Use of dinuclear manganese complex in treating and / or preventing corneal neovascular disease

Abstract

Disclosed is use of a dinuclear manganese complex in treating and / or preventing a corneal neovascular disease. The dinuclear manganese metal complex exhibits excellent catalytic activity. It can catalyze the generation of a large amount of oxygen from hydrogen peroxide and can inhibit neovascularization.

Description

Use of binuclear manganese complexes for treating and / or preventing corneal neovascular diseases Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to the application of a binuclear manganese metal complex as a new drug for treating corneal neovascular diseases. Background Art

[0002] Corneal neovascularization (CNV) is usually caused by a variety of causes, such as alkali burns, contact lens wear, dry eye syndrome, corneal infection and other ocular surface diseases. It is a major vision-threatening disease worldwide that can lead to reduced corneal transmittance and even blindness.

[0003] Currently, the treatment methods for corneal neovascularization diseases are mainly local applications of immunomodulators, corticosteroids, collagenase activity inhibitors, etc. In severe cases, there are also surgical treatments such as amniotic membrane coverage combined with anti-VEGF, corneal transplantation, etc.

[0004] However, the use of local drugs may cause complications, such as steroid-induced intraocular hypertension and steroid-induced cataracts. Immunomodulators such as tacrolimus are also toxic and can damage normal corneal tissue cells. Corneal transplantation is expensive and has the possibility of rejection and dissolution.

[0005] Therefore, further research and development of convenient, safe and effective drugs for the treatment of corneal neovascular diseases is needed in clinical practice. Summary of the Invention

[0006] To solve the above problems, the inventors have discovered that a binuclear manganese metal complex with manganese as the metal center and other small molecule ligands has excellent catalytic activity, can catalyze hydrogen peroxide to produce a large amount of oxygen, and can inhibit the formation of new blood vessels, thereby completing the present invention.

[0007] The present invention aims to provide a use of a binuclear manganese metal complex in the preparation of a drug for preventing and / or treating corneal neovascularization diseases. The binuclear manganese metal complex is a binuclear manganese metal complex with manganese as the metal center and formed with other small molecule ligands.

[0008] Manganese complex refers to a metal complex with manganese as the metal center and other small molecule ligands.

[0009] The binuclear manganese complex of the present invention is selected from Mn10, Mn11, Mn16, Mn31, Mn36, Mn40, Mn54, Mn75, Mn79, Mn87, Mn89, Mn100, Mn103, Mn104, Mn105, Mn114, and Mn177. These complexes can catalyze hydrogen peroxide to efficiently produce oxygen, thereby inhibiting angiogenesis.

[0010] Since ocular inflammation can lead to the accumulation of a large amount of hydrogen peroxide, angiogenesis is accompanied by a hypoxic environment. The binuclear manganese complex provided by the present invention can catalyze hydrogen peroxide to produce oxygen, consume harmful hydrogen peroxide, increase oxygen concentration, alleviate hypoxia and inflammation characteristics, and has the potential to be used as a drug for ocular diseases.

[0011] These dinuclear manganese complexes can be prepared by known methods, for example, see: Kal, S.; Ayensu-Mensah, L.; Dinolfo, PH, Evidence for catalytic water oxidation by a dimanganese tetrakis-Schiff base macrocycle. Inorg Chim Acta 2014, 423, 201-206, or: Drew, MGB; Harding, CJ; McKee, V.; Morgan, GG; Nelson, J., Geometric Control of Manganese Redox State. J Chem Soc Chem Comm 1995, (10), 1035-1038. These documents are incorporated herein by reference in their entirety.

[0012] The present invention has the following beneficial effects:

[0013] (1) Binuclear manganese complexes can efficiently catalyze hydrogen peroxide to produce a large amount of oxygen.

