NLG919 and metal conjugate as well as preparation method and application thereof

Abstract

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

Description

Technical Field

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

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

[0003] At present, the main means of clinical treatment of tumors are surgery, chemotherapy, radiotherapy, gene therapy, targeted therapy, immunotherapy, etc. Immunotherapy is a very promising cancer treatment method, but the strong immunosuppressive microenvironment of tumors greatly limits the effectiveness of immunotherapy. Indoleamine-2,3-dioxygenase (IDO) is a key immunosuppressive enzyme that regulates tumor immune responses and participates in tumor immune escape through multiple pathways. IDO inhibitors can activate T cell-mediated anti-tumor immune responses to kill tumor cells through different immune signaling pathways. NLG919 is one of the few IDO inhibitors that have entered the clinical research stage. However, clinical trial results show that the existing IDO inhibitors have limited efficacy as a single drug. Research progress has shown that a synergistic effect can be observed when IDO inhibitors are used in combination with chemotherapeutic agents.

[0004] Although platinum drugs represented by cisplatin have made great achievements in anti-tumor, their own toxic side effects and drug resistance have limited their long-term clinical use. Summary of the invention

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

[0006] The technical solution of the present invention is as follows:

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

[0008]

[0009] in:

[0010]

[0011] Anion is PF 6 - .

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

[0013]

[0014] The present invention also provides a method for preparing the IDO inhibitor NLG919 and a metal iridium conjugate. The method for preparing the NLG919 and a metal iridium conjugate comprises the following specific steps:

[0015] S1.IrCl 3 ·3H 2 O and C^N ligand Ⅰ react in a mixed solvent of ethylene glycol ethyl ether and deionized water at high temperature under nitrogen protection, and then cool, filter, wash and dry to obtain the iridium precursor [Ir(C^N) 2 Cl] 2 ; [Ir(C^N) 2 Cl] 2 Then with silver hexafluorophosphate (AgPF 6 ) in acetonitrile, heated under nitrogen protection, cooled and filtered through diatomaceous earth, the filtrate was concentrated and added dropwise to ether, and the precipitated solid was the Ir(III) intermediate [Ir(C^N) 2 (CH 3 CN) 2 ]PF 6 ;

[0016] S2.[Ir(C^N) 2 (CH 3 CN) 2 ]PF 6 and NLG919 in a mixed solvent of methanol and dichloromethane, N 2 The reaction was heated under protection, and the solid was concentrated by rotary evaporation to obtain a solid. The solid was dissolved with a small amount of dichloromethane, and then separated and purified by silica gel column chromatography using dichloromethane / methanol as eluent, and dried to obtain NLG919 and metal Ir (III) conjugates Ir-NLG-1 and Ir-NLG-2.

[0017] IrCl in step S1 3 ·3H 2The molar ratio of O and C^N ligand I is 1:2-2.2, C^N ligand I is 2-(2-thiophene)pyridine (thpy) ​​or 2-(2,4-difluorophenyl)pyridine (dfppy); the conditions of the high temperature reaction are: temperature 110-135°C, time 20-24h, and the mixed solvent of ethylene glycol ethyl ether and deionized water is obtained by mixing ethylene glycol ethyl ether and deionized water in a volume ratio of 3:1; [Ir(C^N) 2 Cl] 2 and silver hexafluorophosphate (AgPF 6 ) is in a molar ratio of 1:2 to 2.5; the conditions for the heating reaction are: temperature 80 to 85°C, time 20 to 24h.

[0018] In step S2, [Ir(C^N) 2 (CH 3 CN) 2 ]PF 6 The molar ratio of methanol to NLG919 is 1:3-8, and the heating reaction conditions are: temperature 65-69°C, time 5.5-6h; the mixed solvent of methanol and dichloromethane is obtained by mixing methanol and dichloromethane in a volume ratio of 1:1.

