not only can energy be provided for the animals, but also the growth of the animals can be promoted, and the growth of the animals can be promoted. Moreover, the phytol and the phytanic acid metabolite of the phytol can also be used as signal molecules to participate in glycolipid metabolism and adipocyte differentiation regulation processes, but people cannot directly take the phytol and the phytanic acid from the nature. The effect of phytol in the generation and development of tumors still has huge unknown property. According to the research, the influence of the phytol and the derivative thereof on the growth of colon cancer, breast cancer and melanoma is detected through an in-vitro experiment CCK-8 (Cell Count Kit 8) and an in-vivo experiment such as a mouse subcutaneous tumor model, and the phytol and the derivative thereof are found to be capable of remarkably inhibiting the growth of colon cancer, breast cancer and melanoma and have no obvious toxic and side effects on heart, liver and kidney functions.

Description

TECHNICAL FIELD

[0001] The present application relates to a small molecule compound, phytol, for treating tumors, and use of the composition in the preparation of a medicament for treating cancer. Specifically, the pharmaceutical composition of the present application comprises phytol and derivatives thereof. BACKGROUND

[0002] Colorectal cancer is a common digestive system tumor, which poses a great threat to human survival and health. In China, the incidence and mortality of colorectal cancer are increasing year by year, ranking fourth in the incidence and fifth in the mortality among various malignant tumors [1, 2]. Melanoma is a highly malignant skin tumor, accounting for more than 80% of skin cancer deaths. Breast cancer is a common type of malignant tumor in women, with an incidence rate currently ranking first in the world and China, and is the main cause of cancer-related deaths in women worldwide. According to GLOBOCAN 2018 statistics, more than 2 million new breast cancer patients were diagnosed in 2018, accounting for 1.6% of the total number of new cancer patients, and more than 600,000 women died of breast cancer, accounting for 6.6% of the total number of cancer-related deaths. Although surgical resection is the most common treatment, about 30% of patients die from distant metastasis 2-3 years after surgery. Traditional surgical resection is difficult to completely cure the tumor, and postoperative radiotherapy and chemotherapy are also difficult to achieve the goal of completely removing residual tumor cells, and chemotherapy has little or no effect on distant metastasis. In addition, repeated surgery + radiotherapy and chemotherapy not only seriously affect the quality of life of patients, but also easily cause tumor drug resistance [3, 4]. Not only is it related to the occult onset of tumors and the lack of reliable screening and diagnostic markers, which makes it difficult to detect early, but it is also related to the insufficient effectiveness of current treatment methods. Therefore, it is particularly urgent to find other means to enhance the effectiveness of tumor treatment. SUMMARY

[0003] Based on the above technical background,

[0004] Based on the above technical effects, the present application provides the following technical solutions:

[0005] According to one aspect of the present application, a compound and a pharmaceutically acceptable salt thereof are disclosed, the compound having a structure shown in formula (I):

[0006]

[0007] wherein R is -OH, -N2C2H6, -NOC4H4, -NO3C2H3, or -NOC4H5.

[0008] According to certain embodiments of the present application, the compound of formula (I) is selected from:

[0009]

[0010]

[0011] According to one aspect of the present application, the present application discloses a method for preparing the compound, characterized in that the method comprises:

[0012] Step one: synthesis of (7R, 11R, E)-3, 7, 11, 15-tetramethylhexadec-2-enal

[0013]

[0014] 20-30 mmol of chlorophyllol is dissolved in 20-100 mL of dichloromethane, and des martin oxidant C is added under ice bath 13 H 13 IO8 (20-100 mmol) is stirred at room temperature until the reaction is complete; after the reaction is complete, 5-30 mL of sodium thiosulfate and sodium bicarbonate mixed solution is added, and stirred for 2-10 min; the organic phase is separated, the aqueous phase is washed with dichloromethane (10-30 mL, 2-5 times), and the organic phases are combined; the organic phase is washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent is removed by reduced pressure evaporation to obtain a crude product; the crude product is purified by column chromatography to obtain the product (7R, 11R, E)-3, 7, 11, 15-tetramethylhexadec-2-enal;

[0015] Step two: synthesis of the compound of formula (I)

[0016]

[0017] wherein R-NH2, R is -C4H2O, -C3H2O2 or -C5H3O;

[0018] (7R, 11R, E)-3, 7, 11, 15-tetramethylhexadec-2-enal obtained in step one is dissolved in 5-30 mL of dichloromethane, and primary or secondary amine (1-5 mmol) is added; after stirring at room temperature for 2-10 min, triacetoxy sodium borohydride (5-30 mmol) is added in batches; stirring is performed at room temperature for 1-5 h; after the reaction is complete, saturated sodium bicarbonate solution is added to the reaction system; the organic phase is separated, the inorganic phase is extracted with dichloromethane (2-10 mL, 2-5 times), and the organic phases are combined; the organic phase is washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent is removed by reduced pressure evaporation to obtain a crude product; the crude product is purified by column chromatography (0%-30% ethyl acetate / petroleum ether, V / V) to obtain the final product.

[0019] According to one aspect of the present application, the present application discloses the use of the compound or chlorophyllol and its pharmaceutically acceptable salt in the preparation of a drug for treating tumors.

[0020] According to some embodiments of the present application, the tumor is selected from colorectal tumor, melanoma, lung metastatic cancer, liver cancer, uterine tumor, leukemia, lymphoma, multiple myeloma, etc.

[0021] According to one aspect of the present application, a pharmaceutical composition for treating tumor is disclosed, comprising the compound of formula (I) or phytol and pharmaceutically acceptable salts thereof and a second therapeutic agent.

[0022] According to some embodiments of the present application, the second therapeutic agent is selected from 5-fluorouracil, oxaliplatin, an immune checkpoint inhibitor.

[0023] According to some embodiments of the present application, the immune checkpoint inhibitor is selected from a PD1 antibody or a PDL1 antibody.

[0024] According to one aspect of the present application, the use of the pharmaceutical composition in the preparation of a medicament for treating tumor is disclosed.

[0025] According to some embodiments of the present application, the tumor is selected from colorectal tumor, melanoma, lung metastatic cancer, liver cancer, uterine tumor, leukemia, lymphoma, multiple myeloma, etc.

[0026] Previous studies have shown that it is an ideal model for treating tumors to find compounds that enhance the anti-tumor effect from traditional drugs [5]. Traditional drugs on the market have been evaluated by clinical trials, and the toxicity and pharmacological effects of the drugs are clear. For example, metformin, a classic drug for diabetes, has been confirmed to have an inhibitory effect on a variety of tumors [6-8], non-steroidal drugs have been confirmed to have killing power on colorectal, breast and head and neck tumors [9, 10], and curcumin, a chemical component extracted from the rhizomes of some plants in the ginger family and araceae family, can not only directly inhibit tumor growth, but also enhance the effect of immunotherapy, thereby indirectly killing tumor cells [11, 12]. The combination of these drugs with other treatment methods is expected to improve the effect of tumor treatment and is more likely to be used in clinical practice. Therefore, we tried to find compounds with anti-cancer activity from non-tumor drugs.