[0014] (2) Binuclear manganese complexes can significantly inhibit angiogenesis in vitro and in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG1 shows a mass spectrum of the binuclear manganese complex obtained in Example 1;

[0016] FIG2 shows the mass spectrum of the binuclear manganese complex obtained in Example 2;

[0017] FIG3 shows the mass spectrum of the binuclear manganese complex obtained in Example 3;

[0018] FIG4 shows a mass spectrum of the binuclear manganese complex obtained in Example 4;

[0019] FIG5 shows the mass spectrum of the binuclear manganese complex obtained in Example 5;

[0020] FIG6 shows the mass spectrum of the binuclear manganese complex obtained in Example 6;

[0021] FIG7 shows the mass spectrum of the binuclear manganese complex obtained in Example 7;

[0022] FIG8 shows the mass spectrum of the binuclear manganese complex obtained in Example 8;

[0023] FIG9 shows the mass spectrum of the binuclear manganese complex obtained in Example 9;

[0024] FIG10 shows the mass spectrum of the binuclear manganese complex obtained in Example 10;

[0025] FIG11 shows the mass spectrum of the binuclear manganese complex obtained in Example 11;

[0026] FIG12 shows the mass spectrum of the binuclear manganese complex obtained in Example 12;

[0027] FIG13 shows the mass spectrum of the binuclear manganese complex obtained in Example 13;

[0028] FIG14 shows the mass spectrum of the binuclear manganese complex obtained in Example 14;

[0029] FIG15 shows the mass spectrum of the binuclear manganese complex obtained in Example 15;

[0030] FIG16 shows the mass spectrum of the binuclear manganese complex obtained in Example 16;

[0031] FIG17 shows the mass spectrum of the binuclear manganese complex obtained in Example 17;

[0032] FIG18 is a schematic diagram showing the binuclear manganese complex obtained in Example 1 catalyzing hydrogen peroxide to produce oxygen;

[0033] FIG19 shows the results of inhibiting in vitro neovascularization in Experimental Example 2;

[0034] FIG20 shows a slit lamp photograph of blood vessels in the eye of a black mouse in Experimental Example 3;

[0035] FIG21 shows the change in the length of new blood vessels in black mice over the days of Experimental Example 3;

[0036] FIG22 shows the change in the area of ​​new blood vessels in black mice over time in Experimental Example 3;

[0037] FIG23 shows the single crystal diffraction results of the binuclear manganese complex in Example 1. Example

[0038] The present invention is further described below through specific examples. However, these examples are merely exemplary and do not constitute any limitation to the scope of protection of the present invention.

[0039] Example 1 Preparation of binuclear manganese complex Mn10

[0040] 20.6 mg of 5-(1,1-dimethylethyl)-2-hydroxy-1,3-benzenedicarboxaldehyde and 11.7 mg of 2,2'-diamino-N-methyldiethylamine were dissolved in 10 mL of methanol. 36.1 mg of manganese perchlorate was added and the mixture was reacted at 65°C for 24 hours. The reaction mixture was then dried by vortexing and washed three times with anhydrous ether. The mixture was dissolved in 10 mL of methanol and 8.2 mg of sodium acetate was added and allowed to react at room temperature for 24 hours. The solvent was then dried by vortexing, washed three times with anhydrous ether, and recrystallized from a methanol / ether (1:10) mixture to obtain a yellow powder, Mn10. The mass spectrum shows Mw = 741.28408, with a theoretical molecular weight of 741.27.

[0041] The yellow powder was grown in a mixed solvent of methanol / acetonitrile / diethyl ether (volume ratio 1:1:10) to obtain a crystal. The single crystal diffraction results are shown in FIG23 , and the single crystal parameters are shown in Table 1 below.

[0042] Note: Since perchlorate exists in the environment where the single crystal is obtained, the crystal molecule contains one perchlorate (ClO4, molecular weight 99.88), so the crystal shows a molecular weight of 841.15.

[0043] Example 2 Preparation of binuclear manganese complex Mn11

[0044] 20.6 mg of 5-(1,1-dimethylethyl)-2-hydroxy-1,3-benzenedicarboxaldehyde and 11.7 mg of 2,2'-diamino-N-methyldiethylamine were dissolved in 10 mL of methanol, and 36.1 mg of manganese perchlorate was added, and the mixture was reacted at 65°C for 24 hours.

[0045] The reaction mixture was dried and washed three times with anhydrous ether. The mixture was dissolved in 10 mL of methanol, and 16.6 mg of p-phthalic acid was added. The mixture was incubated at room temperature for 24 hours. The solvent was then dried and washed three times with anhydrous ether. Mn11 was recrystallized from a methanol / ether (1:10) mixture to yield a yellow powder. The mass spectrum is shown in Figure 2.

