Hexokinase inhibitor, hexokinase inhibitor ruthenium complex and use thereof in preparing antitumor drugs

Abstract

The present invention provides a hexokinase inhibitor, a hexokinase inhibitor ruthenium complex and its application in the preparation of anti-tumor drugs, belonging to the technical field of anti-tumor drugs. The present invention uses 2-deoxy-d-ribose as a parent nucleus to replace the 2-deoxy-D-glucose structure for modification. The present invention adopts deoxyribose and amino acids, which are endogenous substances in the human body, to construct a hexokinase inhibitor with a structure shown in Formula I with low drug toxicity and high anti-tumor biological activity. At the same time, the present invention introduces the concept of ruthenium complex on the basis of the hexokinase inhibitor with a structure shown in Formula I, and the obtained hexokinase inhibitor ruthenium complex can undergo molecular self-assembly in an aqueous solution to form self-assembled nanoparticles, thereby improving the targeting of tumors. At the same time, the hexokinase inhibitor ruthenium complex can act on the aerobic respiration and glycolysis pathways of tumor cells, effectively inhibit the energy metabolism pathways of tumor cells, and further enhance the anti-tumor activity.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-tumor drugs, and in particular to a hexokinase inhibitor, a hexokinase inhibitor ruthenium complex and applications in the preparation of anti-tumor drugs. Background Art

[0002] Cancer is a disease caused by the loss of normal cellular regulation and excessive proliferation. The "Warburg effect" explains that cancer cells, whether under hypoxic or aerobic conditions, do not utilize mitochondrial oxidative phosphorylation to generate energy. Instead, they rely on aerobic glycolysis, generating a large number of glycolytic intermediates for biosynthesis. Furthermore, cancer cells generate large amounts of ATP through high-rate glycolysis, which fuels their rapid proliferation. Therefore, targeted inhibition of cancer cell glucose metabolism is of great significance for cancer treatment.

[0003] Hexokinase, a key rate-limiting enzyme in glucose metabolism, plays a vital role in metabolic processes. Hexokinase is divided into five isoforms: HK1, HK2, HK3, HK4, and the hexokinase domain-containing protein HKDC1. HK1, HK2, and HKDC1 are located on the outer mitochondrial membrane and bind to the mitochondrial voltage-dependent anion channel (VDAC), thereby exerting their physiological functions. HK2 is highly expressed in gastric cancer, lymphoma, esophageal cancer, and central nervous system cancers, and high HK2 expression is associated with poor cancer prognosis. 50 Studies have also demonstrated that HK2 gene deletion significantly inhibits cancer cell proliferation in vivo, indicating that HK2 plays a crucial role in tumor development and progression. In addition to glycolysis, HK2 also inhibits the opening of the mitochondrial permeability transition pore, helping to maintain mitochondrial membrane potential and prevent excessive ROS production. HK2 inhibition or deficiency can promote mitochondrial ROS production and reduce mitochondrial membrane potential, prompting cell apoptosis.

[0004] 2-Deoxy-D-glucose (2-DG) is a hexokinase inhibitor widely used to study glucose metabolism and related diseases, such as cancer and diabetes. 2-DG competes with glucose for binding to the active site of hexokinase, leading to a blockage of intracellular energy metabolism. 2-DG competitively inhibits glucose transport and phosphorylation via hexokinase, forming 2-DG-6-phosphate, which reduces the production of glucose-6-phosphate and, consequently, glycolysis and the pentose phosphate pathway. Because 2-DG effectively inhibits glycolysis and glucose metabolism in cancer cells, it is widely used to interfere with tumor metabolism and is being studied as a therapeutic approach in clinical trials. However, 2-DG must be used at relatively high concentrations to compete with glucose, and its short half-life limits its use as a monotherapy in the clinic.

[0005] Currently, it is clinically believed that combining 2-DG with other chemotherapy drugs can maximize its effectiveness, potentially offering a therapeutic approach that simultaneously targets both hypoxic and aerobic cells in tumors. Clinical trial results have shown that 2-DG combined with paclitaxel can delay tumor growth compared to docetaxel alone, and that paclitaxel enhances 2-DG uptake by cancer cells. In addition to its anti-tumor effects, 2-DG also has therapeutic potential for inflammation, viral infections, epilepsy, and immune system disorders. While 2-DG has potential clinical applications, its inherent characteristics limit its effectiveness. Therefore, the design and development of 2-DG analogs to improve its pharmacokinetics and enhance its anti-tumor activity are of great significance.

[0006] Summary of the Invention

[0007] In view of this, the object of the present invention is to provide a hexokinase inhibitor, a hexokinase inhibitor ruthenium complex and use thereof in the preparation of anti-tumor drugs. The hexokinase inhibitor and the hexokinase inhibitor ruthenium complex provided by the present invention have low drug toxicity and good anti-tumor biological activity.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a hexokinase inhibitor having the structure shown in Formula I:

[0010]

[0011] In Formula I, R is One of them.

[0012] The present invention provides a method for preparing the above-mentioned hexokinase inhibitor, comprising the following steps:

[0013] Under weak alkaline conditions, 2-deoxy-D-ribose having the structure shown in Formula 1 and amino acid benzyl ester having the structure shown in Formula 2 undergo a condensation reaction to obtain a compound having the structure shown in Formula 3;

[0014]

[0015] The compound having the structure shown in Formula 3 is subjected to a reduction reaction with a reducing agent to obtain a compound having the structure shown in Formula 4;

[0016]

[0017] Under the action of a catalyst, the compound having the structure shown in Formula 4 undergoes a deprotection reaction with H2 to obtain a hexokinase inhibitor having the structure shown in Formula I.

[0018] Preferably, the pH value of the weakly alkaline environment is 9;

[0019] The condensation reaction is carried out in an ice bath, and the condensation reaction time is 12 to 28 hours.

[0020] Preferably, the molar ratio of the compound having the structure shown in Formula 3 to the reducing agent is 1:1.2-3.6;

[0021] The reduction reaction is carried out in an ice bath, and the reduction reaction time is 24 to 50 hours.

[0022] Preferably, the catalyst used in the deprotection reaction is a palladium-carbon catalyst;

[0023] The temperature of the deprotection reaction is 18-28° C., and the time is 2-6 hours.

[0024] The present invention provides a hexokinase inhibitor ruthenium complex having the structure shown in Formula II:

[0025]

[0026] In formula I, R is One of them.

[0027] Preferably, the particle size of the hexokinase inhibitor ruthenium complex is 20 to 120 nm.

[0028] The present invention provides a method for preparing the above-mentioned hexokinase inhibitor ruthenium complex, comprising the following steps:

[0029] The hexokinase inhibitor is subjected to a complex reaction with bis(2,2-bipyridine)ruthenium dichloride to obtain a hexokinase inhibitor ruthenium complex having a structure shown in Formula II.

[0030] The present invention provides the use of the hexokinase inhibitor or the hexokinase inhibitor ruthenium complex in the preparation of anti-tumor drugs.

[0031] Preferably, the anti-tumor drug is one or more of an anti-breast cancer drug, an anti-lymphoma drug, an anti-leukemia drug, an anti-colon cancer drug, and an anti-lung cancer drug.

[0032] The present invention provides a hexokinase inhibitor having a structure shown in Formula I. 2-Deoxy-D-ribose is a pentose derivative naturally present in all cells and is a component of polynucleotide deoxyribose and acid. 2-Deoxy-D-ribose is a reducing sugar present in the body and is used to study oxidative stress in vivo and in vitro, inducing cell apoptosis by inhibiting the synthesis of glutathione and increasing its excretion. Because its structure is similar to 2-deoxy-D-glucose, the present invention uses 2-deoxy-D-ribose as the parent nucleus to replace the 2-deoxy-D-glucose structure for modification. Amino acids are the basic building blocks of biological functional macromolecular proteins and play an important role in the life activities of the body. The present invention uses deoxyribose and amino acids, which are endogenous substances of the human body, to construct a hexokinase inhibitor with low drug toxicity and high anti-tumor biological activity.

[0033] The present invention provides a ruthenium complex of a hexokinase inhibitor having the structure represented by Formula II. Based on the hexokinase inhibitor having the structure represented by Formula I, the present invention introduces the concept of a ruthenium complex. The resulting hexokinase inhibitor ruthenium complex can undergo molecular self-assembly in aqueous solution to form self-assembled nanoparticles, improving tumor targeting. Furthermore, the hexokinase inhibitor ruthenium complex can act on the aerobic respiration and glycolysis pathways of tumor cells, effectively inhibiting their energy metabolism and further enhancing their anti-tumor activity.

[0034] The present invention provides methods for preparing the aforementioned hexokinase inhibitor and hexokinase inhibitor ruthenium complex. The method uses 2-deoxy-D-ribose as a raw material, reacts it with three amino acid benzyl esters under weakly alkaline conditions, condenses the amino groups of the amino acids with the aldehyde groups of the deoxyribose, reduces it with potassium borohydride, and deprotects it to obtain the hexokinase inhibitor. The method also complexes the hexokinase inhibitor with bis(2,2-bipyridine)ruthenium dichloride (II) to obtain the hexokinase inhibitor ruthenium complex. The preparation method provided by the present invention has a short synthetic route, is simple to operate, is low-cost, and is easily scalable for industrial mass production. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 A synthetic route for hexokinase inhibitors;

[0036] Figure 2 A synthetic route for ruthenium complexes of hexokinase inhibitors;

[0037] Figure 3 is the nanoparticle size of compounds 5a and 6a;

[0038] Figure 4 Transmission electron microscopy images of compounds 5a and 6a;

[0039] Figure 5 The pro-apoptotic effects of different compounds on THP-1;

[0040] Figure 6 This is a statistical diagram of apoptosis of THP-1 cells;

[0041] Figure 7 The effect of different compounds on THP-1 mitochondrial membrane potential;

[0042] Figure 8 This is a statistical diagram of the effects of different compounds on mitochondrial membrane potential;

[0043] Figure 9 The effects of different compounds on intracellular G6P;

[0044] Figure 10 The effects of different compounds on intracellular ATP;

[0045] Figure 11 The effect of different compounds on the apoptotic protein Bax;

[0046] Figure 12 Statistical diagram of the effects of different compounds on Bax;

[0047] Figure 13 The effects of different compounds on HK2 protein;

[0048] Figure 14 This is a statistical graph showing the effects of different compounds on HK2 protein;

[0049] Figure 15 AnnexinV-FITC / PI cell apoptosis images;

