Metal complex tailed with immunologically active molecules and its preparation method and application

Abstract

The present invention discloses a metal complex tailed with an immunologically active molecule, as well as its preparation method and application. The metal complex tailed with an immunologically active molecule exhibits significant anticancer activity. On the one hand, the metal complex exerts cytotoxicity against rapidly growing cancer cells through its active ingredient with anti-tumor immune efficacy. On the other hand, it can also induce immunogenic cell death, enabling the host immune system to attack tumor cells and induce a tumor-specific immune response. (The metal complex of the present invention has been shown to effectively induce immunogenic cell death in non-small cell lung cancer cells and successfully activate anti-tumor immunity in mice in vaccine experiments.) Therefore, the metal complex of the present invention has outstanding effects in anti-cancer, anti-inflammatory, inducing immunogenic cell death, and achieving anti-tumor immunity, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to a metal complex tailed with an immunologically active molecule, and also relates to a preparation method and application of the metal complex. Technical Background

[0002] Metal anti-tumor drugs, represented by cisplatin, have been widely used in clinical practice. However, the "indiscriminate attack" of platinum drugs on cells and inherent drug resistance have limited their development. Therefore, the research on new metal anti-cancer drugs is of great significance. Among them, aryl ruthenium and iridium metal complexes have the advantages of high activity, multiple targets, and low drug resistance, and are the most promising non-platinum metal chemotherapy drugs. However, the efficacy of chemotherapy drugs is often greatly reduced due to tumor heterogeneity and acquired drug resistance. Chemotherapeutic drugs with anti-tumor immune efficacy can not only exert cytotoxicity to rapidly growing cancer cells to produce a "first strike", but also achieve a "second strike" against tumor cells by inducing immune cell death, thereby inducing tumor-specific immune responses. At present, research on metal drugs as immunogenic cell death inducers and achieving anti-tumor immunity is very scarce. Summary of the Invention

[0003] Purpose of the invention: The purpose of the present invention is to provide a metal complex tailed with an immunologically active molecule. By tailing with the immunologically active molecule betulin or triptolide, the metal complex obtained has anti-tumor immune efficacy and can become a metal anticancer active ingredient for inducing immunogenic cell death; at the same time, it solves the problems of poor lipid solubility and large dosage when the immunologically active molecule is used alone as an antitumor drug or an antitumor drug component; another purpose of the present invention is to provide a preparation method of the above-mentioned metal complex and its use in preparing antitumor drugs or antitumor drug components.

[0004] Technical solution: The metal complex tailed with an immunologically active molecule of the present invention has a general structural formula as shown in Formula (I) or Formula (II):

[0005]

[0006]

[0007] in, is p-cymene, biphenyl, pentamethylcyclopentadiene or phenyltetramethylcyclopentadiene; M is Ru or Ir.

[0008] The preparation method of the metal complex tailed with an immunoactive molecule is specifically as follows: first, the immunoactive molecule betulin or triptolide is subjected to an esterification reaction or an amidation reaction with a bipyridine ligand to obtain an organic compound tailed with an immunoactive molecule; and then, the organic compound tailed with an immunoactive molecule is subjected to a coordination reaction with an aryl / metallocene dimer to obtain a metal complex.

[0009] The method for preparing the metal complex tailed with an immunologically active molecule specifically comprises the following steps:

[0010] (1) Under an inert atmosphere, betulin, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (activation reagent for carboxyl groups), 4-dimethylaminopyridine (condensation reaction catalyst) and bipyridine ligand are dissolved in an organic solvent, and after the reaction, the resulting crude product is separated and purified by column chromatography to obtain an organic compound Bet-Bpy modified with a tailed immunoactive molecule; or under an inert atmosphere, celastrol, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (condensation reagent), N,N-diisopropylethylamine and bipyridine ligand are dissolved in an organic solvent, and after the reaction, the resulting crude product is separated and purified by column chromatography to obtain an organic compound Cel-Bpy modified with a tailed immunoactive molecule;

[0011] (2) dissolving the aryl / metallocene dimer in an organic solvent under a noble gas atmosphere, adding an organic compound Bet-Bpy or Cel-Bpy tailed with an immunoreactive molecule, and refluxing with stirring. The crude product after the coordination reaction is separated and purified by column chromatography to obtain an aryl / metallocene complex;

[0012] Among them, the structural formula of the organic compound Bet-Bpy is as follows:

[0013]

[0014] The structural formula of the organic compound Cel-Bpy is shown below:

[0015]

[0016] Wherein, in step (1), the molar ratio of betulin, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine and bipyridine ligand added is 1:1:1:1.

[0017] Wherein, in step (1), the molar ratio of celastrol, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, N,N-diisopropylethylamine and bipyridine ligand added is 19:23:23:19-20.

[0018] Wherein, in step (2), the molar ratio of the added aryl metal dimer to the organic compound tailed with the immunologically active molecule is 1:2 to 2.5.

[0019] The preparation method of the metal complex tailed with the immunologically active molecule is specifically as follows: first, an aryl / metallocene dimer is reacted with a bipyridine ligand to obtain an aryl / metallocene complex precursor; then, the aryl / metallocene complex precursor is reacted with the immunologically active molecule betulin or triptolide to obtain the metal complex.