[0046] Example 3 Preparation of binuclear manganese complex Mn16

[0047] 20.6 mg of 5-(1,1-dimethylethyl)-2-hydroxy-1,3-benzenedicarboxaldehyde and 10.3 mg of diethylenetriamine were dissolved in 10 mL of methanol, and 36.1 mg of manganese perchlorate was added and reacted at 65°C for 24 hours.

[0048] The reaction mixture was dried and washed three times with anhydrous ether. The mixture was dissolved in 10 mL of methanol, and 24.4 mg of biotin was added. The mixture was incubated at room temperature for 24 hours. The solvent was then dried and washed three times with anhydrous ether. Mn16 was recrystallized from a methanol / ether (1:10) mixture to yield a yellow powder. The mass spectrum is shown in Figure 3.

[0049] Example 4 Preparation of binuclear manganese complex Mn31

[0050] 20.6 mg of 5-(1,1-dimethylethyl)-2-hydroxy-1,3-benzenedicarboxaldehyde and 11.7 mg of 2,2'-diamino-N-methyldiethylamine were dissolved in 10 mL of methanol, and 36.1 mg of manganese perchlorate was added, and the mixture was reacted at 65°C for 24 hours.

[0051] The reaction mixture was dried and washed three times with anhydrous ether. The mixture was dissolved in 10 mL of methanol, and 12.2 mg of benzoic acid was added. The mixture was incubated at room temperature for 24 hours. The solvent was then dried and washed three times with anhydrous ether. Mn31 was recrystallized from a methanol / ether (1:10) mixture to yield a yellow powder. The mass spectrum is shown in Figure 4.

[0052] Example 5 Preparation of binuclear manganese complex Mn36

[0053] 18.3 mg of 5-chloro-2-hydroxy-1,3-benzenedicarboxaldehyde and 11.7 mg of 2,2'-diamino-N-methyldiethylamine were dissolved in 10 mL of methanol, and 36.1 mg of manganese perchlorate was added. The mixture was reacted at 65°C for 24 hours.

[0054] The reaction mixture was dried and washed three times with anhydrous ether. The mixture was dissolved in 10 mL of methanol, and 12.2 mg of benzoic acid was added. The mixture was incubated at room temperature for 24 hours. The solvent was then dried and washed three times with anhydrous ether. Mn36 was recrystallized from a methanol / ether (1:10) mixture to yield a yellow powder. The mass spectrum is shown in Figure 5.

[0055] Example 6 Preparation of binuclear manganese complex Mn40

[0056] 18 mg of 5-methoxy-2-hydroxy-1,3-benzenedicarboxaldehyde and 11.7 mg of 2,,2'-diamino-N-methyldiethylamine were dissolved in 10 mL of methanol, and 36.1 mg of manganese perchlorate was added and reacted at 65°C for 24 hours.

[0057] The reaction mixture was dried and washed three times with anhydrous ether. The mixture was dissolved in 10 mL of methanol, and 12.2 mg of benzoic acid was added. The mixture was incubated at room temperature for 24 hours. The solvent was then dried and washed three times with anhydrous ether. Mn40 was recrystallized from a methanol / ether (1:10) mixture to yield a yellow powder. The mass spectrum is shown in Figure 6.

[0058] Example 7 Preparation of binuclear manganese complex Mn54

[0059] Dissolve 20.6 mg of 5-(1,1-dimethylethyl)-2-hydroxy-1,3-benzenedicarboxaldehyde and 14.6 mg of tris(2-aminoethyl)amine in 10 mL of methanol. Add 36.1 mg of manganese perchlorate and react at 65°C for 24 hours. The solvent is dried by spin drying, washed three times with anhydrous ether, and recrystallized from methanol / ether (1:10) to obtain a yellow powder, Mn54. The mass spectrum is shown in Figure 7.

[0060] Example 8 Preparation of binuclear manganese complex Mn75

[0061] 20.6 mg of 5-(1,1-dimethylethyl)-2-hydroxy-1,3-benzenedicarboxaldehyde and 10.3 mg of diethylenetriamine were dissolved in 10 mL of methanol, and 36.1 mg of manganese perchlorate was added and reacted at 65° C. for 24 hours.

