Brucea javanica picrosol derivative with low-oxygen activation prodrug property as well as preparation method and application of brucea javanica picrosol derivative

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a nitroimidazole brucea javanica bitter alcohol derivative with a low-oxygen activation prodrug property in dry fruits of a medicinal plant brucea javanica, and a preparation method and an application of the nitroimidazole brucea javanica bitter alcohol derivative. According to the invention, a series of novel brucea javanica bitter alcohol derivatives or pharmaceutically acceptable salts thereof are designed and synthesized by carrying out necessary structural modification on brucea javanica bitter alcohol. Anti-tumor drug screening on digestive system cancer cell proliferation through pharmacological experiments shows that the brucea javanica bitter alcohol derivative or the pharmaceutically acceptable salt thereof has good colon cancer cell inhibition activity, so that the brucea javanica bitter alcohol derivative or the pharmaceutically acceptable salt thereof can be developed into a new anti-colon cancer drug; and the method has very high potential research value and clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a preparation method and use of brucea brucea in Brucea javanica, and in particular to a nitroimidazole brucea brucea derivative with hypoxia-activated prodrug properties in the dried fruit of the medicinal plant Brucea javanica, and a preparation method and use thereof. Background Art

[0002] The cancer report released in 2024 shows that the top five malignant tumors are lung cancer, colorectal cancer, thyroid cancer, liver cancer, and gastric cancer. The top five malignant tumors that cause death are lung cancer, liver cancer, gastric cancer, colorectal cancer, and esophageal cancer. Except for lung cancer, the other four are digestive tract cancers, so finding a specific drug that has an anti-digestive system cancer effect has become a top priority.

[0003] Due to the rapid growth of tumor tissue, the high expansion of volume and the incomplete vascular system inside the tumor tissue, the oxygen supply in the tumor tissue is insufficient, and the tumor microenvironment presents the characteristics of overall hypoxia, which leads to low pH, high glutathione (GSH) concentration, high reactive oxygen species (ROS) level, unique enzyme activity, high adenosine triphosphate (ATP) content, etc. Therefore, targeted anti-tumor drugs can be designed through the special hypoxic environment of tumor tissue, and these drugs are converted into active drugs with cytotoxicity in a highly reducing environment.

[0004] Bruce ajavanica (L.) Merr. is a plant of the genus Brucea in the family Simulaceae. Brucea ajavanica is used as medicine with its fruit and leaves, and is a commonly used Chinese medicine. Brucea ajavanica is cold in nature and bitter in taste. It belongs to the large intestine meridian and the liver meridian. It has the effects of clearing away heat and detoxifying, stopping malaria, stopping dysentery, and corroding warts. Studies have shown that quasin compounds are the main components of Brucea ajavanica. This type of compound includes an unsaturated six-membered ring (marked as A), two cyclohexane rings (marked as B and C), a six-membered lactone ring (marked as D), and a tetrahydrofuran ring (marked as E). In their molecular structures, hydroxyl groups usually exist at positions C-3, 11, 12, and 15, and the hydroxyl group at position 15 often appears in the form of an ester. Among them, brucea brucea picroside is a quasin compound with a relatively high content (content of about 0.3%) and a stable source.

[0005]

[0006] Brucea brucea has significant biological activity, and semi-synthetic modification using this compound as a starting material has broad application prospects in the pharmaceutical industry. The inventors have synthesized a series of brucea brucea derivatives based on the design idea of ​​hypoxia-activated prodrugs by researching literature and using brucea brucea as the parent core. Summary of the invention

[0007] In order to solve the above technical problems, the present invention uses brucein as the mother core to prepare a series of hypoxia-activated prodrug derivatives or pharmaceutically acceptable salts thereof, and further provides a pharmaceutical composition containing the derivative or pharmaceutically acceptable salts thereof, and a method for preparing the derivative or pharmaceutically acceptable salts thereof. In addition, the present invention also provides the use of the derivative or pharmaceutically acceptable salts thereof or pharmaceutical compositions thereof in the preparation of drugs targeting digestive system tumors.

[0008] Specifically, the present invention is realized through the following technical solutions:

[0009] In a first aspect, the present invention provides a brucein derivative or a pharmaceutically acceptable salt thereof having hypoxia-activated prodrug properties, wherein the brucein derivative has a chemical structure of formula (I), (II) or (III),

[0010]

[0011] R in formula (I), (II) and (III) is selected from:

[0013] More preferably, R in formula (I) is selected from:

[0014] R in formula (II) is selected from:

[0015] R in formula (III) is selected from:

[0016] Preferably, the brucein derivative or a pharmaceutically acceptable salt thereof having hypoxia activated prodrug properties is selected from the following:

[0017]

[0018]

[0019] In a second aspect, the present invention provides a pharmaceutical composition comprising the brucea bruceiol derivative or a pharmaceutically acceptable salt thereof as described in the first aspect, and a pharmaceutically acceptable carrier.

[0020] The dosage form of the pharmaceutical composition is an oral dosage form or an injection dosage form, wherein the oral dosage form is a capsule, a tablet, a granule, an oral liquid, a sustained-release preparation or a controlled-release preparation.

[0021] Preferably, the pharmaceutically acceptable carrier is selected from one or more of a diluent, a lubricant, a binder, a disintegrant, a stabilizer or a solvent.

[0022] More preferably, the diluent of the present invention is selected from starch, microcrystalline cellulose, sucrose, dextrin, lactose, powdered sugar, glucose; the lubricant is selected from magnesium stearate, stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate, poloxamer; the binder is selected from water, ethanol, starch slurry, syrup, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, sodium alginate, polyvinyl pyrrolidone; the disintegrant is selected from starch effervescent mixture, i.e. sodium bicarbonate and citric acid, tartaric acid, low-substituted hydroxypropyl cellulose; the stabilizer is selected from polysaccharides such as acacia gum, agar, alginic acid, cellulose ether and carboxymethyl chitosan; the solvent is selected from water and a balanced salt solution.

