JP6086576B2 - Antioxidant - Google Patents

Description

  The present invention relates to an antioxidant, and more particularly to a novel antioxidant using a porphyrin complex.

  Oxidative stress is caused by excessive generation of active oxygen in the body. Among the cells present in the body, nerve cells are particularly vulnerable to oxidative stress, and it is suggested that oxidative stress due to excessively generated active oxygen is involved in the development of various neurodegenerative diseases. Has been. The elimination of active oxygen, which is a mediator that leads to these diseases, is extremely effective in the treatment of these diseases.

From such a viewpoint, the present inventors have proposed a porphyrin complex as an antioxidant in Patent Documents 1 and 2, and a compound represented by the following formula as an antioxidant.

On the other hand, in recent studies, in diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS), there is a large amount of accumulated metal in the brain. It is considered as one of the causes causing these diseases (for example, Patent Document 1 or 2). However, the development of a drug capable of scavenging active oxygen and capable of eliminating metal has not been carried out so far.

JP 2008-255011 A

JP 2007-75058 A

Bush.Al et al, Nature Medicine (2012)

Kiaei et al. J. Neurosci. (2004)

As described above, the functions required of recent antioxidants, high O ₂ ^- in addition to free metal chelate of scavenging activity (SOD activity) - DOO ability, H ₂ O ₂ scavenging activity (catalase - peptidase activity) is, Antioxidants that satisfy all of these requirements have not yet been provided, and there is a demand for the development of antioxidants having all of these functions.

Accordingly, an object of the present invention, high O ₂ ^- in addition to free metal chelate of scavenging activity (SOD activity) - DOO ability, H ₂ O ₂ scavenging activity - with all (Catalá zero activity), a novel antioxidant Is to propose.

  As a result of intensive studies on an antioxidant using porphyrin for removal of active oxygen in order to solve the above problems, the present inventors synthesized various compounds obtained by linking two porphyrin molecules with various substituents. As a result of finding out that the above problem can be solved in the case of a specific substituent, and further researching it, the present invention has been completed.

That is, the present invention provides the following inventions.
1. An antioxidant represented by the following formula (1).
A- (B) ₂ (1)
(In the formula, A represents a group capable of capturing a metal ion.)
2. 2. The antioxidant according to 1, wherein A in the formula (1) is a compound represented by the following formula (2).
_{-C 2 H 4 - (NH-} CH 2 CH 2) n - ····· (2)
(In the formula, n represents an integer of 1 to 10)
3. 3. The antioxidant according to 1 or 2, wherein B in the formula (1) is a phthalocyanine complex, a porphyrin complex or a derivative thereof.
4). 3. The antioxidant according to 2, wherein n in the formula (2) is an integer of 3 to 5.

Antioxidants of the present invention, high O ₂ ^- in addition to free metal chelate of scavenging activity (SOD activity) - DOO ability, H ₂ O ₂ scavenging activity - has all the functionality of (Catalá zero activity), outstanding It exhibits an antioxidant function.

FIG. 1 is a chart showing the 1H-NMR spectrum (in CDCl 3) of the product obtained by mesylation of Ts-triaminediol (Ts-triaminediol). FIG. 2 is a chart showing the 1H-NMR spectrum (in CDCl 3) of the product obtained by mesylation of Ts-tetramine-diol. FIG. 3 is a chart showing the 1H-NMR spectrum (in CDCl3) of the porphyrin monomer. FIG. 4 is a chart showing a 1H-NMR spectrum (in CDCl3) of a porphyrin dimer. FIG. 5 is a chart showing a 1H-NMR spectrum (in CDCl3) of a product obtained by Ts group deprotection of a porphyrin dimer. FIG. 6 is a chart showing the result of tracking the long wavelength shift of the maximum absorption wavelength (Soret band) and the Q band by UV-vis spectrum when Mn is introduced into the triamine dimer. FIG. 7 is a chart showing the fluorescence spectrum of the ZnPD-N3 solution and the fluorescence spectrum when an excessive amount of FeCl2 is added to the ZnPD-N3 solution.

Hereinafter, the present invention will be described in more detail.
The antioxidant of the present invention is represented by the following formula (1).
A- (B) ₂ (1)
(In the formula, A represents a group capable of capturing a metal ion.)