[0062] The reaction mixture was dried and washed three times with anhydrous ether. Dissolved in 10 mL of methanol, 8.2 mg of sodium acetate was added, and the mixture was incubated at room temperature for 24 hours. The solvent was then dried and washed three times with anhydrous ether. Mn75 was recrystallized from a methanol / ether (1:10) mixture to yield a yellow powder. The mass spectrum is shown in Figure 8.

[0063] Example 9 Preparation of binuclear manganese complex Mn79

[0064] 20.6 mg of 5-(1,1-dimethylethyl)-2-hydroxy-1,3-benzenedicarboxaldehyde and 13.1 mg of 3,3'-diaminodipropylamine were dissolved in 10 mL of methanol, and 36.1 mg of manganese perchlorate was added, and the mixture was reacted at 65°C for 24 hours.

[0065] The reaction mixture was dried and washed three times with anhydrous ether. The mixture was dissolved in 10 mL of methanol, and 8.2 mg of sodium acetate was added. The mixture was incubated at room temperature for 24 hours. The solvent was then dried and washed three times with anhydrous ether. Mn79 was recrystallized from a methanol / ether (1:10) mixture to yield a yellow powder. The mass spectrum is shown in Figure 9.

[0066] Example 10 Preparation of binuclear manganese complex Mn87

[0067] 20.6 mg of 5-(1,1-dimethylethyl)-2-hydroxy-1,3-benzenedicarboxaldehyde and 11.7 mg of 2,2'-diamino-N-methyldiethylamine were dissolved in 10 mL of methanol, and 36.1 mg of manganese perchlorate was added, and the mixture was reacted at 65°C for 24 hours.

[0068] The reaction mixture was dried and washed three times with anhydrous ether. The mixture was dissolved in 10 mL of methanol, and 8.2 mg of sodium acetate was added. The mixture was incubated at room temperature for 24 hours. The solvent was then dried and washed three times with anhydrous ether. Mn87 was recrystallized from a methanol / ether (1:10) mixture to yield a yellow powder. The mass spectrum is shown in Figure 10.

[0069] Example 11 Preparation of binuclear manganese complex Mn89

[0070] 20.6 mg of 5-(1,1-dimethylethyl)-2-hydroxy-1,3-benzenedicarboxaldehyde and 10.4 mg of 2,2'-oxybis(ethylamine) were dissolved in 10 mL of methanol, and 36.1 mg of manganese perchlorate was added, and the mixture was reacted at 65°C for 24 hours.

[0071] The reaction mixture was dried and washed three times with anhydrous ether. The mixture was dissolved in 10 mL of methanol, and 8.2 mg of sodium acetate was added. The mixture was incubated at room temperature for 24 hours. The solvent was then dried and washed three times with anhydrous ether. Mn89 was recrystallized from a methanol / ether (1:10) mixture to yield a yellow powder. The mass spectrum is shown in Figure 11.

[0072] Example 12 Preparation of binuclear manganese complex Mn100

[0073] 15.0 mg of 2-hydroxy-1,3-benzenedicarboxaldehyde and 11.7 mg of 2,2'-diamino-N-methyldiethylamine were dissolved in 10 mL of methanol, and 36.1 mg of manganese perchlorate was added and reacted at 65°C for 24 hours.

[0074] The reaction mixture was dried by vortexing and washed three times with anhydrous ether. The mixture was dissolved in 10 mL of methanol, and 8.2 mg of sodium acetate was added and incubated at room temperature for 24 hours. The solvent was then dried by vortexing, washed three times with anhydrous ether, and recrystallized from a methanol / ether (1:10) mixture to yield a yellow powder, Mn100. The mass spectrum is shown in Figure 12.

[0075] Example 13 Preparation of binuclear manganese complex Mn103

[0076] 16.4 mg of 5-methyl-2-hydroxy-1,3-benzenedicarboxaldehyde and 11.7 mg of 2,,2'-diamino-N-methyldiethylamine were dissolved in 10 mL of methanol, and 36.1 mg of manganese perchlorate was added, and the mixture was reacted at 65°C for 24 hours.