[0023] In a third aspect, the present invention provides a method for preparing the brucein derivative or a pharmaceutically acceptable salt thereof according to the first aspect, the preparation method comprising the following steps:

[0024]

[0025]

[0026] Using brucea stearoyl alcohol (BST) as the starting material, compound BO-1-6 is prepared through step a, compound BO-7-9 is prepared through step b, compound BO-10 is prepared through steps c and d, and compound BO-11 is prepared through step e;

[0027] Specifically, the preparation method comprises the following steps:

[0028] (5) Synthesis of BO-1-6: Dissolve the corresponding hydroxy compound in a solvent, add 1 times the amount of coupling agent, 1 times the amount of organic base and 3 times the amount of acid binding agent, stir for 30 minutes, add 1.2 times the amount of brucea stearoyl alcohol (BST), and react at 25°C. Remove the solvent, extract, wash, dry, filter, and purify;

[0029] (6) Synthesis of BO-7-9 and BST-6C: Dissolve BST, condensing agent and acid binding agent in a solvent, stir at room temperature, add the corresponding carboxylic acid fragment, react at room temperature, and perform post-treatment in the same manner as step (1);

[0030] (7) Synthesis of BO-10: Dissolve BST-6C and an inorganic base in a solvent, stir at room temperature, add p-nitrobenzyl bromide, react at room temperature, and perform post-treatment in the same manner as step (2);

[0031] (8) Synthesis of BO-11: Dissolve BST and an inorganic base in a solvent, stir at room temperature, add p-nitrobenzyl bromide, react at room temperature, and perform post-treatment in the same manner as step (2).

[0032] The above preparation method, wherein:

[0033] The coupling agent is p-nitrophenyl chloroformate, carbonyldiimidazole, carbonyltriazole, phosgene or triphosgene;

[0034] The condensing agent is dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI);

[0035] The acid binding agent is N,N-diisopropylethylamine, pyridine or 4-dimethylaminopyridine;

[0036] The organic base is triethylamine, N,N-diisopropylethylamine, pyridine, 4-dimethylaminopyridine or N-methylmorpholine;

[0037] The inorganic base is potassium carbonate, cesium carbonate, sodium carbonate, potassium hydroxide, sodium hydroxide, sodium hydride, potassium methoxide, sodium methoxide, sodium tert-butoxide or potassium tert-butoxide.

[0038] In a fourth aspect, the present invention provides use of the brucea javanica derivative or a pharmaceutically acceptable salt thereof as described in the first aspect, or the pharmaceutical composition as described in the second aspect, in the preparation of a drug targeting digestive system tumors.

[0039] Preferably, the digestive system tumor is gastric cancer, colon cancer or liver cancer.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The present invention designs and synthesizes a series of novel brucein derivatives or pharmaceutically acceptable salts thereof by making necessary structural modifications to brucein. Through pharmacological experiments, anti-tumor drug screening for digestive system cancer cell proliferation is conducted and it is found that the brucein derivatives or pharmaceutically acceptable salts thereof of the present invention have good colon cancer cell inhibition activity, and thus can be developed into new anti-colon cancer drugs, which have very high potential research value and clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 : HPLC monitoring of the release process of BO-1 under the action of Na2S2O4 (mobile phase: 40 / 60 acetonitrile / water-70 / 30 acetonitrile / water (v / v), 20 min, 0.8 mL / min);

[0043] Figure 2: HPLC monitoring of BO-1 release process under the action of nitroreductase (mobile phase: 40 / 60 acetonitrile / water-70 / 30 acetonitrile / water (v / v), 20 min, 0.8 mL / min);

[0044] Figure 3 : Hypoxia selectivity (IC) of the brucein derivatives of the present invention on HepG2, MGC-803 and HCT116 50 (Normal oxygen) / IC 50 (hypoxia));

[0045] Figure 4 : The cell selectivity of the brucein derivatives of the present invention for HepG2, MGC-803, and HCT116;

[0046] Figure 5 :The relationship curve between survival rate and concentration of BO-1 after intraperitoneal injection. DETAILED DESCRIPTION

[0047] The present invention is further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention. If specific techniques or conditions are not specified in the embodiments, they are carried out according to the techniques or conditions described in the literature in this field, or according to the product instructions. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0048] Example 1: Preparation of compound BO-1:

[0049]

[0050] Take a 10mL reaction bottle, take a1 (10mg, 58.43μmol, 1eq) and dissolve it in 1mL dry DCM, add p-nitrophenyl chloroformate (11.78mg, 58.43μmol, 1eq), TEA (16.24μL, 116.85μmol, 2eq) and DMAP (21.41mg, 175.28μmol, 3eq) at 0℃ and stir for 30 minutes, add BST (36.50mg, 70.11μmol, 1.2eq) and, react at 25℃ for 12h. TLC monitors the completion of the reaction, extracts twice with 5mL saturated ammonium chloride aqueous solution and once with 5mL saturated sodium chloride aqueous solution, collects the organic phase, and removes the solvent under reduced pressure after drying over anhydrous sodium sulfate. Purify by RP-HPLC to obtain BO-1: white solid, the structural identification data are as follows:

[0051] BO-1: white solid, 1H NMR(600MHz,Chloroform-d)δ7.33(d,J=1.1Hz,1H),

[0052] 7.19(d,J=1.1Hz,1H),6.27(s,1H),5.63(s,1H),4.82(s,1H),4.74(d,J=8.0Hz,1H),4.66-4.56(m,2H),4. 31-4.26(m,2H),4.25-4.22(m,1H),4.21(s,1H),3.84-3.76(m,4H),3.31(s,1H),3.09(d,J=12.6Hz,1H),3 .01(d,J=16.1Hz,1H),2.46(d,J=16.1Hz,1H),2.42(dt,J=14.6,3.0Hz,1H),2.31(p,J=6.5Hz,2H),2.20(d ,J=1.3Hz,3H),2.12(s,1H),1.94(d,J=1.3Hz,3H),1.89(d,J=1.4Hz,3H),1.87-1.84(m,1H),1.51(s,3H); 13 C NMR(150MHz,Chloroform-d)δ189.4,172.0,167.1,164.6,161.3,152.7,145.9,144.8,142.3,128.8,127.1,114.1,82.1,81.4, 75.9,74.0,71.0,65.9,65.0,53.2,51.8,50.1,46.8,45.5,42.9,41.8,40.9,29.6,28.9,27.8,20.8,15.6,14.6; HR-ESI-MS: m / z calcd forC 33 H 40 N3O 14 [M+H] + 718.2454, found718.2454.

[0053] Example 2: Preparation of compound BO-2:

[0054]

[0055] The preparation method of BO-2 is the same as above, and the structural identification data are as follows:

[0056] BO-2: white solid, 1 H NMR(600MHz,Chloroform-d)δ7.48(d,J=1.1Hz,1H),

[0057] 7.20(d,J=1.1Hz,1H),6.26(s,1H),5.63(s,1H),4.80(s,1H),4.73(m,1 H),4.27(d,J=4.5Hz,1H),4.20(s,1H),3.87(s,2H),3.83-3.74(m,4H),3 .16(s,1H),3.08(m,3H),3.01(d,J=16.0Hz,1H),2.54-2.35(m,2H),2.19 (s,3H),2.11(s,1H),1.94(s,3H),1.89-1.78(m,4H),1.53-1.41(m,3H); 13 C NMR (150MHz, Chloroform-d) δ189.1,172.1,167.0,164.5,161.3,153.6,152.6,145.3,142.3,128.9,128.0,114.1,82.2,81. 4,75.9,74.1,71.1,66.7,65.7,55.8,53.2,51.8,50.2,45.5,42.9,40.9,40.3,29.8,27.8,20.8,15.6,14.5; HR-ESI-MS: m / z calcd for C 32 H 38 N3O 15 [M+H] + 704.2297, found 704.2284.

[0058] Example 3: Preparation of compound BO-3:

[0059]

[0060] The preparation method of BO-3 is the same as above, and the structural identification data are as follows:

[0061] BO-3: white solid, 1H NMR(600MHz,Chloroform-d)δ7.16-7.11(m,2H),6.24(s,1H),5.62(s,1H),4.81(s,1H),4.73(d,J =7.9Hz,1H),4.42(t,J=7.4Hz,2H),4.27-4.18(m,4H),3.82-3.75(m,4H),3.15(s,1H),3.06(d,J= 13Hz,1H),2.97(d,J=16.0Hz,1H),2.49-2.35(m,2H),2.19(d,J=1.3Hz,3H),2.10(s,1H),1.93(d, J=1.3Hz,3H),1.89-1.81(overlap,6H),1.73(q,J=7.0Hz,2H),1.48(s,3H),1.41(q,J=7.0Hz,2H). 13 C NMR (150MHz, Chloroform-d) δ189.2,172.0,167.1,164.6,161.3,153.0,145.3,144.9,142.3,128.5,126.2,114.2,81.8,81.4,75.9,74. 0,71.0,68.8,65.9,53.2,51.6,50.3,50.2,45.5,42.9,41.9,40.9,29.0,28.4,27.8,26.0,25.2,20.8,15.6,14.5; HR-ESI-MS: m / zcalcd for C 35 H 44 N3O 15 [M+H] + 746.2767, found746.2762.

[0062] Example 4: Preparation of compound BO-2:

[0063]

[0064] The preparation method of BO-4 is the same as above, and the structural identification data are as follows:

[0065] BO-4: white solid, 1 H NMR(600MHz,Chloroform-d)δ8.19(d,J=8.6Hz,2H),

[0066] 7.45(d,J=8.6Hz,2H),6.27(s,1H),5.63(s,1H),4.80(s,1H),4.72(d,J=7.9Hz,1H), 4.49-4.43(m,2H),4.26(d,J=3.4Hz,1H),4.20(s,1H),3.82-3.76(m,4H),3.27(s,1H) ,3.17-3.11(m,3H),3.06(d,J=12.7Hz,1H),2.99(d,J=16.0Hz,1H),2.44-2.36(m,2H) ,2.20(d,J=1.3Hz,3H),2.10(s,1H),1.94(d,J=1.3Hz,3H),1.82(m,4H),1.46(s,3H); 13 C NMR(150MHz,Chloroform-d)δ189.1,172.0,167.0,164.6,161.3,152.8,147.2,145.6,145.1,142.2,130.1×2,124.0×2,114.1,82.2, 81.4,75.9,74.0,71.0,68.3,65.9,53.2,51.8,50.1,45.4,42.9,41.8,40.9,34.9,28.9,27.8,20.8,15.5,14.5; HR-ESI-MS: m / zcalcd for C 35 H 39 NO 15 [M+H] + 714.2393, found 714.2396.