The group capable of capturing the metal ion represented by A is preferably a compound represented by the following formula (2).
_{-C 2 H 4 - (NH-} CH 2 CH 2) n - ····· (2)
(In the formula, n preferably represents an integer of 1 to 10, more preferably n is an integer of 3 to 5).
Specific examples of the compound represented by the above formula (2) include the following substituents.
_{-C 2 H 4 - (NH-} CH 2 CH 2) 3 -
_{-C 2 H 4 - (NH-} CH 2 CH 2) 4 -
_{-C 2 H 4 - (NH-} CH 2 CH 2) 5 -
In addition, as a group capable of capturing the metal ion represented by A,
_{-C 2 H 4 - (NH-} CH 2 CH 2 CH 2) n -NH-CH 2 CH 2 -
_{-C 3 H 6 - (NH-} CH 2 CH 2 CH 2) n -NH-CH 2 CH 2 CH 2 -
(In the formula, n represents an integer of 3 to 5)
Can also be preferably used.

The group capable of capturing oxygen represented by B is preferably a phthalocyanine complex, a porphyrin complex, or a derivative thereof.
Examples of the metal forming the complex include Mn, Zn, Fe, Cu and the like.
Specific examples of this group include the following substituents.

Specific examples of the antioxidant of the present invention include the following compounds.

  The antioxidant of the present invention can obtain a desired effect sufficiently with the above-mentioned compound alone, but it contains various components that are usually used in the formulation of this kind of medicine within the range that does not impair the effect of the above-mentioned compound. May be.

  The antioxidant of the present invention is synthesized by (1) synthesis of polyamine block, (2) synthesis of porphyrin monomer, (3) porphyrin dimerization, (4) Ts group deprotection, (5) Mn introduction. It can be manufactured by performing 5 steps. Details of each step are shown in the examples.

The antioxidant of the present invention has a super-oxide dismutase (SOD) activity and a peroxynitrite (ONOO ⁻ ) scavenging activity, and also has a metal ion scavenging function and a catalase function. . A confirmation method for these functions will be described.
The SOD activity and ONOO ^- erase activity can be measured by the stopped flow method.
The stopped-flow method is a technique used for rapid mixing of solutions and reaction tracking. By mixing the solution of two reacting species faster than the reaction time scale, a concentration non-equilibrium state can be instantaneously generated, and subsequent dynamic changes in the concentration composition in the reaction system can be observed. Thus, the reaction rate can be determined. The law, unlike the indirect method, such as cytochrome C method, O ₂ · ^- - Stop Tofuro you are possible to observe the attenuation directly high versatility.
The method for calculating the reaction rate constant by the stopped flow method is shown below.
Disproportionation reaction of superoxide anion radical (O ₂ ·) (2O ₂ · + 2H ⁺ → H ₂ O ₂ + O ₂ ) can be expressed as follows.
-D [O ₂ ·] / dt = k _self [O ₂ ·] ² + k _obs [O ₂ ]-(4-1)
Since k _self [O ₂ ·] ² is very small and negligible under the conditions of this reaction (pH = 8.1, temperature 37.0 ° C.), it is written as the following equation (3-2) be able to.
-D [O ₂ ·] / dt = k _obs [O ₂ ·]-(4-2)
When KO ₂ and a porphyrin solution (solvent HEPES / HEPESNa buffer solution) in DMSO are rapidly mixed and the peak of O ₂ · is traced at an absorbance of 270 nm, the initial reaction appears to be a primary reaction. This decay curve is determined for each porphyrin concentration. Next, when (4-2) is transferred and integrated,
-D [O ₂ ·] / [O ₂ ·] = k _obs dt-(4-3)
ln [O ₂ ·] ₀ / [O ₂ ·] _t = −k _obs × t − (4-4)
Next, the measured O ₂ · erasing rate constant (k _obs) is the plotting the natural logarithm (ln) of the absorbance of O ₂ · to the reaction time pseudo-first order straight line. Calculate k _obs at each concentration. The following conditional expression holds for the second-order reaction rate constant (k _cat ) and the previous k _obs .
k _cat = k _obs × [SOD model] − (4-5)
Therefore, the graph obtained by plotting the concentration on the horizontal axis and k _obs on the vertical axis has a linear relationship, and the reaction rate constant (k _cat : M ⁻¹ s ⁻¹ ) is obtained from the slope.

