Alpha-linolenic acid-metal conjugate, and preparation method and application thereof

Abstract

The application discloses an alpha-linolenic acid and metal iridium coupling compound, a preparation method and application thereof, and relates to the field of medicine, in particular to an alpha-linolenic acid and metal iridium coupling compound, which has good antitumor activity on human cervical cancer cells, human hepatoma cells and mouse breast cancer cells. The preparation method is simple and easy to operate, and the product has good antitumor activity.

Description

Technical Field

[0001] This invention belongs to the field of chemical biology and relates to α-linolenic acid (ALA) and iridium metal conjugates, their preparation methods, and anti-tumor applications. Background Technology

[0002] Phosphorescent iridium(III) complexes exhibit irreplaceable advantages in cancer treatment due to their unique photophysical properties (long phosphorescence lifetime, large Stokes shift), easily modifiable structure, and good redox activity. Their core innovation lies in achieving precise anchoring of specific organelles through ligand regulation, while simultaneously integrating multiple functions such as diagnosis, treatment, and efficacy monitoring. ALA is an essential fatty acid extracted from perilla, which cannot be endogenously produced by the body due to a lack of the key enzyme system for its synthesis. Previous studies have confirmed that ALA can exert biological effects by regulating the PI3K-AKT signaling pathway. Summary of the Invention

[0003] This invention provides a method for preparing an ALA-iridium conjugate and its research and application in terms of antitumor activity. The prepared ALA-Ir(III) conjugate has good antitumor activity against HeLa, HepG2, and 4T1 cell lines.

[0004] The technical solution of this invention is as follows: The ALA-iridium coupling compound ALA-Ir(III) has the following structural formula:

[0005] The anion is PF6. - ;

[0006] Specifically, the ALA-iridium coupling compound ALA-Ir(III) has the following structure:

[0007] This invention also provides a method for preparing the ALA-iridium coupling compound ALA-Ir(III), the specific steps of which are as follows: S1.IrCl·3H2O and C^N ligands were reacted at high temperature under nitrogen protection in a mixed solvent of ethylene glycol ethyl ether and deionized water. After cooling, filtration, washing and drying, the iridium precursor [Ir(C^N)2Cl]2 was obtained. S2,4'-Me-bpy-4-CH2OH, ALA, and catalysts DMAP and EDCI were reacted in dichloromethane solvent at room temperature for 24 hours overnight. The mixture was concentrated by rotary evaporation to obtain an oily mixture. The mixture was then separated and purified by silica gel column chromatography using a dichloromethane / methanol mixed solvent as the eluent to obtain the ligand 4'-Me-bpy-4-CH2O-ALA. S3. [Ir(C^N)2Cl]2 and 4'-Me-bpy-4-CH2O-ALA were reacted in a dichloromethane / methanol mixture under N2 protection by heating. After the reaction was completed, a saturated NH4PF6 aqueous solution was added to the reaction solution to replace the anions. The mixture was concentrated by rotary evaporation to obtain a solid. The solid was dissolved in dichloromethane and purified by silica gel column chromatography using a dichloromethane / methanol mixed solvent as the eluent. The ALA-Ir(III) conjugate was obtained by freeze drying.

[0008] In step S1, the molar ratio of IrCl3·3H2O to C^N ligands is 1:2.2, and the C^N ligands are 2-(2-thiophene)pyridine (thpy), 2-(2,4-difluorophenyl)pyridine (dfppy), and 2-phenylpyridine (ppy). The reaction temperature is 135℃, the reaction time is 24h, and the mixed solvent of ethylene glycol ethyl ether and deionized water is obtained by mixing ethylene glycol ethyl ether and deionized water at a volume ratio of 3:1.

[0009] In step S2, the molar ratio of 4'-Me-bpy-4-CH2OH, ALA, DMAP, and EDCI is 1:1.2:1.5:1.5.