[0020] The method for preparing the metal complex tailed with an immunologically active molecule specifically comprises the following steps:

[0021] (1) Preparing an aryl / metallocene complex precursor: dissolving an aryl / metallocene dimer in an organic solvent under a noble gas atmosphere, adding a bipyridine ligand, and stirring under reflux. After the reaction, the resulting crude product is separated and purified by column chromatography to obtain an aryl / metallocene complex precursor; the molar ratio of the aryl metal dimer to the bipyridine ligand is 1:2 to 2.5;

[0022] (2) Under an inert atmosphere, a metal complex precursor, an immunologically active molecule betulin, and 4-dimethylaminopyridine are dissolved in an organic solvent, reacted in an ice bath, and after returning to room temperature, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is added to the above mixture. After the reaction is completed, the crude product is separated and purified by column chromatography to obtain a metal complex; or under an inert atmosphere, celastrol, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine are dissolved in an organic solvent, the metal complex precursor is added thereto, and the mixture is stirred overnight. After the reaction, the crude product is separated and purified by column chromatography to obtain a metal complex.

[0023] Wherein, in step (2), the molar ratio of the metal complex precursor, the immunoactive molecule betulin, 4-dimethylaminopyridine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1:1:1-1.5; the molar ratio of celastrol, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine to the metal complex precursor is 19:23:23:19-20.

[0024] Wherein, the bipyridine ligand is 4-methyl-4'-aminomethyl-2,2'-bipyridine or 4-methyl-4'-carboxyl-2,2'-bipyridine.

[0025] The structure of 4-methyl-4'-aminomethyl-2,2'-bipyridine is:

[0026] It was prepared using the synthesis method reported in the literature Bioorthogonal “Labeling after Recognition” Affording an FRET-Based Luminescent Probe for Detecting and Imaging Caspase-3 via Photoluminescence Lifetime Imaging.

[0027] The structure of 4-methyl-4'-carboxyl-2,2'-bipyridine is:

[0028] It was prepared using the synthesis method reported in the literature Synthesis and Characterization of Oligoproline-Based Molecular Assemblies for Light Harvesting.

[0029] The metal dimer of the present invention is dichlorobis(4-methylisopropylphenyl)ruthenium(II) dimer ([Ru(η 6 -p-cymene)Cl2]2), dichlorobis(biphenylruthenium(II) dimer ([Ru(η 6 -p-bip)Cl2]2), dichloro(pentamethylcyclopentadienyl)iridium(III) dimer ([Ir(η 5 -Cp * )Cl2]2) and dichloro(phenyltetramethylcyclopentadienyl)iridium(III) dimer ([Ir(η 5 -Cp xph )Cl2]2).

[0030] The structural formulas of the above metal dimers are

[0031] Metal dimer [Ru(η 6 -p-cymene)Cl2]2 were purchased from Aladdin Biochemical Co., Ltd., with CAS number 52462-29-0; [Ru(η 6 -p-bip)Cl2]2 was purchased from Leyan Reagent Co., Ltd., with CAS number 128972-45-2; [Ir(η 5 -Cp * )Cl2]2 was purchased from Xiens Biochemical Technology Co., Ltd., with a CAS number of 12354-84-6; [Ir(η 5 -Cp xph)Cl2]2 was prepared by the synthesis method reported in the literature Regiodivergent Iridium(III)-Catalyzed Diamination of AlkenylAmides with Secondary Amines: Complementary Access toγ-orδ-Lactams.

[0032] Application of the metal complex tailed with an immunologically active molecule in the preparation of anti-tumor drugs or anti-tumor drug components.

[0033] The mechanism of the metal complex synthesis method of the present invention is:

[0034] (1) The metal dimer is coordinated with an organic compound tailed with an immunologically active molecule:

[0035]

[0036] The organic compound Bet-Bpy or the organic compound Cel-Bpy.

[0037] (2) Synthesized from metal complex precursors and immunoactive molecules betulin or tripterygium wilfordii:

[0038]

[0039] in, is p-cymene, biphenyl, pentamethylcyclopentadiene or phenyltetramethylcyclopentadiene; M is Ru or Ir; R2 is -COOH or -NH2;

[0040]

[0041] Beneficial effects: The metal complex tailed with an immunoactive molecule of the present invention exhibits significant anti-cancer activity. On the one hand, it exerts cytotoxicity against rapidly growing cancer cells through active ingredients with anti-tumor immune efficacy. On the other hand, it can also induce immunogenic cell death, thereby enabling the host immune system to attack tumor cells and induce tumor-specific immune responses (the metal complex of the present invention has been proven to effectively induce immunogenic cell death in non-small cell lung cancer cells and successfully activate anti-tumor immunity in mice in vaccine experiments). Therefore, the metal complex of the present invention has outstanding effects in anti-cancer, anti-inflammatory, inducing cell immunogenic death and achieving anti-tumor immunity, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1Betulin-tailed arylruthenium (Bet-Ru) or iridium (Bet-Ir) complexes induce immunogenic cell death in non-small cell lung cancer cells;

[0043] Figure 2 These are photos of the corresponding mice's tumors after the vaccine experiment;

[0044] Figure 3 This is the change in tumor volume of the corresponding mice after the vaccine experiment;

[0045] Figure 4 This is the weight change of the corresponding mice after the vaccine experiment;

[0046] Figure 5 These are the results of hematoxylin and eosin staining of tumor sections after the vaccine experiment. DETAILED DESCRIPTION

[0047] The technical solution of the present invention is further described below in conjunction with specific embodiments.