The metal ion trapping function can be measured by chelate titration or ¹ H-NMR titration of Zn complex. However, in the antioxidant of the present invention, quenching due to exciplex formation should occur in the fluorescence spectrum. Therefore, it can be confirmed by tracking the change in the fluorescence spectrum.

Catalase activity means that H ₂ O ₂ is disproportionated and activated to cause a reaction in which H ₂ O and O ₂ are generated.

For this reason, there are _two catalase activity measurement methods: a method of measuring the amount of disproportionated H ₂ O ₂ or a method of measuring the amount of O ₂ generated by disproportionation.
The measurement of the amount of H ₂ O ₂ includes a method of directly measuring absorbance and a method of indirectly measuring by peroxidase or the like. However, since the absorbance method is easily influenced by other compounds present in the mixture, it is difficult to perform accurate measurement, and the indirect method tends to have a larger error than the direct method. If you want to measure the disproportionation of the above _H 2 _{O 2,} the radical cleavage of the _{H 2 O 2 (H 2 O} 2 → · OH + OH -) because the effect of not considered, it is hard to say that very accurate way.
Whereas O ₂ measurement, for measuring the O ₂ generated only when the H ₂ O ₂ was disproportionated, more accurate, if you want to precise evaluation of such enzyme activity, usually employed here how It is common. The Clark-type dissolved oxygen electrode is the most accurate method because it can be measured with a sensitivity of about several nmol and the oxygen is specifically detected although the device itself is expensive.
The Clark-type dissolved oxygen electrode is, for example, a commercially available apparatus (manufactured by HANSATECH, UK, OXY-1), and the following reaction proceeds at both electrodes of the anode silver electrode and the cathode platinum electrode. To do.
Cathode - de ^{_{O 2 + 4H + + 4e -}} → 2H 2 O
Anode 4Ag + 4Cl ⁻ → 4AgCl + 4e ⁻
When the solution has O _2, the electrode reaction as described above proceeds, in terms of electrochemical potential of that time the amount of O ₂ to measure the amount of O _2.

The antioxidant of the present invention is used as a therapeutic and / or prophylactic composition for various disorders and diseases such as aging, carcinogenesis, inflammation, ischemic organ damage, arteriosclerosis caused in the body by the influence of active oxygen. Can do. The administration route of the antioxidant composition includes oral administration, intravenous administration, intraperitoneal administration, subcutaneous administration, intramuscular administration, and the like, and preparations suitable for oral administration include tablets, capsules, powders, granules, Examples include solutions and syrups. For therapeutic and / or prophylactic compositions, as a pharmaceutically acceptable carrier, suitable excipients, binders, disintegrants, lubricants, flavoring agents, coloring agents, solubilizing aids known in the art. Agents, suspensions, coating agents and the like may be included.
It can also be used as a topical pharmaceutical preparation or cosmetic for transdermal administration for treating or ameliorating various skin or mucosal symptoms such as skin or mucosal aging, carcinogenesis or inflammation, or skin tanning. Good forms suitable for topical medicines or cosmetics include solutions, poultices, patches and the like. External preparations may contain excipients, flavoring agents, coloring agents, solubilizing agents, suspending agents and the like as pharmaceutically acceptable carriers.
Furthermore, it can be provided in the form of food or beverage. Preferred food forms include powders, granules, pastes, jellies and the like. When making food, other additives and the like can be added as appropriate. Foods or beverages include those in the category of food for specified health use, food for the sick, and the like.
In addition, the usage-amount of the antioxidant of this invention is arbitrary according to a use purpose, a dosage form, a user's physique, etc., and is not restrict | limited in particular.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.

[Synthesis example]
A novel Mn porphyrin dimer “MnPD-N3” having a metal ion chelating ability was synthesized.