[0050] Figure 16 AnnexinV-FITC / PI cell apoptosis statistics;

[0051] Figure 17 is the effect of the compound on intracellular G6P;

[0052] Figure 18 is the effect of the compound on intracellular ATP;

[0053] Figure 19 The effects of different compounds on 4T1 cell migration;

[0054] Figure 20 Statistical graph showing the effects of different compounds on 4T1 cell migration;

[0055] Figure 21 To observe the changes of mouse tumors for in vivo imaging;

[0056] Figure 22 This is a picture of a tumor in a 4T1 tumor-bearing mouse;

[0057] Figure 23This is a statistical chart of mouse tumor weight;

[0058] Figure 24 Changes in tumor volume in mice after 14 days of drug administration;

[0059] Figure 25 The changes in the body weight of mice within 14 days of administration;

[0060] Figure 26 is the weight change of mouse organs;

[0061] Figure 27 These are pictures of lung tissues from different groups;

[0062] Figure 28 The effect on metastatic nodules after drug administration;

[0063] Figure 29 is the expression of hexokinase in tumor tissue;

[0064] Figure 30 is the expression of lactate in tumor tissue;

[0065] Figure 31 is the expression of G6P in tumor tissue;

[0066] Figure 32 This is the HE staining result of tumor tissue sections;

[0067] Figure 33 HE staining of normal mouse organ sections;

[0068] Figure 34 This is the TUNEL staining picture of tumor tissue;

[0069] Figure 35 The changes of TUNEL-positive mean fluorescence intensity by different compounds;

[0070] Figure 36 for the uptake of ruthenium complexes in tumors and other organs;

[0071] Figure 37 is the mass spectrum of compound 5a;

[0072] Figure 38 Compound 5a 1 H NMR spectrum;

[0073] Figure 39 is the mass spectrum of compound 5b;

[0074] Figure 40 For compound 5b 1 H NMR spectrum;

[0075] Figure 41 is the mass spectrum of compound 5c;

[0076] Figure 42 For compound 5c 1 H NMR spectrum;

[0077] Figure 43 is the mass spectrum of compound 6a;

[0078] Figure 44 For compound 6a 1 H NMR spectrum;

[0079] Figure 45 is the mass spectrum of compound 6b;

[0080] Figure 46 For compound 6b 1 H NMR spectrum;

[0081] Figure 47 is the mass spectrum of compound 6c;

[0082] Figure 48 For compound 6c 1 H NMR spectrum;

[0083] Figure 49 is the infrared spectra of compounds 5a and 6a;

[0084] Figure 50 is the infrared spectra of compounds 5b and 6b;

[0085] Figure 51 IR spectra of compounds 5c and 6c. DETAILED DESCRIPTION

[0086] The present invention provides a hexokinase inhibitor having the structure shown in Formula I:

[0087]

[0088] In formula I, R is One of the following, where the dotted line indicates the attachment site.

[0089] In the present invention, the hexokinase inhibitor has a structure shown in any one of Formulas 5a to 5c:

[0090]

[0091] In the present invention, the preparation method of the hexokinase inhibitor comprises the following steps:

[0092] Under weak alkaline conditions, 2-deoxy-D-ribose having the structure shown in Formula 1 and amino acid benzyl ester having the structure shown in Formula 2 undergo a condensation reaction to obtain a compound having the structure shown in Formula 3;

[0093]

[0094] The compound having the structure shown in Formula 3 is subjected to a reduction reaction with a reducing agent to obtain a compound having the structure shown in Formula 4;

[0095]

[0096] Under the action of a catalyst, the compound having the structure shown in Formula 4 undergoes a deprotection reaction with H2 to obtain a hexokinase inhibitor having the structure shown in Formula I.

[0097] In the present invention, under weakly alkaline conditions, 2-deoxy-D-ribose having the structure represented by Formula 1 and an amino acid benzyl ester having the structure represented by Formula 2 undergo a condensation reaction to obtain a compound having the structure represented by Formula 3. In the present invention, the pH value of the weakly alkaline environment is preferably 9, and the alkaline reagent providing the weakly alkaline environment is preferably one or more of N-methylmorpholine, Na2CO3, and NaHCO3.

[0098] In the present invention, the molar ratio of 2-deoxy-D-ribose having the structure represented by Formula 1 to the amino acid benzyl ester having the structure represented by Formula 2 is preferably 1:0.8 to 1.5, more preferably 1:1. In the present invention, the amino acid benzyl ester is preferably tryptophan benzyl ester, phenylalanine benzyl ester, or tyrosine benzyl ester.

[0099] In the present invention, the condensation reaction is preferably carried out in an organic solvent, and the organic solvent is preferably one or more of methanol, ethanol and acetonitrile.

[0100] In the present invention, the condensation reaction is preferably carried out under stirring conditions; the condensation reaction is preferably carried out under ice bath conditions, and the time is preferably 12 to 28 hours, more preferably 24 hours.

[0101] After the condensation reaction, the present invention preferably does not perform post-treatment, and directly uses the obtained condensation reaction liquid as a raw material for subsequent reactions.

[0102] After obtaining the compound having the structure shown in Formula 3, the compound having the structure shown in Formula 3 is subjected to a reduction reaction with a reducing agent to obtain a compound having the structure shown in Formula 4. In the present invention, the reducing agent is preferably one or more of potassium borohydride, sodium borohydride and lithium aluminum hydride, more preferably potassium borohydride. In the present invention, the molar ratio of the compound having the structure shown in Formula 3 to the reducing agent is preferably 1:1.2 to 3.6, more preferably 1:3. In the present invention, the reducing agent is preferably added to the reaction solution in small amounts and multiple times to avoid the generation of a large amount of hydrogen.

[0103] In the present invention, the reduction reaction is preferably carried out in an ice bath; the reduction reaction time is preferably 24 to 50 hours, more preferably 48 hours.

[0104] After the reduction reaction, the present invention preferably performs post-treatment on the obtained reduction reaction solution, and the post-treatment preferably includes the following steps:

[0105] The pH value of the reduction reaction liquid is adjusted to neutral, solid-liquid separation is performed, the organic solvent of the obtained liquid phase is removed, the obtained liquid phase is redissolved in pure water, and the reactant dissolved in the obtained pure water is purified by cation exchange resin.

[0106] In the present invention, the reagent for adjusting the pH value to neutral is preferably a 5wt% potassium hydrogen sulfate solution. In the present invention, the solid-liquid separation is preferably vacuum filtration; and the method for removing the organic solvent is preferably rotary evaporation.

[0107] In the present invention, the cation exchange resin is preferably 732 cation exchange resin. In the present invention, the method for purifying the cation exchange resin preferably comprises the following steps:

[0108] Soak 732 cation exchange resin in a 10% NaCl solution for 24 hours. Rinse with clean water until the wash water is colorless. Place the resin into the column using the wet packing method. Slowly flow 1 mol / L dilute hydrochloric acid solution through the resin, using a dosage of 2-3 times the resin volume, at a drip rate of 1 d / s. After acid immersion, soak for at least one hour. Then rinse with pure water until the effluent pH is 5-6. After acid treatment, flow 1 mol / L NaOH solution through the resin, using the same dosage and flow rate as for the acid treatment. After the alkali solution is added, soak for at least one hour. Rinse with water until the pH is around 8. After the alkali treatment, treat with 1 mol / L dilute hydrochloric acid solution again, converting the resin to H-form, using a dosage of 3-5 times the resin volume. After the addition of the solution, rinse with pure water until the effluent pH is around 6-7 before use.

[0109] Add the reactant solution dissolved in pure water to the resin column and slowly inject the sample. After the sample solution is completely injected, rinse with plenty of pure water to remove any unreacted 2-deoxy-D-ribose. Then, slowly elute the target product with 1 mol / L dilute hydrochloric acid. After thorough elution and collection, rinse with pure water until the effluent pH is 5-6. Add 1 mol / L NaOH solution to elute any unreacted amino acids. After thorough elution, rinse the cationic resin column to regenerate it for the next use. Concentrate the collected target product eluate by rotary evaporation and lyophilize.

[0110] After obtaining the compound having the structure represented by Formula 4, the compound having the structure represented by Formula 4 is subjected to a deprotection reaction with H2 in the presence of a catalyst to obtain a hexokinase inhibitor having the structure represented by Formula I. In the present invention, the catalyst is preferably a palladium-carbon catalyst; the mass of the palladium-carbon catalyst is preferably 10 to 25% of the mass of the compound having the structure represented by Formula 4, and more preferably 15 to 20%.

[0111] In the present invention, the deprotection reaction is preferably carried out in an organic solvent, and the organic solvent is preferably methanol.

[0112] In the present invention, the temperature of the deprotection reaction is preferably 18 to 28° C., more preferably 20 to 25° C.; the time is preferably 2 to 6 hours, more preferably 5 hours.

[0113] After the deprotection reaction, the present invention preferably performs post-treatment on the obtained deprotection reaction solution, and the post-treatment preferably includes the following steps:

[0114] The obtained deprotection reaction liquid is subjected to solid-liquid separation, the obtained liquid phase is subjected to rotary evaporation to remove the organic solvent, and the residual phase after rotary evaporation is recrystallized to obtain a pure hexokinase inhibitor having the structure shown in Formula I.

[0115] In the present invention, the solid-liquid separation method is preferably suction filtration. In the present invention, the recrystallization preferably includes: using ethanol to dissolve the residual phase after rotary evaporation, and adding ethyl acetate to precipitate the target product.

[0116] In the present invention, the hexokinase inhibitor having the structure shown in Formula I and the synthetic route are as follows: Figure 1 shown. Figure 1 Medium, i.MeOH / NMM; ii.KBH4; iii.H2, Pd / C.

[0117] The present invention provides a hexokinase inhibitor ruthenium complex having the structure shown in Formula II:

[0118]

[0119] In formula I, R is One of them.

[0120] In the present invention, the hexokinase inhibitor ruthenium complex has a structure shown in any one of Formulas 6a to 6c:

[0121]

[0122] In the present invention, the particle size of the hexokinase inhibitor ruthenium complex is preferably 20 to 120 nm, more preferably 50 to 100 nm.

[0123] The present invention provides a method for preparing the above-mentioned hexokinase inhibitor ruthenium complex, comprising the following steps:

[0124] The hexokinase inhibitor is subjected to a complex reaction with bis(2,2-bipyridine)ruthenium dichloride to obtain a hexokinase inhibitor ruthenium complex having a structure shown in Formula II.