[0067] Example 5: Preparation of compound BO-5:

[0068]

[0069] Take a 10mL reaction bottle, dissolve BST (19.88mg, 38.19μmol, 1.2eq) in 1mL dry DCM, add p-nitrophenyl chloroformate (6.41mg, 31.82μmol, 1eq), TEA (5.31μL, 38.19μmol, 2eq) and DMAP (11.66mg, 95.46μmol, 3eq) at 0℃, stir for 30 minutes, add p-nitrobenzyl alcohol (5mg, 31.82μmol, 1eq), and react at 25℃ for 12h. TLC monitors the completion of the reaction, extracts twice with 5mL saturated ammonium chloride aqueous solution, and once with 5mL saturated sodium chloride aqueous solution, collects the organic phase, and removes the solvent under reduced pressure after drying over anhydrous sodium sulfate. Purify by RP-HPLC to obtain BO-5: white solid, the structural identification data are as follows:

[0070] BO-5: white solid, 1 H NMR(600MHz,Chloroform-d)δ8.26(d,J=8.7Hz,2H),

[0071] 7.59(d,J=8.7Hz,2H),6.26(s,1H),5.63(d,J=1.8Hz,1H),5.33(s,2H),4.81(s,1H),4.73(d,J =8.0Hz,1H),4.27(d,J=3.7Hz,1H),4.20(s,1H),3.82-3.76(m,4H),3.15(s,1H),3.08(d,J=12 .9Hz,1H),3.01(d,J=15.9Hz,1H),2.44(d,J=15.9Hz,1H),2.40(dt,J=14.5,3.0Hz,1H),2.20( d,J=1.3Hz,3H),2.11(s,1H),1.94(d,J=1.3Hz,3H),1.88-1.81(m,4H),1.48(d,J=1.1Hz,3H); 13 C NMR(150MHz,Chloroform-d)δ189.1,172.0,167.0,164.6,161.4,152.7,148.1,145.8,142.3,142.1,128.4×2,124.1×2,114.1, 82.1,81.4,75.9,74.0,71.0,68.9,65.8,53.2,51.7,50.1,45.4,42.9,41.8,40.9,28.9,27.8,20.8,15.6,14.6; HR-ESI-MS: m / z calcd for C 34 H 37 NO 15Na[M+Na] + 722.2055, found 722.2050.

[0072] Example 6: Preparation of compound BO-6:

[0073]

[0074] The preparation method of BO-6 is the same as above, and the structural identification data are as follows:

[0075] BO-6: white solid, 1 H NMR(600MHz,Chloroform-d)δ8.27(s,1H),8.21(d,J=8.3Hz,1H),7.76(d,J=7.6Hz,1H),7.59(t, J=7.9Hz,1H),6.24(s,1H),5.62(s,1H),5.32(s,2H),4.80(s,1H),4.72(d,J=7.9Hz,1H),4.26(s, 1H),4.19(s,1H),3.84-3.73(m,4H),3.15(s,1H),3.08(d,J=12.8Hz,1H),3.00(d,J=16.0Hz,1H) ,2.48-2.37(m,2H),2.19(s,3H),2.14-2.09(m,1H),1.93(s,3H),1.89-1.80(m,4H),1.47(s,3H); 13 C NMR(150MHz,Chloroform-d)δ189.0,172.0,167.0,164.6,161.4,152.7,148.5,145.6,142.3,137.0,133.9,129.9,123.7,122.9,11 4.1,82.1,81.4,75.9,74.1,71.0,69.0,65.8,53.2,51.8,50.1,45.5,42.9,41.9,40.9,28.9,27.8,20.8,15.6,14.6; HR-ESI-MS: m / z calcd forC 34 H 37 NO 15 Na[M+Na] + 722.2055, found 722.2045.

[0076] Example 7: Preparation of compound BO-7:

[0077]

[0078] BST (20.0 mg, 38.42 μmol, 1 eq), DMAP (9.39 mg, 76.84 μmol, 2 eq), EDCI (27.6 mg, 211.32 μmol, 5.5 eq) and p-nitrophenylacetic acid (8.35 mg, 46.11 μmol, 1.2 eq) were placed in a 10 mL eggplant-shaped bottle, dissolved in 2 mL of anhydrous DCM, and reacted at 25°C for 12 h. The solvent was evaporated under reduced pressure at 50°C, extracted three times with 5 mL of DCM, washed with 5 mL of saturated ammonium chloride solution and 5 mL of saturated NaCl solution, dried over anhydrous Na2SO4 and concentrated, and purified by high performance liquid chromatography to obtain BO-7: a white solid with the following structural identification data:

[0079] BO-7: white solid, 1 H NMR(600MHz,Chloroform-d)δ8.21(d,J=8.64Hz,

[0080] 2H),7.55(d,J=8.64Hz,2H),6.25(s,1H),5.62(s,1H),4.79(s,1H),4.72(d,J=7 .9Hz,1H),4.25(d,J=4.4Hz,1H),4.19(s,1H),4.03-3.93(m,2H),3.78(s,4H),3. 14(s,1H),3.04(d,J=12.8Hz,1H),2.97(d,J=16.0Hz,1H),2.44-2.30(m,3H),2.1 9(s,3H),2.10(s,1H),1.93(s,3H),1.83-1.78(m,1H),1.73(s,3H),1.48(s,3H); 13 C NMR(150MHz,Chloroform-d)δ188.8,172.1,168.3,167.0,164.6,161.4,147.5,145.7,142.4,140.8,130.6×2,124.0×2,114.1, 82.2,81.4,75.9,74.1,71.0,65.7,53.2,51.8,50.2,45.5,43.0,41.9,40.9,40.3,29.0,27.9,20.8,15.6,14.6; HR-ESI-MS: m / z calcd for C 34 H 38 NO 14 [M+H] + 684.2287, found 684.2282.

