JP2006008623A - Porphyrin compound having succinimidyl group or its metal complex, albumin bonded with porphyrin metal complex and oxygen infusion containing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a porphyrin metal complex capable of forming an oxygen complex stable over a longer period and effectively acting as an oxygen infusion or a porphyrin compound as a precursor of the complex. <P>SOLUTION: The porphyrin compound is expressed by formula [I] (R<SB>1</SB>is a (substituted) straight-chain or alicyclic hydrocarbon group; R<SB>2</SB>is an alkylene; R<SB>3</SB>is a group allowing the coordination of imidazolyl group to the center transition metal ion M in the case of coordinating a transition metal ion M of the 4-5 period; and R is methylene group or ethylene group). The invention further provides a metal complex of the compound coordinated with a transition metal ion of the 4-5 period. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a porphyrin compound having a succinimidyl group capable of binding and dissociating oxygen reversibly and directly binding to a protein or a metal complex thereof, a porphyrin metal complex-bound albumin obtained by covalently binding it to albumin, and a porphyrin thereof The present invention relates to an oxygen infusion solution (artificial oxygen carrier) containing metal complex-bound albumin.

  Heme, which is a prosthetic group of hemoglobin and myoglobin that plays a role in oxygen transport and storage in vivo, that is, porphyrin iron (II) complex, reversibly binds and dissociates molecular oxygen in response to oxygen partial pressure. Research that attempts to reproduce the same oxygen adsorption / desorption ability as that of natural heme with a synthetic porphyrin iron (II) complex has been reported since the 1970s. Patent Document 2 and the like are representative, and recent development trends are systematically described in Non-Patent Document 3, Non-Patent Document 4, and the like.

  Examples of porphyrin iron (II) complexes that can form stable oxygen complexes under room temperature conditions include 5,10,15,20-tetrakis (α, α, α, α-o-pivalamidophenyl) porphyrin iron (II). Complexes (hereinafter referred to as FeTpivPP complexes) are known (Non-Patent Document 5). The FeTpivPP complex can be used in an organic solvent such as benzene, toluene, dichloromethane, tetrahydrofuran, N, N-dimethylformamide at room temperature when an axial base such as 1-alkylimidazole, 1-alkyl-2-methylimidazole, etc. coexists in excess. Can reversibly bond and dissociate molecular oxygen. Further, when this complex is embedded in a bilayer vesicle composed of phospholipid, the same oxygen adsorption / desorption function is exhibited even under physiological conditions (aqueous phase system, pH 7.4, 37 ° C.) (for example, Non-patent document 6). In order for the FeTpivPP complex to reversibly bind and dissociate oxygen, it is indispensable to add an excess molar number of axial base molecules from the outside as described above. However, some imidazole derivatives widely used as axial bases have a pharmacological action and are often highly toxic in the body. In addition, when phospholipid endoplasmic reticulum is used, an excessively coexisting imidazole derivative may be a factor destabilizing the form. The method of extremely reducing the amount of the axial base added is none other than introducing an imidazole derivative through a covalent bond in the molecule.

  Our group is able to supply a stable oxygen carrier without external addition of an axial base if, for example, an alkylimidazole derivative is covalently bonded as a substituent into the porphyrin iron (II) complex molecule. The FeTpivPP analogue already having a substituent at the 2-position of the porphyrin ring was synthesized, and the reversible oxygen adsorption / desorption reaction was clarified in a system in which this was included in phospholipid endoplasmic reticulum or human serum albumin. (Patent Document 1, Patent Document 2, Patent Document 3).

  However, since the oxidation reaction of central iron (II) is generally accelerated in an aqueous solution, the stability of the obtained oxygen complex is extremely low. In other words, reversible oxygen coordination activity can be expressed only when the central iron of the porphyrin iron complex is in a divalent state, and when the central iron is oxidized and becomes an iron (III) complex, the oxygen coordination is achieved. Position activity is completely lost. In the above phospholipid endoplasmic reticulum, in which the porphyrin iron (II) complex developed by the present inventors group is dispersed in water, and in the prior art for inclusion in human serum albumin, the porphyrin iron (II) complex is uniformly introduced into water. In addition to dissolving, providing a minute hydrophobic space in the vicinity of the oxygen coordination site has the effect of extending the stability of the oxygen complex.