[0027] Advantages of the present application:

[0028] 1) Phytol is a metabolic product of chlorophyll in green plants, and is widely available and easy to obtain.

[0029] 2) Phytol has low hepatotoxicity, nephrotoxicity and cardiotoxicity.

[0030] 3) Phytol can inhibit the growth of various tumor cells such as intestinal cancer and breast cancer cells in vitro, and has significant anti-cancer activity.

[0031] 4) Phytol has significant anti-cancer activity in mice and can inhibit the growth of intestinal cancer.

[0032] 5) Phytol derivatives can inhibit the growth of various tumor cells such as intestinal cancer, melanoma cells in vitro and in vivo, and have significant anticancer activity;

[0033] 6) Phytol derivatives have significant anticancer activity and can inhibit the growth of intestinal cancer;

[0034] Definitions

[0035] Phytol (PHY)

[0036] Phytol, also known as phytenol, is derived from a branch of a chlorophyll molecule in plants, and is a chain-like diterpene composed of four isoprene units. It is reported that phytol is a raw material for the synthesis of vitamin K, vitamin E and other growth, and due to its aromatic components, it has applications in many aromatic compounds, such as cosmetics

[13] . Phytol also has the effects of regulating fat differentiation

[14] , antibacterial

[15] , anti-malformation

[16] , antioxidant

[17] , immunomodulation

[18] , etc. Ruminants eat plants rich in chlorophyll, and the phytol group of chlorophyll is digested and released by special microorganisms in the rumen to produce phytol and its metabolite phytanic acid, which is absorbed into the blood through the small intestine and stored in the body. The phytol ingested by animals is oxidized and metabolized in the body, not only providing energy for animals, but also phytol and its metabolite phytanic acid can act as a signal molecule to participate in the regulation of sugar and lipid metabolism and fat cell differentiation. According to a retrospective study, the cancer rate of ruminants is significantly lower than that of carnivores and primates. It is not yet clear whether phytol plays an important role in the occurrence of cancer in animals. On the other hand, humans cannot ingest phytol from green plants, but can only ingest the metabolite phytanic acid from animal meat products and dairy products, and only about 3% of phytanic acid can be finally stored in the body. The role of phytol in human anticancer is still largely unknown. In general, phytol is widely distributed and easy to obtain, and exploring the role of phytol in the occurrence and development of tumors may have important significance for the development of new tumor drugs.

[0037] Compounds of the structure of formula (I)

[0038] According to one aspect of the present application, the present application discloses a compound of the structure of formula (I) and a pharmaceutically acceptable salt thereof:

[0039]

[0040] According to certain embodiments of the present application, R is -OH, -N2C2H6, -NOC4H4, -NO3C2H3 or -NOC4H5.

[0041] According to certain embodiments of the present application, the compound of formula (I) is selected from:

[0042]

[0043] Process for preparing the compound of formula (I)

[0044] According to one aspect of the present application, the present application discloses a process for preparing a compound of formula (I), the process comprising:

[0045] Step one: synthesis of (7R,11R,E)-3,7,11,15-tetramethylhexadec-2-enal

[0046]

[0047] 20-30 mmol of chlorophyllol is dissolved in 20-100 mL of dichloromethane, and Dess-Martin oxidant C is added under ice bath 13 H 13 IO8 (20-100 mmol), and stirred at room temperature until the reaction is complete; after the reaction is complete, 5-30 mL of a mixed solution of sodium thiosulfate and sodium bicarbonate is added, and stirred for 2-10 min; the organic phase is separated, the aqueous phase is washed with dichloromethane (10-30 mL, 2-5 times), the organic phases are combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent is removed by evaporation under reduced pressure to obtain a crude product; the crude product is purified by column chromatography to obtain the product (7R,11R,E)-3,7,11,15-tetramethylhexadec-2-enal;

[0048] Step two: synthesis of the compound of formula (I)

[0049]

[0050] wherein R-NH2, R is -C4H2O, -C3H2O2 or -C5H3O;

[0051] (7R,11R,E)-3,7,11,15-tetramethylhexadec-2-enal obtained in step one is dissolved in 5-30 mL of dichloromethane, and primary or secondary amine (1-5 mmol) is added; after stirring at room temperature for 2-10 min, triacetoxy sodium borohydride (5-30 mmol) is added in batches, and stirred at room temperature for 1-5 h; after the reaction is complete, saturated sodium bicarbonate solution is added to the reaction system, the organic phase is separated, the inorganic phase is extracted with dichloromethane (2-10 mL, 2-5 times), the organic phases are combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent is removed by evaporation under reduced pressure to obtain a crude product; the crude product is purified by column chromatography (0%-30% ethyl acetate / petroleum ether, V / V) to obtain the final product.

[0052] According to certain embodiments of the application, the phytol is 20-25 mmol or 25-30 mmol. According to certain embodiments of the application, the phytol is 20 mmol, 21 mmol, 22 mmol, 23 mmol, 24 mmol, 25 mmol, 26 mmol, 27 mmol, 28 mmol, 29 mmol, or 30 mmol.

[0053] According to certain embodiments of the application, the phytol is dissolved in 20-100 mL of dichloromethane, for example 20-80 mL, 20-60 mL, 20-40 mL, 40-100 mL, 40-80 mL, 40-60 mL, 60-100 mL, 60-80 mL, or 80-100 mL of dichloromethane. According to certain embodiments of the application, the phytol is dissolved in 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, 50 mL, 55 mL, 60 mL, 65 mL, 70 mL, 75 mL, 80 mL, 85 mL, 90 mL, 95 mL, or 100 mL of dichloromethane.

[0054] According to certain embodiments of the application, the Dess-Martin periodinane C 13 H 13 IO8 is 20-100 mL, for example 20-80 mL, 20-60 mL, 20-40 mL, 40-100 mL, 40-80 mL, 40-60 mL, 60-100 mL, 60-80 mL, or 80-100 mL. According to certain embodiments of the application, the Dess-Martin periodinane C 13 H 13 IO8 is 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, 50 mL, 55 mL, 60 mL, 65 mL, 70 mL, 75 mL, 80 mL, 85 mL, 90 mL, 95 mL, or 100 mL.

[0055] According to certain embodiments of the application, the sodium thiosulfate, sodium bicarbonate mixed solution is 5-30 mL, for example 5-20 mL, 5-10 mL, 10-30 mL, 10-20 mL, or 20-30 mL. According to certain embodiments of the application, the sodium thiosulfate, sodium bicarbonate mixed solution is 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL, 21 mL, 22 mL, 23 mL, 24 mL, 25 mL, 26 mL, 27 mL, 28 mL, 29 mL, or 30 mL.

[0056] According to certain embodiments of the application, the ratio of mixing of the sodium thiosulfate, sodium bicarbonate mixed solution is 1:1, 1:2, 1:3, 1:4, 1:5, 2:1, 3:1, 4:1 or 5:1.