[0081] Example 8: Preparation of compound BO-8:

[0082]

[0083] BST (20.0 mg, 38.42 μmol, 1 eq), DMAP (9.39 mg, 76.84 μmol, 2 eq), EDCI (27.6 mg, 211.32 μmol, 5.5 eq) and p-nitrobenzoic acid (7.71 mg, 46.11 μmol, 1.2 eq) were placed in a 10 mL eggplant-shaped bottle, dissolved in 2 mL of anhydrous DCM, and reacted at 25°C for 12 h. The solvent was evaporated under reduced pressure at 50°C, extracted three times with 5 mL of DCM, washed with 5 mL of saturated ammonium chloride solution and 5 mL of saturated NaCl solution, dried over anhydrous Na2SO4 and concentrated, and purified by high performance liquid chromatography to obtain BO-8: a white solid with the following structural identification data:

[0084] BO-8: white solid, 1 H NMR(600MHz,Chloroform-d)δ8.38-8.26(overlap,4H),6.28(s,1H),5.64(s,1H),4.83(s,1H),4.76(d,J=7.9Hz,1H),4.29(d,J=4.5Hz,1H),4.21(s,1H),3.8 5-3.75(m,4H),3.20-3.08(m,2H),3.04(d,J=16.0Hz,1H),2.49-2.42(m,2H),2 .20(s,3H),2.15(s,1H),1.94(d,J=1.3Hz,3H),1.90-1.84(m,4H),1.59(s,3H); 13 C NMR (150MHz, Chloroform-d) δ188.6,172.1,167.3,164.9,164.3,151.1,146.0,142.4,134.3,131.6×2,123.9×2,114. 1,82.2,81.4,76.0,74.1,71.0,66.0,55.5,53.2,50.3,45.5,43.2,42.0,41.0,29.0,27.9,15.7,14.8; HR-ESI-MS:m / z calcd for C 33 H 36 NO 14 [M+H] + 670.2130, found 670.2120.

[0085] Example 9: Preparation of compound BO-9:

[0086]

[0087] BST (20.0 mg, 38.42 μmol, 1 eq), DMAP (9.39 mg, 76.84 μmol, 2 eq), EDCI (27.6 mg, 211.32 μmol, 5.5 eq) and p-nitrobenzoic acid (7.71 mg, 46.11 μmol, 1.2 eq) were placed in a 10 mL eggplant-shaped bottle, dissolved in 2 mL of anhydrous DCM, and reacted at 25°C for 12 h. The solvent was evaporated under reduced pressure at 50°C, extracted three times with 5 mL of DCM, washed with 5 mL of saturated ammonium chloride solution and 5 mL of saturated NaCl solution, dried over anhydrous Na2SO4 and concentrated, and purified by high performance liquid chromatography to obtain BO-9: a white solid with the following structural identification data:

[0088] BO-9: white solid, 1 H NMR(600MHz,Chloroform-d)δ8.96(t,J=1.9Hz,1H),

[0089] 8.59-8.36(m,2H),7.70(t,J=8.0Hz,1H),6.26(s,1H),5.63(s,1H),4.84(s, 1H),4.76(d,J=7.9Hz,1H),4.28(s,1H),4.21(s,1H),3.88-3.71(m,4H),3.3 8(d,J=3.3Hz,1H),3.16(s,1H),3.03(d,J=16.0Hz,1H),2.49(d,J=7.9Hz,1H ),2.43(dt,J=14.6,3.0Hz,1H),2.19(m,4H),1.98-1.85(m,7H),1.67(s,3H); 13 C NMR (150MHz, Chloroform-d) δ189.1,172.1,167.1,164.6,164.2,161.3,145.7,142.6,133.8,130.4×2,128.9,128.7×2,114. 1,82.4,81.4,76.0,74.1,71.0,65.9,53.2,52.2,50.4,45.5,43.1,42.0,41.0,29.1,27.8,20.8,15.7,14.7; HR-ESI-MS: m / z calcd for C 33 H 35 NO 14 Na[M+Na] + 692.1950, found 692.1948.

[0090] Example 10: Preparation of compound BO-10:

[0091]

[0092] BST (20.0 mg, 38.42 μmol, 1 eq), DMAP (9.39 mg, 76.84 μmol, 2 eq), EDCI (27.6 mg, 211.32 μmol, 5.5 eq) and p-nitrobenzoic acid (7.71 mg, 46.11 μmol, 1.2 eq) were placed in a 10 mL eggplant-shaped bottle, dissolved in 2 mL of anhydrous DCM, and reacted at 25°C for 12 h. The solvent was evaporated under reduced pressure at 50°C, extracted three times with 5 mL of DCM, washed with 5 mL of saturated ammonium chloride solution and 5 mL of saturated NaCl solution, dried over anhydrous Na2SO4 and concentrated, and purified by high performance liquid chromatography to obtain BST-6C: a white solid. Take a 10mL eggplant-shaped bottle, put 2-nitroimidazole (1.35mg, 11.95μmol, 1eq) and K2CO3 (3.3mg, 23.89μmol, 2eq) in it, add 2mL of anhydrous acetonitrile to dissolve, stir at 25℃ for 30 minutes, add BST-6C (10mg, 14.34μmol, 1eq), and react at 75℃ for 24 hours. Evaporate the solvent under reduced pressure, extract three times with 5mL DCM, wash with 5mL saturated NaHCO3 solution and 5mL saturated NaCl solution, dry with anhydrous Na2SO4 and concentrate, purify with high performance liquid chromatography to obtain BO-10: white solid, the structural identification data are as follows:

[0093] BO-10: white solid, 1 H NMR(600MHz,Chloroform-d)δ7.14(d,J=1.0Hz,

[0094] 1H),7.12(d,J=1.0Hz,1H),6.27(s,1H),5.63(s,1H),4.80(s,1H),4.74(d,J=7.9Hz,1H),4.44(t,J=7.3Hz,2H), 4.27(s,1H),4.20(s,1H),3.83-3.76(m,4H),3.15(s,1H),3.05(d,J=12.7Hz,1H),2.97(d,J=16.0Hz,1H),2.55(t d,J=16.1,15.2,8.1Hz,2H),2.40(ddd,J=13.8,8.6,5.7Hz,2H),2.32(d,J=8.1Hz,2H),2.19(d,J=1.3Hz,3H),2. 11(s,1H),1.93(d,J=1.3Hz,3H),1.92-1.85(m,2H),1.84(d,J=2.2Hz,1H),1.81-1.74(m,5H),1.54-1.45(m,5H); 13 C NMR(150MHz,Chloroform-d)δ189.2,172.1,171.3,167.1,164.6,161.4,145.4,144.6,142.4,128.5,126.2,114.1,82.3,81.4,76.0, 74.1,71.0,65.6,53.2,51.8,50.3,50.2,45.5,43.0,41.9,40.9,33.2,30.3,29.0,27.9,25.7,24.3,20.8,15.6,14.6; HR-ESI-MS: m / z calcd for C 35 H 43 N3O 14 Na[M+Na] + 752.2637, found 752.2628.

[0095] Example 11: Preparation of compound BO-11:

[0096]

[0097] BST (20.0 mg, 38.42 μmol, 1 eq), K2CO3 (15.93 mg, 115.27 μmol, 3 eq) and catalytic amount of 18-crown ether-6 were placed in a 10 mL eggplant-shaped bottle, 2 mL of anhydrous acetonitrile was added to dissolve, stirred at 25 ° C for 30 minutes, p-nitrobenzyl bromide (9.96 mg, 46.11 μmol, 1.2 eq) was added dropwise, and reacted at 75 ° C for 24 hours. The solvent was evaporated under reduced pressure, extracted three times with 5 mL of DCM, washed with 5 mL of saturated NaHCO3 solution and 5 mL of saturated NaCl solution respectively, dried with anhydrous Na2SO4 and concentrated, and purified by high performance liquid chromatography to obtain BO-11: a white solid with the following structural identification data:

[0098] BO-11: white solid, 1 H NMR(600MHz,Chloroform-d)δ8.22(d,J=8.7Hz,

[0099] 2H),7.60(d,J=8.7Hz,2H),6.27(s,1H),5.63(s,1H),5.04(d,J=12.2Hz,1H),4.91(d, J=12.2Hz,1H),4.78(s,1H),4.71(d,J=8.0Hz,1H),4.27(s,1H),4.20(s,1H),3.79(s, 4H),3.12(s,1H),3.00-2.94(m,2H),2.37(m,2H),2.20(d,J=1.3Hz,3H),2.08(s,1H), 1.93(d,J=1.3Hz,3H),1.85(d,J=1.6Hz,3H),1.78-1.73(m,1H),1.36(d,J=1.1Hz,3H); 13 C NMR (150MHz, Chloroform-d) δ192.4,172.1,167.1,164.6,161.4,147.8,147.7,144.9,144.3,128.7×2,123.8×2,114.1,82. 3,81.5,75.9,74.2,72.3,71.0,65.6,53.2,51.9,51.4,45.4,43.0,42.1,40.7,29.2,27.9,20.8,15.5,14.6; HR-ESI-MS: m / z calcdfor C 33 H 38 NO 13 [M+H] + 656.2338, found 656.2328.

[0100] Example 12: Na2S2O4 reduction experiment of BO-1

[0101] In order to prove that the brucein derivative of the present invention has the potential release effect under hypoxic conditions, the prepared compound BO-1 was subjected to a Na2S2O4 reduction experiment. The specific method and results are as follows:

[0102] 13.9 mg of Na2S2O4 was accurately weighed and dissolved in 4 mL of PBS solution (pH = 7.4) to obtain a Na2S2O4 reduction stock solution.

[0103] Next, take 1 mg of BO-1 and dissolve it in 50 μL of methanol. Then add 450 μL of Na2S2O4 reduction stock solution. Take 20 μL of the mixture, add 180 μL of methanol, and analyze by HPLC to measure the chromatogram at time 0. After the determination is completed, incubate at 37°C. Take samples in the same way at different time points during the reaction to monitor the release of the compound.

[0104] The results showed that the content of compound BO-1 gradually decreased over time, while the content of BST gradually increased.

[0105] Example 13: Nitroreductase assay of BO-1

[0106] In order to prove that the brucein derivative of the present invention has the potential release effect under hypoxic conditions, the prepared compound BO-1 was subjected to a nitroreductase test. The specific method and results are as follows:

[0107] Take 20 μg of nitroreductase and dissolve it in 1 mL of PBS solution (pH=7.4) to obtain a nitroreductase stock solution.

[0108] Next, 1 mg of the target compound was dissolved in 50 μL of DMSO. Then 450 μL of nitroreductase stock solution was added. To quench the reaction and prepare for HPLC analysis, 50 μL of the mixture was sampled, 150 μL of methanol was added, mixed well, and sonicated. Subsequently, 100 μL was accurately measured and analyzed by HPLC, and the chromatogram at time 0 was measured.

[0109] After the determination, 1 μmol of reduced nicotinamide adenine dinucleotide phosphate (NADPH) was added to the samples and incubated at 37°C. Samples were taken in the same way at different time points (10 minutes, 20 minutes, 30 minutes, 1 hour, and 2 hours) during the reaction to monitor the release of the compound ( Figure 2 ).