[0125] In the present invention, the molar ratio of the hexokinase inhibitor to bis(2,2-bipyridine)ruthenium dichloride is preferably 1:0.5-2, more preferably 1:1.

[0126] In the present invention, the complexation reaction is preferably carried out in a solvent, and the solvent is preferably a mixture of water and ethanol; the volume ratio of water to ethanol in the mixture is preferably 1:0.5-2, more preferably 1:1-1.5.

[0127] In the present invention, the complexation reaction is preferably carried out under stirring, the temperature of the complexation reaction is preferably 15 to 28° C., more preferably 22° C. or room temperature, and the time is preferably 5 hours.

[0128] After the complexation reaction, the present invention preferably freeze-dries the obtained complexation reaction product for storage.

[0129] The present invention provides the use of the hexokinase inhibitor or the hexokinase inhibitor ruthenium complex in the preparation of anti-tumor drugs.

[0130] In the present invention, the anti-tumor drug is preferably one or more of an anti-breast cancer drug, an anti-lymphoma drug, an anti-leukemia drug, an anti-colon cancer drug, and an anti-lung cancer drug. In the present invention, the leukemia preferably includes human monocytic leukemia or human myeloid leukemia.

[0131] The hexokinase inhibitor, the hexokinase inhibitor ruthenium complex and the application in the preparation of anti-tumor drugs provided by the present invention are described in detail below with reference to the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0132] In the following examples, 2-deoxy-D-glucose and 2-deoxy-D-ribose were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and 732 cation exchange resin was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0133] Example 1 Synthesis of Compound 5a

[0134] (1) Synthesis: Weigh 5 mmol of 2-deoxy-D-ribose and tryptophan benzyl ester Trp-OBzl respectively, dissolve them in an appropriate amount of anhydrous methanol, react on a magnetic stirrer, adjust the pH of the reaction solution to 9 with N-methylmorpholine, and react in an ice bath for 24 hours. After sufficient reaction, add 3 times the amount of potassium borohydride (KBH4) to the reaction solution in small amounts and multiple times to avoid the generation of large amounts of hydrogen gas, and react in an ice bath for 48 hours. After sufficient reaction, add a small amount of 5% potassium bisulfate solution to neutralize the pH of the reaction solution to neutral, terminate the reaction, remove the white precipitate by vacuum filtration, remove the solvent by rotary evaporation, and redissolve with deionized water.

[0135] (2) Purification of intermediate products:

[0136] Weigh 200g of 732 cation exchange resin and soak it in a 10% NaCl solution for 24 hours. Rinse with clean water until the wash water is colorless. Place the resin into the column using the wet packing method. Slowly flow 1 mol / L dilute hydrochloric acid solution through the resin, using a dosage of 2-3 times the resin volume, at a drip rate of 1 d / s. After acid immersion, soak for at least one hour. Then rinse with pure water until the effluent pH is approximately 5-6. After acid treatment, flow 1 mol / L NaOH solution through the resin, using the same dosage and flow rate as for the acid treatment. After the alkali solution is added, soak for at least one hour. Rinse with water until the pH is approximately 8. After the alkali treatment, treat with 1 mol / L dilute hydrochloric acid solution again, using a dosage of 3-5 times the resin volume, to convert the resin to the H-type. After the addition of the solution, rinse with pure water until the effluent pH is approximately 6-7. It is then ready for use.

[0137] Add the reactant solution dissolved in pure water to the resin column and slowly inject the sample. After the sample solution has been completely injected, rinse with plenty of pure water to remove any unreacted 2-deoxy-D-ribose. Then, slowly elute the target product with 1 mol / L dilute hydrochloric acid. After thorough elution and collection, rinse with pure water until the effluent pH is approximately 5-6. Add 1 mol / L NaOH solution to elute any unreacted amino acids. After thorough elution, rinse the cationic resin column to regenerate it for the next use. Concentrate the collected target product eluate by rotary evaporation and lyophilize.

[0138] (3) Product Deprotection: Weigh 12% palladium on carbon and add it to the sample solution dissolved in anhydrous methanol. Under the reaction of H2, remove the benzyl ester protecting group. React for 5 hours, remove the palladium on carbon by suction, and remove the solvent by rotary evaporation.

[0139] (4) Recrystallization: Dissolve the sample in a small amount of ethanol and precipitate the target product with a large amount of ethyl acetate. Collect the product by vacuum filtration, weigh, and record. 731.8 mg (yield 45.3%) of a yellow-brown solid was obtained. ESI-MS: [M+H] - 323.1, 1HNMR (300MHz, DMSO-d6): δ (ppm) = 11.06-10.93 (m, 1H), 8.45 (s, 1H), 7.59 (dd, J = 7.8, 3 .0Hz,1H),7.33(d,J=8.0Hz,1H),7.23(d,J=2.5Hz,1H),7.03(dt,J=20.9,13.4,7.0Hz ,3H),4.05(d,J=7.1Hz,1H),3.35(d,J=5.7Hz,2H),3.26-3.20(m,2H),3.09(d,J=7.2H z,2H),2.90-2.73(m,2H),1.99(s,1H),1.84-1.53(m,2H),1.17(t,J=7.1Hz,1H).IR(cm -1 ):3248.36cm -1 ,2926.79cm -1 ,1588.11cm -1 ,1455.99cm -1 ,1392.98cm -1 ,1351.83cm -1 ,1072.88cm -1 ,742.66cm -1 .Mp:137.9-138.3℃.

[0140] Example 2 Synthesis of Compound 5b

[0141] 5 mmol of each of 2-deoxy-D-ribose and phenylalanine benzyl ester (Phe-OBzl) was weighed and dissolved in an appropriate amount of anhydrous methanol. The reaction solution was adjusted to pH 9 with N-methylmorpholine and allowed to react in an ice bath for 24 hours. After sufficient reaction, potassium borohydride (KBH4) was added in three equal amounts, in small, multiple additions to the reaction solution to avoid the generation of large amounts of hydrogen gas. The reaction was allowed to react in an ice bath for 48 hours. After sufficient reaction, a small amount of 5% potassium bisulfate solution was added to neutralize the pH of the reaction solution to terminate the reaction. The white precipitate was removed by filtration under reduced pressure, and the solvent was removed by rotary evaporation. After redissolving in deionized water, the reaction product was purified, deprotected, and recrystallized according to the post-processing method described in Example 1 to obtain 852.3 mg (yield 60.0%) of a yellow-brown solid. ESI-MS: [M+1] - =284.1, 1HNMR (300MHz, DMSO-d6): δ (ppm) = 7.32-7.10 (m, 5H), 3.47 (dd, J = 10.9, 4.0Hz, 2H), 3.37-3.33 (m, 2H), 3.30 (d, J = 6.0Hz, 1H), 3.2 8-3.14(m,2H),3.04(t,J=7.0Hz,1H),2.86(dd,J=18.9,13.7,6.9Hz,2H),2.73(dd,J=12.2,6.9Hz,1H),1.90-1.44(m,2H).IR(cm -1 ):3295.67cm -1 ,2921.69cm -1 ,2866.75cm -1 ,1581.90cm -1 ,1494.12cm -1 ,1453.56cm -1 ,1386.20cm -1 ,1074.89cm -1 ,697.48cm -1 ,Mp:199.5-199.9℃.

[0142] Example 3 Synthesis of Compound 5c

[0143] 5 mmol of each of 2-deoxy-D-ribose and tyrosine benzyl ester Tyr-OBzl was weighed and dissolved in an appropriate amount of anhydrous methanol. The reaction solution was adjusted to pH 9 with N-methylmorpholine and allowed to react in an ice bath for 24 hours. After sufficient reaction, potassium borohydride (KBH4) was added in three equal amounts, in small, multiple additions to the reaction solution to avoid the generation of large amounts of hydrogen gas. The reaction was allowed to react in an ice bath for 48 hours. After sufficient reaction, a small amount of 5% potassium bisulfate solution was added to neutralize the pH of the reaction solution to terminate the reaction. The white precipitate was removed by filtration under reduced pressure, and the solvent was removed by rotary evaporation. After redissolving in deionized water, the reaction product was purified, deprotected, and recrystallized according to the post-processing method in Example 1 to obtain 817.5 mg (54.5% yield) of a light yellow solid. ESI-MS: [M+1] - =300.4, 1HNMR (300MHz, DMSO-d6): δ (ppm) = 7.04 (d, J = 8.0Hz, 2H), 6.81-6.52 (m, 2H), 3.47-3.40 (m, 3H), 3.36-3.26 (m, 3H), 3. 16(d,J=4.4Hz,2H),2.97(dd,J=14.1,5.4Hz,2H),2.87-2.79(m,2H),1.89-1.46(m,2H),1.06(t,J=7.0Hz,1H).IR(cm -1 ):3174.07cm -1 ,2928.14cm -1 ,1586.11cm -1 ,1515.66cm -1 ,1437.06cm -1 ,1381.53cm -1 ,1323.83cm -1 ,1248.94cm -1 ,1176.34cm -1 ,1105.99cm -1 ,1077.18cm -1 ,827.84cm -1 ,Mp:145.1-145.5℃.

[0144] Example 4 Synthesis of Compound 6a

[0145] 0.2 mmol of compound 5a and 0.2 mmol of bis(2,2-bipyridine)ruthenium dichloride (II) were weighed and stirred in a pure water / ethanol mixture for 5 hours. The mixture was lyophilized to obtain 135.6 mg (92.1% yield) of a purple-brown solid. ESI-MS: [M+H] - =736.2, 1 HNMR (300MHz, DMSO-d6): δ (ppm) = 8.90 (d, J = 8.1Hz, 4H), 8.18 (td, J = 7.9, 1.5Hz, 4H), 7.74 (dd, J = 5.6, 1.4Hz, 4H), 7.63-7.49 (m, 5H), 7.11-7.01 (m, 1 H),6.96(t,J=7.5Hz,1H),3.59-3.48(m,6H),3.48-3.40(m,5H),3.30-3.1 1(m,4H),3.09(s,1H),2.88(d,J=7.4Hz,1H),1.05(t,J=7.0Hz,1H).IR(cm -1 ):3324.92cm -1 ,3068.13cm-1 ,1622.32cm -1 ,1458.45cm -1 ,1442.78cm -1 ,1417.42cm -1 ,1262.17cm -1 ,1017.64cm -1 ,761.67cm -1 ,726.32cm -1 ,Mp:212.4-212.8℃.