[0048] Example 1

[0049] The organic compound Bet-Bpy of the present invention is prepared by the following method, specifically:

[0050]

[0051] 0.25 mmol of betulin, 0.25 mmol of 4-methyl-4'-carboxy-2,2'-bipyridine, and 0.25 mmol of 4-dimethylaminopyridine were placed in a vacuum reaction flask filled with argon. The mixture was dissolved in 10 mL of N,N-dimethylformamide and rapidly stirred in an ice bath for 15 minutes. After returning to room temperature, 0.25 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added to the mixture, and stirring was continued for 24 hours. After completion of the reaction, the reaction solution was removed by vacuum rotary evaporation, and the crude product was purified by flash column chromatography to obtain a yellow powder product with a yield of 85%, which was named Bet-Bpy. 1 H NMR(400MHz,DMSO-d6)(ppm)δ8.90(s,1H),8.83(s,1H),8.59(s,1H),8.28(s,1H),7.91(s,1H),7. 34(s,1H),4.75(s,1H),4.67(s,2H),4.54(s,2H),2.44(s,3H),0.5-3.0(m,44H,6CH3,10CH2,6CH).

[0052] Example 2

[0053] The organic compound Cel-Bpy of the present invention is prepared by the following method, specifically:

[0054]

[0055] 0.38 mmol of tripterygium wilfordii, 0.46 mmol of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, and 0.46 mmol of N,N-diisopropylethylamine were dissolved in 10 mL of N,N-dimethylformamide, and the mixture was stirred at room temperature for 30 minutes. Then, 0.38 mmol of 4-methyl-4'-aminomethyl-2,2'-bipyridine was added and stirred overnight. After the reaction was completed, the solution was diluted with dichloromethane, then washed with water, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude product was further purified by column chromatography to obtain a red powder product with a yield of 77%, which was named Cel-Bpy. 1 H NMR (400MHz, CDCl3) (ppm) δ8.12 (dd, J = 7.5, 0.6Hz, 1H), 7.96-7.93 (m, 1H), 7.71 (s, 1H), 7. 55(d,J=1.6Hz,1H),7.52(d,J=1.7Hz,1H),7.17(dd,J=7.5,1.5Hz,1H),6.96(d,J=6.8Hz,1 H),6.42(s,1H),6.34(d,J=6.8Hz,1H),2.37(s,3H),2.17(s,3H),1.99-1.40(m,15H,7CH2, 1CH), 1.46 (s, 3H), 1.14 (d, J = 1.5Hz, 3H), 1.04 (d, J = 1.5Hz, 3H), 0.99 (s, 3H), 0.93 (s, 3H).

[0056] Example 3

[0057] The method for preparing the metal complex tailed with an immunologically active molecule of the present invention comprises: performing a coordination reaction between the organic compound Bet-Bpy tailed with an immunologically active molecule and a metal dimer to obtain the metal complex, specifically:

[0058]

[0059] Under argon protection, 0.25 mmol of metal dimer [Ru(η 6 -p-cymene)Cl2]2, [Ru(η 6 -p-bip)Cl2]2, [Ir(η 5 -Cp * )Cl2]2 or [Ir(η 5 -Cp xph)Cl2]2 and 0.50 mmol of the organic compound Bet-Bpy were dissolved in 10 mL of dichloromethane, and the reaction mixture was refluxed with stirring at 50°C for 12 hours. After the reaction was completed, the solvent was removed by vacuum rotary evaporation, and the crude product was purified by flash column chromatography. The products obtained based on the above four metal dimers were named Bet-Ru, Bet-bipRu, Bet-Ir, and Bet-xphIr, and the yields of the four products were 75%, 68%, 78%, and 72%, respectively.

[0060] Bet-Ru: 1 H NMR (400MHz, DMSO-d6) (ppm) δ9.72 (d, J = 5.9Hz, 1H), 9.39

[0061] (d,J=5.8Hz,1H),8.91(s,1H),8.78(s,1H),8.12-8.02(m,1H),7.69(d,J=6.6Hz,1H),6.2 4(dd,J=13.1,6.2Hz,2H),6.00(dd,J=10.5,6.3Hz,2H),4.77-4.67(m,2H),4.60(s,1H),4 .31(d,J=4.6Hz,1H),4.14(dd,J=10.7,7.3Hz,1H),3.02-2.94(m,1H),2.59(s,3H),2.17( s,3H),0.95(d,J=6.9Hz,6H),3.00-0.50(m,44H,6CH3,10CH2,6CH).ESI-MS:[Bet-Ru-Cl] + Theoretical value: m / z = 909.66, actual value: m / z = 909.67.

[0062] Bet-bipRu: 1H NMR(400MHz,DMSO-d6)(ppm)δ8.98(s,1H),8.96(s,1H),8.64(s,1H),8.51(s,1H),8.47(s,1H),8.44(s,1H),7.38-7.35(m,4H),7.35( s,1H),7.31-7.29(m,7H),4.85(d,J=12.4Hz,1H),4.75(d,J=12.4Hz,1H),4.26(d,J=12.3Hz,1H),4.23(d,J=12.5Hz,1H),4.15(s,1H), 3.61(s,6H),3.32(s,1H),2.38(s,3H),2.28(s,1H),1.71(s,3H),1.64(d,J=13.3Hz,1H),1.57(s,1H),1.55(s,1H),1.48(s,1H),1.39( s,3H),1.38(s,2H),1.14(d,J=12.3Hz,1H),1.00(s,3H),0.96(s,3H),0.92(s,3H),0.87(s,3H),0.84(s,3H).ESI-MS:[Bet-bipRu-Cl] + Theoretical value: m / z = 945.43, actual value: m / z = 945.71.