On the other hand, in these systems, the inclusion driving force of porphyrin iron (II) complex is hydrophobic interaction, that is, non-covalent bond, so the porphyrin metal complex that is oxygen binding site is dissociated from the hydrophobic field. There is also a possibility of end. When the porphyrin metal complex aqueous solution or dispersion is administered into the body as an artificial oxygen carrier, for example, as a substitute for red blood cells, it is desirable that the porphyrin metal complex aqueous solution or dispersion stay in the bloodstream for a certain period of time and serve as an oxygen transporter. In fact, when an albumin-porphyrin metal complex complex is administered into blood, the porphyrin metal complex dissociates from albumin. In other words, in order to achieve a longer blood half-life, the design and synthesis of a porphyrin metal complex that can be immobilized on albumin in a strong binding mode has been awaited.
JP Collman, Acc. Chem. Res., 10, 265 (1977) F. Basolo, BM Hoffman, JA Ibers, Acc. Chem. Res., 8, 384 (1975) Momentau et al., Chem. Rev., 110, 7690 (1994) JP Collman, Chem. Rev., 104, 561 (2004) JP Collman, et al., J. Am. Chem. Soc., 97, 1427 (1975) E. Tsuchida et al., J. Chem. Soc., Dalton Trans., 1984, 1147 (1984) JP 59-164791 A JP 59-162924 A JP-A-8-301873

  Accordingly, the present invention provides a porphyrin metal complex or a porphyrin compound as a precursor thereof that eliminates the above-mentioned problems of the prior art, forms a stable oxygen complex for a longer period of time, and can effectively act as an oxygen infusion solution. With the goal.

  As a result of intensive studies on the molecular design and functional expression of porphyrin metal complexes that can be immobilized on albumin with a stronger binding mode, the present inventors have obtained substituents necessary for effectively exerting oxygen adsorption ability at the 2-position. That is, albumin is attached to the side chain portion of a 5,10,15,20-tetrakis (α, α, α, α-o-substituted amidophenyl) porphyrin metal complex bound with an imidazole derivative which is a basic axial ligand. Introducing a succinimide ester group that can form a covalent bond specifically with the amino acid residue of the above, and linking the active group and albumin with a covalent bond, the conventional non-covalent bond is included in the hydrophobic region of albumin Compared to the above, it is possible to synthesize albumin in which the porphyrin metal complex is fixed with a much stronger force (that is, porphyrin metal complex-bound albumin). Found to be able to provide new oxygen infusion is capable of forming an oxygen complex, and have completed the present invention.

That is, according to the first aspect of the present invention, the formula [I]:

(Here, R ₁ is a linear or alicyclic hydrocarbon group which may have a substituent, R ₂ is an alkylene group, and R ₃ is a transition metal ion M in the 4th to 5th periods of the periodic table.) Porphyrin compounds represented by a group that allows coordination of an imidazolyl group to the central transition metal ion M when coordinated, R is a methylene group or an ethylene group), or transitions in the 4th to 5th periods of the periodic table The metal complex coordinated with a metal ion is provided.

  Moreover, according to the 2nd side surface of this invention, the porphyrin metal complex binding albumin obtained by making the succinimidyl group of the porphyrin metal complex of this invention react with albumin is provided.

  Furthermore, according to the 3rd side surface of this invention, the oxygen infusion containing the porphyrin metal complex binding albumin of this invention is provided.

  The porphyrin metal complex of the present invention has an imidazolyl group that functions as a basic axial ligand at the 2-position of the tetraphenylporphyrin metal complex and a succinimidyl group as an active side chain substituent that can be covalently bound to a protein. By forming an amide bond with a lysine residue, an oxygen complex with high stability can be formed. That is, the porphyrin metal complex of the present invention can be firmly bonded to albumin by covalent bond while maintaining oxygen binding ability. A novel oxygen infusion solution containing this can be provided as a highly stable preparation that is practically used and does not dissociate the porphyrin metal complex even after administration into blood. The porphyrin metal complex of the present invention is useful as a gas adsorbent, oxygen adsorbent / desorbent, oxidation-reduction catalyst, oxygen oxidation reaction catalyst, etc. in addition to the oxygen infusion described above.