The synthesis was performed in five steps: (1) synthesis of polyamine block, (2) synthesis of porphyrin monomer, (3) porphyrin dimerization, (4) Ts group deprotection, and (5) Mn introduction. Hereinafter, each step will be specifically described.
(1) Synthesis of polyamine block As starting materials, diethylenetriamine (diethylenetriamine) protected with a tosyl group (Ts group), N, N ′, N ″ -tris (p-toluenesulfonyl) diethylenetriamine (N, N ′, N ′) '-Tris (p-toluenesulfonyll) diethylenetriamine) (Tokyo Kasei) and triethylenetetramine (triethylenetetramine) (Aldrich) were used. In all syntheses, the progress of the reaction was followed by silica TLC (chloroform: methanol = 9: 1 etc.).
(A) Both-end alkyldiolation of Ts-triamine (Ts-triamine)

8.487 g (15 mmol) of “N, N ′, N” -tri (p-toluenesulfonyll) diethylenetriamine (Ts-triamine), 3.00 g (34.1 mmol) of ethylene carbonate, 0.03 g (0 .58 mmol) of KOH was added and heated and stirred at 165 ° C. under nitrogen.
After stirring with heating for 4 hours, the reaction mixture was cooled to 90 ° C., 20 ml of ethanol was added, and the mixture was stirred with heating for 1 hour.
Chloroform: Separation with water, the chloroform phase was recovered, the solvent was removed, and the solid was recovered.
The white solid obtained by recrystallization was collected by suction filtration and dried in vacuo at 50 ° C. to obtain Ts-triaminediol. The yield was 5.13 g, and the yield was 52%. Assignment of ¹ H-NMR spectrum of the obtained product was 7.68 ppm 6H phenyl H, 7.43 ppm 6H phenyl H, 4.83 ppm 2H NH, 3. It was 50-3.10 ppm 12H-CH2-, 2.42 ppm 9H Ts-Me.
(B) Mesylation of Ts-triaminediol (Ts-triaminediol)

1.006 g (1.54 mmol) of Ts-triamine-diol was added and dissolved in 10 ml of chloroform.
The mixture was cooled to -15 to -20 ° C, 250 µl (3.23 mmol) of methanesulfonyl chloride was added, and the mixture was stirred for 30 minutes. Thereafter, a mixture of 5 ml of ice (5 g) and 2.5 ml of 10% HCl was added for liquid separation, and the organic layer was recovered.
After dehydration and filtration, the solvent was removed, and then recrystallized with ethyl acetate to obtain a mesylated product. The ¹ H-NMR spectrum (in CDCl ₃ ) of the obtained product is shown in FIG. Attribution 7.78Ppm 6H phenyl H, 7.38ppm 6H phenyl _{H, 4.43-3.38ppm 16H -CH 2 -,} 3.08ppm 6H Me, a 2.47ppm 9H Ts-Me, yield 0.1796g The yield was 23%.
(C) Ts group protection of triethylenetetramine (triethylenetetramine)

25 g (0.131 mol) of p-toluenesulfonic acid chloride was added and dissolved in 65 ml of pyridine and brought to 50-60 ° C. under nitrogen.
5 ml of a pyridine solution of 4.8 g (0.0328 mol) of triethylenetetramine was dropped using a dropping funnel, and stirring was continued for 1 hour after the dropping.
After cooling the solution, 32 ml of water was added and stirred overnight.
The mixture was cooled in an ice bath for 2 hours, and the precipitate was collected by suction filtration to obtain Ts-tetramine. Washing was performed with chilled 95% ethanol. The yield was 7.441 g, and the yield was 30%.
The assignment of ¹ H-NMR spectrum (in CDCl ₃ ) of the obtained product is 7.77 ppm 8H phenyl H, 7.34 ppm 8H phenyl H, 5.50 ppm 2H NH, 3.44-3.18 ppm 12H—CH _2. -2.45 ppm 12H Ts-Me.
(D) Both ends alkyldiolation of Ts-tetramine

1.00 g (1.31 mmol) of Ts-tetramine and 1.81 g (13.1 mmol) of potassium carbonate were added and dissolved in 20 ml of dimethylformamide (DMF).
214 μl (3.0 mmol) of 2-bromoethanol was added, and the mixture was heated and stirred at 75 ° C. for 2 days under nitrogen.
The potassium carbonate was removed by filtration, and DMF was evaporated by vacuum evaporation to obtain a yellow solid.
When methanol was added to the obtained yellow solid, Ts-tetraminediol was obtained as a white precipitate.
The yield was 0.28797 g, and the yield was 26%. The assignment of ¹ H-NMR spectrum (in CDCl ₃ ) of the obtained product is 7.75 ppm 8H phenyl H, 7.42 ppm 8H phenyl H, 2.49 ppm 12H Ts-Me, 3.90-2.60 ppm 20H −. CH ₂ - it was.
(E) Mesylation of Ts-tetramine-diol