[0063] Bet-Ir: 1 H NMR (400MHz, CDCl3) (ppm) δ9.18 (t, J = 5.6Hz, 1H), 8.92-8.77

[0064] (m,2H),8.40-8.25(m,2H),7.82(d,J=3.6Hz,1H),4.79-4.59(m,3H),4.22(dd,J=15.7,11.3Hz,1H),3.20(dd ,J=10.5,3.9Hz,1H),2.74(s,3H),1.81(s,15H),3.00-0.50(m,44H,6CH3,10CH2,6CH).ESI-MS:[Bet-Ir-Cl] + Theoretical value: m / z = 1001.82, actual value: m / z = 1001.83.

[0065] Bet-xphIr: 1 H NMR (400MHz, DMSO-d6) (ppm) δ7.97 (d, J = 8.3Hz, 2H), 7.63

[0066] (d,J=0.9Hz,1H),7.51(s,1H),7.45(d,J=11.2Hz,2H),7.35(d,J=1.0Hz,1H),7.33-7.19(m,6H),4.85(d,J =12.4Hz,1H),4.75(d,J=12.4Hz,1H),4.24(d,J=5.2Hz,2H),4.15(d,J=5.0Hz,1H),3.61(s,6H),3.32(d,J= 5.0Hz,1H),2.38(s,3H),2.30(s,1H),1.84(s,6H),1.77(s,6H),1.71-1.62(m,4H),1.62-1.30(m,18H),1. 15(d,J=12.8Hz,1H),0.98(d,J=14.3Hz,6H),0.92(s,3H),0.85(d,J=14.3Hz,6H).ESI-MS:[Bet-xphIr-Cl] + Theoretical value: m / z = 1079.54, actual value: m / z = 1079.95. Example 4

[0067] The method for preparing the metal complex tailed with an immunologically active molecule of the present invention comprises: performing a coordination reaction between the organic compound Cel-Bpy tailed with an immunologically active molecule and a metal dimer to obtain the metal complex, specifically:

[0068]

[0069] Under argon protection, 0.25 mmol of metal dimer [Ru(η 6 -p-cymene)Cl2]2, [Ru(η 6 -p-bip)Cl2]2, [Ir(η 5 -Cp * )Cl2]2 or [Ir(η 5 -Cp xph )Cl2]2 and 0.50 mmol of the organic compound Cel-Bpy were dissolved in 10 mL of dichloromethane, and the reaction mixture was refluxed with stirring at 50°C for 12 hours. After the reaction was completed, the solvent was removed by vacuum rotary evaporation, and the crude product was purified by flash column chromatography. The products obtained based on the above four metal dimers were named Cel-Ru, Cel-bipRu, Cel-Ir, and Cel-xphIr, and the yields of the four products were 81%, 63%, 76%, and 75%, respectively.

[0070] Cel-Ru: 1 H NMR (400MHz, CDCl3) (ppm) δ8.46-8.43 (m, 1H), 8.34 (d, J=

[0071] 7.5Hz,1H),7.99-7.98(m,1H),7.53(dd,J=7.5,1.5Hz,2H),7.16(dd,J=7.6,1.0Hz,2H),7.13(dd,J=7.5,1.5Hz,1H),7.10(dq,J=7.6,1.0H z,2H),6.98(d,J=6.8Hz,1H),6.42(s,1H),6.34(d,J=6.8Hz,1H),2.97-2.89(m,1H),2.34(s,3H),2.31(t,J=1.0Hz,3H),2.19(s,3H),1.96 -1.45(m,15H,7CH2,1CH),1.43(s,3H),1.05(d,J=1.4Hz,3H),0.99(s,6H),0.96(d,J=1.5Hz,3H),0.94(d,J=6.9Hz,6H).ESI-MS:[Cel-Ru-Cl] + Theoretical value: m / z = 888.34, actual value: m / z = 888.50.

[0072] Cel-bipRu: 1 H NMR (400MHz, DMSO-d6) (ppm) δ8.89 (s, 1H), 8.33 (d, J = 8.7

[0073] Hz,1H),8.25(d,J=9.1Hz,1H),7.98(t,J=8.7Hz,1H),7.64-7.59(m,1H),7.54-7.45(m,3H),7.29(dd,J =9.1,2.3Hz,1H),7.17-7.08(m,2H),6.76(d,J=6.8Hz,1H),6.23(s,1H),6.11(tt,J=6.8,2.0Hz,2H),5 .18-5.09(m,4H),4.73-4.65(m,4H),4.38(d,J=8.7Hz,2H),2.40(s,3H),2.25(s,3H),2.10(dddt,J=8. 8,7.4,2.9,1.4Hz,1H),1.96-1.38(m,17H),1.08(s,3H),1.03-0.94(m,12H).ESI-MS:[Cel-bipRu-Cl] + Theoretical value: m / z = 1000.35, actual value: m / z = 1001.13.

[0074] Cel-Ir: 1H NMR (400MHz, CDCl3) (ppm) δ8.11 (dd, J=7.5, 0.6Hz, 1H), 7.99-

[0075] 7.98(m,1H),7.71(s,1H),7.54(d,J=1.6Hz,1H),7.52(d,J=1.7Hz,1H),7.13(dd,J=7.5,1.5Hz,1H),6.98 (d,J=6.8Hz,1H),6.42(s,1H),6.34(d,J=6.8Hz,1H),2.35(s,3H),2.27-2.24(s,15H),2.17(s,3H),1.96 -1.43(m,15H,7CH2,1CH),1.46(s,3H),1.14(d,J=1.5Hz,3H),1.04(d,J=1.5Hz,3H),0.99(s,3H),0.93(s,3H).ESI-MS:[Cel-Ir-Cl] + Theoretical value: m / z = 980.41, actual value: m / z = 980.72.