[0146] Example 5 Synthesis of Compound 6b

[0147] 0.2 mmol of compound 5b and 0.2 mmol of bis(2,2-bipyridine)ruthenium dichloride (II) were weighed and stirred in a pure water / ethanol mixture for 5 hours. The mixture was lyophilized to obtain 125.3 mg (90.0% yield) of a purple-brown solid. ESI-MS: [M+H] - =696.2, 1 HNMR (300MHz, DMSO-d6): δ (ppm) = 8.88 (d, J = 8.2Hz, 4H), 8.18 (td, J = 7.8, 1.5Hz, 4H ),7.74(dd,J=5.6,1.4Hz,4H),7.54(dd,J=7.6,6.1Hz,4H),7.32-7.14(m,2H),3.3 4(t,J=5.4Hz,4H),3.30(s,2H),3.25-3.17(m,2H),3.17-3.03(m,2H),2.96(dd,J= 14.2,6.7Hz,1H),2.83(q,J=6.7,6.2Hz,1H),2.35(s,1H),1.94-1.51(m,1H).IR(cm -1 ):3350.14cm -1 ,3063.85cm -1 ,1599.15cm -1 ,1457.99cm -1 ,1478.48cm -1 ,1442.57cm -1 ,1417.21cm -1 ,1308.11cm -1 ,1262.12cm -1 ,1242.90cm -1 ,1017.25cm -1 ,762.33cm -1 ,726.25cm -1,687.66cm -1 ,656.84cm -1 ,Mp:205.5-205.8℃.

[0148] Example 6 Synthesis of Compound 6c

[0149] 0.2 mmol of compound 5b and 0.2 mmol of bis(2,2-bipyridine)ruthenium dichloride (II) were weighed and stirred in a pure water / ethanol mixture for 5 hours. The mixture was lyophilized to obtain 134.3 mg (94.4% yield) of a purple-brown solid. ESI-MS: [M+1] - =711.1, 1 HNMR (300MHz, DMSO-d6): δ (ppm) = 8.89 (d, J = 8.2Hz, 4H), 8.18 (t, J = 7.8Hz, 4H ),7.74(d,J=5.5Hz,4H),7.54(t,J=6.6Hz,4H),7.06(dd,J=8.5,1.9Hz,1H),6 .72-6.61(m,1H),3.34(s,5H),3.24(s,3H),3.16(s,2H),3.05-2.93(m,2H),2 .84(dt,J=14.1,6.7Hz,3H),1.79(s,1H),1.59(dd,J=13.8,6.7Hz,1H).IR(cm -1 ):3178.49cm -1 ,2949.64cm -1 ,1589.36cm -1 ,1515.66cm -1 ,1459.89cm -1 ,1441.89cm -1 ,1418.38cm -1 ,1382.17cm -1 ,1324.32cm -1 ,1249.43cm -1 ,1106.03cm -1 ,1077.90cm -1 ,1019.47cm -1 ,764.25cm -1 ,Mp:183.5-183.7℃.

[0150] Using 2-deoxy-D-ribose as the raw material, it reacts with amino acids under weak alkaline conditions to obtain intermediate product 3(ac), which is then reduced with a sufficient amount of potassium borohydride and purified by a cation exchange resin column to obtain intermediate product 4(ac). Hydrogen reduction reaction removes the protecting group to obtain the target compound 5(ac). Complexation reaction gives compound 6(ac). The structures of all compounds are shown in Figure 2. 1 The structure of the compound was identified by HNMR, MS and IR. The identification results showed that the structure of the compound was correct. The infrared spectrum of the ruthenium complex was 700-800 cm -1 The characteristic peak of ruthenium appears, 1400-1500cm -1 The characteristic peak of νC=N was displayed, indicating the successful complexation of the ruthenium complex.

[0151] Structural characterization

[0152] (1) Nanoparticle size

[0153] The nanoparticle sizes of compounds 5a and 6a are as follows Figure 3 The nanoparticle sizes of different compounds are shown in Table 1.

[0154] Table 1 Nanoparticle size of different compounds

[0155]

[0156] As can be seen, the nanoparticle size of 6(ac) is significantly reduced compared to 5(ac), and the nanoparticle size of all three ruthenium complexes is less than 100 nm. Therefore, the results show that the morphology of the compounds changes after complexing with ruthenium, and the three ruthenium complexes are able to form nanostructures in aqueous solution.

[0157] (2) Transmission electron microscopy analysis

[0158] The particle morphology of the compound and the changes in the morphology after ruthenium complexation were observed using transmission electron microscopy. The transmission electron microscopy images of compounds 5a and 6a are shown in Figure 2. Figure 4 As shown in the figure, the particles of compound 5a are irregular or elongated, while the particles of ruthenium complex 6a are regular and spherical. The experimental results show that the morphology of the nanoparticles changes after ruthenium complexation.

[0159] Test Example 1 Evaluation of in vitro anti-tumor cell activity and the effect of glycolysis

[0160] Preparation of experimental reagents:

[0161] (1) Preparation of complete culture medium: DMEM, 1640 culture medium (containing double antibody) and fetal bovine serum were mixed evenly at a ratio of 10:1 to obtain a complete culture medium containing 10% fetal bovine serum. The complete culture medium was sealed with sealing film and stored in a refrigerator at 4°C.

[0162] (2) Compound solution preparation: Dissolve 5 μmol of the target compound in 250 μL of dimethyl sulfoxide to prepare a compound stock solution. Take an appropriate amount of the compound stock solution, dilute it to a target compound concentration gradient, and store in a refrigerator at 4°C until use.

[0163] (3) Preparation of MTT solution: Accurately weigh 250 mg of MTT solid and dissolve it in 50 mL of PBS to prepare MTT solution. After thorough sonication, filter the solution through a 0.22 μm pore size microporous filter to sterilize it and store it in a refrigerator at 4°C away from light. Shake well before use.

[0164] (I) In vitro anti-tumor cell proliferation activity of target compounds

[0165] MTT and CCK-8 assays were used to detect the effects of six compounds 5(ac) and 6(ac) on the activity of seven tumor cells, including human myeloid leukemia cells (HL-60), human monocytic leukemia cells (THP-1), colon cancer cells (SW480), breast cancer cells (4T1), mouse Lewis lung cancer cells (LLC), and human highly metastatic lung cancer cells (95D).

[0166] Here’s how:

[0167] (1) Cell inoculation: Take a cell line in logarithmic growth phase and in good growth condition. Digest the adherent cells with 1 mL of trypsin for 4 min. Stop the digestion with 2 mL of complete medium, collect and centrifuge. Discard the supernatant and make a single cell suspension with complete medium. Adjust the cell concentration to 5 × 10 4 / mL (the cell concentration can be adjusted appropriately due to differences in cell growth rate and volume of different cell lines), the cell suspension was evenly inoculated into a 96-well plate with a volume of 100 μL per well, and 150 μL PBS solution was added to the outer wells of the 96-well plate to seal the liquid, and the plate was placed in a 37°C, 5% CO2 incubator for culture.

[0168] (2) Cell administration stimulation: After the plate is plated, when the cells in the wells are cultured to about 50%, the compound solution to be tested is added. The compound solution concentration is set to 7 concentrations, with 6 replicates for each concentration. The compound solution is added at a volume of 25 μL per well and the drug administration stimulation is carried out for 48 hours. The final concentrations of the compound solution after dilution are 200, 100, 50, 12.5, 6.25, and 3.12 μM, respectively. 2-Deoxy-D-ribose (2-DG) is used as the positive control drug, and an equal volume of complete culture medium is added to the blank control group.

[0169] (3) Colorimetric determination of IC 50Value: After 48 hours of drug action, add 25 μL of MTT solution (5 mg / mL) to each well and incubate in an incubator for 4 hours. After incubation, discard the solution in the well and add 200 μL of dimethyl sulfoxide, place it on a shaker and shake for 10 minutes to allow the formazan to be fully dissolved by DMSO. The absorbance values ​​of each well at dual wavelengths of 490 and 570 nm were measured by a microplate reader, and the cell growth inhibition rate was calculated according to the public. The final IC value of the drug to be tested was calculated using Prism software. 50 All experiments were repeated three times and the average value was taken.

[0170] Growth inhibition rate (%) = (OD value of experimental group - OD value of blank group) / OD value of control group × 100%.

[0171] MTT assay to evaluate the IC value of compounds on different tumor cells 50 The values ​​are shown in Table 2. CCK-8 method was used to evaluate the IC values ​​of the compounds against different tumor cells. 50 The values ​​are shown in Table 3.

[0172] Table 2 Evaluation of IC values ​​of compounds on different tumor cells by MTT assay 50 value

[0173]

[0174] Table 3 IC values ​​of compounds evaluated on different tumor cells by CCK-8 method 50 value

[0175]

[0176] In Table 3, nd means >100 μM.

[0177] The results of the six compounds on tumor cells showed that the antitumor activity of compound 5(ac) was not statistically different from that of the positive control 2-DG, while ruthenium complex 6(ac) showed stronger inhibitory activity against various tumor cells. Cell screening results showed that ruthenium complex 6(ac) exhibited good antitumor activity, with the highest antitumor activity against human monocytic leukemia THP-1 cells.

[0178] (II) Colorimetric determination of the enzyme inhibitory activity of the target compound

[0179] In order to explore the in vitro inhibitory activity of the six target compounds on hexokinase, the six target compounds were directly incubated and combined with recombinant hexokinase protein, and the NADH generated by the coupling reaction with glucose-6-phosphate dehydrogenase (G6PDH) was detected at a wavelength of 340 nm to reflect the inhibitory ability of the compounds on hexokinase.

[0180] The specific method is as follows:

[0181] (1) Preparation of compound solution: Accurately weigh 5 μmol of compound and dissolve it in 250 μL of DMSO to prepare a compound stock solution. Take an appropriate amount of the compound stock solution and dilute it according to the concentration gradient. The final concentrations of the compound solution after dilution are 100, 50, 12.5, 6.25, 3.12, and 1.56 μM, respectively.

[0182] (2) Preparation of reaction substrate: Prepare the reaction substrate according to Table 4.

[0183] Table 4 Substrate concentration and required volume for enzyme inhibition activity assay

[0184]

[0185] (3) Incubation: 20 μL of HK protein was incubated with 30 μL of compound at 37°C for 15 min.

[0186] (4) Determination: 85 μL of reaction substrate solution was added to each well of a 96-well plate. After incubation, 15 μL of the mixture was added to each well of the 96-well plate. The mixture was quickly pipetted and evenly mixed. The change in NADH absorbance was immediately detected continuously at a wavelength of 340 nm for 5 minutes.

