A metal complex of nlg919 and its preparation method and application

Abstract

The application discloses a NLG919 and metal conjugate, a preparation method and application thereof, wherein the metal is iridium or rhenium, and the conjugate is a NLG919 and metal iridium conjugate and a NLG919 and rhenium conjugate. The several conjugates have good anti-tumor activity on 4T1, MDA-MB-231 and MCF-7 cells.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of chemical biology, and particularly relates to an IDO inhibitor NLG919 and a metal conjugate, a preparation method thereof and anti-tumor application. BACKGROUND

[0002] Cyclo-metal Ir(III) complexes have superior optical properties, such as high quantum yield, large Stokes shift, and optical and photochemical properties that can be changed with ligands, and are widely used in biological imaging and sensing. Nowadays, many metal anti-tumor drugs target subcellular organelles such as endoplasmic reticulum, mitochondria and lysosomes, which can effectively avoid the side effects of platinum drugs.

[0003] The main means for clinical treatment of tumors at present are surgery, chemotherapy, radiotherapy, gene therapy, targeted therapy, immunotherapy and the like. Immunotherapy is a promising cancer treatment method, but due to the strong immunosuppressive microenvironment of tumors, the effect of immunotherapy is greatly limited. Indoleamine-2,3-dioxygenase (IDO) is a key immunosuppressive enzyme that regulates tumor immune response and participates in tumor immune escape through multiple pathways. IDO inhibitors can activate T cell-mediated anti-tumor immune response to kill tumor cells through different immune signal pathways. NLG919 is one of the few IDO inhibitors that have entered the clinical research stage. However, the clinical trial results show that the existing IDO inhibitors have limited single-agent efficacy. Research progress shows that the combination of IDO inhibitors and chemotherapy agents can observe a synergistic effect.

[0004] Although platinum drugs represented by cisplatin have made great achievements in anti-tumor, due to their own toxic side effects and drug resistance, their long-term use in clinical has been limited. SUMMARY

[0005] The present application provides an IDO inhibitor NLG919 and a rare metal iridium or rhenium conjugate, a preparation method thereof and research and application in anti-tumor activity. The prepared IDO inhibitor NLG919 and metal iridium / rhenium conjugate have good anti-tumor activity on 4T1, MDA-MB-231 and MCF-7 cell lines.

[0006] The technical scheme of the present application is as follows:

[0007] A NLG919 and metal conjugate, the metal being iridium or rhenium, the structural formula of the NLG919 and metal iridium conjugate or the NLG919 and rhenium conjugate being as follows:

[0008]

[0009] Among them:

[0010]

[0011] Anion is PF6 - .

[0012] Specifically, the NLG919 and metal iridium or rhenium conjugate has the following complex structure:

[0013]

[0014] The present application also provides a preparation method of the IDO inhibitor NLG919 and metal iridium conjugate, and the specific steps are as follows:

[0015] S1. IrCl3·3H2O and C^N ligand I are reacted in a mixed solvent of ethylene glycol ether and deionized water under high temperature and nitrogen protection, and then the reaction product iridium precursor [Ir(C^N)2Cl]2 is obtained by cooling, filtering, washing and drying; [Ir(C^N)2Cl]2 is further reacted with silver hexafluorophosphate (AgPF6) in acetonitrile under heating and nitrogen protection, and then the filtrate is filtered by diatomite after cooling, and the solid separated by dropwise adding the filtrate into ether is the Ir(III) intermediate [Ir(C^N)2(CH3CN)2]PF6;

[0016] S2. [Ir(C^N)2(CH3CN)2]PF6 and NLG919 are reacted in a mixed solvent of methanol and dichloromethane under heating and N2 protection, and then the solid is obtained by rotary evaporation and concentration, the solid is dissolved in a small amount of dichloromethane, and then the dichloromethane / methanol is used as an eluent for separation and purification by silica gel column chromatography, and then NLG919 and metal Ir(III) conjugate Ir-NLG-1 and Ir-NLG-2 are obtained by drying.