[0076] Cel-xphIr: 1 H NMR (400MHz, DMSO-d6) (ppm) δ8.89 (s, 1H), 8.33 (d, J = 8.7

[0077] Hz,1H),8.25(d,J=9.1Hz,1H),7.98(t,J=8.7Hz,1H),7.64-7.59(m,1H),7.54-7.45(m,3H),7.29(dd,J=9.1,2.3Hz,1H ),7.17-7.08(m,2H),6.76(d,J=6.8Hz,1H),6.23(s,1H),6.00(tt,J=6.8,2.0Hz,1H),5.61-5.53(m,2H),5.47-5.38(m ,2H),4.38(d,J=8.7Hz,2H),2.40(s,3H),2.25(s,3H),2.09(ddp,J=10.4,8.8,1.4Hz,1H),1.91-1.88(m,2H),1.88-1. 84(m,2H),1.84-1.75(m,2H),1.75-1.40(m,11H),1.13(d,J=9.5Hz,12H),1.10-0.94(m,15H).ESI-MS:[Cel-xphIr-Cl] + Theoretical value: m / z = 1134.47, actual value: m / z = 1135.37.

[0078] Example 5

[0079] The method for preparing the metal complex tailed with an immunologically active molecule of the present invention first reacts a metal dimer with a bipyridine ligand to obtain a metal complex precursor, specifically:

[0080]

[0081] Under argon protection, 0.25 mmol of metal dimer [Ru(η 6 -p-cymene)Cl2]2, [Ru(η 6 -p-bip)Cl2]2, [Ir(η 5 -Cp * )Cl2]2 or [Ir(η 5 -Cp xph )Cl2]2 and 0.50 mmol 4-methyl-4'-carboxy-2,2'-bipyridine were dissolved in 10 mL of dichloromethane, and the reaction mixture was refluxed with stirring at 50°C for 12 hours. After the reaction was completed, the solvent was removed by vacuum rotary evaporation, and the crude product was purified by flash column chromatography. The products obtained based on the above four metal dimers were named Ru-COOH, bipRu-COOH, Ir-COOH, and xphIr-COOH, and the yields of the four products were 79%, 85%, 83%, and 78%, respectively.

[0082] Ru-COOH: 1 H NMR(400MHz, DMSO-d6)(ppm)δ9.65(d,J=5.7Hz,1H),9.38(d,J=5.8Hz,1H),8.87(s,1H),8.76(s,1H),8.04(s,1H),7.66(d,J=5.5Hz,1H),6 .23(d,J=2.3Hz,2H),5.99(t,J=6.5Hz,2H),2.58(s,3H),2.55(d,J=3.4Hz,1H),2.18(s,3H),0.94(d,J=5.6Hz,6H).ESI-MS:[Ru-COOH-Cl] + Theoretical value: m / z = 485.06, actual value: m / z = 485.10.

[0083] bipRu-COOH: 1H NMR(400MHz, DMSO-d6)(ppm)δ12.79(s,1H),8.54-8.49(m,1H),8.45-8.38(m,1H),8.36(d,J=7.5Hz,1H),7.75-7.68(m,2H),7.68-7.64(m,2H) ,7.61-7.49(m,6H),7.48-7.39(m,3H),7.34(ddt,J=8.1,6.3,1.5Hz,2H),7.25(dd,J=7.5,1.5Hz,1H),2.35(s,3H).ESI-MS:[bipRu-COOH-Cl] + Theoretical value: m / z = 505.03, actual value: m / z = 504.96.

[0084] Ir-COOH: 1 H NMR(400MHz,DMSO-d6)(ppm)δ9.04(d,J=5.7Hz,1H),8.97(s,1H),8.87(s,1H),8.81(d,J=5.8Hz,1 H),8.12(d,J=5.5Hz,1H),7.70(d,J=5.4Hz,1H),2.64(s,3H),1.65(s,15H).ESI-MS:[Ir-COOH-Cl] + Theoretical value: m / z = 577.12, actual value: m / z = 577.20.

[0085] xphIr-COOH: 1 H NMR(400MHz, DMSO-d6)(ppm)δ12.77(s,1H),8.52(d,J=1.4Hz,1H),8.32-8.26(m,1H),7.77-7.68(m,2H),7.59(d,J=1.5Hz,1H),7.51(s, 1H),7.48-7.29(m,6H),7.23(dd,J=7.5,1.5Hz,1H),2.52(d,J=0.8Hz,6H),2.36(s,3H),2.15(d,J=0.8Hz,6H).ESI-MS:[xphIr-COOH-Cl] + Theoretical value: m / z = 639.14, actual value: m / z = 639.19.

[0086] Example 6

[0087] The method for preparing the metal complex tailed with an immunologically active molecule of the present invention comprises: performing a coordination reaction between a metal complex precursor and the immunologically active molecule betulin to obtain the metal complex, specifically:

[0088]

[0089] 0.23 mmol of betulin, 0.23 mmol of the metal carboxylic acid precursor prepared in Example 5, and 0.23 mol of 4-dimethylaminopyridine were placed in a vacuum reaction flask filled with argon. The mixture was dissolved in 10 mL of N,N-dimethylformamide and rapidly stirred in an ice bath for 15 minutes. After returning to room temperature, 0.23 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added to the mixture, and stirring was continued for 24 hours. After the reaction was completed, the reaction solution was removed by vacuum rotary evaporation, and the crude product was purified by flash column chromatography to obtain the same metal complexes Bet-Ru, Bet-bipRu, Bet-Ir, and Bet-xphIr as in Example 3. The yields of the four products were 84%, 80%, 87%, and 79%, respectively.