[0019] The present invention also provides a method for preparing the IDO inhibitor NLG919 and metal rhenium conjugate. The method for preparing the NLG919 and metal rhenium conjugate comprises the following specific steps:

[0020] (1) Re(CO) 5 Cl and N^N ligand II were heated in toluene solvent under nitrogen protection to obtain Re(N^N)(CO) 3 Cl, Re(N^N)(CO) 3 Cl is then reacted with silver trifluoromethanesulfonate (AgOTf) in acetonitrile solvent and heated under nitrogen protection for dechlorination reaction; a large amount of saturated NH 4 PF 6 The aqueous solution gave a solid [Re(N^N)(CO) 3 (CH 3 CN)]PF 6 ;

[0021] (2)[Re(N^N)(CO) 3 (CH 3 CN)]PF 6 and NLG919 in tetrahydrofuran solvent, N 2 The reaction was heated under protection, and the solid was concentrated by rotary evaporation to obtain a solid. The solid was dissolved with a small amount of dichloromethane, and purified by silica gel column chromatography using dichloromethane / methanol as eluent. The NLG919 and metal rhenium conjugates Re-NLG-1 and Re-NLG-2 were obtained after drying.

[0022] Re(CO) in step (1) 5 The molar ratio of Cl to 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°C, time 6-8h; Re(N^N)(CO) 3 The molar ratio of Cl to silver trifluoromethanesulfonate (AgOTf) is 1:1-1.2, and the conditions for the heating dechlorination reaction are: temperature 80-85° C., time 20-24 h.

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

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

[0025] The present invention forms a coordination compound with NLG919, metal iridium and rhenium, in order to improve the chemotherapy and immunotherapy effects of the compound. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The synthetic route of the NLG919 and metal iridium / rhenium conjugate of the present invention;

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

[0028] Figure 3 is the Ir-NLG-1 in Example 1 1 HNMR spectrum;

[0029] Figure 4 is the Ir-NLG-1 in Example 1 13 CNMR spectrum;

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

[0031] Figure 6 is the Ir-NLG-2 in Example 2 1 HNMR spectrum;

[0032] Figure 7 is the Ir-NLG-2 in Example 2 13 CNMR spectrum;

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

[0034] Fig. 9 is the Re-NLG-1 in Example 3 1 HNMR spectrum;

[0035] Fig.10 is the Re-NLG-1 in Example 3 13 CNMR spectrum;

[0036] Fig.11 is the ESI-HRMS spectrum of Re-NLG-2 in Example 4;

[0037] Fig.12 is the Re-NLG-2 in Example 4 1 HNMR spectrum;

[0038] Fig.13 is the Re-NLG-2 in Example 4 13 CNMR spectrum;

[0039] Fig.14 This is the uptake of the complex by 4T1 cells in Example 6. DETAILED DESCRIPTION

[0040] The following is a further description of the IDO inhibitor NLG919 and metal iridium / rhenium conjugates and their preparation methods and anti-tumor applications in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents and methods used in the examples are commonly used reagents in the art, and can be purchased from the market or prepared by conventional methods unless otherwise specified.

[0041] Example 1

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

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

[0044] IrCl 3 ·3H 2O (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), N 2 The mixture was heated to 135°C under protection and stirred for 24 hours, cooled and filtered, and the solid and liquid were separated using a suction filtration device. The solid was washed with water, ethanol, and ether in sequence, and vacuum dried to obtain the iridium precursor [Ir(thpy) 2 Cl] 2 ;

[0045] (2)[Ir(thpy) 2 (CH 3 CN) 2 ]PF 6 Synthesis of:

[0046] [Ir(thpy) 2 Cl] 2 (0.150 g, 0.136 mmol) and AgPF 6 (0.073 g, 0.287 mmol) was added to 60 mL of acetonitrile solvent, N 2 The mixture was heated to 85°C under protection and stirred for 24 h, cooled to room temperature, covered with diatomaceous earth on the Buchner funnel, filtered to remove AgCl, 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 a solid, which was then dried in vacuo to obtain [Ir(thpy) 2 (CH 3 CN) 2 ]PF 6 ;

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

[0048] [Ir(thpy) 2 (CH 3 CN) 2 ]PF 6 (0.150 g, 0.203 mmol) and NLG919 (0.229 g, 0.811 mmol) were added to a 60 mL mixed solvent of methanol and dichloromethane (1:1, v / v). 2 The mixture was heated to 69°C under protection and stirred for 6 h. The mixture was concentrated by rotary evaporation at 45°C to obtain a solid. The solid was dissolved with a small amount of dichloromethane. Dichloromethane / methanol (200:1, v / v) was used as the eluent. The mixture was separated and purified by silica gel column chromatography to obtain the conjugate of NLG919 and metal Ir(III) Ir-NLG-1. The yield was 0.126 g, 62.3%.