  The porphyrin compound of the present invention is represented by the above formula [I].

In the formula [I], R ₁ is a linear or alicyclic hydrocarbon group which may have a substituent. R ₁ is preferably a linear or alicyclic hydrocarbon group having a substituent at the 1-position. Examples of such linear or alicyclic hydrocarbon groups include 1,1-disubstituted C ₁ -C ₁₈ alkane groups, 1-substituted cyclopropyl groups, 1-substituted cyclopentyl groups, 1-substituted cyclohexyl groups. , 2-substituted norbornyl groups (the substituents in these groups are methyl group, alkylamide group (R′CONH—), alkyl ester group (R′OOC—), alkyl ether group (R′O—), 1-methyl -2-cyclohexenyl group, 1-adamantyl group, etc. Here, the alkyl group represented by R ′ is preferably a C ₁ -C ₆ alkyl group.

In the formula [I], R ₂ is an alkylene group, preferably a C ₁ -C ₁₀ alkylene group.

In the formula [I], R ₃ allows coordination of the imidazolyl group to the central transition metal ion M when the transition metal ion M in the 4th to 5th periods of the periodic table is coordinated (inhibition of coordination). Not) group. Examples of such R ₄ are a hydrogen atom, a methyl group, an ethyl group or a propyl group.

  In the formula [I], R represents a methylene group or an ethylene group.

The present invention also provides a porphyrin metal complex in which transition metal ions M in the 4th to 5th periods of the periodic table are coordinated to the porphyrin compound of the formula [I]. As the transition metal ion M, Fe or Co is preferable. The valence of Fe can be +2 or +3, and the valence of Co can be +2. This porphyrin metal complex can be represented by the formula [II].

In the formula [II], X ⁻ represents a halide ion such as a chloride ion or a bromide ion, ^and the number n of X ⁻ is a value obtained by subtracting 2 from the valence of the transition metal ion M.

When M is a +2 valent transition metal ion such as Fe (II) or Co (II), this porphyrin metal complex has an imidazole group bonded in the porphyrin molecule as shown in the following formula [III]. The state is coordinated to the transition metal ion M.

  Such a porphyrin metal complex can exhibit an oxygen binding ability only by the molecule. In addition, since the porphyrin metal complex of the present invention has a succinimidyl group that can be covalently bonded to a protein as a side chain substituent, the porphyrin metal complex that is an oxygen binding site can be obtained by simply mixing this with albumin at room temperature. Forms a covalent bond with the lysine amino group of albumin and is immobilized inside albumin via an amide bond. That is, even when administered into the body, the porphyrin metal complex does not dissociate from albumin in the blood circulation system, and a longer residence time in the blood is expected.

  In addition to resuscitation fluid for hemorrhagic shock (blood substitute for blood for transfusion), oxygen transfusion is used for preoperative blood dilution, extracorporeal circulation such as cardiopulmonary bypass, perfusion fluid for transplanted organs, ischemic sites Oxygen supply solution (myocardial infarction, cerebral infarction, respiratory failure, etc.), chronic anemia treatment agent, liquid ventilation reflux solution, cancer treatment sensitizer, regenerative tissue cell culture solution, and use for rare blood group patients, It is expected to respond to patients who refuse to receive blood transfusions for religious reasons and to be applied to animal medicine. The oxygen infusion is obtained by dispersing the porphyrin metal complex-bound albumin of the present invention in physiological saline. The concentration of the porphyrin metal complex-bound albumin varies depending on the application, but as a blood substitute, a heme concentration of about 9.2 mM / L can be used, and other concentrations can be used.

  In addition, when the porphyrin is, for example, a complex of metal ions belonging to the 4th to 5th periods, the added value as a catalyst for the oxidation-reduction reaction, oxygen oxidation reaction, or oxygen addition reaction is also high. Therefore, the porphyrin metal complex of the present invention has characteristics as a gas adsorbent, a redox catalyst, an oxygen oxidation reaction catalyst, and an oxygen addition reaction catalyst in addition to oxygen infusion.

Although there is no restriction | limiting in particular in the manufacturing method of the porphyrin compound of this invention, For example, following formula [IV]:

2-hydroxymethyl-5,10,15,20-tetrakis (α, α, α, α-o-substituted amidophenyl) porphyrin can be synthesized as a starting material. In the formula [IV], R ₁ is as defined above. This porphyrin can be synthesized, for example, using the method described in Tsuchida et al., J. Chem. Soc. Perkin Trans 2, 1995, 747 (1995).