11.286 g (13.3 mmol) of Ts-tetramine-diol (M.W = 851.08), 4 ml of triethylamine were added and dissolved in 65 ml of dichloromethane.
The mixture was cooled to −15 to −20 ° C., and 3.242 g (28.3 mmol) of methanesulfonyl chloride was slowly added.
The solution was stirred for 30 minutes, 43 ml (43 g) of ice and 22 ml of 10% HCl were added and separated, and the organic layer was collected.
After dehydration, the solvent was removed to obtain a mesylated product as a white solid. The ¹ H-NMR spectrum (in CDCl ₃ ) of the obtained product is shown in FIG. The assignments are 7.79 ppm 8H phenyl H, 7.37 ppm 8H phenyl H, 4.43 ppm 4H, 3.38 ppm 4H, 3.43 ppm 4H —CH ₂ —, 3.06 ppm 6H Me, 2.47 ppm 12H Ts-Me. The yield was 6.275 g, and the yield was 47%.
(F) Ns group protection of triethylenetetramine

12.18 g (0.0549 mol) of o-nitrobenzenesulfonic acid chloride is dissolved in 30 ml of pyridine, and 5 ml of a dehydrated pyridine (3 ml) solution of 2 ml of triethylenetetramine (triethylenetetramine) is heated and stirred at 50 ° C. under nitrogen using a dropping funnel Added while. After 1 hour, it was cooled and 20 ml of water was slowly added and stirred overnight. The black gel-like liquid was dissolved in a small amount of dimethyl sulfoxide (DMSO) and added dropwise to water for precipitation purification to obtain a brown solid. The yield was 3.66 g, and the yield was 43%. The assigned ¹ H-NMR spectrum (in CDCl ₃ ) of the obtained product is 7.79 ppm 8H phenyl H, 7.37 ppm 8H phenyl H, 4.43 ppm 4H, 3.38 ppm 4H, 3.43 ppm 4H —CH. _2- , 3.06 ppm 6H Me, 2.47 ppm 12H Ts-Me.
(2) Synthesis of porphyrin monomer (a) Synthesis of H ₂ PyP ₃ P

Benzaldehyde (14 ml), 4-pyridinecarboxaldehyde (6 ml) and pyrrole (12.5 ml) were dissolved in propionic acid (500 ml), heated and refluxed for 1.5 hours, added with ethylene glycol (350 ml), allowed to cool, and left overnight in a refrigerator. did. The solution was filtered and a purple solid was collected.
H ₂ PyP ₃ P was isolated by silica column chromatography (developing solvent: chloroform). Note that TPP comes out first, and the target porphyrin monomer comes out second.
The ¹ H-NMR spectrum (in CDCl ₃ ) of the obtained product is shown in FIG. The assignments are A: -2.81 (2H, inner H), B: 9.04 (2H, pyridyl), C: 8.90, 8.79 (8H, β-pyrrole), D: 8.20. (8H, pyridyl and m-phenyl), E: 7.78 (9H, o- and p-phenyl), yield 1.78 g, yield 2%.

(3) Synthesis of porphyrin dimerized Ts-triamine dimer (a) Method of quaternization with mesyl group

