Ruthenium complex as well as preparation method and application thereof

Abstract

The invention discloses a ruthenium complex as well as a preparation method and application thereof, and belongs to the technical field of organic synthesis. The polypyridine ligand and the ruthenium dimer are combined through the substitution reaction, the chelate of the polypyridine ligand and the ruthenium aromatic hydrocarbon is obtained, the synthesis process is simple, the cost is low, and the chelate can be combined with an existing medicine to improve and enhance the medication curative effect. The prepared ruthenium complex can effectively inhibit cancer cell proliferation, and especially shows an excellent anti-proliferation effect on cervical cancer cells. And the liposome can improve the lipophilicity / lipophilicity of the ruthenium medicines and increase the stability of the medicines, and has better anti-tumor activity compared with platinum medicines and unmodified ruthenium medicines.

Description

Technical Field

[0001] The invention belongs to the technical field of organic synthesis, and specifically relates to a ruthenium complex and a preparation method and application thereof. Background Art

[0002] Endometrial cancer is a very common disease among women. It is an epithelial malignant tumor that occurs in the endometrium. In recent years, the incidence of endometrial cancer has continued to rise and the incidence is getting younger. Currently, the treatment of endometrial cancer is mainly surgery, and there are few treatment options and treatment progress.

[0003] Ruthenium metal complexes are a potential alternative to platinum drugs, and have good therapeutic effects on a variety of malignant tumors, such as epidermal cancer, colorectal cancer, breast cancer, ovarian cancer, etc., but their poor water solubility and large toxic side effects limit their clinical application and development. Related studies have shown that some binuclear polypyridyl ruthenium (II) metals have also been reported to have good anti-tumor activity and good anti-tumor effects in gynecological tumors, but the anti-tumor effects are mainly focused on colon cancer, breast cancer, etc., and there are fewer studies on endometrial cancer in gynecological diseases. At the same time, there are fewer reports on the semi-sandwich ruthenium complex hexafluorophosphate in gynecological tumors. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a ruthenium complex and a preparation method and application thereof, so as to solve the technical problems that the existing ruthenium metal complexes have great toxic side effects and poor therapeutic effects.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is: to provide a ruthenium complex, the general structural formula of which is:

[0006]

[0007] Among them, the structural formula of the NN group is:

[0008]

[0009]

[0010] On the basis of the above technical solution, the present invention can also be improved as follows:

[0011] Further, the structural formula of the ruthenium complex is:

[0012]

[0013] The present invention also discloses a method for preparing the ruthenium complex, comprising the following steps:

[0014] S1. Mixing a ruthenium salt solution with phellandrene, and then placing it under reflux reaction at 110-130°C for 3-5 hours to generate a ruthenium dimer complex of p-cymene; then freezing it at -30--10°C for 7-9 hours, filtering it, and drying it to obtain a ruthenium dimer; the molar ratio of Ru element to phellandrene in the ruthenium salt solution is 0.8-1.2:2-2.2;

[0015] S2. Dissolve the ruthenium dimer in an organic solvent, then add a polypyridine ligand containing a NN group, and react at 30-50°C for 3-5h. The cyclic conjugated π ligand of the ruthenium dimer is replaced by the polypyridine σ-ligand, and post-treat with saturated NH4PF6 to generate a semi-sandwich type ruthenium complex; the molar ratio of the ruthenium dimer to the polypyridine ligand is 1.8-2.2:1.5-2.5.

[0016] On the basis of the above technical solution, the present invention can also be improved as follows:

[0017] Furthermore, the molar ratio of Ru element to phellandrene in the ruthenium salt solution is 1:2; the molar ratio of ruthenium dimer to polypyridine ligand is 1:1.

[0018] Furthermore, the ruthenium salt is RuCl3·3H2O.

[0019] Furthermore, the organic solvent is a mixture of chloroform and anhydrous methanol in a volume ratio of 1-2:1-2.