  Specifically, the formula [A]: R "OOCRC (NH-Fmoc) COOH (wherein R is as defined above, Fmoc is 9-fluorenylmethyloxycarbonyl, R" is, for example, Aspartic acid or glutamic acid derivative represented by (t-butyl) is dissolved in an appropriate dry solvent (for example, dichloromethane, benzene, dimethylformamide, etc.), dicyclohexylcarbodiimide (DCC) is added as a condensing agent, and the mixture is stirred at room temperature for 1 to 12 hours. To do. As the reaction proceeds, a white precipitate is formed. The reaction solution is cooled in an ice bath for 2 to 30 minutes, and the precipitated N, N′-dicyclohexylurea (DCU) is removed by filtration. To the filtrate is added 2-hydroxymethyl-5,10,15,20-tetrakis (α, α, α, α-o-substituted amidophenyl) porphyrin of formula [IV] and stirred for 1 to 12 hours in the dark. . The progress of the reaction is followed by thin layer chromatography (TLC), and an appropriate base (pyridine, dimethylaminopyridine, triethylamine, etc.) is added if necessary. The solvent is removed under reduced pressure, the residue is dissolved in benzene, and the precipitated DCU is removed again by filtration. After removing the solvent under reduced pressure, cold hexane is added to precipitate porphyrin. The resulting mixture was purified by silica gel column chromatography to give 2- (N-Fmoc-t-butoxycarbonyl-aminoacyl) methyl-5,10,15,20-tetrakis (α, α, α, α-o-substituted). Amidophenyl) porphyrin is obtained. Here, needless to say, aminoacyl is asparagyl or glutamyl (hereinafter the same).

  The obtained porphyrin is dissolved in dimethylformamide, piperidine is added, and the mixture is stirred at room temperature for 6 to 24 hours under light shielding. After removing dimethylformamide and piperidine under reduced pressure, the residue was purified by silica gel column chromatography. 2- (t-Butoxycarbonyl-aminoacyl) methyl-5,10,15,20-tetrakis (α, α, α, α- o-substituted amidophenyl) porphyrin is obtained.

Next, the hydrochloride salt of ω-imidazolylalkanoic acid represented by the following formula [B] (in the formula [B], R ₂ and R ₃ are as defined above) is dissolved in distilled dimethylformamide, and DCC is used as appropriate. A simple base (for example, pyridine, dimethylaminopyridine, triethylamine, etc.) is added and stirred at room temperature for 30 minutes to 4 hours.

  DCU produced as the reaction progresses is removed by filtration, and 2- (t-butoxycarbonyl-aminoacyl) methyl-5,10,15,20-tetrakis (α, α, α, α-o-substituted amide is added to the filtrate. Phenyl) porphyrin is added and stirred for 15 minutes to 2 hours in the dark. The solvent is removed under reduced pressure, and the residue is redissolved in a mixed solvent of dichloromethane and triethylamine, and stirred for 6 to 24 hours at room temperature under light shielding. After the DCU is removed by filtration, the residue is dissolved in benzene, the precipitate is filtered off, and the solvent is removed under reduced pressure. The residue was dissolved in chloroform, washed with pure water and an aqueous sodium hydrogen carbonate solution, dehydrated, and then the chloroform was removed to give 2- (N- (ω-imidazolylalkanoyl) -t-butoxycarbonyl-aminoacyl) methyl-5, 10,15,20-Tetrakis (α, α, α, α-o-substituted amidophenyl) porphyrin is obtained. Since this compound is decomposed by light and separation using a silica gel column, it is used in the next reaction as it is.

  The porphyrin thus obtained is dissolved in a suitable dry solvent (dichloromethane, chloroform, benzene, etc.), trifluoroacetic acid is added, and the mixture is stirred at room temperature for 1 to 6 hours under light shielding. Following the progress of the reaction by TLC, the solvent was removed under reduced pressure, benzene was added to the residue, and 2- (N- (ω-imidazolylalkanoyl) -aminoacyl) methyl-5,10,15,20-tetrakis (α, α, α, α-o-substituted amidophenyl) porphyrin is obtained. Since this compound is decomposed by light and separation using a silica gel column, it is used in the next reaction as it is.