[0017] In step S1, the molar ratio of IrCl3·3H2O and C^N ligand I is 1:2-2.2, and the C^N ligand I is 2-(2-thiophene)pyridine (thpy) or 2-(2,4-difluorophenyl)pyridine (dfppy); the high temperature reaction condition is that the temperature is 110-135℃, the time is 20-24h, and the mixed solvent of ethylene glycol ether and deionized water is obtained by mixing ethylene glycol ether and deionized water in a volume ratio of 3:1; the molar ratio of [Ir(C^N)2Cl]2 and silver hexafluorophosphate (AgPF6) is 1:2-2.5; and the heating reaction condition is that the temperature is 80-85℃, and the time is 20-24h.

[0018] In step S2, the molar ratio of [Ir(C^N)2(CH3CN)2]PF6 and NLG919 is 1:3-8, and the heating reaction condition is that the temperature is 65-69℃, and the time is 5.5-6h; and the mixed solvent of methanol and dichloromethane is obtained by mixing methanol and dichloromethane in a volume ratio of 1:1.

[0019] The present application also provides a preparation method of the IDO inhibitor NLG919 and metal rhenium conjugate, and the specific steps are as follows:

[0020] (1) Re(CO)5Cl and N^N ligand II are heated to react to obtain Re(N^N)(CO)3Cl in toluene solvent under nitrogen protection, and Re(N^N)(CO)3Cl is heated to react with silver trifluoromethyl sulfonate (AgOTf) in acetonitrile solvent under nitrogen protection to carry out dechlorination reaction; a large amount of saturated NH4PF6 aqueous solution is added to obtain solid [Re(N^N)(CO)3(CH3CN)]PF6;

[0021] (2) [Re(N^N)(CO)3(CH3CN)]PF6 and NLG919 are heated to react in tetrahydrofuran solvent under N2 protection, and the solid obtained by rotary evaporation is dissolved in a small amount of dichloromethane, and then purified by silica gel column chromatography with dichloromethane / methanol as eluent, and dried to obtain NLG919 and metal rhenium conjugates Re-NLG-1 and Re-NLG-2.

[0022] In step (1), the molar ratio of Re(CO)5Cl and N^N ligand II is 1:1-1.2, N^N ligand II is 4,7-diphenyl-1,10-phenanthroline (dip) or 1,10-phenanthroline (phen), and the heating reaction conditions are: temperature 100-110℃, time 6-8h; the molar ratio of Re(N^N)(CO)3Cl and silver trifluoromethyl sulfonate (AgOTf) is 1:1-1.2, and the heating dechlorination reaction conditions are: temperature 80-85℃, time 20-24h.

[0023] In step (2), the molar ratio of [Re(N^N)(CO)3(CH3CN)]PF6 and NLG919 is 1:2.5-3, and the heating reaction conditions are: temperature 60-66℃, time 5.5-6h.

[0024] The present application also provides the application of the IDO inhibitor NLG919 and metal iridium or rhenium conjugate in antitumor drugs, and the IDO inhibitor NLG919 and metal iridium conjugate and NLG919 and rhenium conjugate have good antitumor activity on 4T1, MDA-MB-231 and MCF-7 cell lines.

[0025] The present application forms a coordination compound by combining NLG919 with metal iridium and rhenium, so as to improve the chemotherapeutic and immunotherapeutic effects of the compound. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 It is a synthetic route diagram of the NLG919 and metal iridium / rhenium conjugate of the present application.

[0027] Figure 2 ESI-HRMS spectrum of Ir-NLG-1 in Example 1;

[0028] Figure 3 ESI-HRMS spectrum of Ir-NLG-1 in Example 1; 1 HNMR spectrum;

[0029] Figure 4 ESI-HRMS spectrum of Ir-NLG-1 in Example 1; 13 CNMR spectrum;

[0030] Figure 5 ESI-HRMS spectrum of Ir-NLG-2 in Example 2;

[0031] Figure 6 ESI-HRMS spectrum of Ir-NLG-2 in Example 2; 1 HNMR spectrum;

[0032] Figure 7 ESI-HRMS spectrum of Ir-NLG-2 in Example 2; 13 CNMR spectrum;

[0033] Figure 8 ESI-HRMS spectrum of Re-NLG-1 in Example 3;