[0187] IC values ​​of six compounds for hexokinase inhibition 50 See Table 5 for values.

[0188] Table 5 IC values ​​of six compounds for hexokinase inhibition 50 value

[0189]

[0190] The experimental results showed that compared with the positive control group 2-DG, the enzyme inhibition IC 50 There were no significant statistical differences, indicating that all six compounds exhibited potent hexokinase inhibitory activity. Furthermore, ruthenium complex 6(ac) showed no statistically significant difference compared to ligand 5(ac). Therefore, these results suggest that the ruthenium complex significantly enhances the antitumor activity of the ligand while not interfering with its inhibitory effect on hexokinase.

[0191] (III) In vitro apoptosis-promoting effect of target compound 6b

[0192] In order to explore whether the target compound 6b induces apoptosis in THP-1 cells, the present invention uses the Annexin V-FITC method to detect the early and late apoptotic changes in THP-1 cells after treatment with 6b. Phosphatidylserine (PS) is mainly distributed on the inner side of the cell membrane. In the early stages of apoptosis, different types of cells will externalize phosphatidylserine to the cell surface, that is, the outer side of the cell membrane. When phosphatidylserine is exposed to the cell surface, it promotes coagulation and inflammatory responses. Annexin V selectively binds to phosphatidylserine and can block the pro-coagulant and pro-inflammatory activity of phosphatidylserine after binding. Using Annexin V labeled with the fluorescent probe FITC with green fluorescence, the important feature of apoptosis, phosphatidylserine externalization, can be simply and directly detected by flow cytometry.

[0193] The experimental steps are as follows:

[0194] (1) Seed plate: THP-1 cells were plated at 1×10 6 / mL concentration, add 2mL per well into a 6-well plate and spread evenly on the 6-well plate. Let it stand for a while before administering the stimulus.

[0195] (2) Administration: The final concentration of the compound drug was diluted to 200 and 100 μM, and 500 μL was added to each well of a 6-well plate. After gently shaking to mix, the plate was placed in an incubator for incubation and administration stimulation was performed for 48 hours.

[0196] (3) Staining: After 48 hours of cell apoptosis stimulation, centrifuge at 1000 g for 5 minutes, discard the supernatant, collect the cells, gently resuspend the cells in PBS and count them. Take 100,000 resuspended cells, centrifuge at 1000 g for 5 minutes, discard the supernatant, add 195 μL Annexin V-FITC conjugate solution and gently resuspend the cells. Add 4 μL Annexin V and 10 μL propidium iodide staining solution in sequence and mix gently. Incubate at room temperature in the dark for 10 minutes, then place in an ice bath.

[0197] (4) Analysis on the flow cytometer: Use a pipette to draw about 200 μL of the mixed solution through the filter membrane and inject it into the flow cytometer tube for analysis (if the number of cells is too large, add a small amount of PBS to dilute it before analysis).

[0198] The pro-apoptotic effects of different compounds on THP-1 Figure 5 The apoptosis statistics of THP-1 cells are shown in Figure 6 shown. Figure 5 and Figure 6 CON: blank control group; 2-DG: 2-deoxyglucose positive control group; ***P<0.001 vs. CON. Figure 5 、 6As shown, compared with the blank control group, at a concentration of 200 μM, there was no significant statistical difference in the distribution of 2-DG and 5b in the Q2 and Q4 regions compared with the blank control group, while at the same concentration, the distribution of 6b ruthenium complex in the Q2 and Q4 regions was significantly increased (P<0.001), indicating that ruthenium complex 6b can significantly induce apoptosis of human monocytic leukemia cells THP-1.

[0199] (IV) Effect of target compound 6b on THP-1 mitochondrial membrane potential

[0200] A decrease in mitochondrial membrane potential is a hallmark of early apoptosis. Using a fluorescent probe like JC-1, the mitochondrial membrane potential of cells and tissues can be monitored. The principle is that when the mitochondrial membrane potential is high, JC-1 aggregates in the mitochondrial matrix, forming aggregates that produce red fluorescence. However, when the mitochondrial membrane potential is low, JC-1 cannot aggregate in the mitochondrial matrix and forms monomers, producing green fluorescence. The shift from red to green fluorescence of JC-1 indicates a decrease in intracellular membrane potential.

[0201] The experimental steps are as follows:

[0202] (1) Seed plate: See “(III) (1) Seed plate” above for details.

[0203] (2) Administration: See above “(III) (2) Administration” for details.

[0204] (3) Staining: After 48 hours of cell apoptosis stimulation, centrifuge at 1000g for 5 minutes, discard the supernatant, resuspend in 0.5mL cell culture medium, add 0.5mL JC-1 staining working solution, and mix by inverting several times. Incubate in a cell culture incubator at 37℃ for 20 minutes. After incubation, centrifuge at 600g for 3 minutes to pellet the cells. Discard the supernatant, wash twice with 1mL JC-1 staining buffer (1X), resuspend with an appropriate amount of 1mL JC-1 staining buffer (1X), incubate in the dark at room temperature for 10 minutes, and then place in an ice bath.

[0205] (4) Computer: See “(III)(4) Computer” above for details.

[0206] Effects of different compounds on THP-1 mitochondrial membrane potential Figure 7 The statistical diagram of the effects of different compounds on mitochondrial membrane potential is shown in Figure 8 shown. Figure 7 and Figure 8 CON: blank control group; 2-DG: 2-deoxyglucose positive control group; **P<0.01, ***P<0.001 vs. CON.

[0207] like Figure 7As shown in Figure 2, flow cytometric analysis of the stained cells showed that the blank control group showed strong red fluorescence, and only 1.1% of the THP-1 cells showed green fluorescence. Figure 8 As shown, compared with the blank control group, the green fluorescence of 2-DG and 5b at a concentration of 200 μM increased slightly, but the difference was not statistically significant (P>0.05). However, at the same concentration, the distribution of ruthenium complex 6b in the Q4 region increased significantly compared with the blank control group (P<0.001), showing strong green fluorescence, indicating that ruthenium complex 6b can significantly reduce the mitochondrial membrane potential of THP-1 cells. Therefore, the results indicate that ruthenium complex 6b can induce a decrease in the mitochondrial membrane potential of THP-1 cells and induce early apoptosis in THP-1 cells.

[0208] (V) Effect of target compound 6b on intracellular glucose-6-phosphate

[0209] Glucose-6-phosphate (G-6-P) is a molecule produced by the phosphorylation of the hydroxyl group on the 6th carbon of glucose under the catalysis of hexokinase. It is a common small molecule in sugar metabolism in cells and participates in biochemical pathways such as glycolysis and pentose phosphate. In the first step of glycolysis, glucose is catalyzed by hexokinase to produce glucose-6-phosphate, which is then catalyzed by phosphoglucose isomerase to form fructose-6-phosphate to continue glycolysis. This experiment detects the changes in the content of intracellular glucose-6-phosphate after stimulation with different drugs, reflecting the effects of different stimuli on the intracellular glycolysis rate, and indirectly reflects the effects of different stimuli on the catalytic effect of hexokinase. The experiment uses a glucose-6-phosphate (G6P) content detection kit to evaluate the intracellular glycolysis process. The experimental steps are as follows:

[0210] (1) Seed plate: See “(III) (1) Seed plate” above for details.

[0211] (2) Administration: See “(III) (2) Administration” above for details.

[0212] (3) Sample preparation: 72 hours after drug stimulation, collect cells, centrifuge at 1000g for 5 minutes, discard the supernatant, gently resuspend the cells with PBS, wash once, centrifuge at 1000g for 5 minutes, discard the supernatant, gently flick the cells, add 200μL of ice-cold G6P lysis buffer, mix thoroughly to promote cell lysis. The lysis process is performed on ice. After lysis, centrifuge at 12000g for 10 minutes at 4℃, take the supernatant, and store on ice for testing. Pipette 50μL of the sample or standard to be tested into a 96-well plate, then add 50μL of G6P detection solution to each well and gently pipette to mix. Incubate at 37℃ in the dark for 10 minutes, then measure the absorbance at 450nm.

[0213] Effects of different compounds on intracellular G6P Figure 9 shown. Figure 9 CON: blank control group; *P<0.05, **P<0.01, ***P<0.001 vs. CON. It can be seen that compared with group 5b, the G6P content in THP-1 cells was significantly reduced after treatment with ruthenium complex 6b at the same concentration (P<0.05).

[0214] (VI) Effect of target compound 6b on intracellular ATP

[0215] ATP is a crucial energy molecule in cellular energy metabolism and plays a crucial role in various physiological and pathological processes. Changes in ATP levels can affect cellular function. A decrease in ATP levels indicates impaired or decreased mitochondrial function. During apoptosis, a decrease in ATP levels often coincides with a decrease in mitochondrial membrane potential. An ATP assay kit was used to detect changes in ATP levels in human monocytic leukemia THP-1 cells stimulated by target compounds. The experimental steps are as follows:

[0216] (1) Seed plate: See “(III) (1) Seed plate” above for details.

[0217] (2) Administration: See “(III) (2) Administration” above for details.

[0218] (3) Sample preparation: After 72 hours, collect cells and centrifuge at 1000 g for 5 minutes. Discard the supernatant and wash once with PBS. Centrifuge at 1000 g for 5 minutes and discard the supernatant. Gently flick the cells and add 200 μL of lysis buffer per well of a 6-well plate to lyse the cells. Mix thoroughly. After lysis, centrifuge at 12000 g for 5 minutes at 4°C. Remove the supernatant and store on ice until assayed.

[0219] (4) Assay: Add 100 μL of ATP assay working solution to each well of a 96-well plate. Incubate at room temperature for 3 minutes. Add 20 μL of sample or standard to the plate and quickly mix using a pipette. Measure the RLU value of the sample using a chemiluminescence analyzer.

[0220] Effects of different compounds on intracellular ATP Figure 10 shown. Figure 10 CON: blank control; *P < 0.05, **P < 0.01 vs. CON. As can be seen, compared with group 5b, treatment with ruthenium complex 6b at the same concentration reduced ATP content in THP-1 cells (P < 0.05). These results suggest that ruthenium complex 6b can inhibit the production of G6P and ATP in THP-1 cells, thereby inhibiting glycolysis in THP-1 cells and suppressing tumor cell growth.

[0221] (VII) Western blotting analysis of related proteins

[0222] In order to explore whether the six compounds can inhibit the expression of hexokinase protein and induce apoptosis of cancer cells, the present invention uses Western Blot method to detect the expression level of hexokinase protein and the expression level of apoptosis-related proteins in THP-1 cells after treatment with the six compounds.