[0020] Furthermore, the preparation steps of the polypyridine ligand are as follows: 1,10-phenanthroline-5,6-dione and raw material one are mixed, and then refluxed at 70-90°C for 1.5-2.5 hours, and then filtered and dried to obtain the polypyridine ligand; the raw material one is a para-derivative of o-phenylenediamine, and the molar ratio of 1,10-phenanthroline-5,6-dione to raw material one is 0.8-1.2:1.4-1.6.

[0021] Furthermore, the raw material one is 3-fluoro-1,2-o-phenylenediamine, 3,4-diaminobenzonitrile, 3,4-diaminobenzoic acid, 3,4-diaminobenzoic acid methyl ester or 4,5-difluoro-1,2-o-phenylenediamine.

[0022] Furthermore, the drying temperature is 25 to 35° C., and the drying time is 8 to 12 hours.

[0023] The invention also discloses the application of the ruthenium complex in the preparation of anti-tumor drugs.

[0024] Further, the tumor is endometrial cancer.

[0025] The beneficial effects of the present invention are:

[0026] 1. The salt uses ruthenium metal ions as the core, aromatic ruthenium ligands as the skeleton, and o-DPPZ-F, DPPZ-CN, p-FC-DPPZ, p-MF-DPPZ, DPPZ-F2 and Bphen as ligands. The polypyridine ligands are obtained by condensation reaction, and then the polypyridine ligands are combined with ruthenium dimers by substitution reaction to obtain a chelate of the polypyridine ligand and ruthenium aromatic hydrocarbon. The prepared product has stable structure and properties, simple synthesis process, low cost, and is suitable for large-scale production.

[0027] 2. The ruthenium complex prepared by the present invention can effectively inhibit the proliferation of cancer cells, especially showing excellent anti-proliferation effects on cervical cancer cells. Aromatic hydrocarbon ligands (benzene and its derivatives) are strongly coordinated with ruthenium, and ruthenium is stabilized in the +2 oxidation state, which can reduce the toxicity and side effects of ruthenium-based drugs and make the complex lipophilic. The type of substituent on the ring determines the electron distribution on the ruthenium (II) complex molecule, increases the stability of the drug, and facilitates the complex to enter the cell. Compared with platinum drugs and unmodified ruthenium drugs, it has better anti-tumor activity. The complex has good anti-tumor activity against endometrial cancer cells, can be combined with existing drugs, and can be prepared into anti-tumor drugs with pharmaceutical carriers to improve and enhance the efficacy of medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a comparison of tumors after administration of ruthenium complex and cisplatin. DETAILED DESCRIPTION

[0029] The specific embodiments of the present invention are described below to facilitate the understanding of the present invention by those skilled in the art. If the specific conditions are not specified in the examples, the methods are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturers are not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention defined and determined by the attached claims, these changes are obvious, and all inventions and creations using the concept of the present invention are protected.

[0030] Example 1

[0031] A method for preparing a ruthenium complex, the reaction equation is as follows:

[0032]

[0033] The specific steps include:

[0034] S1. Dissolve 0.5 g of RuCl3·3H2O in 30 mL of anhydrous ethanol, then reflux with 4 mL of phellandrene in anhydrous ethanol at 110°C for 5 h, then freeze at -30°C for 7 h, filter, and vacuum dry at 30°C for 10 h to obtain a reddish brown solid, i.e., ruthenium dimer;

[0035] S2. Oxidation reaction of 1,10-phenanthroline was carried out at 85°C and concentrated sulfuric acid: concentrated nitric acid = 20 mL.: 10 mL for 4 h, and then filtered and the filter cake was washed to obtain 1,10-phenanthroline-5,6-dione (ptd); 0.51 g of ptd and 0.47 g of 3-fluoro-1,2-o-phenylenediamine (3.7 mmol) were mixed, and then refluxed in a methanol system at 90°C for 1.5 h, and then filtered after cooling to room temperature, and the filter cake was washed with a methanol solution, and vacuum dried at 30°C for 10 h to obtain a white solid, i.e., oF-dppz ligand;