[0034] Figure 9 ESI-HRMS spectrum of Re-NLG-1 in Example 3; 1 HNMR spectrum;

[0035] Figure 10 ESI-HRMS spectrum of Re-NLG-1 in Example 3; 13 CNMR spectrum;

[0036] Figure 11 ESI-HRMS spectrum of Re-NLG-2 in Example 4;

[0037] Figure 12 ESI-HRMS spectrum of Re-NLG-2 in Example 4; 1 HNMR spectrum;

[0038] Figure 13 ESI-HRMS spectrum of Re-NLG-2 in Example 4; 13 CNMR spectrum;

[0039] Figure 14 Uptake of complexes by 4T1 cells in Example 6. DETAILED DESCRIPTION

[0040] The following further illustrates the IDO inhibitor NLG919 and metal iridium / rhenium conjugate, its preparation method and anti-tumor application with reference to the accompanying drawings and specific examples, but the examples do not limit the present application in any way. Unless otherwise specified, the reagents and methods used in the examples are commonly used reagents in the art, which can be purchased on the market or prepared by conventional methods.

[0041] Example 1

[0042] The synthesis of metal Ir(III) complex Ir-NLG-1 is shown in the following process route, and the specific steps are as follows: Figure 1

[0043] (1) Synthesis of precursor [Ir(thpy)2Cl]2:

[0044] IrCl3·3H2O (1.000 g, 2.836 mmol) and 2-(2-thiophene) pyridine thpy (1.005 g, 6.239 mol) were added to 80 mL of a mixed solvent of ethylene glycol ethyl ether and deionized water (3:1, v / v), heated to 135°C under N2protection and stirred for 24 h, cooled and filtered, and the solid-liquid was separated using a suction filtration device, and the solid was washed with water, ethanol and ether in turn, and vacuum dried to obtain the reaction iridium precursor [Ir(thpy)2Cl]2;

[0045] (2) Synthesis of [Ir(thpy)2(CH3CN)2]PF6:

[0046] [Ir(thpy)2Cl]2(0.150 g, 0.136 mmol) and AgPF6(0.073 g, 0.287 mmol) were added to 60 mL of acetonitrile solvent, heated to 85°C under N2protection and stirred for 24 h, cooled to room temperature, and a Buchner funnel was filled with diatomite to remove AgCl by filtration, and the filtrate was concentrated to 3-5 mL under reduced pressure at 45°C, then added dropwise to a large amount of ether to precipitate the solid, and vacuum dried to obtain [Ir(thpy)2(CH3CN)2]PF6;

[0047] (3) Synthesis of Ir-NLG-1:

[0048] ​[Ir(thpy)2(CH3CN)2]PF6(0.150 g, 0.203 mmol) and NLG919 (0.229 g, 0.811 mmol) were added to 60 mL of a mixture of methanol and dichloromethane (1:1, v / v) and stirred at 69 °C under N2protection for 6 h. The reaction mixture was concentrated by rotary evaporation at 45 °C to give a solid, which was dissolved in a small amount of dichloromethane and purified by column chromatography on silica gel using dichloromethane / methanol (200:1, v / v) as eluent to give Ir-NLG-1, yield: 0.126 g, 62.3%.