[0049] Figure 2is the ESI-HRMS spectrum of Ir-NLG-1 in Example 1, Figure 3 is the Ir-NLG-1 in Example 1 1 HNMR spectrum, Figure 4 is the Ir-NLG-1 in Example 1 13 CNMR spectrum, from the figure we can see: 1 H NMR (500 MHz, DMSO-d 6 )δ8.62(d,J=5.8Hz,1H),8.57(d,J=5.7Hz,1H),8.08(d,J=6.2Hz,2H),7.91–7.81(m, 2H),7.70–7.54(m,6H),7.48(dd,J=4.7,1.3Hz,2H),7.37–7.30(m,4H),7.22–7.16(m ,2H),6.95(d,=27.5Hz,2H),6.17(dd,J=4.7,2.2Hz,2H),5.39(q,J=6.6Hz,2H),4.72 (dd,J=21.8,5.4Hz,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-d 6 )δ164.34,153.43,150.69,150.49,145.54,139.23,137.62,136.47,130.19,128.79,128.62,127.92,124.77,121.00,120.71,120.66,1 18.83,118.59,118.21,72.05,71.83,61.36,61.13,44.57,44.54,28.85,27.88,27.81,26.63,26.34,26.33,26.23,26.22, ESI-HRMS (CH 3 OH): m / z 1077.3533 [M-PF 6 ] + .

[0050] Example 2

[0051] The synthesis of metal Ir(III) complex Ir-NLG-2 is as follows: Figure 1 The specific steps are as follows

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

[0053] IrCl 3 ·3H 2 O (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), N 2 The mixture was heated to 135°C under protection and stirred for 24 hours, cooled and filtered, and the solid and liquid were separated using a suction filtration device. The solid was washed with water, ethanol, and ether in sequence, and vacuum dried to obtain the iridium precursor [Ir(dfppy) 2 Cl] 2 ;

[0054] (2)[Ir(dfppy) 2 (CH 3 CN) 2 ]PF 6 Synthesis of:

[0055] [Ir(dffpy) 2 Cl] 2 (0.200 g, 0.164 mmol) and AgPF 6 (0.084 g, 0.344 mmol) was added to 60 mL of acetonitrile solvent, N 2 The mixture was heated to 85°C under protection and stirred for 24 h, cooled to room temperature, covered with diatomaceous earth on the Buchner funnel, filtered to remove AgCl, 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 a solid, which was then dried in vacuo to obtain [Ir(dfppy) 2 (CH 3 CN) 2 ]PF 6 ;

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

[0057] [Ir(dfppy) 2 (CH 3 CN) 2 ]PF 6 (0.100 g, 0.124 mmol) and NLG919 (0.141 g, 0.499 mmol) were added to a 60 mL mixed solvent of methanol and dichloromethane (1:1, v / v). 2 The mixture was heated to 69°C under protection and stirred for 6 h. The mixture was concentrated by rotary evaporation at 45°C to obtain a solid. The solid was dissolved with a small amount of dichloromethane. Dichloromethane / methanol (200:1, v / v) was used as the eluent. The mixture was separated and purified by silica gel column chromatography to obtain the conjugate of NLG919 and metal Ir (III) Ir-NLG-2. The yield was 0.070 g, 68.3%.

[0058] Figure 5 is the ESI-HRMS spectrum of Ir-NLG-2 in Example 2, Figure 6 is the Ir-NLG-2 in Example 2 1 HNMR spectrum, Figure 7 is the Ir-NLG-2 in Example 2 13 CNMR spectrum, from which we can see the complex Ir-NLG-2: 1 H NMR (600 MHz, DMSO-d 6 )δ8.85(dd,J=30.6,5.4Hz,2H),8.25–8.01(m,6H),7.7–7.23(m,10H),7.13(d,J=25.9Hz,2H),6.74(q,J=12.5Hz,2H ),5.75(d,J=8.8Hz,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-d 6 )δ163.93,163.91,150.92,150.72,145.40,139.97,137.93,136.38,128.80,128.66,128.58,124.78,124.42,121.16,120.98,1 18.49,118.42,71.80,71.54,61.36,61.13,44.66,44.55,28.81,27.94,27.82,26.62,26.31,26.30,26.22,26.20, ESI-HRMS (CH 3 OH): m / z 1137.4034 [M-PF 6 ] + .