[0110] The results showed that the content of compound BO-1 gradually decreased over time, while the content of BST gradually increased, which was consistent with the conclusion of Example 12 and had the potential to be a hypoxia-activated prodrug.

[0111] Example 14: Anti-tumor activity screening

[0112] Cell lines: Human hepatoma HepG2, gastric cancer MGC-803, colon cancer HCT116 and human normal liver cells L-02 were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were inoculated in DMEM culture medium containing 10% fetal bovine serum, 10 mg / mL streptomycin, 100 U / mL penicillin and 0.03% glutamine. The normoxic culture environment was a humidified atmosphere of 37°C and 5% carbon dioxide; the hypoxic culture environment was 37°C, and the gas composition included 94% N2, 1% O2 and 5% CO2.

[0113] Instruments: carbon dioxide incubator (Thermo, USA); 96-well culture plate (NestBiotech, USA); microplate reader (BIO-RAD Model 680, Thermo, USA).

[0114] Reagents: Trypsin (Biological Industries, USA); fetal bovine serum (Biological Industries, USA); DMEM medium (HyClon, USA); methylthiazolyl blue (MTT, Sigma, USA); DMSO (Sigma, USA).

[0115] Drugs and their concentration gradients: the concentration gradient of BO-1-11 and brucea javanica (BST) is 10, 1, 0.1, 0.05, 0.01 μM; the concentration gradient of sorafenib (SORA) is 25, 12.5, 6.25, 3.125, 1.1625 μM; the concentration gradient of doxorubicin (DOX) is 1, 0.5, 0.25, 0.125, 0.0625 μM; the concentration gradient of oxaliplatin (OXA) is 50, 25, 12.5, 6.25, 3.125 μM.

[0116] Experimental method: The cytotoxicity of the above drugs on MGC-803, HepG2, HCT116 and L-02 cells was determined by MTT method. 3Cells were seeded at a density of 100 cells / well in a 96-well plate and cultured overnight. Then, cells were treated with different concentrations of the above compounds or dimethyl sulfoxide for 24, 48, and 72 h, and 20 μL of MTT (5 mg / mL) was added to each well and incubated for 4 h. After removing the culture medium, 150 μL of dimethyl sulfoxide was added to the wells, and the results were obtained using a microplate reader (Thermo, Carlsbad, CA, USA) at 490 nm. The experiment was repeated 3 times, and the IC was calculated using GraphPad Prism 6 software. 50 value.

[0117] Calculation formula:

[0118] Cell growth inhibition rate (%) = (A 490(control) -A 490(druggroups )) / (A 490(control) -A 490(blank) )×100%

[0119] The screening results are shown in Table 1.

[0120] (1) Hypoxia selectivity test

[0121] Through screening, the present invention found that p-nitrobenzene and p-nitroimidazole compounds generally have obvious hypoxia selectivity for colon cancer cells, and as the carbon chain is extended, imidazole compounds show hypoxia selectivity for liver cancer cell line HepG2, and its mechanism needs further investigation. However, most of the modified compounds showed hypoxia selectivity and high inhibitory activity for digestive system cancer under hypoxic conditions. The target compound BO-1 showed significant hypoxia selectivity for colon cancer cell HCT116 under hypoxic environment (SI 常氧 / 低氧 =9.62). This means that BO-1 can more effectively inhibit the activity of HCT116 cells under hypoxic conditions ( Figure 3 and Figure 4 ), which may be related to its unique mechanism of action, making it more active in a hypoxic environment or more able to act on the cellular characteristics under hypoxic conditions.

[0122] Table 1: Toxic activity test of the prodrug of the present invention on HepG2, MGC-803, HCT116 and L-02 (72h normoxia and 72h hypoxia)

[0123]

[0124]

[0125] (2) PBMC inhibition rate

[0126] Absorption of plasma layer: Carefully and slowly aspirate the plasma layer and transfer it to a new centrifuge tube. Do not touch the PBMC layer. A small amount of plasma layer can be retained. The plasma layer sample can be used for plasma-related tests later. If you need to obtain plasma with higher purity, it is recommended to only aspirate 2 / 3 of the plasma from top to bottom of the plasma layer. The remaining 1 / 3 of the plasma may contain some PBMC and a small amount of separation fluid components. After centrifugation, it is divided into four layers from top to bottom, namely the plasma layer (containing platelets), the milky white annular layer (ie, the PBMC layer), the transparent separation fluid layer, and the red blood cell / granulocyte layer. After centrifugation, the centrifuge tube must be carefully removed and do not shake or vibrate.

[0127] Experimental method: The cytotoxicity of PBMCs was determined by MTT assay. 3 Cells were inoculated at a density of 1000 μL / well in a 96-well plate and cultured overnight. Then, cells were treated with different concentrations of compounds or dimethyl sulfoxide for 72 h, and 20 μL MTT (5 mg / mL) was added to each well and incubated for 4 h. After removing the culture medium, 150 μL of dimethyl sulfoxide was added to the wells, and the results were obtained using a microplate reader (Thermo, Carlsbad, CA, USA) at 490 nm. The experiment was repeated 3 times, and the inhibition rate was calculated using GraphPad Prism 6 software.

[0128] The data in Table 2 show that when the compound concentration reaches 10 μM, the inhibition rate of BST is 49.15%, which means that nearly half of the normal cell activity is inhibited, showing relatively high cytotoxicity. However, the inhibition rates of compounds BO-1 and BO-5 are about 28%, indicating that their toxicity to normal cells is lower than that of BST, and BO-3 is particularly outstanding, with an inhibition rate of only 5.47%, which is significantly lower than other tested compounds, indicating that it has little effect on normal cells at the current test concentration.