  A central metal M is introduced into the obtained porphyrin. The introduction of the central metal M can be achieved by a general method described in, for example, D. Dolphin edited by The Porphyrin, 1978, Academic Press, etc., and can be obtained as a corresponding porphyrin metal complex. Generally, a porphyrin iron (III) complex is obtained in the case of an iron complex, and a porphyrin cobalt (II) complex is obtained in the case of a cobalt complex.

  Specifically, 2- (N- (ω-imidazolylalkanoyl) -aminoacyl) methyl-5,10,15,20-tetrakis (α, α, α, α-o-substituted amidophenyl) porphyrin in distilled THF Is rapidly added to iron (II) bromide prepared with electrolytic iron and hydrobromic acid under a dry argon atmosphere and reacted at 60 to 80 ° C. for 2 to 24 hours. After removing the solvent under reduced pressure, the residue is dissolved in chloroform and washed thoroughly with pure water. After dehydration and solvent removal, the resulting mixture was fractionated and purified by silica gel column chromatography. As a result, 2- (N- (ω-imidazolylalkanoyl) -aminoacyl) methyl-5,10,15,20-tetrakis ( α, α, α, α-o-substituted amidophenyl) porphinate iron (III) is obtained.

  This porphyrin is dissolved in a suitable dry solvent (dichloromethane, chloroform, benzene, diethyl ether, etc.), DCC is added, and the mixture is stirred at room temperature for 10 minutes to 2 hours. Thereto is added a dichloromethane solution of N-hydroxysuccinimide, and the mixture is allowed to react at room temperature for 2 to 24 hours under light shielding. The progress of the reaction is followed by TLC, the precipitate is filtered off at 0 ° C., and the solvent is removed under reduced pressure. The residue was dissolved in benzene, DCU was removed by filtration again, and then the solvent was removed under reduced pressure to obtain the target compound 2- (N- (ω-imidazolylalkanoyl) -succinimidyl-amino acid ester) methyl-5,10,15,20. -Tetrakis (α, α, α, α-o-pivalamidophenyl) porfinato iron (III) is obtained. Here, needless to say, the amino acid ester is alginate or glutamate.

  Of the above porphyrin metal complexes, when the iron (III) complex is in the form, an appropriate reducing agent (sodium dithionite, ascorbic acid, etc.) is used, and the central metal is trivalent to divalent by a conventional method. If reduced, oxygen binding activity can be imparted.

To bind these porphyrin iron (II) complexes to albumin, first prepare a carbonyl complex ethanol solution of the porphyrin iron (II) complex, mix it with, for example, a phosphate buffered aqueous solution of human serum albumin, and slowly For 30 minutes to 3 hours. The resulting solution is dialyzed against an aqueous phosphate buffer solution for 10 to 24 hours to remove ethanol. The porphyrin iron (II) complex-bound albumin thus obtained has an amide bond with the lysine amino group of albumin formed by the acyl group from which the succinimidyloxy group of porphyrin is released, as shown by the following formula [V]. It is what. Store refrigerated as a carbon monoxide complex in the dark.

  In both cases, a system covalently bound to recombinant human serum albumin and a system covalently bound to albumin multimers, a stable oxygen complex is rapidly formed upon contact with oxygen. These complexes can adsorb and desorb oxygen according to the oxygen partial pressure. This oxygen bond dissociation can be repeated reversibly and acts as an oxygen adsorbing / desorbing agent and oxygen carrier.

  In the case of a gas that is coordinated to a metal other than oxygen, a corresponding coordination complex can be formed (for example, carbon monoxide, nitrogen monoxide, nitrogen dioxide, etc.). For these reasons, the porphyrin metal complex of the present invention functions as an effective oxygen infusion solution, particularly in the case of an iron (II) or cobalt (II) complex, as well as a redox reaction catalyst in homogeneous and heterogeneous systems. And application as a gas adsorbent.

  Hereinafter, the present invention will be described in detail with reference to some examples, but the present invention is not limited to these examples.