[0036] S3, 24 mg of ruthenium dimer was dissolved in 10 mL of organic solvent (chloroform: anhydrous methanol = 1:1, v:v), and then 24 mg of oF-dppz ligand was added, and the mixture was reacted at 30°C for 5 h. After cooling to room temperature, ammonium hexafluorophosphonate and water were added for washing, and the mixture was centrifuged. After separation and purification by silica gel, the liquid was evaporated under reduced pressure, and finally vacuumed to obtain a yellow solid, namely, ruthenium complex 1, named [(η 6 -p-cymene)Ru(oF-dppz)Cl]PF6].

[0037] The structural characterization results of complex 1 are as follows: 1 H NMR (400MHz, Acetonitrile-d3) δ9.52(d,J=5.6Hz,2H),8.64(d,J=7.9Hz,2H),7.92(dd,J=8.0,5.6Hz ,2H),6.06–6.00(m,3H),5.92–5.74(m,4H),2.71(p,J=6.9Hz,1H),2.23(s,3H),1.07(d,J=6.9Hz,6H). 13 C NMR (101MHz, CD3CN) δ175.05,173.39,160.10,154.41,153.77,138.80,130.79,130.43,130.15,129. 56,106.97,104.56,86.68,85.05,33.90,31.49,30.08,29.95,29.64,21.87,18.64.HRMS(ESI):calcd for C 28 H 23 ClFN4Ru(M-PF6) +571.0639, found 571.0641.

[0038] Example 2

[0039] A method for preparing a ruthenium complex, the reaction equation is as follows:

[0040]

[0041] The difference between this embodiment and embodiment 1 is that the 3-fluoro-1,2-o-phenylenediamine in step S2 is replaced by 3,4-diaminobenzonitrile to obtain p-NC-dppz ligand, and the remaining components and preparation conditions are the same as those in embodiment 1, and finally a ruthenium complex 2 is obtained, which is named [(η 6 -p-cymene)Ru(p-NC-dppz)Cl]PF6].

[0042] The structural characterization results of complex 2 are as follows: 1 H NMR(400MHz, Acetonitrile-d3)δ9.77(d,J=5.4Hz,2H),9.66(d,J=1.2Hz,1H), 9.64(t,J=1.6Hz,1H),9.61(d,J=1.1Hz,1H),8.82(d,J=1.7Hz,1H),8.45(d,J= 8.8Hz,1H),8.21(d,J=2.0Hz,1H),8.21–8.14(m,2H),6.32–6.04(m,3H),5.91( d,J=6.3Hz,2H),2.75(h,J=6.9Hz,1H),2.22(s,3H),1.06(d,J=6.9Hz,6H).13C NMR(101MHz,CD3CN)δ158.20,158.07,149.51,149.32,143.99,141.96,141.85,141.47,136.64,136.54,136.44,133.07 ,131.78,130.34,128.58,128.54,118.25,115.86,106.74,103.71,86.59,85.20,31.54,21.85,18.56.HRMS(ESI):calcd forC 29 H 23 ClN5Ru(M-PF6) + 578.0681, found 578.0682.

[0043] Example 3

[0044] A method for preparing a ruthenium complex, the reaction equation is as follows:

[0045]

[0046] The difference between this embodiment and embodiment 1 is that the 3-fluoro-1,2-o-phenylenediamine in step S2 is replaced by 3,4-diaminobenzoic acid to obtain p-FC-dppz ligand, and the remaining components and preparation conditions are the same as those in embodiment 1, and finally a ruthenium complex 3 is obtained, which is named [(η 6 -p-cymene)Ru(p-FC-dppz)Cl]PF6].