[0129] Table 2: Inhibition rate of BST derivatives on PBMC

[0130]

[0131]

[0132] Example 15: Acute toxicity test in mice

[0133] Animal source: Liaoning Changsheng Biotechnology Co., Ltd., KM male mice

[0134] Laboratory unit use license number: SYXK(Liao)2023-0012

[0135] Experimental operation: Before the test, the mice were marked and weighed, and randomly divided into the experimental group and the control group, with 5 mice in each group. The intraperitoneal injection method (IP) was used. This test was carried out in accordance with the method specified in GB / T 16886.11-2011 "Biological Evaluation of Medical Devices Part 11: Systemic Toxicity Test". The doses given to each mouse were 5mg / kg, 9mg / kg, 15mg / kg, 24mg / kg, and 40mg / kg, respectively.

[0136] Observe all behavioral performances of the mice immediately after injection, and then continue to observe the behavioral performance of the mice and whether there is animal death at 4h, 24h, 48h, and 72h after injection, and record the weight of the animals at 24h, 48h, and 72h. If it is necessary to extend the test period, weigh the animals once a week after the first contact, measure the weight at the end of the test, and observe them every day.

[0137] KM mice were intraperitoneally injected with 5mg / kg, 7mg / kg, 10mg / kg, 14mg / kg, and 18mg / kg at a single dose. For the 10mg / kg and 14mg / kg dose groups, on the 6th day of administration, the mice had more secretions in their eyes, were listless, and had slow movements; on the 14th day, their weight and condition had recovered. The mice were then anatomically observed. The results showed that the 14mg / kg dose group had enlarged lung volume accompanied by dark blood spots, suggesting that target organ toxicity may exist. SPSS software was used to calculate its LD 50 The toxicity is significantly lower than that of BST (LD 50 =5.02mg / kg).

[0138] Table 3: Survival status after intraperitoneal injection of BO-1 (n=6)

[0139]

[0140]

[0141] Although the specific embodiments of the present invention have been described in detail, according to all the teachings disclosed, those skilled in the art can make various modifications and substitutions to the details of the technical solution of the present invention, and these changes are within the protection scope of the present invention. The full protection scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. A brucein derivative or a pharmaceutically acceptable salt thereof having hypoxia-activated prodrug properties, characterized in that: The brucein derivative has a chemical structure of formula (I), (II) or (III), R in formula (I), (II) and (III) is selected from:

2. The brucein derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: R in formula (I) is selected from: R in formula (II) is selected from: and / or R in formula (III) is selected from:

3. The brucein derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The brucein derivative or a pharmaceutically acceptable salt thereof is selected from the following:

4. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the brucein derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, and a pharmaceutically acceptable carrier.

5. The pharmaceutical composition according to claim 4, characterized in that: The dosage form of the pharmaceutical composition is an oral dosage form or an injection dosage form, the oral dosage form is a capsule, a tablet, a granule, an oral liquid, a sustained-release preparation or a controlled-release preparation, and the pharmaceutically acceptable carrier is selected from one or more of a diluent, a lubricant, a binder, a disintegrant, a stabilizer or a solvent.

6. The method for preparing the brucein derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: Using brucea stearoyl alcohol (BST) as the starting material, compound BO-1-6 is prepared through step a, compound BO-7-9 is prepared through step b, compound BO-10 is prepared through steps c and d, and compound BO-11 is prepared through step e.

7. The preparation method according to claim 6, characterized in that: The preparation method comprises the following steps: (1) Synthesis of BO-1-6: Dissolve the corresponding hydroxy compound in a solvent, add 1 times the amount of coupling agent, 1 times the amount of organic base and 3 times the amount of acid binding agent, stir for 30 minutes, add 1.2 times the amount of brucea stearoyl alcohol (BST), and react at 25°C. Remove the solvent, extract, wash, dry, filter, and purify; (2) Synthesis of BO-7-9 and BST-6C: Dissolve BST, condensing agent and acid binding agent in a solvent, stir at room temperature, add the corresponding carboxylic acid fragment, react at room temperature, and perform post-treatment in the same manner as step (1); (3) Synthesis of BO-10: Dissolve BST-6C and an inorganic base in a solvent, stir at room temperature, add p-nitrobenzyl bromide, react at room temperature, and perform post-treatment in the same manner as step (2); (4) Synthesis of BO-11: Dissolve BST and an inorganic base in a solvent, stir at room temperature, add p-nitrobenzyl bromide, react at room temperature, and perform post-treatment in the same manner as step (2).

8. The preparation method according to claim 7, characterized in that: The solvent is anhydrous acetonitrile, anhydrous acetone, N,N-dimethylformamide, tetrahydrofuran or dichloromethane; The coupling agent is p-nitrophenyl chloroformate, carbonyldiimidazole, carbonyltriazole, phosgene or triphosgene; The condensing agent is dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI); The acid binding agent is N,N-diisopropylethylamine, pyridine or 4-dimethylaminopyridine; The organic base is triethylamine, N,N-diisopropylethylamine, pyridine, 4-dimethylaminopyridine or N-methylmorpholine; The inorganic base is potassium carbonate, cesium carbonate, sodium carbonate, potassium hydroxide, sodium hydroxide, sodium hydride, potassium methoxide, sodium methoxide, sodium tert-butoxide or potassium tert-butoxide.

9. Use of the brucea javanica derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 4 or claim 5 in the preparation of a drug targeting digestive system tumors.

10. The use according to claim 9, characterized in that: The digestive system tumor is gastric cancer, colon cancer or liver cancer.

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