[0090] Example 7

[0091] The method for preparing the metal complex tailed with an immunologically active molecule of the present invention first reacts a metal dimer with a bipyridine ligand to obtain a metal complex precursor, specifically:

[0092]

[0093] Under argon protection, 0.30 mmol of metal dimer [Ru(η 6 -p-cymene)Cl2]2, [Ru(η 6 -p-bip)Cl2]2, [Ir(η 5 -Cp * )Cl2]2 or [Ir(η 5 -Cp xph )Cl2]2 and 0.60mmol 4-methyl-4'-aminomethyl-2,2'-bipyridine were dissolved in 10mL of dichloromethane, and the reaction mixture was refluxed with stirring at 50°C for 12 hours. After the reaction was completed, the solvent was removed by vacuum rotary evaporation, and the crude product was purified by flash column chromatography. The products obtained based on the above four metal dimers were named Ru-NH2, bipRu-NH2, Ir-NH2, and xphIr-NH2, and the yields of the four products were 88%, 84%, 81%, and 85%, respectively.

[0094] Ru-NH2: 1H NMR(400MHz, CDCl3)(ppm)δ8.42-8.38(m,1H),8.38-8.36(m,1H),7.63-7.62(m ,1H),7.52(s,1H),7.30(dd,J=7.6,1.6Hz,1H),7.18-7.15(m,2H),7.12-7.10( m,1H),7.10-7.07(m,2H),3.94(t,J=6.7Hz,2H),2.98-2.89(m,1H),2.40(d,J= 0.9Hz, 3H), 2.31 (t, J = 1.0Hz, 3H), 0.94 (d, J = 6.8Hz, 6H). ESI-MS: [Ru-NH2-Cl] + Calculated value: m / z = 470.09, actual value: m / z = 470.10.

[0095] bipRu-NH2: 1 H NMR(400MHz,DMSO-d6)(ppm)δ8.38-8.30(m,2H),7.73-7.65(m,4H),7.59(d ,J=1.4Hz,1H),7.52(d,J=1.3Hz,2H),7.49-7.39(m,5H),7.38-7.32(m,2H) ,7.32-7.26(m,1H),7.14(dd,J=7.6,1.6Hz,1H),6.09(q,J=6.8Hz,1H),5.9 3(q,J=6.8Hz,1H),4.29-4.12(m,2H),2.40(s,3H).ESI-MS:[bipRu-NH2-Cl] + Calculated value: m / z = 490.06, actual value: m / z = 489.99.

[0096] Ir-NH2: 1 H NMR (400MHz, CDCl3) (ppm) δ8.16-8.13 (m, 1H), 7.74 (d, J = 7.4

[0097] Hz,1H),7.57(t,J=1.2Hz,1H),7.51(d,J=1.3Hz,1H),7.31(dd,J=7.5,1.5Hz,1H),7.17-7.12( m,1H),3.95(t,J=6.7Hz,2H),2.36(d,J=0.9Hz,3H),2.30-2.26(m,15H).ESI-MS:[Ir-NH2-Cl] + Theoretical value: m / z = 562.16, actual value: m / z = 562.15.

[0098] xphIr-NH2: 1 H NMR(400MHz, DMSO-d6)(ppm)δ8.24-8.19(m,1H),8.14(dd,J=7.7,1.9Hz,2H),7.57(d,J=1.4Hz,1H),7.51(s,1H),7.49-7.29(m,7H),7.24(dd,J=7. 5,1.7Hz,1H),5.96(dq,J=24.7,6.9Hz,2H),4.28-4.10(m,2H),2.52(d,J=0.8Hz,6H),2.35(s,3H),2.15(d,J=0.8Hz,6H).ESI-MS:[xphIr-NH2-Cl] + Theoretical value: m / z = 624.18, actual value: m / z = 624.23.

[0099] Example 8

[0100] The method for preparing the metal complex tailed with an immunologically active molecule of the present invention comprises: obtaining the metal complex by a coordination reaction between a metal complex precursor and the immunologically active molecule tripterine, specifically:

[0101]

[0102] 0.38 mmol of celastrol, 0.46 mmol of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and 0.46 mmol of N,N-diisopropylethylamine were dissolved in 10 mL of N,N-dimethylformamide; the mixture was stirred at room temperature for 30 minutes, and then 0.38 mmol of the metal amino precursor prepared in Example 7 was added and stirred overnight; after the reaction was completed, the solution was diluted with dichloromethane, then washed with water, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude product was further purified by column chromatography to obtain the same metal complexes Cel-Ru, Cel-bipRu, Cel-Ir, and Cel-xphIr as in Example 4, with yields of 80%, 74%, 79%, and 72%, respectively.

[0103] The invention relates to the application of the metal complex in the preparation of anti-tumor drugs.

[0104] Methods: The cytotoxicity of the metal complexes was evaluated using the MTT assay performed as follows. Human non-small cell lung cancer cells (A549), human lung cancer resistant cell line (A549R), mouse lung cancer cells (LLC), human ovarian cancer cells (A2780), human liver cancer cells (HepG2), and human breast cancer cells (MDA-MB-231) were cultured at 5×10 3The cells / well were seeded in a 96-well plate and cultured in a 5% CO2 incubator at 37°C for 24 hours. Three parallel comparative experiments were set up, with five concentrations of the test sample, and the incubation continued for 48 hours. 20 μL of MTT solution (5 mg / mL) was added to each well. After incubation for 4 hours, 150 μL of DMSO was added to each well to dissolve the purple crystals formed. The absorbance of each well at 490 nm was measured by a microplate reader (LabServ K3), and the IC was calculated accordingly. 50 value.