[0049] Figure 2 ESI-HRMS spectrum of Ir-NLG-1 in Example 1, Figure 3 ESI-HRMS spectrum of Ir-NLG-1 in Example 1, 1 HNMR spectrum of Ir-NLG-1 in Example 1, Figure 4 ESI-HRMS spectrum of Ir-NLG-1 in Example 1, 13 CNMR spectrum of Ir-NLG-1 in Example 1, from which it can be seen that: 1 H NMR (500 MHz, DMSO-d6) δ 8.62 (d, J = 5.8 Hz, 1H), 8.57 (d, J = 5.7 Hz, 1H), 8.08 (d, J = 6.2 Hz, 2H), 7.91 - 7.81 (m, 2H), 7.70 - 7.54 (m, 6H), 7.48 (dd, J = 4.7, 1.3 Hz, 2H), 7.37 - 7.30 (m, 4H), 7.22 - 7.16 (m, 2H), 6.95 (d, = 27.5 Hz, 2H), 6.17 (dd, J = 4.7, 2.2 Hz, 2H), 5.39 (q, J = 6.6 Hz, 2H), 4.72 (dd, J = 21.8, 5.4 Hz, 2H), 2.00 - 1.88 (m, 4H), 1.69 - 1.47 (m, 10H), 1.26 - 0.84 (m, 14H), 13 C NMR (150 MHz, DMSO-d6) δ 164.34, 153.43, 150.69, 150.49, 145.54, 139.23, 137.62, 136.47, 130.19, 128.79, 128.62, 127.92, 124.77, 121.00, 120.71, 120.66, 118.83, 118.59, 118.21, 72.05, 71.83, 61.36, 61.13, 44.57, 44.54, 28.85, 27.88, 27.81, 26.63, 26.34, 26.33, 26.23, 26.22, ESI-HRMS (CH3OH): m / z 1077.3533 [M-PF6] +.

[0050] Example 2

[0051] Synthesis of metal Ir(III) complex Ir-NLG-2, the process route is shown as Figure 1 below

[0052] (1) Synthesis of precursor [Ir(dfppy)2Cl]2:

[0053] IrCl3·3H2O (1.000 g, 2.836 mmol) and 2-(2,4-difluorophenyl)pyridine dfppy (0.900 g, 6.069 mmol) were added to 80 mL of a mixed solvent of ethylene glycol ethyl ether and deionized water (3:1, v / v), and heated to 135 °C under N2protection for 24 h of stirring reaction. After cooling and filtration, the solid-liquid was separated using a suction filtration device, and the solid was washed with water, ethanol, and ethyl ether in sequence, and vacuum dried to obtain the reaction iridium precursor [Ir(dfppy)2Cl]2;

[0054] (2) Synthesis of [Ir(dfppy)2(CH3CN)2]PF6:

[0055] [Ir(dfppy)2Cl]2(0.200 g, 0.164 mmol) and AgPF6(0.084 g, 0.344 mmol) were added to 60 mL of acetonitrile solvent, and heated to 85 °C under N2protection for 24 h of stirring reaction. After cooling to room temperature, the Buchner funnel was filled with diatomite, and AgCl was removed by filtration. After the filtrate was concentrated to 3-5 mL under reduced pressure at 45 °C, it was added dropwise to a large amount of ethyl ether to precipitate a solid, which was vacuum dried to obtain [Ir(dfppy)2(CH3CN)2]PF6;

[0056] (3) Synthesis of Ir-NLG-2:

[0057] [Ir(dfppy)2(CH3CN)2]PF6(0.100 g, 0.124 mmol) and NLG919 (0.141 g, 0.499 mmol) were added to 60 mL of a mixed solvent of methanol and dichloromethane (1:1, v / v), and heated to 69 °C under N2protection for 6 h of stirring reaction. After concentration to obtain a solid at 45 °C by rotary evaporation, the solid was dissolved in a small amount of dichloromethane, and eluted with dichloromethane / methanol (200:1, v / v) as the eluent, and separated and purified by silica gel column chromatography to obtain the NLG919 and metal Ir(III) coupling product Ir-NLG-2, with a yield of 0.070 g, 68.3%.

[0058] Figure 5 ESI-HRMS spectrum of Ir-NLG-2 in Example 2, Figure 6Synthesis of Ir-NLG-2 in Example 2 1 HNMR spectrum, Figure 7 Synthesis of Ir-NLG-2 in Example 2 13 CNMR spectrum, from which the complex Ir-NLG-2: 1 H NMR (600 MHz, DMSO-d6) δ 8.85 (dd, J = 30.6, 5.4 Hz, 2H), 8.25 - 8.01 (m, 6H), 7.7 - 7.23 (m, 10H), 7.13 (d, J = 25.9 Hz, 2H), 6.74 (q, J = 12.5 Hz, 2H), 5.75 (d, J = 8.8 Hz, 2H), 5.37 (s, 2H), 4.82 - 4.57 (m, 2H), 2.02 - 1.82 (m, 4H), 1.67 - 1.42 (m, 10H), 1.26 - 0.79 (m, 14H), 13 C NMR (150 MHz, DMSO-d6) δ 163.93, 163.91, 150.92, 150.72, 145.40, 139.97, 137.93, 136.38, 128.80, 128.66, 128.58, 124.78, 124.42, 121.16, 120.98, 118.49, 118.42, 71.80, 71.54, 61.36, 61.13, 44.66, 44.55, 28.81, 27.94, 27.82, 26.62, 26.31, 26.30, 26.22, 26.20, ESI-HRMS (CH3OH): m / z 1137.4034 [M-PF6] + .