0.207 g (0.336 mmol) of H ₂ PyP ₃ P was dissolved in 8 ml of NMP and heated to 100 ° C.
2 ml of an NMP solution in which 0.161 g (0.16 mmol) of Ts-triamine-OSO ₂ CH ₃ was dissolved was slowly added dropwise. After 48 hours, the reaction was stopped and NMP was evaporated by vacuum evaporation. Subsequently, the target porphyrin dimer was isolated by silica column chromatography.
After dissolving in a small amount of methanol, an appropriate amount of NH ₄ PF ₆ was added to obtain a reddish purple precipitate. Washed with water and collected by filtration.
The ¹ H-NMR spectrum (in CDCl ₃ ) of the obtained product is shown in FIG. The assignments are 9.60 ppm 4H ortho-pyridyl, 9.10-8.80 ppm 20H meta-pyridyl and β-pyrrole, 8.20 ppm 12H phenyl, 7.90 ppm 18H phenyl, 7.70 ppm 12H Ts-phenyl, The yield was 2-4%.
(4) Ts group deprotection of porphyrin dimer 0.03 g of Ts-triamine dimer was added, 2 ml of 96% H ₂ SO ₄ was added, and the mixture was heated and stirred at 100 ° C. for 72 hours.
After 72 hours, the mixture was allowed to cool and neutralized with an aqueous NaOH solution.
Solvent water was evaporated and a brown solid was recovered. Then, it processed with TOYOPEARL HW-50F.
The ¹ H-NMR spectrum (in CDCl ₃ ) of the obtained product is shown in FIG. The assignments were 9.60 ppm 4H ortho-pyridyl, 9.40 ppm 4H meta-pyridyl, 9.05-8.80 ppm 16H β-pyrrole, 8.30-8.10 ppm 30H phenyl.

(5) Metal introduction into porphyrin dimer (a) Mn introduction into triamine dimer Triamine dimer was dissolved in 5 ml of methanol. 10 equivalents of manganese acetate tetrahydrate was added to the triamine dimer, and the mixture was heated to reflux at 50 ° C.
The long wavelength shift of the maximum absorption wavelength (Soret band) and the Q band were traced by UV-vis spectrum. (FIG. 6) The reaction was stopped when the peak at 423 nm disappeared and only the peak at 466 nm was reached, and the Q band was changed from four to two. (About 24 hours)
Excess manganese acetate tetrahydrate was removed by evaporating the solvent and dialyzing against a dialysis membrane with a molecular weight cut off of 500 for several days.
Thereafter, it was treated with an ion exchange column (filler: chelate resin (Amberlite IRC-748 or CR11)). By removing the solvent, a Mn introduced product MnPD-N3, which was a purple-green solid, was recovered. The final yield was about 5 MG and the yield in all 4 steps was 0.1%.

(B) Introducing Zn into Triamine Dimer Triamine dimer was dissolved in water, 10 equivalents of ZnCl ₂ was added to the triamine dimer, and the mixture was allowed to stand at 50 ° C. for 1 day in a water bath.
The reaction was stopped when a long wavelength shift in the Soret band and a decrease in the Q band were confirmed in the UV-vis spectrum.
By treating with an ion exchange column (filler: chelate resin (Amberlite IRC-748 or CR11)) and removing the solvent. A Zn-introduced ZnPD-N3 which was a purple solid was recovered.

[Example 1]
Of the obtained compounds, the SOD activity and ONOO ^- erase activity of Mn porphyrin dimer (hereinafter referred to as “MnPD-N3”) were evaluated by the above-mentioned stopped-flow method.

At this time, SOD activity evaluation was performed as follows, and ONOO ^- erase activity evaluation was performed as follows.
(SOD activity evaluation)
O ₂ · ^- as the source using the KO _2. KO ₂ is immediately _{KO 2 + H 2 O → KOH} + O 2 · when mixed with water ^{- +} H +
To produce O ₂ · ⁻ .
In the experiment, a porphyrin complex dissolved in 60MM HEPES / HEPES-Na buffer was set in one syringe with a solution in which KO ₂ was saturated in DMSO, and the other syringe was mixed at 36 ° C. and 25: 1. Te, _{O 2} · generated ^- is a state that is decomposed by the porphyrin complex _{O 2} · ^- was followed by a maximum absorption wavelength ^{245nm (ε = 1640M -1 cm -1} ), was calculated secondary reaction rate constant.

(ONOO-Erasure Activity Evaluation)
As in the SOD activity evaluation, a porphyrin complex in which ONO ^- solution is dissolved in PB is set in one syringe and PB is mixed in the other syringe, mixed at 36 ° C., 25: 1, and decomposed by the porphyrin complex Was tracked at a maximum absorption wavelength of 302 nm of ONOO ⁻ , and a second-order reaction rate constant was calculated. Note that ONOO-erasing activity was measured in the presence of 2 mM ascorbic acid.