[0047] The structural characterization results of complex 3 are as follows: 1 H NMR(400MHz, Acetonitrile-d3)δ9.78(ddd,J=5.5,2.6,1.3Hz,2H),9.65(ddd,J =8.2,4.0,1.4Hz,2H),9.16(d,J=2.5Hz,1H),8.71(dd,J=9.3,2.5Hz,1H),8.52(s ,1H),8.51–8.48(m,1H),8.19(dd,J=8.2,5.4Hz,2H),6.10(d,J=6.4Hz,2H),5.9 1(d,J=6.3Hz,2H),2.75(p,J=6.9Hz,1H),2.23(s,3H),1.07(d,J=6.9Hz,6H).13C NMR (101MHz, DMSO) δ165.34,157.75,157.61,148.51,148.28,143.48,141.10,140.98,140.47,135.72,135.55,132.52 ,131.42,130.77,130.25,129.33,127.81,104.97,102.97,86.01,84.25,53.05,30.52,21.80,18.30.HRMS(ESI):calcd forC 29 H 24 ClN4O2Ru(M-PF6) + 597.0631, found 597.0633.

[0048] Example 4

[0049] A method for preparing a ruthenium complex, the reaction equation is as follows:

[0050]

[0051] The difference between this embodiment and embodiment 1 is that the 3-fluoro-1,2-o-phenylenediamine in step S2 is replaced by 3,4-diaminobenzoic acid methyl ester to obtain p-MF-dppz ligand, and the remaining components and preparation conditions are the same as those in embodiment 1, and finally a ruthenium complex 4 is obtained, which is named [(η 6 -p-cymene)Ru(p-MF-dppz)Cl]PF6].

[0052] The structural characterization results of complex 4 are as follows: 1 H NMR (400MHz, Acetonitrile-d3) δ10.31(d,J=5.3Hz,2H),10.23(d,J=8.2Hz,2H),9.49(d,J=1.8Hz,1H),9.07(dd,J=8.9,1.9Hz,1H),8.96(d,J=8.9 Hz,1H),8.74(dd,J=8.2,5.4Hz,2H),6.65(d,J=6.2Hz,2H),6.46(d,J=6.3 Hz,2H),4.61(s,3H),3.33(s,1H),2.79(s,3H),1.62(d,J=6.8Hz,6H).13C NMR (101MHz, CD3CN) δ166.24,157.90,157.78,149.33,149.11,144.55,142.30,141.30,140.82,136.51,136.33,133.98,132.33,131.66 ,130.67,130.55,129.30,128.40,106.70,103.64,86.55,85.19,65.94,53.23,31.53,30.92,21.86,19.51,18.56.HRMS(ESI):calcdfor C 30 H 26 ClN4O2Ru(M-PF6) + 611.0788, found 611.0789.

[0053] Example 5

[0054] A method for preparing a ruthenium complex, the reaction equation is as follows:

[0055]

[0056] The difference between this embodiment and embodiment 1 is that the 3-fluoro-1,2-o-phenylenediamine in step S2 is replaced by 4,5-difluoro-1,2-o-phenylenediamine to obtain F2-dppz ligand, and the remaining components and preparation conditions are the same as those in embodiment 1, and finally a ruthenium complex 5 is obtained, which is named [(η6 -p-cymene)Ru(F2-dppz)Cl]PF6].

[0057] The structural characterization results of complex 5 are as follows: 1 H NMR(400MHz, Acetonitrile-d3)δ9.74(dd,J=5.5,1.4Hz,2H),9.62(dd,J=8.2,1.3Hz,2H),8.24–8.15(m,4H) ,6.08(d,J=6.3Hz,2H),5.89(d,J=6.2Hz,2H),2.74(hept,J=6.9Hz,1H),2.22(s,3H),1.06(d,J=6.9Hz,6H). 13 CNMR(101MHz,CD3CN)δ158.12,156.50,156.32,153.91,153.72,149.30,141.45,141.39,141.32,140.36,136.56,130.74 ,128.76,118.32,115.97,115.90,115.84,115.77,107.10,104.03,86.97,85.59,31.94,22.26,18.96.HRMS(ESI):calcd for C 28 H 22 ClF2N4Ru(M-PF6) + 589.0545, found 589.0547.