[0059] Example 3

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

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

[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. 2 The mixture was heated to 110°C and stirred under reflux for 6 h under protection, and then cooled and filtered to obtain a solid. The solid was washed with toluene and ether in turn, and then dried under vacuum to obtain the rhenium precursor Re(dip)(CO) 3 Cl;

[0063] (2) [Re(dip)(CO) 3 (CH 3 CN)]PF 6 Synthesis of:

[0064] Re(dip)(CO) 3 Cl (0.200 g, 0.313 mmol) and silver trifluoromethanesulfonate AgOTf (0.089 g, 10.344 mmol) were added to 80 mL of acetonitrile solvent. 2 Heat to 85 °C and stir for 20 h to carry out heating dechlorination reaction. In order to replace the anion, cool and filter to remove AgCl, concentrate the filtrate to a small amount, add saturated NH 4 PF 6 The aqueous solution was stirred for 2 h, concentrated under reduced pressure at 45 °C, and water was added to precipitate a yellow solid, which was filtered and dried in vacuo to obtain the intermediate [Re(dip)(CO) 3 (CH 3 CN)]PF 6 ;

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

[0066] [Re(dip)(CO) 3 (CH 3 CN)]PF 6 (0.100 g, 0.127 mmol) and NLG919 (0.179 g, 0.634 mmol) were added to 40 mL of tetrahydrofuran solvent. 2 The mixture was heated to 66°C under protection and stirred for 6 hours. After cooling and concentration by rotary evaporation, the obtained solid was dissolved with a small amount of dichloromethane and separated and purified by silica gel column chromatography using dichloromethane / methanol (150:1, v / v) as eluent. After drying, the NLG919 and metal rhenium complex Re-NLG-1 was obtained with a yield of 0.081 g, 72.9%.

[0067] Figure 8 is the ESI-HRMS spectrum of Re-NLG-1 in Example 3, Fig. 9 is the Re-NLG-1 in Example 3 1 HNMR spectrum, Fig.10 is the Re-NLG-1 in Example 3 13 CNMR spectrum, from which we can know the complex Re-NLG-1: 1 H NMR (600 MHz, DMSO-d 6 )δ9.70(dd,J=15.4,5.3Hz,2H),8.15(dt,J=10.9,6.2Hz,5H),7.70–7.61(m,10H),7.49(dd,J=48.2,7.5Hz,2H),7.28(dt,J=29.4,7.4Hz,2H),7.1 3(s,1H),5.28(t,J=6.0Hz,1H),4.61(d,J=5.4Hz,1H),1.73(q,J=14.2,1 2.2Hz,2H),1.56–1.45(m,4H),1.34(d,J=11.8Hz,1H),1.09–0.66(m,7H), 13 C NMR (150 MHz, DMSO-d 6 )δ154.79,151.74,147.06,145.26,138.09,136.99,135.55,130.52,130.40,129.66,128.86,128.76,128.74,127. 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 (CH 3 OH):m / z 885.2516[M-PF 6 ] + .

[0068] Example 4

[0069] The synthesis of the metal rhenium complex Re-NLG-2 is as follows: Figure 1 As shown, the specific steps are as follows:

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

[0071] Re(CO) 5 Cl (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. 2 The mixture was heated to 110°C and stirred under reflux for 6 h. The mixture was cooled and filtered to obtain a solid. The solid was washed with toluene and ether in turn and dried under vacuum to obtain the rhenium precursor Re(phen)(CO)3 Cl;

[0072] (2)[Re(phen)(CO) 3 (CH 3 CN)]PF 6 Synthesis of:

[0073] Re(phen)(CO) 3 Cl (0.200 g, 0.411 mmol) and silver trifluoromethanesulfonate AgOTf (0.116 g, 0.452 mmol) were added to 80 mL of acetonitrile solvent. 2 Heat to 85 °C and stir for 20 h to carry out heating dechlorination reaction. In order to replace the anion, cool and filter to remove AgCl, concentrate the filtrate to a small amount, add saturated NH 4 PF 6 The aqueous solution was stirred for 2 h, concentrated under reduced pressure at 45 °C, and water was added to precipitate a light yellow solid, which was filtered and dried in vacuo to obtain [Re(phen)(CO) 3 (CH 3 CN)]PF 6 ;