[0059] Example 3

[0060] Synthesis of metal rhenium complex Re-NLG-1, the process route is shown as Figure 1 The specific steps are as follows

[0061] (1) Synthesis of Re(dip)(CO)3Cl:

[0062] Re(CO)5Cl (0.500 g, 1.382 mmol) and 4,7-diphenyl-1,10-phenanthroline dip (0.459 g, 1.382 mmol) were added to 60 mL of toluene solvent, heated to 110°C under N2protection, stirred and refluxed for 6 h, cooled and filtered to obtain a solid, which was washed with toluene and diethyl ether in turn, and dried under vacuum to obtain the rhenium precursor Re(dip)(CO)3Cl;

[0063] (2) Synthesis of [Re(dip)(CO)3(CH3CN)]PF6:

[0064] Re(dip)(CO)3Cl (0.200 g, 0.313 mmol) and silver trifluoromethylsulfonate AgOTf (0.089 g, 10.344 mmol) were added to 80 mL of acetonitrile solvent, heated to 85 °C under N2protection and stirred for 20 h to carry out the heating dechlorination reaction, which was to replace the anion. AgCl was removed by cooling and filtration, and the filtrate was concentrated to a certain amount. A saturated aqueous NH4PF6 solution was added and stirred for 2 h. After being concentrated under reduced pressure at 45 °C, a yellow solid was precipitated by adding water, suction filtered, and dried under vacuum to obtain the intermediate [Re(dip)(CO)3(CH3CN)]PF6;

[0065] (3) Synthesis of Re-NLG-1:

[0066] [Re(dip)(CO)3(CH3CN)]PF6(0.100 g, 0.127 mmol) and NLG919 (0.179 g, 0.634 mmol) were added to 40 mL of tetrahydrofuran solvent, heated to 66 °C under N2protection and stirred for 6 h to carry out the reaction. After being concentrated by rotary evaporation, the obtained solid was dissolved in a small amount of dichloromethane, and purified by silica gel column chromatography with dichloromethane / methanol (150:1, v / v) as the eluent. After drying, NLG919 and metal rhenium complex Re-NLG-1 were obtained with a yield of 0.081 g, 72.9%.

[0067] Figure 8 ESI-HRMS spectrum of Re-NLG-1 in Example 3, Figure 9 ESI-HRMS spectrum of Re-NLG-1 in Example 3, 1 HNMR spectrum, Figure 10 ESI-HRMS spectrum of Re-NLG-1 in Example 3, 13 CNMR spectrum, from which it can be seen that the complex Re-NLG-1: 1 H NMR (600 MHz, DMSO-d6) δ 9.70 (dd, J = 15.4, 5.3 Hz, 2H), 8.15 (dt, J = 10.9, 6.2 Hz, 5H), 7.70 - 7.61 (m, 10H), 7.49 (dd, J = 48.2, 7.5 Hz, 2H), 7.28 (dt, J = 29.4, 7.4 Hz, 2H), 7.13 (s, 1H), 5.28 (t, J = 6.0 Hz, 1H), 4.61 (d, J = 5.4 Hz, 1H), 1.73 (q, J = 14.2, 12.2 Hz, 2H), 1.56 - 1.45 (m, 4H), 1.34 (d, J = 11.8 Hz, 1H), 1.09 - 0.66 (m, 7H), 13C NMR (150 MHz, DMSO-d6) δ 154.79, 151.74, 147.06, 145.26, 138.09, 136.99, 135.55, 130.52, 130.40, 129.66, 128.86, 128.76, 128.74, 127.81, 127.75, 126.48, 124.82, 121.15, 119.28, 71.60, 61.45, 44.36, 28.83, 27.67, 26.50, 26.23, 26.15, ESI-HRMS (CH3OH): m / z 885.2516 [M-PF6] + .