Example 1
Fmoc-L-glutamic acid (t-butyl ester) (314 mg, 0.74 mmol) was dissolved in 2 mL of dichloromethane, DCC (152 mg, 0.74 mmol) was added, and the mixture was stirred at room temperature for 2 hours. A white precipitate formed as the reaction proceeded. The mixture was cooled in an ice bath for 10 minutes, and the precipitated DCU was removed by filtration. 2-Hydroxymethyl-5,10,15,20-tetrakis (α, α, α, α-o-pivalamidophenyl) porphyrin (76 mg, 73 μmol) was added to the filtrate, and the solution was concentrated to 1 mL. Stir for 2 hours in the dark. Follow the progress of the reaction by TLC and add a solution of dimethylaminopyridine (3.0 mg, 25 μmol) in dichloromethane if necessary. The solvent was removed under reduced pressure, the residue was dissolved in benzene, and the precipitated DCU was removed again by filtration. After removing the solvent under reduced pressure, cold hexane was added to precipitate purple porphyrin. The obtained mixture was purified by a silica gel column (developing solvent: chloroform / ethyl acetate = 10/1), and the target compound 2- (N-Fmoc-t-butoxycarbonyl-L-glutamyl) methyl-5,10,15, 195 mg of 20-tetrakis (α, α, α, α-o-pivalamidophenyl) porphyrin was obtained (yield: 92%).

<Analysis results>
Thin layer chromatography: Rf = 0.71 (CHCl ₃ : CH ₃ OH = 20: 1 (v / v))
Infrared absorption spectrum (ν / cm ^-1 ): 3432 (NH); 1726 (C = O (ester)), 1690 (C = O (amide))
UV-visible absorption spectrum (λmax / nm, CHCl ₃ ): 640, 589, 545, 513, 420
¹ H-NMR spectrum _{(500 MHz, CDCl 3, d} (ppm)), -2.7 (s, 2H, internal H), 0.0-0.3 (m, 36H , Bu t), 1.5 (d, 9H, O-Bu ^t of Glu), 1.9, 2.4 (m, 4H, -CH ₂ CH _2- ), 4.0-4.5 (m, 4H,> CHNHCOOCH ₂ CH <), 5.2-5.5 (m, 2H, -CH ₂ -Por) , 7.1-7.8 (m, 24H, phenyl H), 8.6-8.8 (m, 11H, amide H, pyrrole H)
ESI-MS spectrum (m / z): 1448 [M] +; 1470 [M + Na] +.

Example 2
2- (N-Fmoc-t-butoxycarbonyl-L-glutamyl) methyl-5,10,15,20-tetrakis (α, α, α, α-o-pivalamidophenyl) porphyrin (215 mg, 0.15 mmol) ) Was dissolved in 30 mL of dimethylformamide, piperidine (20 mL) was added, and the mixture was stirred at room temperature for 13 hours in the dark. After removing dimethylformamide and piperidine under reduced pressure, the residue was purified by a silica gel column (developing solvent: chloroform / methanol / acetic acid: 100/5/1) to obtain the target compound 2- (t-butoxycarbonyl-L-glutamyl) methyl- 131 mg of 5,10,15,20-tetrakis (α, α, α, α-o-pivalamidophenyl) porphyrin was obtained (yield: 72%).

<Analysis results>
Thin layer chromatography: Rf = 0.31 (CHCl ₃ : CH ₃ OH / 20: 1 (v / v))
Infrared absorption spectrum (ν / cm ^-1 ): 3432 (NH); 1730 (C = O (ester)), 1689 (C = O (amide))
UV-visible absorption spectrum (λmax / nm, CHCl ₃ ): 646, 589, 545, 514, 420
¹ H-NMR spectrum _{(500 MHz, CDCl 3, d} (ppm)), -2.7 (s, 2H, internal H), 0.0-0.2 (m, 36H , Bu t), 1.4 (d, 9H, Glu of O -Bu ^t ), 2.1, 2.4 (m, 4H, -CH ₂ CH _2- ), 3.5 (s, 1H,> CH), 5.2, 5.4 (d, 2H, -CH ₂ -Por), 7.0-7.8 ( m, 16H, phenyl H), 8.6-8.8 (11H, m, amide H, pyrrole H)
ESI-MS spectrum (m / z): 1226 [M] +; 1248 [M + Na] ⁺ .