[0223] Effects of different compounds on the apoptotic protein Bax Figure 11 The statistical diagram of the effects of different compounds on Bax is shown in Figure 12 shown. Figure 12 CON: blank control; 2-DG: 2-deoxyglucose positive control; *P < 0.05, **P < 0.01 vs. 2-DG. Western blot results showed that compared with the blank control, the 2-DG group significantly inhibited hexokinase protein expression and induced increased expression of the pro-apoptotic protein Bax, as expected. Furthermore, compared with the 2-DG group, the ruthenium complex 6b group significantly increased the expression of the pro-apoptotic protein Bax at the same concentration (P < 0.05), with a particularly significant increase at a concentration of 200 μM (P < 0.01).

[0224] Effects of different compounds on hexokinase type 2 (HK2) protein Figure 13 The statistical graph of the effects of different compounds on HK2 protein is shown in Figure 14 shown. Figure 14 CON: blank control; 2-DG: 2-deoxyglucose positive control; **P<0.01, ***P<0.001 vs. 2-DG. Compared with the 2-DG group, ruthenium complex 6b significantly inhibited hexokinase 2 expression at the same concentration (P<0.01), with the inhibition being particularly significant at 200 μM (P<0.001). These results suggest that ruthenium complex 6b can induce cancer cell apoptosis by inhibiting hexokinase 2 protein expression and promoting the expression of the apoptotic protein Bax.

[0225] In summary, the present invention designed a series of 2-DG analogs (5a-5c, 6a-6c) and used MTT and CCK-8 methods to screen for anti-tumor cell activity. It was found that all six synthesized compounds had good enzyme inhibition effects on hexokinase, among which three ruthenium complexes 6a-6c had strong inhibitory activity against human monocytic leukemia cells THP-1. Ruthenium complex 6b can significantly reduce the mitochondrial membrane potential of cancer cells and increase the expression level of the apoptotic protein Bax, inducing THP-1 cell apoptosis, thereby inhibiting cancer cell proliferation. At the same time, ruthenium complex 6b can inhibit the expression level of HK2 protein in cancer cells, reduce the levels of G6P and ATP in cancer cells, thereby inhibiting the glycolysis process in cancer cells and affecting the energy metabolism of cancer cells.

[0226] Test Example 2 Anti-breast cancer activity of hexokinase inhibitors and hexokinase inhibitor ruthenium complexes

[0227] (I) Annexin V-FITC / PI cell apoptosis assay

[0228] In order to verify whether the compound can induce apoptosis in 4T1 cell line, Annexin V-FITC / PI cell apoptosis detection kit was used for evaluation. 4T1 cells in logarithmic growth phase were taken and 5×10 5 The cells were evenly plated in a 6-well plate at a concentration of 100 μL / mL and cultured for 48 hours after administration. The cell culture medium was aspirated into a centrifuge tube, washed with PBS, and digested with an appropriate amount of 0.25% trypsin for 5 minutes. The digestion was stopped by adding twice the amount of complete medium to prevent cell apoptosis caused by excessive digestion. The cells were gently pipetted and transferred to a centrifuge tube. Centrifuged at 1000 g for 5 minutes, the supernatant was discarded, the cells were collected, washed with PBS, the supernatant was discarded, 195 μL Annexin V-FITC binding solution was added to resuspend the cells, 5 μL Annexin V-FITC was added and gently mixed, followed by the addition of 5 μL propidium iodide staining solution and gently pipetting to mix. Incubate at room temperature in the dark for 15 minutes, then place in an ice bath and detect on an instrument.

[0229] Annexin V-FITC / PI cell apoptosis Figure 15 As shown, the AnnexinV-FITC / PI cell apoptosis statistics are shown in Figure 16 As shown, Figure 16 CON: blank control; 2-DG: 2-deoxyglucose positive control; **P<0.01, ***P<0.001 vs. CON. The results showed that both compounds 5a and 6a promoted apoptosis in 4T1 cells compared to the blank and 2-DG groups (P<0.001). 5a significantly promoted both early and late apoptosis, while 6a primarily promoted late apoptosis.

[0230] (2) Detection of intracellular G-6-P content and ATP content

[0231] In order to evaluate whether the compound treatment could inhibit the glycolysis process in cells, the levels of G6P and ATP in 4T1 cells were measured after administration.

[0232] The method for detecting intracellular G-6-P content is as follows:

[0233] 4T1 cells in the logarithmic growth phase were taken and 5×10 5 / mL concentration was evenly spread in a 6-well plate and cultured for 48 hours after administration. After drug stimulation, the culture medium was aspirated, 200μL of ice-bath pre-cooled G6P extract was added, and the cells were gently pipetted to promote cell lysis. The liquid was then collected, centrifuged at 12000g, 4°C for 10 minutes, and the supernatant was taken as the sample to be tested and stored in an ice bath for later use. In a 96-well plate, 50μL of G6P detection solution was added to each well, followed by 50μL of the sample to be tested, and the mixture was gently pipetted to mix. The cells were incubated at 37°C for 10 minutes, and the absorbance at 450nm was measured.

[0234] The method for detecting intracellular ATP content is as follows:

[0235] 4T1 cells in the logarithmic growth phase were taken and 5×10 5 / mL concentration was evenly spread in a 6-well plate and cultured for 48 hours after administration. The culture medium was aspirated and 200μL lysis solution was added to each well to lyse the cells. During lysis, the cells were blown thoroughly with a pipette to ensure complete cell lysis. After lysis, the liquid was collected into a centrifuge tube, centrifuged at 12000g and 4℃ for 5 minutes, the supernatant was taken and placed on ice for testing. On a 96-well black board, 100μL ATP detection working solution was added to each well and placed at room temperature for 3 minutes. Then 20μL of sample was added to the detection well, and the RLU value was measured by chemiluminescence instrument.

[0236] Effects of compounds on intracellular G6P Figure 17 shown. Figure 17 In the figure, CON: blank control group; 2-DG: 2-deoxyglucose positive control group, 200 μM; 5a, 6a: drug-treated groups, 200 μM; **P<0.01 vs. CON.

[0237] Effects of compounds on intracellular ATP Figure 18 shown. Figure 18 In the figure, CON: blank control group; 2-DG: 2-deoxyglucose positive control group, 200 μM; 5a, 6a: drug-treated groups, 200 μM; *P<0.05, **P<0.01 vs. CON.

[0238] The experimental results showed that compared with the blank control group, the 2-DG group had no significant effect on intracellular G6P levels, but significantly reduced the level of ATP in 4T1 cells. This may be due to the low dosage concentration, at which the competitive ability with glucose is low, making it difficult to inhibit the production of G6P during glycolysis. Compared with the blank control group and the 2-DG group, compound 6a had a significant inhibitory effect on intracellular G6P and ATP levels (P < 0.01). In addition, compared with compound 5a, ruthenium complex 6a can significantly enhance the inhibitory effect on the glycolysis process, suggesting that ruthenium complex 6a has higher anti-tumor activity.

[0239] (III) Transwell cell migration assay

[0240] The present invention uses a Transwell migration assay to examine the effects of different compounds on the migration ability of 4T1 cells. The experimental steps are as follows:

[0241] 4T1 cells in the logarithmic growth phase were collected, the supernatant was removed, and the cells were washed twice with PBS to ensure that there was no serum influence. After digestion with 1 mL of trypsin for 4 minutes, the digestion was terminated with DMEM serum-free medium, and the cells were collected and centrifuged to adjust the cell density to 5×10 5 / mL. 100 μL of cell suspension was added to a Transwell chamber. 600 μL of DMEM complete medium containing 10% FBS was added to the lower chamber of a 24-well plate. 25 μL of compound solution was added to the Transwell chamber to dilute the compound to a final concentration of 200 and 100 μM. Stimulation was continued for 48 hours.

[0242] After drug stimulation, the Transwell chamber was removed, the culture medium in the wells discarded, and cells were fixed with cell fixative for 30 minutes. Following fixation, cells were stained with 0.1% crystal violet solution for 20 minutes. Unmigrated cells in the upper layer were gently removed with a cotton swab, and the cells were washed three times with PBS. The chamber was air-dried. Cells were observed under a fluorescence microscope, counted, and averaged for statistical analysis.

[0243] Effects of different compounds on 4T1 cell migration Figure 19 The statistical graph of the effects of different compounds on 4T1 cell migration is shown in Figure 20 shown. Figure 20 CON: blank control group; 2-DG: 2-deoxyglucose positive control group; ***P<0.001 vs. CON.

[0244] The experimental results showed that after 48 hours of drug treatment, the number of cell migration in the blank control group was significantly higher, such as Figure 19 Compared with the blank control group, the number of cells that migrated in the 2-DG, 5a, and 6a groups was significantly reduced, and the number of cells that migrated in the 6a group was the most significantly reduced, and the difference was statistically significant (P<0.001). Figure 20 The results showed that 2-DG, 5a and 6a could effectively inhibit cancer cell migration, among which ruthenium complex 6a had the most significant inhibitory effect.

[0245] (IV) Evaluation of anti-breast cancer activity in vivo

[0246] The 4T1 mouse breast cancer cell line used in this experiment was obtained from KeyGen Biotech Co., Ltd. Eighty female BALB / c mice, 4 - 6 weeks old and weighing 17 - 20 g, were selected for this experiment. They were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., with the certificate number SYXK(Beijing)2022 - 0049. The mice were ensured to be raised under SPF - level conditions, with the indoor temperature at 25°C and the relative humidity at 50 - 60%. The experiment complied with the animal ethics review requirements of the Capital Medical University.

[0247] Experimental methods:

[0248] Construction of the 4T1 mouse breast cancer model and administration of drugs:

[0249] The 4T1 cells were cultured to the logarithmic growth phase, digested with 0.25% trypsin for 5 minutes, and after stopping digestion with complete medium, they were collected by centrifugation. The supernatant was discarded, and the cells were washed twice with PBS buffer to avoid the influence of fetal bovine serum (FBS) on tumor formation. Finally, the cells were resuspended with PBS, and the cell suspension was diluted to 1×10 7 / mL. 0.1 mL was subcutaneously injected at the position of the third mammary pad on the right side of the mice. The tumor volume was observed and measured daily for subsequent experiments.