[0057] According to certain embodiments of the application, the stirring in step one is for 2-10 min, for example 2-8 min, 2-6 min, 2-4 min, 4-10 min, 4-8 min, 4-6 min, 6-10 min, 6-8 min or 8-10 min. According to certain embodiments of the application, the stirring in step one is for 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min or 10 min. ～ 8min, 4-6min, 6-10min, 6-8min or 8-10min. According to certain embodiments of the application, the stirring in step one is for 2min, 3min, 4min, 5min, 6min, 7min, 8min, 9min or 10min

[0058] According to certain embodiments of the application, the dichloromethane is 10-30 mL, for example 10-20 mL or 20-30 mL. According to certain embodiments of the application, the dichloromethane is 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL, 21 mL, 22 mL, 23 mL, 24 mL, 25 mL, 26 mL, 27 mL, 28 mL, 29 mL or 30 mL.

[0059] According to certain embodiments of the application, the dichloromethane is washed 2-5 times. According to certain embodiments of the application, the dichloromethane is washed 2 times, 3 times, 4 times or 5 times.

[0060] According to certain embodiments of the application, the organic phase is washed with saturated brine 2-5 times. According to certain embodiments of the application, the organic phase is washed with saturated brine 2 times, 3 times, 4 times or 5 times.

[0061] According to certain embodiments of the application, the primary or secondary amine is 1-5 mmol. According to certain embodiments of the application, the primary or secondary amine is 1 mmol, 2 mmol, 3 mmol, 4 mmol or 5 mmol.

[0062] According to certain embodiments of the application, the dichloromethane in step two is 5-30 mL, for example 5-20 mL, 5-10 mL, 10-30 mL, 10-20 mL or 20-30 mL. According to certain embodiments of the application, the dichloromethane in step two is 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL, 21 mL, 22 mL, 23 mL, 24 mL, 25 mL, 26 mL, 27 mL, 28 mL, 29 mL or 30 mL.

[0063] According to certain embodiments of the application, the stirring at room temperature in step two is for 2-10 min, such as 2-8 min, 2-6 min, 2-4 min, 4-10 min, 4-8 min, 4-6 min, 6-10 min, 6-8 min, or 8-10 min. According to certain embodiments of the application, the stirring at room temperature in step two is for 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, or 10 min.

[0064] According to certain embodiments of the application, the sodium triacetoxyborohydride is 5-30 mL, such as 5-20 mL, 5-10 mL, 10-30 mL, 10-20 mL, or 20-30 mL. According to certain embodiments of the application, the sodium triacetoxyborohydride is 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL, 21 mL, 22 mL, 23 mL, 24 mL, 25 mL, 26 mL, 27 mL, 28 mL, 29 mL, or 30 mL.

[0065] According to certain embodiments of the application, the stirring at room temperature in step two is for 1-5 h. According to certain embodiments of the application, the stirring at room temperature in step two is for 1 h, 2 h, 3 h, 4 h, or 5 h.

[0066] According to certain embodiments of the application, the inorganic phase is extracted with dichloromethane, which is 2-10 mL, such as 2-8 mL, 2-6 mL, 2-4 mL, 4-10 mL, 4-8 mL, 4-6 mL, 6-10 mL, 6-8 mL, or 8-10 mL. According to certain embodiments of the application, the inorganic phase is extracted with dichloromethane, which is 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, or 10 mL.

[0067] According to certain embodiments of the application, the inorganic phase is extracted with dichloromethane 2-5 times. According to certain embodiments of the application, the inorganic phase is extracted with dichloromethane 2 times, 3 times, 4 times, or 5 times.

[0068] According to some embodiments of the present application, the column chromatography is 0%-30% ethyl acetate / petroleum ether, V / V, for example 10%-30% ethyl acetate / petroleum ether or 20%-30% ethyl acetate / petroleum ether. According to some embodiments of the present application, the column chromatography is 0% ethyl acetate / petroleum ether, 1% ethyl acetate / petroleum ether, 2% ethyl acetate / petroleum ether, 3% ethyl acetate / petroleum ether, 4% ethyl acetate / petroleum ether, 5% ethyl acetate / petroleum ether, 6% ethyl acetate / petroleum ether, 7% ethyl acetate / petroleum ether, 8% ethyl acetate / petroleum ether, 9% ethyl acetate / petroleum ether, 10% ethyl acetate / petroleum ether, 11% ethyl acetate / petroleum ether, 12% ethyl acetate / petroleum ether, 13% ethyl acetate / petroleum ether, 14% ethyl acetate / petroleum ether, 15% ethyl acetate / petroleum ether, 16% ethyl acetate / petroleum ether, 17% ethyl acetate / petroleum ether, 18% ethyl acetate / petroleum ether, 19% ethyl acetate / petroleum ether, 20% ethyl acetate / petroleum ether, 21% ethyl acetate / petroleum ether, 22% ethyl acetate / petroleum ether, 23% ethyl acetate / petroleum ether, 24% ethyl acetate / petroleum ether, 25% ethyl acetate / petroleum ether, 26% ethyl acetate / petroleum ether, 27% ethyl acetate / petroleum ether, 28% ethyl acetate / petroleum ether, 29% ethyl acetate / petroleum ether or 30% ethyl acetate / petroleum ether, V / V.

[0069] According to some embodiments of the present application, the synthesis route of the compound of formula (I) is as follows:

[0070]

[0071] Phytol (7.88 g, 26.57 mmol) was dissolved in 50 mL of dichloromethane, and Dess-Martin oxidant C was added under ice bath. 13 H 13 IO8 (22.54 g, 53.15 mmol) was stirred at room temperature until the reaction was complete. After the reaction was complete, 20 mL of a mixed solution of sodium thiosulfate and sodium bicarbonate (1:1, V / V) was added, stirred for 3-5 min, the organic phase was separated, the aqueous phase was washed with dichloromethane (20 mL*3), the organic phases were combined, the organic phase was washed with saturated brine 3 times, dried over anhydrous sodium sulfate, and the solvent was removed by evaporation under reduced pressure to obtain a crude product. The product (7R, 11R, E)-3,7,11,15-tetramethylhexadec-2-enal (2.20 g, yield 28.1%) was obtained after column chromatography purification of the crude product.

[0072]

[0073] Note: R-NH2, R represents different groups (compound No. 3 is -C4H2O; compound No. 4 is -C3H2O2; compound No. 6 is -C5H3O)

[0074] (7R, 11R, E)-3,7,11,15-tetramethylhexadec-2-enal (1.0 g, 3.40 mmol) was dissolved in 10 mL of dichloromethane, and stirred at room temperature for 5 min. Then, sodium triacetoxyborohydride (3.60 g, 16.98 mmol) was added in batches, and stirred at room temperature for 2 h. After the reaction was completed, saturated sodium bicarbonate solution was added to the reaction system, and the organic phase was separated. The inorganic phase was extracted with dichloromethane (5 mL*3), and the combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (0%-30% ethyl acetate / petroleum ether, V / V) to obtain the final product (yield 35-40%).