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

[0075] [Re(phen)(CO) 3 (CH 3 CN)]PF 6 (0.100 g, 0.157 mmol) and NLG919 (0.221 g, 0.784 mmol) were added to 40 mL of tetrahydrofuran solvent. 2 The mixture was heated to 66°C under protection and stirred for 6 hours. After cooling and concentration by rotary evaporation, the obtained solid was dissolved with a small amount of dichloromethane and separated and purified by silica gel column chromatography using dichloromethane / methanol (100:1, v / v) as eluent. After drying, the NLG919 and metal rhenium complex Re-NLG-2 was obtained with a yield of 0.086 g, 74.3%.

[0076] Fig.11 is the ESI-HRMS spectrum of Re-NLG-2 in Example 4, Fig.12 is the Re-NLG-2 in Example 4 1 HNMR spectrum, Fig.13 is the Re-NLG-2 in Example 4 13 CNMR spectrum, from which we can know the complex Re-NLG-2: 1 H NMR (500 MHz, DMSO-d 6)δ9.67(ddd,J=8.9,5.1,1.3Hz,2H),9.04(d,J=8.3Hz,2H),8.32(s,2H),8.22(ddd,J=8.3,5.1 ,1.2Hz,2H),8.10(s,1H),7.49(d,J=7.4Hz,1H),7.44(d,J=7.5Hz,1H),7.28(dt,J=21.5,7.5H z,2H),6.98(s,1H),5.73(d,J=3.6Hz,1H),5.26(t,J=6.2Hz,1H),4.58(d,J=5.4Hz,1H),1.85– 1.68(m,2H),1.68–1.48(m,4H),1.36(d,J=12.2Hz,1H),1.10–0.96(m,4H),0.85–0.75(m,2H). 13 C NMR (125 MHz, DMSO-d 6 )δ155.17,146.12,145.17,140.76,138.03,136.91,131.17,128.79,128.56,128.21,127.76,127. 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(CH 3 OH): m / z 733.1916 [M-PF 6 ] + .

[0077] Example 5

[0078] The anti-tumor activity test of IDO inhibitor NLG919 and metal iridium / rhenium conjugates was conducted by using the 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 in medium containing 10% fetal bovine serum, and the cells were seeded into 96-well plates and incubated at 37°C and 5% CO. 2The cells were cultured overnight in a cell culture incubator, and a drug-treated group, a control group without drugs, and a blank group without drugs and cells were set up. Different concentrations of drugs diluted with culture medium were added to the drug-treated group, and the concentrations were 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, and 1.5625 μM, respectively. The cells were then cultured in an incubator for 44 h, and then 20 μL of MTT reagent (5 mg / mL) was added to each well. The cells were incubated for another 4 hours, the supernatant was discarded, 150 μL of DMSO was added to each well, and the cells were shaken for 15 min. The absorbance at 590 nm was measured, and the inhibition rate formula was as follows:

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

[0081] Find IC 50 Value (drug concentration when the inhibition rate is equal to 50%), test results NLG919 and metal iridium / rhenium conjugates against different cell lines IC 50 Values, as shown in Table 1:

[0082] Table 1

[0083]

[0084] The anti-proliferative activity of the complexes against 4T1, MDA-MB-231, MCF-7, A549 and BEAS-2B was evaluated by the 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 against mouse breast cancer 4T1 cells.

[0085] IC against 4T1 cells 50 The values ​​were sorted from low to high: Re-NLG-1>Ir-NLG-1>Ir-NLG-2>Re-NLG-2, among which Ir-NLG-1, Ir-NLG-2, and Re-NLG-1 had toxicity to BEAS-2B normal cells 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:

[0088] Mouse breast cancer 4T1 cells in the logarithmic growth phase were cultured in a 35 mm Corning laser confocal culture dish and incubated overnight. 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 using a confocal microscope.

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

[0090] The specific implementation modes of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above implementation modes, and various changes can be made within the knowledge scope of ordinary technicians in this field without departing from the purpose of the present invention.