[0058] Example 6

[0059] A method for preparing a ruthenium complex, the reaction equation is as follows:

[0060]

[0061] The specific steps include:

[0062] S1. Dissolve 0.5 g of RuCl3·3H2O in 10 mL of anhydrous ethanol, then reflux with 4 mL of phellandrene in anhydrous ethanol at 120°C for 4 h, then freeze at -20°C for 8 h, filter, and vacuum dry at 30°C for 10 h to obtain a reddish brown solid, i.e., ruthenium dimer;

[0063] S2. Dissolve 24 mg of ruthenium dimer in 10 mL of organic solvent (chloroform: anhydrous methanol = 1:1, v:v), then add 40 mg of polypyridine ligand (4,7-diphenyl-1,10-phenanthroline, purchased from Adamas), react at 40°C for 4 h, add ammonium hexafluorophosphonate and water for washing after cooling to room temperature, centrifuge, separate and purify on silica gel, evaporate the liquid under reduced pressure, and finally evacuate to obtain a yellow solid, i.e., ruthenium complex 6, named [(η 6 -p-cymene)Ru(Bphen)Cl]PF6].

[0064] The structural characterization results of complex 6 are as follows: 1 H NMR(400MHz, Acetonitrile-d3)δ9.72(d,J=5.6Hz,2H),8.07(s,2H),7.99(d,J=5.5Hz,2H),7.69–7.60(m,1 0H), 6.08 (d, J = 6.2Hz, 2H), 5.89 (d, J = 6.2Hz, 2H), 2.73 (h, J = 6.9Hz, 1H), 2.23 (s, 3H), 1.05 (d, J = 6.9Hz, 6H). 13 CNMR(101MHz,CD3CN)δ158.74,158.59,150.32,150.03,149.77,145.17,142.65,142.18,142.11,137.11,136.87,132.53 ,130.66,130.61,129.03,128.98,126.75,126.29,107.22,104.11,86.99,85.63,31.96,22.28,18.98.HRMS(ESI):calcd for C 34 H 30 ClN2Ru(M-PF6) + 603.1141, found 603.1142.

[0065] The structures of the products prepared in Examples 1-6 are shown in Table 1.

[0066] Table 1 Structural formula of complexes 1-6

[0067]

[0068]

[0069] Experimental Example 1 In vitro antitumor activity

[0070] The in vitro antitumor activity of six ruthenium complexes containing polypyridine ligands (complexes 1-6) was determined by MTT (3-(4,5-dimethyl-2-thiazolidine)-2,5-diphenyltetrazolium bromide) method, and cisplatin was used as a control group. Endometrial cancer cell lines (Ishikawa) were used for cytotoxicity experiments. The above cell lines were cultured in a cell incubator at 37°C and 5% CO2 saturated humidity. The specific experimental steps are as follows: collect cells in the logarithmic growth phase, disperse the cells evenly with complete culture medium, count them under a microscope, and adjust the cell concentration to 5×10 4 / mL was inoculated into a 96-well plate; after the cells adhered to the wall, the culture medium in the well plate was removed, and complete culture medium and drugs (cisplatin for the control group and ruthenium complex for the experimental group) were added again. After incubation for 24 hours, 10μL of MTT solution (5mg / mL) was added to the well plate, and the 96-well plate was placed in an incubator for 4 hours. After the incubation, the supernatant in the well plate was aspirated with a pipette, 100μL of dimethyl sulfoxide was added to the well plate, and the formazan was gently shaken to dissolve, and the OD value at λ=570nm was measured with an enzyme marker. Three independent repeated experiments were performed to ensure the reliability of the experimental results, and the IC value of the complex for endometrial cancer cell line (Ishikawa) was measured. 50 The numerical results are shown in Table 2.