<SOD activity measurement conditions>
Temperature: 36 ° C
Optical path length: 2mm
Wavelength: 245nm
Irradiation time: 100 μs
Sample mixing ratio:
Porphyrin solution / _{O 2} ^- solution = 25: 1
Buffer: HEPES (pH 8.1)
Porphyrin concentration: 0-5 μM
Measurement _{sample: MnPD-N3, Mn-monoM4PyP} 3 P, MnM4Py 4 P

<ONOO ^- erasing activity measurement conditions>
Temperature: 36 ° C
Optical path length: 2mm
Wavelength: 302nm
Irradiation time: 100 μs
Sample mixing ratio:
Porphyrin solution / ONOO ^- solution = 25: 1 (v / v)
Buffer: PB (pH 7.4) (containing 2 mM ascorbic acid)
Porphyrin concentration: 0-5 μM
Measurement _{sample: MnPD-N3, Mn-monoM4PyP} 3 P, MnM4Py 4 P
[ONOO]: 2 mM (final concentration)

<Preparation of KO ₂ saturated solution>
A spoonful of KO ₂ was placed in a brown vial, dissolved in DMSO, stirred for 1.5 hours and allowed to stand for 1 hour.

<ONOO ^- Preparation of>
The maximum absorption wavelength of ONOO ⁻ is λ = 302 nm, the molar extinction coefficient is 1670 M ⁻¹ cm ⁻¹ , and the concentration was adjusted by correcting the concentration with a UV-vis spectrum.
1) Dilute 2.22 ml of 30% H ₂ O ₂ to 25 ml, dissolve 0.8 MH ₂ O ₂ , 1.38 g NaNO ₂ in 25 ml, 0.8 M NaNO ₂ , 1.5 g NaOH Dissolved in 25 ml, each prepared 1.5M NaOH.
2) The three solutions were cooled in an ice bath.
3) With 0.8M H ₂ O ₂ 0.8M NaNO ₂ was mixed in an ice bath.
4) After mixing, 1M HCl was added with stirring, and at the same time, 1.5M NaOH was added within 1 second to obtain a yellow solution.
5) Dilution with water was then performed to adjust the ONOO ⁻ concentration.

As a result, the SOD activity was 6.7, and the ONOO ^- digestion activity was 0.42. This value is almost the same as the activity of the porphyrin monomer corresponding to the MnPD-N3 monomer body, and it was confirmed that it has sufficient SOD activity and ONOO ^- erase activity.

Further, whether or not ZnPD-N3 has a metal ion capturing function was evaluated by fluorescence spectrum measurement.
The fluorescence spectrum of ZnPD-N3 was measured as follows.
<Experimental operation>
A sample solution was prepared so that the total amount of the solution was 2 ml, and this was transferred to a quartz cell and measured.
<Experimental conditions>
Porphyrin: 1 μM (final concentration)
Metal ion: 5 mM (final concentration)
Fe ²⁺ (FeCl ₂ )
Cu ²⁺ (CuCl ₂ )
Cd ²⁺ (CdCl ₂ )
Zn ²⁺ (ZnCl ₂ )
Mn ²⁺ (MnCl ₂ )
Al ³⁺ (Al ₂ Cl ₃ )
(Et) ₄ N ⁺ Cl ⁻ (TEAC)
Solvent: Water excitation wavelength: 423 nm (maximum absorption wavelength of ZnPD-N3)
Fluorescence wavelength: 433-750nm

FIG. 7 shows the fluorescence spectrum of the ZnPD-N3 solution and the fluorescence spectrum when an excessive amount of FeCl ₂ is added to the ZnPD-N3 solution. Although ZnPD-N3 exhibited fluorescence in the absence of Fe ions, a significant decrease in fluorescence intensity was observed in the presence of Fe ions. This change is suggested to be quenching due to the formation of exciplex accompanying the Fe ion chelate of ZnPD-N3.

  When the same evaluation was performed in the presence of Zn, Mn, Cd, Al, and Cu ions in addition to Fe ions, a remarkable decrease in fluorescence intensity was observed as with Fe ions. On the other hand, in the mononuclear porphyrin ZnM4PyP, no change in fluorescence intensity was observed in the presence of various metal ions. (FIG. 7) From the above, it was suggested that ZnPD-N3 chelates these metal ions and that the two porphyrins are prone to form exciplexes due to the accompanying structural changes.