[0075] colon cancer

[0076] Colon cancer is a common malignant tumor in the digestive system, and its incidence and mortality rate rank among the top in malignant tumors. According to a recently released global cancer data statistics report, the incidence of colon cancer ranks third among malignant tumors, only next to lung cancer and breast cancer, and the mortality rate ranks second. In China, the incidence and mortality rate of colorectal cancer are increasing year by year, ranking fourth in the incidence and fifth in the mortality rate among various malignant tumors. Colon cancer often occurs at the junction of the rectum and the colon, and can invade multiple internal organs and tissues, causing serious damage to the patient's body and seriously threatening people's life safety and health.

[0077] cardiac, liver, and renal function indicators

[0078] Cardiac function indicators include:

[0079] (1) Creatine kinase, which is usually present in the cytoplasm and mitochondria of tissues such as the heart, muscle, and brain of animals, is an important kinase directly related to intracellular energy operation, muscle contraction, and ATP regeneration. When myocardial cells are damaged, such as myocardial infarction, creatine kinase in the blood can rapidly increase, which is an important indicator of cardiac function.

[0080] (2) Lactate dehydrogenase is an important enzyme involved in glycolysis and gluconeogenesis, which catalyzes the oxidation-reduction reaction between lactic acid and pyruvic acid in the cytoplasm of cells including myocardial cells. When myocardial damage occurs, lactate dehydrogenase is released into the blood, and blood tests rapidly increase, which is an important indicator of cardiac function.

[0081] Liver function indicators include:

[0082] (1) Aspartate aminotransferase is a transaminase present in the mitochondria of tissue cells including liver cells. However, when the liver cells of the body are damaged, intracellular aspartate aminotransferase is released into the blood, and blood tests rapidly increase, which is an important clinical indicator of liver function.

[0083] (2) Alanine aminotransferase, a transaminase existing in the cytoplasm of hepatocytes, can sensitively respond to liver cell damage.

[0084] (3) Direct bilirubin, indirect bilirubin and total bilirubin. Indirect bilirubin refers to bilirubin that is not combined with glucuronic acid. Indirect bilirubin is difficult to dissolve in water and cannot be excreted through the kidney with urine. Direct bilirubin is soluble in water and can be excreted through the kidney with urine. Total bilirubin is the sum of direct bilirubin and indirect bilirubin. Liver is an important organ for bilirubin metabolism. After liver cell damage, bilirubin metabolism is abnormal, which can cause the increase of various bilirubins in blood test and urine test.

[0085] The renal function indicators include:

[0086] (1) Creatinine, a product of muscle tissue decomposition and metabolism in the human body, mainly filtered through the glomerulus of the human kidney and excreted out of the body with urine, is one of the main indicators commonly used to understand the renal function;

[0087] (2) Urea nitrogen, a nitrogen-containing compound in the blood plasma, is the main product of human protein metabolism, mainly filtered through the glomerulus of the human kidney and excreted out of the body with urine, is one of the main indicators commonly used to understand the renal function.

[0088] Pharmaceutical compositions and kits

[0089] The term "pharmaceutical composition" means a mixture of a therapeutically effective amount of one or more of the compounds and its pharmaceutically acceptable tautomers, solvates, hydrates or salts, with other pharmaceutically acceptable carriers. The purpose of preparing the compounds into pharmaceutical compositions is to make it more convenient to administer to the subject.

[0090] The terms "kit" or "kit of parts" are used interchangeably in the present application. The present application discloses a kit comprising a therapeutically effective amount of the therapeutic agent or pharmaceutical composition. According to certain embodiments of the present application, the kit further comprises one or more other therapeutic agents. According to certain embodiments of the present application, the kit further comprises instructions for use. According to certain embodiments of the present application, the kit further comprises a device for the corresponding administration mode, for example but not limited to a needle.

[0091] According to one aspect of the present application, the present application discloses a pharmaceutical composition for treating tumors, comprising the compound of formula (I) or phytol and its pharmaceutically acceptable salts and a second therapeutic agent.

[0092] According to certain embodiments of the present application, the second therapeutic agent is selected from 5-fluorouracil, oxaliplatin, an immune checkpoint inhibitor.

[0093] According to certain embodiments of the present application, the immune checkpoint inhibitor is selected from a PD1 antibody or a PDL1 antibody.

[0094] According to certain embodiments of the present application, the ratio of the compound or phytol and pharmaceutically acceptable salts thereof to the second therapeutic agent is 1:1-20, such as 1:1-15, 1:1-10, or 1:1-5. According to certain embodiments of the present application, the ratio of the compound or phytol and pharmaceutically acceptable salts thereof to the second therapeutic agent is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20.

[0095] Method of making a pharmaceutical composition

[0096] According to one aspect of the present application, the present application provides a method of making the pharmaceutical composition, the method mixing the composition with a pharmaceutically acceptable excipient.

[0097] Dosage form

[0098] The pharmaceutical composition of the present application can be prepared in any one of the pharmaceutically acceptable dosage forms, including but not limited to tablets, oral agents, powders, injections, liposomes, targeted injection pills, capsules, granules, powders, suppositories, powders, ointments, patches, injection solutions, solutions, suspensions, sprays, lotions, drops, rubs, etc. The pharmaceutical composition can be prepared in the form of a dry powder, and mixed with sterile water or buffer to prepare a solution before administration. The pH of the buffer is usually 3-11, preferably 5-9, more preferably 7-8.

[0099] The term "administration" or "administering" or "administered" means the delivery of a dose of a compound or a pharmaceutical composition to a subject by an appropriate mode of administration.

[0100] The "mode of administration" includes but is not limited to oral administration, intravenous administration, intrapulmonary administration, sublingual administration, topical administration, intramuscular administration, intraocular administration, transdermal absorption, parenteral administration, intraperitoneal administration, vaginal administration, buccal administration, rectal administration, and any other mode of administration known in the art. It will be appreciated by those skilled in the art that the mode of administration to a subject depends on a number of factors, including the location of the disease, the age of the subject, the severity of the disease, and the ingredients of the pharmaceutical composition, etc.

[0101] Method of treatment and pharmaceutical use

[0102] According to one aspect of the present application, there is disclosed a method of treating a tumor, said method administering said phytol or compound of formula (I) and pharmaceutically acceptable salts thereof.

[0103] According to certain embodiments of the present application, said tumor is selected from the group consisting of colorectal tumor, melanoma, lung metastatic cancer, liver cancer, uterine tumor, leukemia, lymphoma, multiple myeloma, and the like.

[0104] According to one aspect of the present application, there is disclosed a method of treating a tumor, said method administering said phytol or compound of formula (I) and pharmaceutically acceptable salts thereof in combination with a second therapeutic agent.

[0105] According to certain embodiments of the present application, said second therapeutic agent is selected from the group consisting of 5-fluorouracil, oxaliplatin, an immune checkpoint inhibitor.

[0106] According to certain embodiments of the present application, said immune checkpoint inhibitor is selected from the group consisting of a PD1 antibody or a PDL1 antibody.

[0107] According to certain embodiments of the present application, the ratio of said compound or phytol and pharmaceutically acceptable salts thereof to said second therapeutic agent is 1:1-20, for example 1:1-15, 1:1-10 or 1:1-5. According to certain embodiments of the present application, the ratio of said compound or phytol and pharmaceutically acceptable salts thereof to said second therapeutic agent is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19 or 1:20.