[0068] Example 4

[0069] Synthesis of metal rhenium complex Re-NLG-2, the process route is shown as Figure 1 , and the specific steps are as follows:

[0070] (1) Synthesis of Re(phen)(CO)3Cl:

[0071] Re(CO)5Cl (0.3 g, 0.829 mmol) and 1,10-phenanthroline phen (0.164 g, 1.382 mmol) were added to 60 mL of toluene solvent, heated to 110°C under N2protection, stirred and refluxed for 6 h, cooled and filtered to obtain a solid, which was washed with toluene and diethyl ether in turn, and dried under vacuum to obtain the rhenium precursor Re(phen)(CO)3Cl;

[0072] (2) Synthesis of [Re(phen)(CO)3(CH3CN)]PF6:

[0073] Re(phen)(CO)3Cl (0.200 g, 0.411 mmol) and silver trifluoromethyl sulfonate AgOTf (0.116 g, 0.452 mmol) were added to 80 mL of acetonitrile solvent, heated to 85°C under N2protection, and stirred for 20 h to carry out the heating dechlorination reaction. In order to replace the anion, AgCl was removed by cooling and filtering, and the filtrate was concentrated to a certain amount, and a saturated NH4PF6aqueous solution was added and stirred for 2 h. After being concentrated under reduced pressure at 45°C, a yellowish solid was precipitated by adding water, filtered, and dried under vacuum to obtain [Re(phen)(CO)3(CH3CN)]PF6;

[0074] (3) Synthesis of Re-NLG-2:

[0075] [Re(phen)(CO)3(CH3CN)]PF6(0.100 g, 0.157 mmol) and NLG919 (0.221 g, 0.784 mmol) were added to 40 mL of tetrahydrofuran solvent, heated to 66 °C under N2protection, stirred for 6 h, concentrated by rotary evaporation after cooling, the obtained solid was dissolved with a small amount of dichloromethane, purified by silica gel column chromatography with dichloromethane / methanol (100:1, v / v) as eluent, dried to obtain NLG919 and metal rhenium complex Re-NLG-2, yield: 0.086 g, 74.3%.

[0076] Figure 11 ESI-HRMS spectrum of Re-NLG-2 in Example 4, Figure 12 ESI-HRMS spectrum of Re-NLG-2 in Example 4, 1 HNMR spectrum of Re-NLG-2 in Example 4, Figure 13 ESI-HRMS spectrum of Re-NLG-2 in Example 4, 13 CNMR spectrum of Re-NLG-2 in Example 4, from which the complex Re-NLG-2: 1 H NMR (500 MHz, DMSO-d6) δ 9.67 (ddd, J = 8.9, 5.1, 1.3 Hz, 2H), 9.04 (d, J = 8.3 Hz, 2H), 8.32 (s, 2H), 8.22 (ddd, J = 8.3, 5.1, 1.2 Hz, 2H), 8.10 (s, 1H), 7.49 (d, J = 7.4 Hz, 1H), 7.44 (d, J = 7.5 Hz, 1H), 7.28 (dt, J = 21.5, 7.5 Hz, 2H), 6.98 (s, 1H), 5.73 (d, J = 3.6 Hz, 1H), 5.26 (t, J = 6.2 Hz, 1H), 4.58 (d, J = 5.4 Hz, 1H), 1.85 - 1.68 (m, 2H), 1.68 - 1.48 (m, 4H), 1.36 (d, J = 12.2 Hz, 1H), 1.10 - 0.96 (m, 4H), 0.85 - 0.75 (m, 2H). 13 C NMR (125 MHz, DMSO-d6) δ 155.17, 146.12, 145.17, 140.76, 138.03, 136.91, 131.17, 128.79, 128.56, 128.21, 127.76, 127.66, 124.80, 121.06, 118.89, 71.31, 61.28, 44.33, 28.87, 27.73, 26.59, 26.28, 26.20. ESI-HRMS (CH3OH): m / z 733.1916 [M-PF6] + .