Example 3
8- (2-Methylimidazolyl) octanoic acid hydrochloride (106 mg, 0.48 mmol) is dissolved in distilled dimethylformamide, DCC (84 mg, 0.48 mmol) and triethylamine (85 mL, 0.612 mmol) are added, and at room temperature. Stir for 2 hours. DCU produced as the reaction progresses is removed by filtration, and 2- (t-butoxycarbonyl-L-glutamyl) methyl-5,10,15,20-tetrakis (α, α, α, α-o-) is added to the filtrate. Pivalamidophenyl) porphyrin (50 mg, 40.8 μmol) was added and stirred for 30 minutes in the dark. The solvent was removed under reduced pressure, and the residue was redissolved in 2 mL of dichloromethane and 3 mL of triethylamine, and stirred for 18 hours at room temperature under light shielding. After removing DCU by filtration, the residue was dissolved in 2 mL of benzene, the precipitate was filtered off, and the solvent was removed under reduced pressure. The residue was dissolved in chloroform and washed with pure water and aqueous sodium hydrogen carbonate solution. After dehydration, chloroform was removed and 2- (N- (8- (2-methylimidazolyloctanoyl))-t-butoxycarbonyl-L-glutamyl) methyl-5,10,15,20-tetrakis (α, α, α, α-o-pivalamidophenyl) porphyrin was obtained. Since this compound was decomposed by light and separation using a silica gel column, it was directly used in the next reaction.

<Analysis results>
Thin layer chromatography: Rf = 0.22 (CHCl ₃ : CH ₃ OH = 20: 1 (v / v))
UV-visible absorption spectrum (λmax / nm, CHCl ₃ ): 643, 588, 544, 513, 421
ESI-MS spectrum (m / z): 1432 [M] +.

Example 4
2- (N- (8- (2-Methylimidazolyloctanoyl))-t-butoxycarbonyl-L-glutamyl) methyl-5,10,15,20-tetrakis (α, α, α obtained in Example 3 , Α-o-pivalamidophenyl) porphyrin was dissolved in 5 mL of dichloromethane, 5 mL of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 3 hours under light shielding. Following the progress of the reaction by TLC, the solvent was removed under reduced pressure, 5 mL of benzene was added to the residue, and 2- (N- (8- (2-methylimidazolyloctanoyl))-L-glutamyl) methyl-5,10,15 , 20-tetrakis (α, α, α, α-o-pivalamidophenyl) porphyrin was obtained. Since this compound was decomposed by light and separation using a silica gel column, it was directly used in the next reaction.

<Analysis results>
Thin layer chromatography: Rf = 0.17 (CHCl ₃ : CH ₃ OH = 10: 1 (v / v))
UV-visible absorption spectrum (λmax / nm, CHCl ₃ ): 643, 589, 544, 513, 420
ESI-MS spectrum (m / z): 1376 [M] +.

Example 5
The reaction for introducing iron into porphyrin can be achieved by a general method described in, for example, D. Dolphin, The Porphyrin, 1978, Academic Press.

A hydrobromic acid aqueous solution (1.62 mL) was placed in the three-necked flask, and nitrogen was bubbled for 30 minutes to completely deoxygenate. 107.8 mg (1.93 mmol) of electrolytic iron was quickly added, the temperature was raised to 80 ° C., and the mixture was stirred for 1 hour. When the electrolytic iron dissolved and became a transparent light green solution, the temperature was raised to 130 ° C., and hydrobromic acid and water were removed by evaporation. To the obtained pale white solid FeBr ₂ , 2- (N- (8- (2-methylimidazolyloctanoyl))-L-glutamyl) methyl-5,10,15,20-tetrakis (synthesized in Example 4) α, α, α, α-o-pivalamidophenyl) porphyrin (50 mg) and 2,6-lutidine (50 mL) in dry tetrahydrofuran (5 mL) were added dropwise under a nitrogen atmosphere, and the mixture was refluxed at the boiling point for 17 hours. When the reaction was completed, the solvent was removed under reduced pressure, and this was extracted with chloroform. After the solvent was removed under reduced pressure, the residue was dissolved in chloroform and washed thoroughly with pure water. After dehydration and solvent removal, the resulting mixture was fractionated and purified on a silica gel column (CHCl ₃ / CH ₃ OH = 10/1). The obtained component was vacuum-dried to give 2- (N- (8- (2-methylimidazolyloctanoyl))-t-butoxycarbonyl-L-glutamyl) methyl-5,10,15,20-tetrakis as a brown solid. 22 mg of (α, α, α, α-o-pivalamidophenyl) porphinate iron (III) was obtained (2- (t-butoxycarbonyl-L-glutamyl) methyl-5,10,15,20-tetrakis ( (α, α, α, α-o-pivalamidophenyl) porphyrin yield 37%).