[0250] The tumor - bearing mice were randomly divided into 8 groups: blank control group, positive drug control group (2 - deoxyglucose group), and drug - administration groups (including 5a, 5b, 5a, 6a, 6b, 6c), with 10 mice in each group. The treatments and drug - administration doses for each group were as follows:

[0251] Blank control group (n = 10): Intraperitoneally injected with 0.9% normal saline according to body weight (10 g / 0.1 mL);

[0252] Positive control group (n = 10): The drug - administration dose was 20 mg / kg, and 2 - deoxyglucose solution was intraperitoneally injected according to body weight (10 g / 0.1 mL);

[0253] Drug - administration group (n = 10): The drug - administration dose was 15 mg / kg, and six compound solutions were intraperitoneally injected respectively according to body weight (10 g / 0.1 mL);

[0254] Drugs were administered every other day for 14 days. The body weight and tumor volume of the mice were recorded. On the 15th day of drug administration, the mice were dissected, tumor tissues were taken, and the weights of the tumor tissues and internal organs of the mice were measured. On the 1st day and the 14th day of drug administration, in - vivo imaging of the mice was performed to visually observe the changes in tumors in the mice. The in - vivo imaging of the mice to observe tumor changes is as Figure 21 shown. The tumor pictures of 4T1 tumor - bearing mice are as Figure 22 shown, the statistical chart of mouse tumor weight is as Figure 23 shown, and the change in the in - vivo tumor volume of the mice after 14 days of drug administration is as [[ID=?]]<00 Figure 24 shown. Figure 23NS: normal saline group; 2-DG: 2-deoxyglucose positive control group; **P<0.01, ***P<0.001 vs. NS.

[0255] The experimental results showed that after 14 days of administration, the tumor volume of mice in the saline group increased significantly. Compared with the saline group, the tumor growth of the positive drug 2-DG group slowed down, but the difference was not statistically significant (P>0.05). However, the tumor growth of the 5(ac) and 6(ac) groups was significantly reduced (P<0.01). It can be seen that in the 4T1 murine breast cancer mouse model, the tumor volume of the positive drug 2-DG group, 5(ac), and 6(ac) groups was significantly suppressed compared with the saline group. The tumor weight of the tumor-bearing mice in the normal saline group was (0.85±0.25) g. Compared with the normal saline group, the tumor weight of the tumor-bearing mice in the positive drug 2-DG group was (0.50±0.14) g (P<0.01), and the tumor inhibition rate was 40.6%; the tumor weight of the tumor-bearing mice in the 5a group was (0.52±0.19) g (P<0.01), and the tumor inhibition rate was 38.3%; the tumor weight of the tumor-bearing mice in the 5b group was (0.38±0.10) g (P<0.001), and the tumor inhibition rate was 54.9%; The tumor weight of mice bearing tumors in group c was (0.44±0.11) g (P<0.01), with a tumor inhibition rate of 48.3%. The tumor weight of mice bearing tumors in group 6a was (0.25±0.11) g (P<0.001), with a tumor inhibition rate of 70.0%. The tumor weight of mice bearing tumors in group 6b was (0.54±0.11) g (P<0.01), with a tumor inhibition rate of 36.9%. The tumor weight of mice bearing tumors in group 6c was (0.32±0.12) g (P<0.001), with a tumor inhibition rate of 62.0%. In summary, the experimental results show that the 5(ac) and 6(ac)-treated groups have good antitumor activity, and the ruthenium complex 6(ac)-treated group has better antitumor activity than the compound 5(ac) group, with the ruthenium complex 6a showing the best antitumor activity. Compared with the positive drug 2-DG group, the anti-tumor activity of ruthenium complex 6a was significantly improved, with statistical difference (P<0.05).

[0256] After the 4T1 breast cancer tumor-bearing mouse model was established, the drug was injected intraperitoneally and the effect of drug administration on the weight change of the mice was observed. Figure 25 As shown in Figure 2, the changes in mouse organ weights are as follows: Figure 26As shown. It can be seen that the body weight of the blank control group and the positive drug 2-DG group decreased, while the body weight of the 5(ac) and 6(ac) drug-treated groups did not show significant changes. In addition, as shown in the figure, the mortality rate of mice in the positive drug 2-DG group increased starting from the 6th day of drug administration. After the completion of drug administration, there was no statistical difference in the body weight of the positive drug 2-DG group and the drug-treated group compared with the normal saline group (P>0.05). The results show that the 5(ac) and 6(ac) drug-treated groups did not cause weight loss in mice, and did not cause mouse death during the drug administration period, and were less toxic than the positive drug 2-DG group.

[0257] (V) Impact on lung metastasis

[0258] The present invention established a mouse 4T1 orthotopic breast cancer model, in which 4T1 cells spontaneously generate highly metastatic tumors and metastasize to the lungs. Figure 27 As shown in Figure 2, the effects of drug administration on metastatic nodules are as follows: Figure 28 As shown, Figure 28 NS: normal saline group; 2-DG: 2-deoxyglucose positive control group; **P<0.01, ***P<0.001 vs. NS.

[0259] As can be seen, the lung tissue of the saline group showed obvious pulmonary metastasis and obvious metastatic nodules. Compared with the saline group, the number of metastatic nodules in the positive drug 2-DG group was slightly reduced, but the difference was not significant (P>0.05). The number of metastatic nodules in the compound 5a and ruthenium complex 6a was significantly reduced (P<0.01), and the lung tissue appeared better than the control group.

[0260] (VI) Expression of hexokinase in tumor tissue

[0261] To verify whether the compound has an inhibitory effect on hexokinase, the mouse hexokinase ELISA detection kit was used to measure the hexokinase expression level in tumor tissue. The specific method is as follows:

[0262] Weigh the tumor tissue from different groups and record the amount. Add a certain amount of PBS (pH 7.4) and thoroughly homogenize the tissue using a homogenizer. Centrifuge for 20 minutes, collect the supernatant, and set aside. Set up blank control wells, standard wells, and sample wells. Add 50 μL of standard at different concentrations to the standard wells. First, add 40 μL of sample diluent to the sample wells to be tested on the enzyme-linked label-coated plate, then add 10 μL of the sample to be tested. Try not to touch the well walls when adding the sample, and gently shake to evenly distribute the sample. Then, seal the plate with a sealing film and incubate at 37°C for 30 minutes. Dilute the 30x concentrated wash solution 30x with distilled water and set aside. Carefully remove the sealing film, discard the liquid, shake dry, fill each well with wash solution, let it sit for 30 seconds, and then discard. Repeat this process five times and pat dry. After washing, add 50 μL of enzyme-linked label to each well, except for the blank well. Repeat the incubation and washing process once. First, add 50 μL of color developer A to each well, then add 50 μL of color developer B, gently shake and mix, and develop at 37°C in the dark for 15 minutes. Add 50 μL of stop solution to each well to terminate the reaction. Within 15 minutes after the reaction is terminated, measure the absorbance of each well at a wavelength of 450 nm. Figure 29 As shown, Figure 29 NS: normal saline group; 2-DG: 2-deoxyglucose positive control group; **P<0.01, ***P<0.001 vs. NS.

[0263] The experimental results showed that compared with the saline group, the positive drug 2-DG group significantly reduced hexokinase levels in tumor tissue (P < 0.01), consistent with the results of in vitro cell experiments. Compared with the saline group, the 6a group significantly reduced hexokinase levels in tumor tissue (P < 0.001), indicating that the drug group has a strong inhibitory effect on hexokinase. Furthermore, compared with the 5a group, the ruthenium complex 6a significantly enhanced the inhibitory effect on hexokinase 2 in tumor tissue.

[0264] (VII) Lactic acid expression in tumor tissue

[0265] In order to verify whether the compound has an inhibitory effect on the glycolysis process in tumors, the lactate content detection kit was used to evaluate the expression level of lactate in tumor tissues. The method is as follows:

[0266] Weigh the tumor tissue from each group and record the amount. Add 1 mL of Extraction Solution 1, homogenize on ice, and centrifuge at 12,000 g for 10 minutes at 4°C. Collect 0.8 mL of the supernatant and add 0.15 mL of Extraction Solution 2. Centrifuge at 12,000 g for 10 minutes at 4°C, and collect the supernatant for analysis. Sample addition follows the instructions for the lactic acid content detection kit. Lactic acid content is calculated based on sample weight:

[0267] LA content (μmol / g) = 1.1875 × x ÷ W.

[0268] Here, x is the measured value, and W is the molecular weight of lactic acid.

[0269] The expression of lactate in tumor tissues Figure 30 As shown, Figure 30 NS: normal saline group; 2-DG: 2-deoxyglucose positive control group; **P<0.01, ***P<0.001 vs. NS.

[0270] The experimental results showed that compared with the saline group, the lactate levels in tumor tissues in the 2-DG, 5a, and 6a groups were significantly reduced (P<0.01), and the reduction in lactate levels in the 5a and 6a groups was more significant than that in the 2-DG group (P<0.001). This suggests that 5a and 6a can effectively inhibit glycolysis in tumor tissues, and their inhibitory effects on glycolysis are stronger than those of 2-DG.

[0271] (8) G6P expression in tumor tissue

[0272] To further verify the compound's inhibitory effect on tumor glycolysis, a G6P content detection kit was used to assess G6P expression in tumor tissue. The G6P content detection kit, purchased from Shanghai Beyotime Biotechnology Co., Ltd., measures G6P content in samples. The specific mechanism is as follows: Glucose-6-phosphate (G6P) is oxidized by glucose-6-phosphate dehydrogenase (G6PDH) to form 6-phosphogluconate (6-PG). In this reaction, NADP+ is reduced to NADPH. The resulting NADPH, in the presence of the electron coupling reagent 1-mPMS (1-Methoxy-5-methylphenazinium Methyl Sulfate), converts WST-8 to form orange-yellow formazan, which has a maximum absorption peak around 450 nm. The amount of formazan generated in the reaction system is directly proportional to the total amount of G6P in the sample.

[0273] The expression of G6P in tumor tissue is as follows Figure 31 As shown, Figure 31 NS: normal saline group; 2-DG: 2-deoxyglucose positive control group; ***P<0.001 vs. NS.

[0274] The experimental results showed that compared with the saline group, there was no significant difference in G6P levels in tumor tissues in the positive drug 2-DG group and the 5a treatment group (P>0.05), while G6P levels in the 6a treatment group were significantly lower than those in the positive drug 2-DG group and the saline group (P<0.001). This indicates that ruthenium complex 6a can effectively inhibit the activity of hexokinase 2 in tumor tissue, reduce the phosphorylation of glucose in tumor tissue, thereby reducing G6P levels in tumor tissue and inhibiting the glycolysis process in tumor tissue.