[0077] The reaction mixture was dried by vortexing and washed three times with anhydrous ether. The mixture was dissolved in 10 mL of methanol, and 8.2 mg of sodium acetate was added and incubated at room temperature for 24 hours. The solvent was then dried by vortexing, washed three times with anhydrous ether, and recrystallized from a methanol / ether (1:10) mixture to yield a yellow powder of Mn103. The mass spectrum is shown in Figure 13.

[0078] Example 14 Preparation of binuclear manganese complex Mn104

[0079] 18.0 mg of 5-methoxy-2-hydroxy-1,3-benzenedicarboxaldehyde and 11.7 mg of 2,2'-diamino-N-methyldiethylamine were dissolved in 10 mL of methanol, and 36.1 mg of manganese perchlorate was added, and the mixture was reacted at 65°C for 24 hours.

[0080] The reaction mixture was dried by vortexing and washed three times with anhydrous ether. The mixture was dissolved in 10 mL of methanol, and 8.2 mg of sodium acetate was added and incubated at room temperature for 24 hours. The solvent was then dried by vortexing, washed three times with anhydrous ether, and recrystallized from a methanol / ether (1:10) mixture to yield a yellow powder of Mn104. The mass spectrum is shown in Figure 14.

[0081] Example 15 Preparation of binuclear manganese complex Mn105

[0082] 18.3 mg of 5-chloro-2-hydroxy-1,3-benzenedicarboxaldehyde and 11.7 mg of 2,2'-diamino-N-methyldiethylamine were dissolved in 10 mL of methanol, and 36.1 mg of manganese perchlorate was added, and the mixture was reacted at 65°C for 24 hours.

[0083] The reaction mixture was dried by vortexing and washed three times with anhydrous ether. The mixture was dissolved in 10 mL of methanol, and 8.2 mg of sodium acetate was added and incubated at room temperature for 24 hours. The solvent was then dried by vortexing, washed three times with anhydrous ether, and recrystallized from a methanol / ether (1:10) mixture to yield a yellow powder of Mn105. The mass spectrum is shown in Figure 15.

[0084] Example 16 Preparation of binuclear manganese complex Mn114

[0085] 15.0 mg of 2-hydroxy-1,3-benzenedicarboxaldehyde and 11.7 mg of 2,2'-diamino-N-methyldiethylamine were dissolved in 10 mL of methanol, and 36.1 mg of manganese perchlorate was added and reacted at 65°C for 24 hours.

[0086] The reaction mixture was dried by vortexing and washed three times with anhydrous ether. The mixture was dissolved in 10 mL of methanol, and 12.2 mg of benzoic acid was added. The mixture was incubated at room temperature for 24 hours. The solvent was then dried by vortexing, washed three times with anhydrous ether, and recrystallized from a methanol / ether (1:10) mixture to yield a yellow powder, Mn114. The mass spectrum is shown in Figure 16.

[0087] Example 17 Preparation of binuclear manganese complex Mn177

[0088] 20.6 mg of 5-(1,1-dimethylethyl)-2-hydroxy-1,3-benzenedicarboxaldehyde and 11.7 mg of 2,2'-diamino-N-methyldiethylamine were dissolved in 10 mL of methanol, and 36.1 mg of manganese perchlorate was added, and the mixture was reacted at 65°C for 24 hours.

[0089] The reaction mixture was dried and washed three times with anhydrous ether. Dissolved in 10 mL of methanol, 51.6 mg of dexamethasone sodium phosphate was added, and the mixture was incubated at room temperature for 24 hours. The solvent was then dried and washed three times with anhydrous ether. Mn177 was recrystallized from a methanol / ether (1:10) mixture to yield a yellow powder. The mass spectrum is shown in Figure 17.

[0090] Test example

[0091] Test Example 1 Catalysis of hydrogen peroxide to produce oxygen

[0092] The binuclear manganese metal complexes of Examples 1-17 were dissolved in PBS solution (pH 7.4) to obtain 3 μmol / L solutions. 1 mL of the above solutions was added to the oxygen electrode working tank. Subsequently, 20 μL of 30% H2O2 was added and the generation of oxygen was monitored. The results are shown in the following table:

[0093] Among them, regarding the binuclear manganese metal complex Mn10 of Example 1, as shown in FIG18 , Mn10 catalyzes hydrogen peroxide to produce a large amount of oxygen within 350 s (maximum oxygen production 532.1 μM).