[0010] In step S3, the molar ratio of [Ir(C^N)2Cl]2 to 4'-Me-bpy-4-CH2O-ALA is 1:2.4, the heating temperature is 65℃, and the time is 5h.

[0011] In steps S2 and S3, the dichloromethane / methanol mixed solvent is obtained by mixing dichloromethane and methanol at a volume ratio of 100:1, and in step S3, the dichloromethane / methanol mixed solution is obtained by mixing methanol and dichloromethane at a volume ratio of 1:2.

[0012] This invention also provides the application of the conjugate in antitumor drug research. The ALA-Ir(III) conjugate has good in vitro antitumor activity against HeLa, HepG2, and 4T1 cancer cell lines.

[0013] The preparation method of this invention is simple, and the product has excellent anti-tumor activity when used in anti-tumor drugs. Attached Figure Description

[0014] Figure 1 The ESI-HRMS spectrum of Ir-ALA-1 in Example 1; Figure 2 For Ir-ALA-1 in Example 1 1 H-NMR spectrum; Figure 3 The ESI-HRMS spectrum of Ir-ALA-2 in Example 2; Figure 4 For Ir-ALA-2 in Example 2 1 H-NMR spectrum; Figure 5 The ESI-HRMS spectrum of Ir-ALA-3 in Example 3; Figure 6 For Ir-ALA-3 in Example 3 1 H-NMR spectrum. Detailed Implementation

[0015] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents or devices used in the embodiments can be obtained from conventional commercial channels or through existing technology.

[0016] Example 1 The synthesis of ALA and the iridium metal conjugate Ir-ALA-1 is carried out through the following steps: (1) Synthesis of the precursor [Ir(dfppy)2Cl]2: IrCl3·3H2O (1.000 g, 2.836 mmol) and dfppy (1.192 g, 6.239 mmol) were added to 80 mL of a mixed solvent of ethylene glycol diethyl ether and deionized water (3:1, v / v). The mixture was heated to 135 °C under N2 protection and stirred for 24 h. After cooling and filtration, the solid and liquid were separated using a vacuum filtration device. The solid was washed with water, ethanol, and diethyl ether in sequence and dried under vacuum to obtain the iridium precursor [Ir(dfppy)2Cl]2. (2) Synthesis of ligand 4'-Me-bpy-4-CH2O-ALA: 4'-Me-bpy-4-CH2OH (0.100 g, 0.500 mmol), ALA (0.167 g, 0.599 mmol), catalyst DMAP (0.091 g, 0.749 mmol), and EDCI (0.144 g, 0.749 mmol) were added to 20 mL of dichloromethane solvent and reacted at room temperature for 24 h. The mixture was concentrated by rotary evaporation to obtain an oily mixture. The mixture was separated and purified by silica gel column chromatography using a dichloromethane / methanol mixed solvent (dichloromethane and methanol volume ratio 100:1) as the eluent to obtain the ligand 4'-Me-bpy-4-CH2O-ALA. (3) Synthesis of the complex Ir-ALA-1: [Ir(dfppy)2Cl]2 (0.100 g, 0.0822 mmol) and 4'-Me-bpy-4-CH2O-ALA (0.091 g, 0.1973 mmol) were added to 60 mL of a mixture of dichloromethane and methanol (2:1, v / v). The mixture was heated to 65 °C and stirred for 5 h under N2 protection. After the reaction was completed, 1 mL of saturated NH4PF6 aqueous solution was added to the reaction solution to displace the anions. The mixture was concentrated by rotary evaporation to obtain a solid. The solid was dissolved in dichloromethane and purified by silica gel column chromatography using a dichloromethane / methanol mixed solvent (dichloromethane and methanol volume ratio 100:1) as the eluent. The ALA-iridium coupling compound Ir-ALA-1 was obtained by lyophilization, with a yield of 0.114 g, 58.9%.