[0105] The anticancer activities of the metal complexes Bet-Ru and Bet-Ir prepared in Example 3 or Example 6 are shown in Table 1.

[0106] Table 1 shows the IC values ​​of the metal complexes Bet-Ru and Bet-Ir, the metal complex precursors Ru-COOH and Ir-COOH, the organic compound Bet-Bpy, betulin, and cisplatin (CCDP). 50 (μM) value

[0107]

[0108] Results showed that the betulin-tailed organic compound Bet-Bpy exhibited moderate anticancer activity against non-cisplatin-resistant lung cancer cells, but showed little activity against cisplatin-resistant cell lines. Furthermore, the betulin-tailed metal complexes Bet-Ru and Bet-Ir exhibited significantly higher antitumor activity against several tumor cell lines than the immunoreactive molecule itself or the metal complex precursor. Furthermore, the metal complexes Bet-Ru and Bet-Ir also exhibited superior anticancer activity against several tumor cell lines, particularly against the drug-resistant lung cancer strain A549R. These results demonstrate that the immunoreactive molecule-based metal complexes Bet-Ru and Bet-Ir can achieve superior anticancer efficacy while effectively reducing the dosage.

[0109] The anticancer activities of the metal complexes Cel-Ru and Cel-Ir prepared in Example 4 or Example 8 are shown in Table 1.

[0110] Table 2 shows the IC values ​​of the metal complexes Cel-Ru and Cel-Ir, the metal complex precursors Ru-NH2 and Ir-NH2, the organic compound Cel-Bpy, Celastrol, and cisplatin (CCDP). 50 (μM) value

[0111]

[0112] The results showed that the organic compound Cel-Bpy, tailed with tripterine, exhibited excellent antitumor activity, reaching nanomolar levels. The metal complexes Cel-Ru and Cel-Ir, tailed with tripterine, exhibited antitumor activity comparable to that of the immunoreactive molecules, or even significantly higher than that of the metal complex precursors, against several tumor cell lines. Furthermore, the metal complexes Cel-Ru and Cel-Ir also demonstrated superior anticancer activity compared to cisplatin against several tumor cell lines, particularly against MDA-MB-231 cells, which are prone to chemotherapy resistance.

[0113] The metal complex of the present invention is used for inducing immunogenic cell death.

[0114] Methods: (1) Calreticulin (CRT) release: A549 cells were cultured at 2×10 5 Cells were seeded in 6-well plates at a density of 100 μg / well and cultured overnight. Cells were treated with different concentrations of metal complexes Bet-Ru and Bet-Ir for 24 hours. A549 cells were collected and washed twice with PBS, and blocking buffer was added and incubated at room temperature for 30 minutes. Calreticulin antibody diluted with blocking buffer was then added, and after incubation at room temperature for 45 minutes, the cells were washed twice with PBS, and a secondary antibody conjugated to FITC diluted with blocking buffer was added and incubated at room temperature for 45 minutes. After incubation, PBS was added for washing, resuspended, and detected by flow cytometry. Data were analyzed using FlowJo 10 software (FITC: λ ex =488nm,λ em =525nm).

[0115] (2) Release of high mobility group protein B1 (HMGB1): A549 cells were cultured at 2×10 5 Cells were seeded at a density of 100 cells / well in 6-well plates and cultured overnight. Cells were treated with different concentrations of the metal complexes Bet-Ru and Bet-Ir for 24 hours. Cell culture supernatants were extracted and stored on ice. Extracellular high-mobility group protein B1 release in the cell culture supernatants was measured using a human HMGB1 ELISA kit.

[0116] (3) Release of adenosine triphosphate (ATP): A549 cells were cultured at a rate of 2×10 5 Cells were seeded at a density of 100 cells / well in 6-well plates and cultured overnight. Cells were treated with different concentrations of the metal complexes Bet-Ru and Bet-Ir for 24 hours. The cell culture supernatant was extracted and stored on ice. Extracellular adenosine triphosphate levels in the cell culture supernatant were measured using an ATP assay kit.

[0117] Three important signs of immunogenic cell death: the release of calreticulin, the release of high-mobility group protein B1 and the release of adenosine triphosphate. Figure 1 The figure shows the successful induction of immunogenic cell death in non-small cell lung cancer cells by the metal complexes Bet-Ru and Bet-Ir, which are tailed with betulin. As shown in the figure, Bet-Ru and Bet-Ir can effectively enhance the fluorescence intensity of calreticulin antibodies (a), the extracellular secretion of high-mobility group protein B1 (b), and the extracellular release of adenosine triphosphate (aDP) in non-small cell lung cancer cells, demonstrating that the prepared metal complexes can effectively induce immunogenic cell death in vitro.

[0118] The metal complex of the present invention is used for activating anti-tumor immunity in mice in vaccine experiments.

[0119] Methods: C57BL / 6J female mice (6-8 weeks old) were randomly divided into 3 groups, with 10 mice in each group. The mice were immunized by intraperitoneal injection of LLC cells pretreated with PBS, Bet-Ru (0.5 μM), and Bet-Ir (1.0 μM). Untreated LLC cells were then injected subcutaneously in the left axilla of the mice. The tumor volume of the mice was measured and recorded every other day for 17 days. Tumor volume was measured with a caliper (formula: volume = length × width). 2 × 0.5) and nude mouse body weight. At the end of the vaccination experiment, mice were sacrificed, and major organs (including heart, kidney, liver, lung, and spleen) and tumors were obtained and fixed with 4% paraformaldehyde. Samples of major organs were then embedded in paraffin and sectioned. Paraffin sections were stained with hematoxylin and eosin to assess histological changes in tumors and major organs.