[0077] Example 5

[0078] The anti-tumor activity of the IDO inhibitor NLG919 and metal iridium / rhenium conjugates was tested by MTT method to evaluate the cytotoxicity of NLG919 and metal iridium / rhenium conjugates on tumor cells 4T1, MDA-MB-231, and MCF-7 cells. The specific steps are as follows:

[0079] The single-cell suspension was resuspended with a culture medium containing 10% fetal bovine serum, and the cells were inoculated into a 96-well plate and cultured overnight in a 37°C, 5% CO2 cell incubator. The drug administration group, the control group without drug, and the blank group without drug and cells were set up. The drug administration group was added with different concentrations of drugs diluted with the culture medium, and the concentrations were 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, and 1.5625 μM, respectively. Then, the plate was incubated in the incubator for 44 h, 20 μL of MTT reagent (5 mg / mL) was added to each well, and the plate was incubated for another 4 h. The supernatant was discarded, 150 μL of DMSO was added to each well, and the plate was shaken for 15 min. The absorbance at 590 nm was measured, and the inhibition rate was calculated according to the following formula:

[0080] Inhibition rate = (average OD value of the control group - average OD value of the drug administration group) / average OD value of the control group x 100%;

[0081] The IC 50 value (the drug concentration when the inhibition rate is 50%) was calculated, and the test results of the IC 50 values of NLG919 and metal iridium / rhenium conjugates on different cell lines are shown in Table 1:

[0082] Table 1

[0083]

[0084] The anti-proliferative activity of the complex on 4T1, MDA-MB-231, MCF-7, A549, and BEAS-2B was evaluated by standard MTT method. The experimental results showed that NLG919 and metal iridium / rhenium conjugates Ir-NLG-(1-2) and Re-NLG-1 had good anti-tumor activity on mouse breast cancer 4T1 cells.

[0085] The IC 50 values of the complex on 4T1 cells were ranked from low to high: Re-NLG-1 > Ir-NLG-1 > Ir-NLG-2 > Re-NLG-2. The cytotoxicity of Ir-NLG-1, Ir-NLG-2, and Re-NLG-1 on BEAS-2B normal cells was in the range of 1.5 μM to 7.5 μM. Finally, 4T1 cells were selected for subsequent related anti-tumor mechanism experiments.

[0086] Example 6

[0087] The uptake of NLG919 and metal iridium / rhenium conjugates in mouse breast cancer 4T1 cells was observed by laser confocal microscopy to observe the uptake of NLG919 and metal iridium / rhenium conjugates:

[0088] The mouse breast cancer 4T1 cells in the logarithmic growth phase were cultured in a 35mm Corning laser confocal culture dish, incubated overnight, and then NLG919 and metal iridium / rhenium conjugates Ir-NLG-1, Ir-NLG-2, and Re-NLG-1 were added and incubated for a certain period of time. The uptake of the complexes in the cells was observed by confocal microscopy.

[0089] The experimental results are shown in Figure 14 The uptake of NLG919 and metal iridium / rhenium conjugates Ir-NLG-1, Ir-NLG-2, and Re-NLG-1 in the cells was observed by confocal microscopy after incubation for a certain period of time. Under an excitation wavelength of 405nm, the fluorescence of the complexes in the cells could be observed, indicating that the complexes could effectively enter the cells within a certain period of time.

[0090] The specific embodiments of the present application are described in detail above in combination with the drawings, but the present application is not limited to the above-described embodiments. Various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present application.

Claims

1. A NLG919-metal complex, the metal being iridium or rhenium, the structure of the NLG919-iridium complex or the NLG919-rhenium complex being as follows: wherein: NLG919 is a compound with the following structure: the preparation method of the NLG919-iridium complex, the specific steps being as follows: 、 ； S1. IrCl3·3H2O and C^N ligand I are reacted in a mixed solvent of ethylene glycol ether and deionized water under nitrogen protection at high temperature, and an iridium precursor [Ir(C^N)2Cl]2 is obtained by cooling, filtering, washing and drying; [Ir(C^N)2Cl]2 and silver hexafluorophosphate are reacted in acetonitrile under nitrogen protection by heating, and the filtrate is filtered by diatomite after cooling, and the solid obtained by adding the filtrate drop by drop into ethyl ether is an Ir(III) intermediate [Ir(C^N)2(CH3CN)2]PF6; ； Anion is PF6 - .