Moreover, the catalase activity evaluation of obtained MnPD-N3 was performed. Catalase activity was measured by quantifying the amount of oxygen produced by disproportionation of H ₂ O ₂ using a Clark-type dissolved oxygen electrode. MnPD-N3 was used in the absence of metal ions, a sample previously adjusted to each concentration, and mixed with 100 μM CuCl ₂ in order to track the change in activity associated with metal chelate.
<Experimental conditions>
Temperature: 20 ° C
Solvent: PB (pH 7.4)
Sample concentration (final concentration): 0, 2, 4, 6, 8, 10 μM
Time: 0-600sec
H ₂ O ₂ : 1 mM (final concentration)
sample:
Mn salen complex (Control)
MnPD-N3
MnPD-N3 + CuCl ₂ (100 μM)

<Operation>
1. Set of equipment Drop a few drops of saturated KCl solution on the electrode section, stack tobacco paper cut into a circle, and 1.5 cm square Teflon (registered trademark) in this order, and then apply an O-ring using an applicator. It set to the electrode part.
Filled with distilled water and kept the apparatus temperature constant.
Aqueous calibration was performed as follows.
The sample chamber of the oxygen electrode was filled with an appropriate amount of water, and was allowed to stand until the output voltage of the oxygen electrode became constant (5 to 10 minutes) while stirring with a stirrer (rotation speed 50). The voltage value was defined as the output voltage of the oxygen concentration of the solution in equilibrium with oxygen in the air.
Thereafter, a small amount of dithionite was added to the sample chamber with a spoon, and the lid was inserted until the bottom surface was in close contact with the water surface. The value when the voltage became constant was taken as the output voltage when the oxygen concentration was 0.
2. Sample preparation 10 mM H ₂ O ₂ was prepared by taking 113 μl of 30% H ₂ O ₂ and diluting to 100 ml.
100 μl of sample solution was prepared so that the final concentration was 0, 2, 4, 6, 8, 10 μM.
3. Measurement: 100 μl of 10 mM H ₂ O ₂ and 800 μl of PB were added. When the rate was stable, 100 μl of sample solution was added. (Total 1ml)
After reacting for 10 minutes, the measurement was stopped, the change in oxygen concentration was analyzed, and the rate constant was calculated.
Table 2 shows the catalase activity of each sample. MnPD-N3 had a low activity value of 38 s ^{−1 in} the absence of metal ions. On the other hand, it was found that in the presence of 100 μM copper ions, it has a high catalase activity (220 s ⁻¹ ) exceeding the Mn salen complex, which is a catalase semimic. When considered together with the results of the metal chelate ability evaluation, the two porphyrins form an associated state along with the copper ion chelate, so that the distance between Mn and Mn is immobilized, and H ₂ O ₂ is considered to be disproportionate in two nuclei. From the above, it was revealed that MnPD-N3 exhibits catalytic activity in response to metal ions.

Table 1: Catalytic activity of cationic Mn porphyrin complexes and Mn salen complexes

In addition, Mn salen complex used as a comparison is Eukarion Inc. The compound having a structure similar to that of “trade name EUK-134” developed by the company was synthesized as follows.
H ₂ (3OMeSalen) was dissolved in methanol, 10 to 40 equivalents of manganese chloride was added in the presence of triethylamine, and the mixture was heated and stirred at 50 ° C. The reaction was followed by UV-vis spectrum, and when the maximum absorption wavelength was changed, the reaction was stopped and purified.

Claims (3)

An antioxidant represented by the following formula (1) ,
A- (B) ₂ (1)
(In the formula, A indicates the capture groups of metal ions,
B is a porphyrin complex or a derivative thereof,
The metal that forms the complex is Mn, Zn, Fe, or Cu . )
The antioxidant in the above formula (1) is a compound represented by the following formula (2).
_{-C 2 H 4 - (NH-} CH 2 CH 2) n- ····· (2)
(In the formula, n represents an integer of 1 to 10) The antioxidant according to claim 1 , wherein the antioxidant represented by the formula (1) is the following compound.

2. The antioxidant according to claim 1, wherein n in the formula (2) is an integer of 3 to 5.

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