[0017] Figure 1 The image shows the ESI-HRMS spectrum of Ir-ALA-1 in Example 1. ESI-HRMS (CH3OH): m / z = 1033.3660 [M-PF6] + .

[0018] Figure 2 For Ir-ALA-1 in Example 1 1 The H-NMR spectrum shows that: 1 H-NMR (600 MHz DMSO- d 6) δ 8.84 (s 1H) 8.77 (s 1H) 8.30 (d, J = 8.5 Hz, 2H), 8.04 (t, J = 7.9 Hz, 2H), 7.89 (d, J = 5.6 Hz, 1H), 7.77 (d, J = 5.7 Hz, 1H), 7.71 (d, J = 5.3 Hz, 2H), 7.65 (d, J = 5.6 Hz, 1H), 7.56 (d, J = 5.7 Hz, 1H), 7.27 – 7.23 (m, 2H), 6.97(ddd, J = 12.0, 9.4, 2.4 Hz, 2H), 5.62 (ddd, J = 12.4, 8.4, 2.4 Hz, 2H), 5.37 –5.23 (m, 6H), 2.78 – 2.71 (m, 2H), 2.57 (s, 3H), 2.46 (t,J = 7.4 Hz, 2H), 2.06 – 1.91 (m, 3H), 1.61 – 1.50 (m, 3H), 1.32 – 1.17 (m, 12H), 0.92 – 0.82 (m, 3H).

[0019] Example 2 The synthesis of ALA and the iridium metal conjugate Ir-ALA-2 is carried out through the following steps: (1) Synthesis of the precursor [Ir(ppy)2Cl]2: IrCl3·3H2O (1.000 g, 2.836 mmol) and ppy (0.968 g, 6.239 mmol) were added to 80 mL of a mixed solvent of ethylene glycol ethyl ether and deionized water (3:1, v / v). The mixture was heated to 135 °C under N2 protection and stirred for 24 h. After cooling and filtration, the solid and liquid were separated using a vacuum filtration device. The solid was washed with water, ethanol, and ether in sequence and dried under vacuum to obtain the iridium precursor [Ir(ppy)2Cl]2. (2) Synthesis of the complex Ir-ALA-2: [Ir(ppy)2Cl]2 (0.100 g, 0.093 mmol) and 4'-Me-bpy-4-CH2O-ALA (0.103 g, 0.224 mmol) prepared in step (2) of Example 1 were added to 60 mL of a mixture of dichloromethane and methanol (2:1, v / v). The mixture was heated to 65 °C and stirred for 5 h under N2 protection. After the reaction was completed, 1 mL of saturated NH4PF6 aqueous solution was added to the reaction solution to replace the anions. The mixture was concentrated by rotary evaporation to obtain a solid. The solid was dissolved in dichloromethane and purified by silica gel column chromatography using a dichloromethane / methanol mixed solvent (dichloromethane and methanol volume ratio 100:1) as the eluent. The ALA-iridium coupling compound Ir-ALA-2 was obtained by freeze drying, with a yield of 0.139 g, 67.5%.

[0020] Figure 3 The image shows the ESI-HRMS spectrum of Ir-ALA-2 in Example 2. ESI-HRMS (CH3OH): m / z = 961.4037 [M-PF6] + .

[0021] Figure 4 For Ir-ALA-2 in Example 2 1 The H-NMR spectrum shows that: 1 H-NMR (600 MHz, DMSO- d 6) δ8.82 (s, 1H), 8.75 (s, 1H), 8.26 (d, J = 8.2 Hz, 2H), 7.95 – 7.91 (m,4H), 7.83 (d, J = 5.7 Hz, 1H), 7.71 (d, J = 5.6 Hz, 1H), 7.63 (t, J = 7.0 Hz, 3H), 7.54 (d, J = 5.6 Hz, 1H), 7.16 (t, J = 4.9 Hz, 2H), 7.02 (t, J = 7.5 Hz, 2H), 6.90 (t, J = 7.4 Hz, 2H), 6.20 (dd, J = 10.5, 7.4 Hz, 2H), 5.37 – 5.24 (m,6H), 2.78 – 2.71 (m, 2H), 2.54 (s, 3H), 2.45 (t, J = 7.4 Hz, 2H), 2.05 – 1.97 (m, 3H), 1.62 – 1.51 (m, 3H), 1.32 – 1.17 (m, 12H), 0.92 – 0.82 (m, 3H).