2. The method for preparing the NLG919-metal coupling compound according to claim 1, characterized in that, S2. [Ir(C^N)2(CH3CN)2]PF6 and NLG919 are reacted in a mixed solvent of methanol and dichloromethane under N2 protection by heating, and a solid is obtained by rotary evaporation and concentration; the solid is dissolved in dichloromethane, and the NLG919-iridium complex is obtained by separation and purification through silica gel column chromatography with dichloromethane / methanol as an eluent and drying. In step S1, the molar ratio of IrCl3·3H2O to C^N ligand I is 1:2-2.2, the C^N ligand I is 2-(2-thiophene)pyridine or 2-(2,4-difluorophenyl)pyridine; the high-temperature reaction is carried out at a temperature of 110-135 ℃ for 20-24 h, and the mixed solvent of ethylene glycol ether and deionized water is obtained by mixing ethylene glycol ether and deionized water at a volume ratio of 3:1; the molar ratio of [Ir(C^N)2Cl]2 to silver hexafluorophosphate is 1:2-2.5; and the heating reaction is carried out at a temperature of 80-85 ℃ for 20-24 h. In step S2, the molar ratio of [Ir(C^N)2(CH3CN)2]PF6 to NLG919 is 1:3-8, the heating reaction is carried out at a temperature of 65-69 ℃ for 5.5-6 h, and the mixed solvent of methanol and dichloromethane is obtained by mixing methanol and dichloromethane at a volume ratio of 1:

1.

3. The method of claim 2, wherein the metal complex is prepared by the method comprising: (a) contacting a compound of Formula (I) with a metal salt to form a mixture; and (b) isolating the metal complex from the mixture. The preparation method of the NLG919-rhenium complex, the specific steps being as follows:

4. The method for preparing the NLG919-metal coupling compound according to claim 2, characterized in that, (1) Re(CO)5Cl and N^N ligand II are reacted in toluene under nitrogen protection by heating to obtain Re(N^N)(CO)3Cl, and Re(N^N)(CO)3Cl and silver trifluoromethylsulfonate are reacted in acetonitrile under nitrogen protection by heating and dechlorination, and a solid [Re(N^N)(CO)3(CH3CN)]PF6 is obtained by adding a large amount of saturated NH4PF6 aqueous solution; 5. The method for preparing the NLG919-metal coupling compound according to claim 1, characterized in that, (2) [Re(N^N)(CO)3(CH3CN)]PF6 and NLG919 are reacted in tetrahydrofuran under N2 protection by heating, and a solid is obtained by rotary evaporation and concentration; the solid is dissolved in dichloromethane, and the NLG919-rhenium complex is obtained by purification through silica gel column chromatography with dichloromethane / methanol as an eluent and drying. ​ ​ 6. The method for preparing the NLG919-metal coupling compound according to claim 5, characterized in that, The molar ratio of Re(CO)5Cl and N^N ligand II in step (1) is 1:1-1.2, and the N^N ligand II is 4,7-diphenyl-1,10-phenanthroline or 1,10-phenanthroline. The heating reaction condition is: temperature 100-110℃, time 6-8h. The molar ratio of Re(N^N)(CO)3Cl and silver trifluoromethyl sulfonate is 1:1-1.

2. The heating reaction condition is: temperature 80-85℃, time 20-24h.

7. The method for preparing the NLG919-metal coupling compound according to claim 5, characterized in that, The molar ratio of [Re(N^N)(CO)3(CH3CN)]PF6 and NLG919 in step (2) is 1:2.5-3. The heating reaction condition is: temperature 60-66℃, time 5.5-6h.

8. The use of NLG919 and metal conjugate in the preparation of an antitumor drug according to claim 1.

Images