[0108] According to certain embodiments of the present application, said tumor is selected from the group consisting of colorectal tumor, melanoma, lung metastatic cancer, liver cancer, uterine tumor, leukemia, lymphoma, multiple myeloma, and the like.

[0109] According to one aspect of the present application, there is disclosed the use of phytol or a compound of formula (I) and pharmaceutically acceptable salts thereof for the preparation of a medicament for the treatment of a tumor.

[0110] According to certain embodiments of the present application, said tumor is selected from the group consisting of colorectal tumor, melanoma, lung metastatic cancer, liver cancer, uterine tumor, leukemia, lymphoma, multiple myeloma, and the like.

[0111] According to one aspect of the present application, there is disclosed the use of phytol or a compound of formula (I) and pharmaceutically acceptable salts thereof in combination with a second therapeutic agent for the preparation of a medicament for the treatment of a tumor.

[0112] According to certain embodiments of this application, the second therapeutic agent is selected from 5-fluorouracil, oxaliplatin, and immune checkpoint inhibitors.

[0113] According to certain embodiments of this application, the immune checkpoint inhibitor is selected from PD1 antibody or PDL1 antibody.

[0114] According to certain embodiments of this application, the ratio of the compound or phytol and its pharmaceutically acceptable salt to the second therapeutic agent is 1:1-20, for example 1:1-15, 1:1-10, or 1:1-5. According to certain embodiments of this application, the ratio of the compound or phytol and its pharmaceutically acceptable salt to the second therapeutic agent is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20.

[0115] According to certain embodiments of the present invention, the tumor is selected from colorectal tumors, melanoma, lung cancer, liver cancer, uterine tumors, leukemia, lymphoma, multiple myeloma, etc.

[0116] In this application, when “about” is used to modify a numerical value, it means that the numerical value can fluctuate within the range of ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%.

[0117] Unless otherwise stated in this application or obviously contradicted by the context, the terms “a,” “an,” “the,” “the,” and “at least one,” and similar designations used in the context of describing this application (including the claims) are to be interpreted to cover both the singular and plural. Unless otherwise stated in this application or obviously contradicted by the context, the terms “comprising,” “having,” “including,” and “containing” used in this application are to be interpreted as open-ended terms (i.e., “including but not limited to”). Unless otherwise stated in this application or obviously contradicted by the context, all methods described in this application may be performed in any suitable order as understood by those skilled in the art.

[0118] All patents, patent applications, and references cited in this application are incorporated herein by reference in their entirety, as if each reference were cited individually. In the event of any conflict between this application and the references provided herein, the content of this application shall prevail. Attached Figure Description

[0119] Figure 1Figure 6 shows that according to certain embodiments of the present application, phytol inhibits the growth of colorectal cancer, breast cancer and melanoma in vitro. A) CCK-8 assay to detect the effect of phytol on the proliferation of colon cancer cells MC38; (B) CCK-8 assay to detect the effect of phytol on the proliferation of breast cancer cells 4T1; (C) CCK-8 assay to detect the effect of phytol on the proliferation of melanoma cells B16F10; *p<0.05, **p<0.01, ***p<0.001. Error bars represent s.e.m.

[0120] Figure 2 Figure 7 shows that according to certain embodiments of the present application, phytol inhibits the growth of colorectal cancer, breast cancer in vivo. (A) Mouse subcutaneous tumor model to detect the effect of phytol on the in vivo growth of 4T1 breast cancer; (B) Mouse subcutaneous tumor model to detect the effect of phytol on the in vivo growth of MC38 colon cancer; (C) Mouse subcutaneous tumor model to detect the effect of phytol on the in vivo growth of human RKO colon cancer; (D) Mouse subcutaneous tumor model to detect the effect of phytol on the in vivo growth of human HCT116 colon cancer; *p<0.05, **p<0.01, ***p<0.001. Error bars represent s.e.m.

[0121] Figure 3 Figure 8 shows that according to certain embodiments of the present application, phytol has no obvious cardiotoxicity, hepatotoxicity and nephrotoxicity. (A) Alanine aminotransferase; (B) Aspartate aminotransferase; (C) Total bilirubin; (D) Direct bilirubin; (E) Indirect bilirubin; (F) Blood creatinine; (G) Urea; (H) Creatine kinase; (I) Lactate dehydrogenase; *p<0.05, **p<0.01, ***p<0.001. Error bars represent s.e.m.

[0122] Figure 4 Figure 9 shows that according to certain embodiments of the present application, phytol and its derivatives exhibit better anticancer activity. (A) Basic structure formula of small molecule compounds; (B) Molecular formula of phytol and its half inhibitory concentration (IC50); (C) Molecular formula of phytol derivative compound 3 and its half inhibitory concentration (IC50); (D) Molecular formula of phytol derivative compound 4 and its half inhibitory concentration (IC50); (E) Molecular formula of phytol derivative compound 6 and its half inhibitory concentration (IC50); (F) Tumor physical map of mouse subcutaneous tumor model to detect the effect of phytol and its derivatives on the in vivo growth of MC38 colon cancer; (G) Tumor growth curve of each group; (H) Tumor weight of each group; *p<0.05, **p<0.01, ***p<0.001. Error bars represent s.e.m.

[0123] Figure 5(A) Subcutaneous tumor model in mice to test phytol (200 mg / kg, 1 time per 2 days) and 5-fluorouracil (50 mg / kg, 2 times per week); (B) Tumor growth curve of each group; (C) Tumor weight of each group; *p<0.05, **p<0.01, ***p<0.001. Error bars represent s.e.m.

[0124] Figure 6 (A) Subcutaneous tumor model in mice to test phytol (200 mg / kg, 1 time per 2 days) and oxaliplatin (3 mg / kg, 2 times per week); (B) Tumor growth curve of each group; (C) Tumor weight of each group; *p<0.05, **p<0.01, ***p<0.001. Error bars represent s.e.m.

[0125] Figure 7 (A) Subcutaneous tumor model in mice to test phytol (200 mg / kg, 1 time per 2 days) and PD1 antibody (200 μg per mouse, 1 time per 2 days); (B) Tumor growth curve of each group; (C) Tumor weight of each group; *p<0.05, **p<0.01, ***p<0.001. Error bars represent s.e.m.

[0126] Figure 8 (A) Subcutaneous tumor model in mice to test phytol (500 mg / kg, 1 time per 2 days) and PD1 antibody (200 μg per mouse, 1 time per 2 days); (B) Tumor growth curve of each group; (C) Tumor weight of each group; *p<0.05, **p<0.01, ***p<0.001. Error bars represent s.e.m. DETAILED DESCRIPTION

[0127] The present application is further illustrated by the following non-limiting examples.