[0022] Example 3 The synthesis of ALA and the iridium metal conjugate Ir-ALA-3 is carried out through the following steps: (1) Synthesis of the precursor [Ir(thpy)2C1]2: IrCl3·3H2O (1.000 g, 2.836 mmol) and thpy (1.005 g, 6.239 mmol) were added to 80 mL of a mixed solvent of ethylene glycol diethyl ether and deionized water (3:1, v / v). The mixture was heated to 135 °C under N2 protection and stirred for 24 h. After cooling and filtration, the solid and liquid were separated using a vacuum filtration device. The solid was washed with water, ethanol, and diethyl ether in sequence and dried under vacuum to obtain the iridium precursor [Ir(thpy)2Cl]2. (2) Synthesis of the complex Ir-ALA-3: [Ir(thpy)2Cl]2 (0.100 g, 0.091 mmol) and 4'-Me-bpy-4-CH2O-ALA (0.101 g, 0.219 mmol) prepared in step (2) of Example 1 were added to 60 mL of a mixture of dichloromethane and methanol (2:1, v / v). The mixture was heated to 65 °C and stirred for 5 h under N2 protection. After the reaction was completed, 1 mL of saturated NH4PF6 aqueous solution was added to the reaction solution to replace the anions. The mixture was concentrated by rotary evaporation to obtain a solid. The solid was dissolved in dichloromethane and purified by silica gel column chromatography using a dichloromethane / methanol mixed solvent (dichloromethane and methanol volume ratio 100:1) as the eluent. The ALA-iridium coupling compound Ir-ALA-3 was obtained by freeze drying, with a yield of 0.124 g, 61.2%.

[0023] Figure 5 The image shows the ESI-HRMS spectrum of Ir-ALA-3 in Example 3. ESI-HRMS (CH3OH): m / z = 973.3166 [M-PF6] + .

[0024] Figure 6 For Ir-ALA-3 in Example 3 1 The H-NMR spectrum shows that: 1 H-NMR (600 MHz, DMSO- d 6) δ 8.81 (s, 1H), 8.73 (s, 1H), 7.83 – 7.78 (m, 3H), 7.75 (d, J = 8.1 Hz,2H), 7.70 – 7.62 (m, 4H),7.59 (d, J = 5.7 Hz, 1H), 7.53 (d, J = 5.8 Hz, 2H), 6.96 (dddd, J = 7.4, 6.0, 3.0, 1.5 Hz, 2H), 6.18 (dd, J = 7.7, 4.7 Hz, 2H),5.35 – 5.28 (m, 6H), 2.80 – 2.71 (m, 3H), 2.56 (s, 3H), 2.46 (t, J = 7.4 Hz,2H), 2.06 – 1.94 (m, 4H), 1.59 – 1.54 (m, 2H), 1.32 – 1.17 (m, 11H), 0.92 –0.82 (m, 3H).

[0025] Example 4 The antitumor activities of Ir-ALA-1, Ir-ALA-2, and Ir-ALA-3 were studied using the MTT assay. Using Ir-ALA-1, Ir-ALA-2, and Ir-ALA-3 obtained in Examples 1-3 as experimental groups and cisplatin as the control group, their cytotoxicity against tumor cells HeLa, HepG2, and 4T1 was measured. The specific measurement methods are as follows: Tumor cells were digested into single-cell suspensions using trypsin and counted using a hemocytometer. Cells were seeded into 96-well plates at 160 μL per well and cultured for 24 h. Then, different concentrations of the drug were added, and the plates were incubated at 37°C for 48 h under ambient oxygen conditions (cells were cultured in an incubator containing 5% CO2). Four h before the end of incubation, 20 μL of MTT was added per well. After 4 h, the supernatant was discarded, and 150 μL of DMSO was added per well. After shaking for 5 minutes, the OD value was measured using a microplate reader at a wavelength of 590 nm. The inhibition rate was calculated using the following formula: Inhibition rate = (mean OD value of control group - mean OD value of treatment group) / mean OD value of control group × 100%.