[0071] Table 2 Activity data of complexes 1-6 against endometrial cancer cell line (Ishikawa) in vitro

[0072] Ruthenium complex name <![CDATA[IC 50 (μM)]]> Complex 1 50.9 Complex 2 28.7 Complex 3 11.4 Complex 4 9.1 Complex 5 >100.0 Complex 6 1.4 Cisplatin 12.0

[0073] As can be seen from Table 2, with the increase of the concentration of the complex, the cytotoxicity gradually increased. Compared with the positive control group cisplatin, complex 1 has the highest inhibitory ability on endometrial cancer Ishikawa cells, which is about 9 times that of cisplatin; complexes 3 and 5 also have good anti-tumor activity.

[0074] Experimental Example 2 Study on the anti-tumor activity of drugs in vivo

[0075] In order to explore the anti-tumor effect of the ruthenium complex prepared by the present invention, the in vivo anti-tumor activity of the drug was further studied. The specific steps are as follows: Ishikawa endometrial cancer cells in the logarithmic growth phase were inoculated on the right back of Balb / c female nude mice to establish an Ishikawa tumor model. The weight and tumor size of the mice were monitored every two days. When the tumor volume reached 40-70 mm 3 The mice were randomly divided into a control group (cisplatin) and a ruthenium complex group (complex 6), with 5 mice in each group. The drugs were injected into the tail vein once every three days for a total of 4 times. The mice were killed one day after the last administration to extract the tumor. Figure 1As shown, the tumor in the ruthenium complex group was significantly smaller than that in the control group. At the same time, the body weight of the mice in the ruthenium complex group remained stable during the experiment without a significant decrease, indicating that the ruthenium complex has good anti-tumor activity in vitro.

Claims

1. A ruthenium complex, characterized in that The general structural formula of the ruthenium complex is: Among them, the structural formula of the NN group is:

2. The method for preparing the ruthenium complex according to claim 1, characterized in that: The following steps are involved: S1, mixing a ruthenium salt solution with phellandrene, then placing it under reflux reaction at 110-130°C for 3-5 hours, then freezing it at -30--10°C for 7-9 hours, filtering it, and drying it to obtain a ruthenium dimer; the molar ratio of Ru element to phellandrene in the ruthenium salt solution is 0.8-1.2:2-2.2; S2. Dissolve the ruthenium dimer in an organic solvent, then add a polypyridine ligand containing an NN group, and react at 30-50° C. for 3-5 hours to obtain a ruthenium complex; the molar ratio of the ruthenium dimer to the polypyridine ligand is 1.8-2.2:1.5-2.

5.

3. The method for preparing a ruthenium complex according to claim 2, characterized in that: The molar ratio of Ru element to phellandrene in the ruthenium salt solution is 1:2; the molar ratio of the ruthenium dimer to the polypyridine ligand is 1:

1.

4. The method for preparing a ruthenium complex according to claim 2 or 3, characterized in that: The ruthenium salt is RuCl3·3H2O.

5. The method for preparing a ruthenium complex according to claim 2, characterized in that: The organic solvent is a mixture of chloroform and anhydrous methanol in a volume ratio of 1-2:1-2.

6. The method for preparing a ruthenium complex according to claim 2, characterized in that: The preparation steps of the polypyridine ligand are as follows: 1,10-phenanthroline-5,6-dione and raw material one are mixed, and then refluxed at 70-90°C for 1.5-2.5h, and then filtered and dried to obtain the polypyridine ligand; the raw material one is a para-derivative of o-phenylenediamine, and the molar ratio of the 1,10-phenanthroline-5,6-dione to the raw material one is 0.8-1.2:1.4-1.

6.

7. The method for preparing a ruthenium complex according to claim 6, characterized in that: The first raw material is 3-fluoro-1,2-o-phenylenediamine, 3,4-diaminobenzonitrile, 3,4-diaminobenzoic acid, 3,4-diaminobenzoic acid methyl ester or 4,5-difluoro-1,2-o-phenylenediamine.

8. The method for preparing a ruthenium complex according to claim 2, characterized in that: The drying temperature is 25-35° C., and the drying time is 8-12 hours.

9. Use of the ruthenium complex according to claim 1 in the preparation of anti-tumor drugs.

10. The use according to claim 9, characterized in that: The tumor is endometrial cancer.

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