[0275] (IX) Hematoxylin and eosin (HE) staining results

[0276] The present invention uses HE staining to investigate the effects of compounds on tumors and other organs. The experimental groups are divided into a normal saline group, a positive drug 2-DG group, a compound 5a, and a ruthenium complex 6a group. The results of HE staining of tumor tissue sections are shown in Figure 2. Figure 32 As shown. It can be seen that the number of tumor cells in the normal saline group is large, and the staining is uniform, the arrangement is tight, the cell structure is intact, and no obvious damage is observed. The number of tumor cells in the 2-DG group, 5a and 6a groups is significantly reduced, the staining becomes lighter, and some cells are degenerated and necrotic. At the same time, large areas of vacuoles can be observed in the tumor tissue, and the cells are sparsely arranged, indicating that the tumor tissue in the drug-treated group is necrotic. At the same time, compared with the 2-DG group and the 5a group, the ruthenium complex 6a has a more obvious killing effect on tumors. Therefore, the experimental results show that the 2-DG group, the 5a and the 6a group have a certain killing effect on tumor tissue, and the effect of the ruthenium complex is more significant.

[0277] HE staining of normal mouse organ sections Figure 33 As shown in the figure, there was no significant difference in the organs between the normal saline group, the positive drug 2-DG group, the compound 5a, and the ruthenium complex 6a group (P>0.05). The cells showed obvious nuclear structures, intact cell morphology, and no obvious damage, indicating that the drug 2-DG group and compounds 5a and 6a had no significant killing effect on normal tissues.

[0278] (10) TUNEL staining results

[0279] The present invention uses TUNEL staining to examine the apoptosis of cells in each group of 4T1 tumor tissue. The experimental groups are divided into a normal saline group, a positive drug 2-DG group, a compound 5a, and a ruthenium complex 6a group. The TUNEL staining of tumor tissue is shown in the figure below. Figure 34 As shown in the figure, a small amount of apoptotic tumor cells were observed in the tumor tissue of the saline group. However, TUNEL fluorescence was significantly higher in the positive drug 2-DG group, compound 5a, and ruthenium complex 6a groups than in the saline group, indicating that these three groups had higher levels of apoptosis. Furthermore, compared with the 2-DG group, compounds 5a and 6a showed a higher percentage of apoptotic cells.

[0280] The changes of TUNEL positive mean fluorescence intensity by different compounds are shown in Figure 2. Figure 35 As shown, Figure 35 NS: saline; 2-DG: 2-deoxyglucose positive control; **P<0.01, ***P<0.001 vs. NS. As can be seen, the TUNEL-positive mean fluorescence intensity was significantly increased in the compound 5a and ruthenium complex 6a groups compared to the saline group (P<0.001). Therefore, these results demonstrate that compounds 5a and 6a can induce apoptosis in breast cancer cells in mice and exhibit greater antitumor activity than 2-DG.

[0281] (XI) ICP-MS determination of tumor tissue targeting of ruthenium complexes

[0282] Inductively coupled plasma mass spectrometry (ICP-MS) is a multi-element analysis method with high sensitivity, wide dynamic range, low spectral interference, low detection limit, rapid analysis, and the ability to handle trace samples. It is widely used in a variety of fields, including chemical engineering, biology, medicine, and environmental science. This study further explores the effects of ruthenium complexes on tumor tissue and other organs through trace metal element analysis and testing. The specific method is as follows:

[0283] (1) Sample pretreatment: Weigh the tissue samples to ensure that the tissue is less than 300 mg and record the weight of each tissue. After weighing, add 3 mL of HNO3 solution to each tube and let it sit overnight. The next day, add 1 mL of 30% H2O2 solution to each tube and digest it in a microwave digester.

[0284] (2) Detection on the instrument: Before loading, centrifuge the sample for ten minutes at 3000g. Prepare standards at 500, 200, 100, 50, 20, 10, 5, 2, and 1 ng / mL and place them in the sample tank from low to high concentration. Place the sample in the sample tank in order and load the instrument for measurement.

[0285] Uptake of ruthenium complexes in tumors and other organs Figure 36 The experimental results showed that compared with the saline group, ruthenium complex 6(ac) showed higher ruthenium accumulation in tumors. Ruthenium complexes also showed high levels of enrichment in other organs, with the liver and kidneys being the most prominent, possibly related to liver and kidney metabolism.

[0286] Summarize

[0287] The present invention uses 2-deoxyribose, which is similar to 2-DG, as the structural basis, combines 2-deoxyribose with different amino acids to obtain three compounds 5(ac), and then conducts a complex reaction with bis(2,2'-bipyridyl)ruthenium dichloride to obtain three compounds 6(ac). Among them, the mass spectrum of compound 5a is as follows Figure 37 As shown, 1 HNMR spectrum Figure 38 As shown; the mass spectrum of compound 5b is as shown Figure 39 As shown, 1 HNMR spectrum Figure 40 As shown; the mass spectrum of compound 5c is as shown Figure 41 As shown, 1 HNMR spectrum Figure 42 As shown; the mass spectrum of compound 6a is as shown Figure 43 As shown, 1 HNMR spectrum Figure 44 As shown; the mass spectrum of compound 6b is as shown Figure 45 As shown, 1 HNMR spectrum Figure 46 As shown; the mass spectrum of compound 6c is as shown Figure 47 As shown, 1 HNMR spectrum Figure 48 As shown; the infrared spectra of compounds 5a and 6a are shown Figure 49 As shown, the infrared spectra of compounds 5b and 6b are as follows Figure 50 As shown, the infrared spectra of compounds 5c and 6c are as follows Figure 51 shown.

[0288] The present invention measures enzyme inhibition IC 50 The inhibitory effects of compound 5(ac) and its ruthenium complex 6(ac) on hexokinase were compared with those of 2-DG. 50 The results showed that compared with 2-DG, the enzyme inhibition IC 50 There was no significant difference in the values. The six compounds had good inhibitory effects on hexokinase. The six compounds could be used as hexokinase inhibitors for subsequent anti-tumor activity evaluation.

[0289] In order to explore the anti-tumor activity of the six compounds, the present invention used MTT and CCK-8 methods to screen the effects of the six compounds on the activity of various tumor cells. The activity evaluation results showed that after 72 hours of administration, the IC value of ruthenium complex 6 (ac) on THP-1 cells was 50Ruthenium complex 6(ac) exhibited significant inhibitory effects on human monocytic leukemia THP-1 cells at concentrations of 39.7±4.6, 41.45±1.3, and 89.96±6.8 μM, respectively. Similar to 2-DG, compound 5(ac) also significantly inhibited THP-1 cells at 100 μM. Compared with 5(ac) and 2-DG, ruthenium complex 6(ac) exhibited superior antitumor activity. Immunoblotting revealed that compounds 5b and 6b both increased the expression of the pro-apoptotic protein Bax and inhibited the expression of hexokinase in cancer cells.

[0290] The present invention measured the expression levels of glucose-6-phosphate and ATP, and experimental results showed that compounds 5b and 6b can significantly inhibit the expression of glucose-6-phosphate and ATP. This suggests that compounds 5b and 6b may work together to achieve anti-tumor effects through two pathways. First, by inhibiting the expression of hexokinase protein, the intracellular glycolysis process is reduced, resulting in reduced ATP production in cancer cells, making it impossible for cancer cells to maintain necessary biosynthesis and energy requirements, thereby inducing cancer cell death. Second, by inducing the expression of apoptotic proteins in cancer cells, activating the apoptotic pathway and inhibiting tumor growth.

[0291] This study used the hexokinase inhibitor 2-DG as a positive control group to compare the anti-breast cancer activities of 2-DG with six compounds. Furthermore, the anti-breast cancer activities of three compounds, 5(ac) and its ruthenium complex 6(ac), were also compared. MTT assay results showed that the six compounds exhibited superior anti-tumor activity against 4T1 cells compared to 2-DG. Annexin V-FITC / PI staining results demonstrated that compound 5a and ruthenium complex 6a induced apoptosis in 4T1 cells.

[0292] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A hexokinase inhibitor having the structure shown in Formula I: Formula I; In formula I, R is 、 、 One of them.

2. The method for preparing the hexokinase inhibitor according to claim 1, comprising the following steps: Under weak alkaline conditions, 2-deoxy-D-ribose having the structure shown in Formula 1 and amino acid benzyl ester having the structure shown in Formula 2 undergo a condensation reaction to obtain a compound having the structure shown in Formula 3; Formula 1; Formula 2; Formula 3; The compound having the structure shown in Formula 3 is subjected to a reduction reaction with a reducing agent to obtain a compound having the structure shown in Formula 4; Formula 4; Under the action of a catalyst, the compound having the structure shown in Formula 4 undergoes a deprotection reaction with H2 to obtain a hexokinase inhibitor having the structure shown in Formula I.

3. The preparation method according to claim 2, characterized in that The pH value of the weakly alkaline environment is 9; The condensation reaction is carried out in an ice bath, and the condensation reaction time is 12 to 28 hours.

4. The preparation method according to claim 2, characterized in that The molar ratio of the compound having the structure shown in Formula 3 to the reducing agent is 1:1.2-3.6; The reduction reaction is carried out in an ice bath, and the reduction reaction time is 24 to 50 hours.

5. The preparation method according to claim 2, characterized in that The catalyst used in the deprotection reaction is a palladium-carbon catalyst; The temperature of the deprotection reaction is 18-28° C., and the time is 2-6 hours.

6. A hexokinase inhibitor ruthenium complex having the structure shown in Formula II: Formula II; In Formula I, R is 、 、 One of them.

7. The hexokinase inhibitor ruthenium complex according to claim 6, characterized in that The particle size of the hexokinase inhibitor ruthenium complex is 20-120 nm.

8. The method for preparing the hexokinase inhibitor ruthenium complex according to claim 6 or 7, comprising the following steps: The hexokinase inhibitor according to claim 1 or the hexokinase inhibitor prepared by the preparation method according to any one of claims 2 to 5 is subjected to a complexation reaction with bis(2,2-bipyridine)ruthenium dichloride to obtain a hexokinase inhibitor ruthenium complex having a structure shown in Formula II.

9. Use of the hexokinase inhibitor according to claim 1 or the hexokinase inhibitor ruthenium complex according to claim 6 or 7 in the preparation of antitumor drugs.

10. The use according to claim 9, characterized in that The anti-tumor drug is one or more of an anti-breast cancer drug, an anti-lymphoma drug, an anti-leukemia drug, an anti-colon cancer drug and an anti-lung cancer drug.

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