[0094] It can be seen that the binuclear manganese metal complexes of Examples 1-17 of the present invention have excellent oxygen production activity and are potentially useful as drugs for eye diseases.

[0095] Experimental Example 2 Inhibition of In vitro Neovascularization

[0096] 100 μL of matrix gel (brand: iCell, product number: iPSMed-iCell-CA004) was added to a 96-well plate and placed in a 37°C incubator for 30 minutes until solidified. Subsequently, human umbilical vein endothelial cells (HUVECs) were added to the 96-well plate (1×10 4The cells were then treated with DMEM containing 80 μM Mn10 and continued to culture (5% CO2, 37°C). A control group was treated with DMEM containing an equal amount of blank solvent (1% DMSO in PBS). After 6 hours, the tube formation of the cells was observed under a microscope, and statistical analysis was performed using Image J. The results are shown in Figure 19.

[0097] Experimental Example 3: Reducing the length and area of ​​new blood vessels in the cornea of ​​animals

[0098] Establishment of an alkali burn neovascularization model in mice: C57BL6j female mice of SPF level 6-8 weeks were used to establish an alkali burn (CoNV) mouse model. The modeling method was as follows: 1% pentobarbital was injected intraperitoneally at a rate of 100uL / 20g. The right eye of the mouse was anesthetized with topical proparacaine eye drops. The mouse was then subjected to alkali burn using Whatman filter paper (2mm diameter) soaked in 1mol / L NaOH. After the filter paper was placed on the eye for 30 seconds, the eye was rinsed with 20ml of 0.9% saline solution, and the mouse cornea was immediately observed and photographed under a slit lamp microscope. The left eye was not treated and was observed for up to 14 days.

[0099] After establishing the mouse alkali burn neovascularization model, the two groups of alkali-burn mice were treated with solvent (20 v% sulfobutyl β-cyclodextrin aqueous solution) and Mn10 (topical eye drops, four times a day) on days 0, 3, 7, 11, and 14, respectively, underwent general anesthesia with 1% pentobarbital at a rate of 100 uL / 20 g intraperitoneally. Slit lamp microscopy was used to observe and obtain photographs of the superior, inferior, temporal, and nasal quadrants of each eye, as shown in Figure 16, to assess the length and area of ​​corneal neovascularization.

[0100] The results are shown in Figures 21 and 22. Compared with mice treated with solvent alone, Mn10 significantly reduced the length (mm) and area (mm) of corneal neovascularization in mice with alkali burns. 2 ).

[0101] Neovascular length L: The length is measured based on the longest vessel with the smallest continuous curvature and facing the center of the cornea, and the average value of the four quadrants is taken; Neovascular area = C / 12×π×[r2-(r-L) 2 ],

[0102] Where C represents the number of circumferential hours of neovascular invasion,

[0103] r represents the corneal radius (measured as 2.4 mm),

[0104] L represents the average length of new blood vessels.

[0105] The present invention has been described in detail above with reference to specific embodiments and / or exemplary examples and the accompanying drawings. However, these descriptions should not be construed as limiting the present invention. Those skilled in the art will appreciate that various equivalent substitutions, modifications, or improvements may be made to the technical solutions and implementations of the present invention without departing from the spirit and scope of the present invention, all of which fall within the scope of the present invention. The scope of protection of the present invention shall be determined by the appended claims.

Claims

1. Use of a binuclear manganese metal complex in the preparation of a medicament for preventing and / or treating corneal neovascularization, wherein the binuclear manganese metal complex is a binuclear manganese metal complex with manganese as the metal center and formed with other small molecule ligands, and is selected from:

2. The method according to claim 1, wherein the binuclear manganese metal complex is:

3. The use according to claim 1, wherein The corneal neovascular diseases include corneal neovascular diseases secondary to corneal inflammation, trauma, chemical injury, bacterial, fungal and viral infections.

4. The use according to claim 3, wherein The chemical injury is a chemical alkali burn.

5. Drugs used to prevent and / or treat corneal neovascular diseases, including: A pharmaceutically effective amount of an active ingredient binuclear manganese metal complex selected from: Pharmaceutically acceptable carriers, such as sulfobutyl beta-cyclodextrin or dimethyl sulfoxide.

6. The drug according to claim 5, wherein the binuclear manganese metal complex is:

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