[0128] Example 1 Cell lines and specific culture methods

[0129] Mouse colon cancer MC38 cells, breast cancer cells 4T1 cells from Professor Yang Wei of Southern Medical University, mouse melanoma B16F10 cells originally from American Cell Culture Bank (ATCC). The frozen cells were quickly taken out from the liquid nitrogen tank or -80°C refrigerator, placed in a 37°C water bath, shaken to dissolve the cells in 1 minute, centrifuged at 800 rpm / min for 5 minutes to precipitate the cells, and then the frozen liquid was discarded with a pipette gun. The cells were resuspended in 1 ml of culture medium, centrifuged and washed once, and the culture medium was removed. New culture medium 5 ml was added, and the cells were taken and placed in a culture bottle, which was incubated in a 37°C, 5% CO2 incubator. After 8 hours, the cell state was observed under a microscope, and the culture medium was replaced after 8 to 12 hours for continuous culture. Mouse colon cancer MC38 cells and 4T1 cells, melanoma cell strain B16F10 were cultured in DMEM medium containing 10% fetal bovine serum in a 37°C, 5% CO2 incubator. When the cells were fully grown, the culture medium was discarded, and PBS was washed 3 times to remove dead cells. An appropriate amount (0.5 ml) of trypsin was added to cover the cell surface, and the cells were observed under a microscope at room temperature or 37°C. When the cells were rounded and the gap between the cells was enlarged, the trypsin digestion was terminated by adding serum-containing medium. The cells were gently blown off the wall with a pipette gun to form a single cell suspension. The cell suspension was transferred to a new culture bottle at a ratio of one to two, and an appropriate amount of serum-containing medium was added. The cells were incubated in a 37°C, 5% CO2 cell incubator. All cell strains were routinely tested for mycoplasma, and the results were negative.

[0130] Example 2 CCK-8 detection

[0131] After the MC38, 4T1 and B16F10 cells in good growth state were digested and counted, phaeophytol and DMSO were added at a concentration of 250 micromoles and 500 micromoles, and 100 cells / well were inoculated in a 96-well plate. The plate was incubated in a 37°C incubator overnight, and the detection was performed on the 2nd to 8th day according to the following method.

[0132] The required mixture was calculated according to the number of wells, and the serum-free medium and CCK8 reagent were mixed at 90 microliters:10 microliters. The mixture was prepared in the dark, and the medium in the 96-well plate was aspirated. 100 microliters of the prepared CCK8 mixture was added to each well, the plate was gently shaken to remove the residual bubbles in the wells, and the culture was continued for 2 hours.

[0133] The absorbance value at 450 nm was measured by enzyme-linked immunoassay instrument. Five repeated wells were set for each group, and the average value was taken to draw a time-growth curve.

[0134] Example 3 animal study

[0135] All experiments related to animal studies were approved by the Southern Medical University Animal Care and Use Committee, and all procedures followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Female C57BL / 6J mice from the Guangdong Medical Laboratory Animal Center were randomly grouped, and on day 2, the left dorsal skin of the mice was routinely depilated, disinfected with 75% alcohol, and then a cell suspension was drawn up with a syringe, the needle tip was parallel to the plane, and it was inserted into the skin 3 cm away in a meandering manner, and then the cell suspension was injected. After a small subcutaneous bump was formed, the needle was slowly withdrawn in a meandering manner. After the inoculation of the mice, observation began after 1 week, and after the appearance of visible small nodules (generally 50-100 cubic millimeters), drug administration began. The mouse body weight was measured, and the phytol was dissolved in DMSO, and the injection volume was 100 μl per mouse. The abdominal skin was first disinfected with 75% alcohol, 100 μl was drawn up with a 1 ml syringe, the mouse was grabbed with its abdomen facing up and its head slightly downward, the dorsal skin was grabbed to make the abdominal skin taut, and the needle was inserted subcutaneously about 1 cm from the intersection of the line connecting the two thigh roots and the midline of the abdomen, and the needle was pushed in parallel to the midline of the abdomen for 3-5 mm, and then it was inserted into the abdominal cavity at a 45° angle, and after the needle tip passed through the abdominal muscle, the resistance disappeared, and no backflow was drawn back, and the drug was slowly pushed in.

[0136] The specific drug regimen was intraperitoneal injection of phytol (50 mg / kg or 200 mg / kg, CAT#T3254, Shanghai Taosu Biological Technology Co., Ltd.), compound No. 3, compound No. 4, and compound No. 6 (50 mg / kg, synthesized by the School of Pharmacy, Southern Medical University), once every 2 days. The long diameter a and the short diameter b of the mouse tumor were measured with a vernier caliper every other day, and the tumor volume was calculated according to the formula 0.5 × ab 2 When the tumor volume was greater than 1500 mm 3 or cachexia, the mice were sacrificed. After the tumor tissue was removed, it was photographed, weighed, and recorded.

[0137] Example 4 Toxic Side Effect Evaluation

[0138] The same age female C57BL / 6J mice were randomly grouped. Phytol was injected at 500 mg / kg once every other day. After 8-10 injections of the drug, the mice were sacrificed, and the eyeball blood was collected. The sample was sent to the Guangdong Animal Testing Center for detection of heart, liver, and kidney function indicators.

[0139] Example 5 Determination of the Half-Inhibitory Concentration of Phytol and Its Derivatives

[0140] After the MC38 cells in good growth condition were digested and counted, phytol and its derivatives were added in concentrations of 1000, 500, 250, 125, 62.25, and 31.125 micromoles in descending order, and 100 cells / well were inoculated in a 96-well plate, and after incubation in a 37°C incubator for 72 hours, the detection was performed according to the following method.

[0141] According to the number of holes, the required mixed solution is calculated, the serum-free medium and the CCK8 reagent are mixed at 90 microliters: 10 microliters, avoid light, and the medium in the 96-well plate is aspirated. 100 microliters of the pre-prepared CCK8 mixed solution is added to each well, the plate is gently shaken, the residual bubbles in the holes are removed, and the culture is continued for 2 hours.

[0142] The absorbance value at 450 nm is measured by an enzyme-linked immunoassay instrument, 5 repeated holes are set for each group, the average value is taken, and the half-inhibitory concentration is calculated.

[0143] Example 6: Statistical analysis

[0144] IBM SPSS 20.0 statistical software and GraphPad Prism 8.0 are used for data analysis, and the measurement data is represented by mean ± standard deviation (standard deviation, SD). The growth curve results of mouse subcutaneous tumors are tested by factorial design variance analysis and one-way analysis of variance. The number of positive cells in immunohistochemistry and the weight of mouse tumors are statistically analyzed by one-way analysis of variance or two independent sample t test.

[0145] Example 7: Inhibition of growth of colon cancer, breast cancer and melanoma by phytol in vitro

[0146] The cell lines and specific culture methods and CCK-8 detection are shown in Examples 1 and 2.

[0147] Figure 1 The results show that in the in vitro experiment, after the MC38, 4T1 and B16F10 cells in good growth state are counted and digested, phytol and DMSO are added at a concentration of 250 micromoles and 500 micromoles, 100 cells / well are inoculated in a 96-well plate, and the cell viability is detected by CCK-8 reagent from the 2nd day to the 8th day.