[0026] The results are shown in Table 1. The experimental results show that the ALA-Ir(III) conjugate has good anti-tumor activity against HeLa, HepG2, and 4T1 cells.

[0027] Table 1. IC50 of ALA-Ir(III) conjugate on different cell lines 50 value

[0028] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An α-linolenic acid-iridium coupling compound, the structural formula of which is as follows: ； in: The anion is PF6-; 。 2. The method for preparing the α-linolenic acid-iridium coupling compound according to claim 1, characterized in that, Includes the following steps: S1.IrCl·3H2O and C^N ligands were reacted at high temperature under N2 protection in a mixed solvent of ethylene glycol ethyl ether and deionized water. After cooling, filtration, washing and drying, the iridium precursor [Ir(C^N)2Cl]2 was obtained. S2,4'-Me-bpy-4-CH2OH, ALA, and catalysts DMAP and EDCI were reacted in dichloromethane solvent at room temperature for 24 h. The mixture was concentrated by rotary evaporation to obtain an oily mixture. The mixture was separated and purified by silica gel column chromatography using a dichloromethane / methanol mixed solvent as the eluent to obtain the ligand 4'-Me-bpy-4-CH2O-ALA. S3.[Ir(C^N)2Cl]2 and 4'-Me-bpy-4-CH2O-ALA were reacted in a mixture of dichloromethane and methanol under N2 protection. After the reaction was completed, a saturated NH4PF6 aqueous solution was added to the reaction solution to replace the anions. The solid was concentrated by rotary evaporation and dissolved in dichloromethane. The solid was separated and purified by silica gel column chromatography using a dichloromethane / methanol mixed solvent as the eluent. The ALA-Ir(III) conjugate was obtained by freeze drying.

3. The method for preparing the α-linolenic acid-iridium coupling compound according to claim 2, characterized in that, In step S1, the molar ratio of IrCl3·3H2O to C^N ligands is 1:2.2, and the C^N ligands are 2-(2-thiophene)pyridine, 2-(2,4-difluorophenyl)pyridine, and 2-phenylpyridine. The high-temperature reaction temperature is 135℃, the time is 24h, and the mixed solvent of ethylene glycol ethyl ether and deionized water is obtained by mixing ethylene glycol ethyl ether and deionized water at a volume ratio of 3:

1.

4. The method for preparing the α-linolenic acid-iridium coupling compound according to claim 2, characterized in that, In step S2, the molar ratio of 4'-Me-bpy-4-CH2OH, ALA, DMAP, and EDCI is 1:1.2:1.5:1.

5.

5. The method for preparing the α-linolenic acid-iridium coupling compound according to claim 2, characterized in that, In step S3, the molar ratio of [Ir(C^N)2Cl]2 to 4'-Me-bpy-4-CH2O-ALA is 1:2.4, the heating temperature is 65℃, and the time is 5h.

6. The method for preparing the α-linolenic acid-iridium coupling compound according to claim 2, characterized in that, In steps S2 and S3, the dichloromethane / methanol mixed solvent is obtained by mixing dichloromethane and methanol at a volume ratio of 100:1, and in step S3, the dichloromethane / methanol mixed solution is obtained by mixing methanol and dichloromethane at a volume ratio of 1:

2.

7. The use of the α-linolenic acid-iridium conjugate of claim 1 in antitumor drugs.

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