Claims

1. A conjugate of NLG919 and a metal, wherein the metal is iridium or rhenium, and the structural formula of the conjugate of NLG919 and metal iridium or the conjugate of NLG919 and rhenium is as follows: in: Anion is PF6 - .

2. The method for preparing the NLG919-metal conjugate according to claim 1, characterized in that: The preparation method of the NLG919 and metal iridium conjugate comprises the following specific steps: S1. IrCl3·3H2O and C^N ligand Ⅰ react in a mixed solvent of ethylene glycol ether and deionized water at high temperature under nitrogen protection, and then cool, filter, wash and dry to obtain the iridium precursor [Ir(C^N)2Cl]2. [Ir(C^N)2Cl]2 and silver hexafluorophosphate react in acetonitrile under nitrogen protection, and then cool and filter with diatomaceous earth. The filtrate is concentrated and then added dropwise to ether. The precipitated solid is the Ir(III) intermediate [Ir(C^N)2(CH3CN)2]PF6. S2.[Ir(C^N)2(CH3CN)2]PF6 and NLG919 are heated to react in a mixed solvent of methanol and dichloromethane under N2 protection, and the solid is concentrated by rotary evaporation. The solid is dissolved in dichloromethane, and separated and purified by silica gel column chromatography using dichloromethane / methanol as eluent, and dried to obtain a conjugate of NLG919 and metal Ir(III).

3. The method for preparing the NLG919-metal conjugate according to claim 2, characterized in that: In step S1, the molar ratio of IrCl3·3H2O and C^N ligand I is 1:2~2.2, and C^N ligand I is 2-(2-thiophene)pyridine or 2-(2,4-difluorophenyl)pyridine; the temperature of the high-temperature reaction is 110~135℃, the time is 20~24h, and the mixed solvent of ethylene glycol ethyl ether and deionized water is obtained by mixing ethylene glycol ethyl ether and deionized water in a volume ratio of 3:1; the molar ratio of [Ir(C^N)2Cl]2 and silver hexafluorophosphate is 1:2~2.5; the conditions of the heating reaction are: temperature 80~85℃, time 20~24h.

4. The method for preparing the NLG919-metal conjugate according to claim 2, characterized in that: In step S2, the molar ratio of [Ir(C^N)2(CH3CN)2]PF6 to NLG919 is 1:3-8, the heating reaction temperature is 65-69°C, and the time is 5.5-6h; the mixed solvent of methanol and dichloromethane is obtained by mixing methanol and dichloromethane in a volume ratio of 1:

1.

5. The method for preparing the NLG919-metal conjugate according to claim 1, characterized in that: The preparation method of the NLG919-metal rhenium conjugate comprises the following specific steps: (1) Re(CO)5Cl and N^N ligand II are heated in toluene under nitrogen protection to obtain Re(N^N)(CO)3Cl. Re(N^N)(CO)3Cl and silver trifluoromethanesulfonate are heated in acetonitrile under nitrogen protection for dechlorination reaction. A large amount of saturated NH4PF6 aqueous solution is added to obtain solid [Re(N^N)(CO)3(CH3CN)]PF6. (2) [Re(N^N)(CO)3(CH3CN)]PF6 and NLG919 were heated in tetrahydrofuran under N2 protection and concentrated by rotary evaporation to obtain a solid. The solid was dissolved in dichloromethane and purified by silica gel column chromatography using dichloromethane / methanol as eluent. The solid was dried to obtain a conjugate of NLG919 and metal rhenium.

6. The method for preparing the NLG919-metal conjugate according to claim 5, characterized in that: In step (1), the molar ratio of Re(CO)5Cl to N^N ligand II is 1:1-1.2, N^N ligand II is 4,7-diphenyl-1,10-phenanthroline or 1,10-phenanthroline, and the conditions for the heating reaction are: temperature 100-110°C, time 6-8h; the molar ratio of Re(N^N)(CO)3Cl to silver trifluoromethanesulfonate is 1:1-1.2, and the conditions for the heating dechlorination reaction are: temperature 80-85°C, time 20-24h.

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

8. Use of the NLG919 and metal conjugates according to claim 1 in anti-tumor drugs, wherein the IDO inhibitor NLG919 and metal iridium conjugates or NLG919 and rhenium conjugates have good anti-tumor activity against 4T1, MDA-MB-231, and MCF-7 cell lines.

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