[0148] The results show that different concentrations (250, 500 micromoles) of phytol can not only significantly inhibit the growth of mouse colon cancer cells MC38 ( Figure 1 A, P<0.05), but also significantly inhibit the growth of mouse breast cancer cells 4T1 ( Figure 1 B, P<0.05) and melanoma B16F10 ( Figure 1 C, P<0.05).

[0149] Example 8: Inhibition of growth of colon cancer and breast cancer by phytol in vivo

[0150] The cell lines and specific culture methods and animal experiments are shown in Examples 1 and 3.

[0151] A homologous mouse colon cancer model is constructed, and phytol is given in the homologous mouse colon cancer model.

[0152] First, the well-grown intestinal cancer cells and breast cancer cells were counted, and 0.5-1*10 6 / 100 micro liter injection dose of cell suspension, female C57BL / 6J or nude mice from Guangdong Medical Laboratory Animal Center were randomly divided into groups, C57BL / 6J mice need to be routinely depilated on the left back skin, and after disinfection with 75% alcohol, the cell suspension was taken up with a syringe, the needle tip was parallel to the skin, and the needle was inserted into the skin 3 cm away from the skin, and then the cell suspension was injected. After a small package was formed under the skin, the needle was slowly withdrawn. When the mice developed visible tumors under the skin, the mice were randomly divided into groups, and the mice were injected intraperitoneally with chlorophyll (50 mg / kg, CAT#T3254, Shanghai Taos Biological Technology Co., Ltd.). Every 2 days, 1 time. In the same mouse breast cancer model and xenotransplantation colon cancer model experiments, every 2 days, 1 time. The length a and the short diameter b of the mouse tumor were measured with a vernier caliper, and the tumor volume was calculated according to the formula 0.5*ab 2 When the tumor volume was greater than 1500mm 3 or cachexia, the mice were sacrificed. After the tumor tissue was removed, it was photographed, and the tumor volume of each mouse was input into the software Graphpad prism

[0153] 8.0, two independent sample analysis of variance design of mouse tumor volume.

[0154] As shown in Figure 2 A, chlorophyll can significantly inhibit the growth of 4T1 breast cancer and slow down the tumor progression.

[0155] As shown in Figure 2 B-2D, chlorophyll can significantly inhibit the growth of mouse MC38 colon tumors, human RKO colon tumors and human HCT116 colon tumors, and slow down the tumor progression. It is shown that chlorophyll can inhibit the growth of colon cancer and breast cancer in vivo, and has good antitumor activity.

[0156] Example 9: Chlorophyll has no obvious cardiotoxicity, hepatotoxicity and nephrotoxicity

[0157] The operation method for evaluating the side effects of chlorophyll is shown in Example 4.

[0158] In wild-type C57BL / 6J mice of the same age, chlorophyll was injected at a dose of 500 mg / kg once a day. After 8-10 times of drug injection, the mice were sacrificed, and the eyeball blood was collected. The blood biochemical machine (iCARE-2000, San Nuo Biological Sensor Co., Ltd.) was used to detect the heart, liver and kidney function indexes.

[0159] As shown in Figure 3As shown in A-3E, liver function indicators such as alanine aminotransferase (ATL), aspartate aminotransferase (ASL), total bilirubin (TBIL), direct bilirubin (DBIL), and indirect bilirubin (IBIL) did not change significantly after phytol treatment (P<0.05).

[0160] like Figure 3 As shown in F-3G, indicators reflecting renal function, such as blood urea nitrogen (BUN) and creatinine (CREA), did not change significantly after phytol treatment (P<0.05).

[0161] like Figure 3 As shown in H-3I, indicators reflecting cardiac function, such as creatine kinase (CK) and lactate dehydrogenase (LDH), did not change significantly after phytol treatment (P<0.05).

[0162] The above indicates that phytol did not exhibit significant toxic side effects in the body.

[0163] Example 10: Phytophthol and its derivatives exhibit better anticancer activity

[0164] Cell lines and specific culture methods, animal experiments, and determination of half-inhibitory concentrations of phytol and its derivatives are described in Examples 1, 3, and 5.

[0165] The School of Pharmacy of Southern Medical University was commissioned to conduct research based on the original structural foundation of phytol ( Figure 4 A) was modified for synthesis. When R was substituted with -H, the compound phytol was obtained. Figure 4 B); When R is substituted with -NOC4H4, compound 3 is obtained ( Figure 4 C); When R is substituted with -NO3C2H3, compound 4 is obtained ( Figure 4 D); When R is substituted with -NOC4H5, compound 6 is obtained ( Figure 4 E).

[0166] The half-inhibitory concentrations of phytol and its derivatives were determined using mouse colon cancer cells MC38.

[0167] Select the growth of MC38 cells count, according to 1000, 500, 250, 125, 62.25, 31.125 micro-molar concentration of phaeophytol and its derivatives, and 100 cells per hole in the 96 well plate, 37℃ incubator culture 72 hours after the following method detection. According to the number of wells required to mix the solution, serum-free medium and CCK8 reagent according to 90 microliters: 10 microliters mixed, avoid light configuration, 96 well plate of the medium is absorbed, each hole is added to the pre prepared CCK8 mixed solution 100 microliters, gently shake the plate, remove the residual bubbles in the hole, continue to cultivate 2 hours. The enzyme linked immunoassay instrument was used to determine the absorbance value at 450 nm, 5 repeated holes were set in each group, the average value was taken, and the half inhibition concentration was calculated. For example Figure 4 As shown in A-4D, the half inhibition concentration of phaeophytol on mouse colon cancer cells MC38 was 345.02 micromole, the half inhibition concentration of compound No. 3 was 134.1 micromole, the half inhibition concentration of compound No. 4 was 45.66 micromole, and the half inhibition concentration of compound No. 6 was 22.5 micromole. This shows that after structural modification, the anti-cancer activity of phaeophytol derivatives is significantly improved.

[0168] Further, phaeophytol and its derivatives were used to treat mouse MC38 colon cancer in vivo, and the length a and the short diameter b of the tumor were measured by vernier caliper, and the tumor volume calculation formula was 0.5×ab 2 When the tumor volume was greater than 1500mm 3 or cachexia, the mice were sacrificed. After the tumor tissue was removed, the tumor was photographed, weighed and recorded, and the tumor volume and weight of each mouse were input into the software Graphpad prism8.0, and the mouse tumor volume was analyzed by two independent sample analysis of variance design, and the mouse tumor weight was analyzed by two independent sample t test.

[0169] As Figure 5 As shown in E-4G, compared with the control group, phaeophytol and its derivatives can significantly inhibit tumor growth in vivo (P<0.05).

[0170] Compared with the phaeophytol group, the derivatives of compounds 3, 4 and 6 can significantly inhibit the growth of tumors (P<0.05), and the anti-cancer activity of compound No. 6 is the strongest.