[0120] Figure 2 and Figure 3 The images show tumors and changes in tumor volume in the corresponding mice after the vaccine experiment. In the PBS-treated control group, tumors gradually increased, while in the Bet-Ru or Bet-Ir complex-treated groups, tumor volume decreased significantly, indicating that the betulin-tailed metal complexes, Bet-Ru or Bet-Ir, inhibited tumor growth in mice by activating anti-tumor immunity. Figure 4 The results showed that there was no significant change in the weight of mice before and after treatment, proving that vaccine experiments using Bet-Ru or Bet-Ir are very safe and reliable. Figure 5 Figure 3 shows hematoxylin and eosin staining of tumor sections after the vaccine experiment. The results showed that the proportion of dead cells in the Bet-Ru and Bet-Ir groups was significantly higher than that in the control group.

[0121] Experiments have shown that the prepared Bet-Ru and Bet-Ir not only exhibit significant anti-cancer activity against lung cancer cells, but can also effectively induce immunogenic cell death in non-small cell lung cancer cells. They also successfully activated anti-tumor immunity in mice in vaccine experiments.

Claims

1. A metal complex tailed with an immunologically active molecule, characterized in that: Its structural formula is:

2. The method for preparing the metal complex tailed with an immunologically active molecule according to claim 1, characterized in that: The specific steps include: (1) Under an inert atmosphere, betulin, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine, and a bipyridine ligand are dissolved in an organic solvent. After the reaction, the resulting crude product is separated and purified by column chromatography to obtain an organic compound Bet-Bpy modified with a tailed immunoactive molecule; the bipyridine ligand is 4-methyl-4'-carboxyl-2,2'-bipyridine; The structural formula of the 4-methyl-4'-carboxyl-2,2'-bipyridine is: The structural formula of the organic compound Bet-Bpy modified with a tailed immunoactive molecule is: (2) dissolving the aryl / metallocene dimer in an organic solvent under a noble gas atmosphere, adding the organic compound Bet-Bpy tailed with an immunologically active molecule, and stirring under reflux. The crude product after the coordination reaction is separated and purified by column chromatography to obtain a metal complex tailed with an immunologically active molecule; The aryl / metallocene dimer is dichlorobis(4-methylisopropylphenyl)ruthenium(II) dimer [Ru(η 6 -p-cymene)Cl2]2 or dichloro(pentamethylcyclopentadienyl)iridium(III) dimer [Ir(η 5 -Cp * )Cl2]2; Dichlorobis(4-methylisopropylphenyl)ruthenium(II) dimer [Ru(η 6 The structural formula of [-p-cymene)Cl2]2 is: Dichloro(pentamethylcyclopentadienyl)iridium(III) dimer [Ir(η 5 -Cp * )Cl2]2 has the structural formula:

3. The method for preparing a metal complex tailed with an immunologically active molecule according to claim 2, wherein: In step (1), the molar ratio of betulin, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine and 4-methyl-4'-carboxyl-2,2'-bipyridine is 1:1:1:1-1.

5.

4. The method for preparing a metal complex tailed with an immunologically active molecule according to claim 2, wherein: In step (2), the molar ratio of the aryl / metallocene dimer to the organic compound Bet-Bpy tailed with an immunologically active molecule is 1:2 to 2.

5.

5. The method for preparing the metal complex tailed with an immunologically active molecule according to claim 1, characterized in that: The specific steps include: (1) Preparing an aryl / metallocene complex precursor: dissolving an aryl / metallocene dimer in an organic solvent under a noble gas atmosphere, adding a bipyridine ligand, and stirring under reflux. After the reaction, the resulting crude product is separated and purified by column chromatography to obtain an aryl / metallocene complex precursor; the molar ratio of the aryl / metallocene dimer to the bipyridine ligand is 1:2 to 2.5; Wherein, the bipyridine ligand is 4-methyl-4'-carboxyl-2,2'-bipyridine; The structural formula of the 4-methyl-4'-carboxyl-2,2'-bipyridine is: The aryl / metallocene dimer is dichlorobis(4-methylisopropylphenyl)ruthenium(II) dimer [Ru(η 6 -p-cymene)Cl2]2 or dichloro(pentamethylcyclopentadienyl)iridium(III) dimer [Ir(η 5 -Cp * )Cl2]2; Dichlorobis(4-methylisopropylphenyl)ruthenium(II) dimer [Ru(η 6 The structural formula of [-p-cymene)Cl2]2 is: Dichloro(pentamethylcyclopentadienyl)iridium(III) dimer [Ir(η 5 -Cp * )Cl2]2 has the structural formula: The structural formula of the aryl / cyclopentadienyl metal complex precursor is: (2) Under an inert atmosphere, an aryl / cyclopentadienyl metal complex precursor, an immunologically active molecule betulin, and 4-dimethylaminopyridine are dissolved in an organic solvent and reacted in an ice bath. After returning to room temperature, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is added to the mixture. After the reaction is completed, the crude product is separated and purified by column chromatography to obtain a metal complex tailed with an immunologically active molecule.

6. The method for preparing a metal complex tailed with an immunologically active molecule according to claim 5, wherein: In step (2), the molar ratio of the aryl / cyclopentadienyl metal complex precursor, the immunologically active molecule betulin, 4-dimethylaminopyridine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1:1:1 to 1.

5.

7. Use of the metal complex tailed with an immunologically active molecule according to claim 1 in the preparation of anti-tumor drugs.

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