[0171] Example 11: Phaeophytol combined with 5-fluorouracil inhibits the growth of intestinal cancer

[0172] Phaeophytol (200 mg / kg, 2 times a day) combined with 5-fluorouracil (75 mg / kg, 2 times a week) was used to treat mouse MC38 colon cancer in vivo, and the length a and the short diameter b of the tumor were measured by vernier caliper, and the tumor volume calculation formula was 0.5×ab 2When the tumor volume is greater than 1500mm3or cachexia, the mice are sacrificed. After the tumor tissue is removed, the tumor is photographed, weighed and recorded, and the tumor volume and weight of each mouse are input into the software Graphpad prism 8.0, and the mouse tumor volume is subjected to two independent sample analysis of variance design, and the mouse tumor weight is subjected to two independent sample t test statistics.

[0173] As shown in Figure 6 As shown in A-5C, compared with the control group, phytol and 5-fluorouracil alone can significantly inhibit tumor growth in vivo. Compared with the 5-fluorouracil alone treatment group, the phytol + 5-fluorouracil treatment group has better treatment effect, which can significantly slow down tumor growth and reduce tumor weight (P<0.05).

[0174] Example 12: Phytol combined with oxaliplatin inhibits the growth of intestinal cancer

[0175] Phytol (200 mg / kg, 2 days once) combined with oxaliplatin (3 mg / kg, 2 times a week) is used to treat MC38 colon cancer in mice in vivo, and the long diameter a and short diameter b of the tumor are measured by vernier caliper, and the tumor volume is calculated by the formula 0.5×ab 2 When the tumor volume is greater than 1500mm 3 When the tumor volume is greater than 1500mm

[0176] As shown in Figure 7 As shown in A-6C, compared with the control group, phytol alone can significantly inhibit tumor growth in vivo. Compared with the oxaliplatin alone treatment group, the phytol + oxaliplatin treatment group has better treatment effect, which can significantly slow down tumor growth and reduce tumor weight (P<0.05).

[0177] Example 13: Phytol combined with immune checkpoint inhibitor PD1 antibody inhibits the growth of intestinal cancer

[0178] Phytol (200 mg / kg, 2 days once) combined with PD1 antibody (200 μg / each, 2 days once) is used to treat MC38 colon cancer in mice in vivo, and the long diameter a and short diameter b of the tumor are measured by vernier caliper, and the tumor volume is calculated by the formula 0.5×ab 2 When the tumor volume is greater than 1500mm 3Or cachexia, mice were sacrificed. After the tumor tissue was removed, the tumor was photographed, weighed and recorded, and the tumor volume and weight of each mouse were input into the software Graphpad prism 8.0, and the mouse tumor volume was analyzed by two independent sample analysis of variance design, and the mouse tumor weight was analyzed by two independent sample t test.

[0179] As shown in Figure 8 As shown in A-7C, compared with the control group, phytol and PD1 antibody alone can significantly inhibit tumor growth in vivo. Compared with the PD1 antibody alone treatment group, the phytol + PD1 antibody treatment group has better treatment effect, which can significantly slow down tumor growth and reduce tumor weight (P<0.05).

[0180] Example 14: Phytol combined with immune checkpoint inhibitor PD1 antibody inhibits melanoma growth

[0181] Phytol (500 mg / kg, 2 times a day) combined with PD1 antibody (200 μg / mouse, 2 times a day) was used to treat mouse melanoma in vivo, and the long diameter a and short diameter b of the mouse tumor were measured by vernier caliper, and the tumor volume was calculated by the formula 0.5×ab 2 When the tumor volume is greater than 1500 mm 3 Or cachexia, mice were sacrificed. After the tumor tissue was removed, the tumor was photographed, weighed and recorded, and the tumor volume and weight of each mouse were input into the software Graphpad prism 8.0, and the mouse tumor volume was analyzed by two independent sample analysis of variance design, and the mouse tumor weight was analyzed by two independent sample t test.

[0182] As shown in ​ As shown in A-8C, compared with the control group, phytol alone can significantly inhibit tumor growth in vivo. Compared with the PD1 antibody alone treatment group, the phytol + PD1 antibody treatment group has better treatment effect, which can significantly slow down tumor growth and reduce tumor weight (P<0.05).

[0183] Reference

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Claims

1. A compound and a pharmaceutically acceptable salt thereof, said compound having the structure shown in formula (I): in, R can be -OH, -N2C2H6, -NOC4H4, -NO3C2H3, or -NOC4H5.

2. The compound and its pharmaceutically acceptable salt according to any one of claims 1-2, characterized in that, The compound of formula (I) is selected from:

3. A method for preparing the compound according to any one of claims 1-2, characterized in that, The method includes: Step 1: Synthesis of (7R,11R,E)-3,7,11,15-tetramethylhexadecyl-2-enal Dissolve 20-30 mmol of phytol in 20-100 mL of dichloromethane, and add Dysmartin oxidant C under ice bath conditions. 13 H 13 IO8 (20-100 mmol) was added and stirred at room temperature until the reaction was complete. After the reaction was complete, 5-30 mL of a mixed solution of sodium thiosulfate and sodium bicarbonate was added and stirred for 2-10 min. The organic phase was separated and the aqueous phase was washed with dichloromethane (10-30 mL, 2-5 times). The organic phases were combined and washed with saturated brine. The mixture was dried over anhydrous sodium sulfate and the solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by column chromatography to obtain the product (7R,11R,E)-3,7,11,15-tetramethylhexadecyl-2-enal. Step 2: Synthesize compound (I) Where R-NH2, R is -C4H2O, -C3H2O2 or -C5H3O; The (7R,11R,E)-3,7,11,15-tetramethylhexadecyl-2-enal obtained in step one is dissolved in 5-30 mL of dichloromethane with a primary or secondary amine (1-5 mmol). The mixture is stirred at room temperature for 2-10 min, and then sodium triacetoxyborohydride (5-30 mmol) is added in portions. The mixture is stirred at room temperature for 1-5 h. After the reaction is complete, a saturated sodium bicarbonate solution is added to the reaction system to separate the organic phase. The inorganic phase is extracted with dichloromethane (2-10 mL, 2-5 times). The combined organic phases are washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent is removed under reduced pressure to obtain the crude product. The crude product is purified by column chromatography (0%-30% ethyl acetate / petroleum ether, V / V) to obtain the final product.

4. Use of the compound according to any one of claims 1-2 or phytol and its pharmaceutically acceptable salt in the preparation of a medicament for treating tumors.

5. The use according to claim 4, characterized in that, The tumors are selected from colorectal tumors, melanoma, lung metastases, liver cancer, uterine tumors, leukemia, lymphoma, multiple myeloma, etc.

6. A pharmaceutical composition for treating tumors, characterized in that, The pharmaceutical composition comprises the compound according to any one of claims 1-2 or phytol and its pharmaceutically acceptable salt, and a second therapeutic agent.

7. The pharmaceutical composition of claim 6, wherein the second therapeutic agent is selected from 5-fluorouracil, oxaliplatin, and immune checkpoint inhibitors; wherein the immune checkpoint inhibitor is selected from PD1 antibody or PDL1 antibody.

8. Use of the pharmaceutical composition according to any one of claims 6-7 in the preparation of a medicament for treating tumors.

9. The use according to claim 8, characterized in that, The tumors are selected from colorectal tumors, melanoma, lung metastases, liver cancer, uterine tumors, leukemia, lymphoma, multiple myeloma, etc.

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