<Analysis results>
Thin layer chromatography: Rf = 0.64 (CHCl ₃ : CH ₃ OH = 1: 1 (v / v))
Infrared absorption spectrum (ν / cm ^-1 ): 3429 (NH); 1740 (C = O (ester)), 1683 (C = O (amide))
UV-visible absorption spectrum (λmax / nm, CHCl ₃ ): 574.5, 417.5
ESI-MS spectrum (m / z): 1430 [M-Br] +.

Example 6
2- (N- (8- (2-methylimidazolyloctanoyl))-t-butoxycarbonyl-L-glutamyl) methyl-5,10,15,20-tetrakis (α, α, α, α-o-pi Valamidophenyl) porfinatoiron (III) (11 mg, 7.7 μmol) was dissolved in 1.5 mL of dichloromethane, DCC (4.2 mg, 20 μmol) was added, and the mixture was stirred at room temperature for 30 minutes. Thereto was added a dichloromethane solution (1 mL) of N-hydroxysuccinimide (2.1 mg), and the mixture was reacted at room temperature for 14 hours under light shielding. The progress of the reaction was followed by TLC, the precipitate was filtered off at 0 ° C., and the solvent was removed under reduced pressure. The residue was dissolved in benzene and DCU was removed again by filtration. The solvent was removed under reduced pressure, and the target compound 2- (N- (8- (2-methylimidazolyloctanoyl))-succinimidyl-L-glutamate) methyl-5,10,15,20-tetrakis (α, α, α , Α-o-pivalamidophenyl) porfinatoiron (III) complex / bromide was obtained in an amount of 12 mg (yield: 100%). The analysis result of this porphyrin is as follows.

Thin layer chromatography: Rf = 0.38 (CHCl ₃ : MeOH = 10: 1)
Infrared absorption spectrum (ν / cm ^-1 ): 3428 (NH); 1740 (C = O (ester)), 1685 (C = O (amide))
UV-visible absorption spectrum (λmax / nm, EtOH): 421, 568 nm
ESI-MS spectrum (m / z): 1527 [M-Br] +.

Example 7
Instead of 2-hydroxymethyl-5,10,15,20-tetrakis (α, α, α, α-o-pivalamidophenyl) porphyrin in Example 1, 2-hydroxymethyl-5,10,15,20- Tetrakis (α, α, α, α-o- (1-methylcyclohexanoyl) aminophenyl) porphyrin was used in Example 3, instead of 8- (2-methylimidazolyl) octanoic acid / hydrochloride, 8-imidazolyloctane 2- (N- (8- (imidazolyloctanoyl))-succinimidyl-L-glutamate) methyl-5,10,15,20 following exactly the same procedure as in Examples 1-6 except that acid / hydrochloride was used. -Tetrakis (α, α, α, α-o- (1-methylcyclohexanoyl) aminophenyl) porphyrin iron (III) complex / bromide was quantitatively synthesized.

<Analysis results>
Thin layer chromatography (chloroform / methanol: 10/1 (volume / volume): Rf: 0.45 (monospot))
Infrared absorption spectrum (cm ^-1 ): 1741 (νC = O (ester)), 1686 (νC = O (amide))
UV-visible absorption spectrum (CHCl ₃ , λmax: 421, 505, 581, 647, 682 nm)
ESI-MS spectrum (m / z): 1688 [M-Br] +.

Example 8
Instead of 2-hydroxymethyl-5,10,15,20-tetrakis (α, α, α, α-o-pivalamidophenyl) porphyrin in Example 1, 2-hydroxymethyl-5,10,15,20- Tetrakis (α, α, α, α-o- (1-methylpentanoyl) aminophenyl) porphyrin was used, and instead of 8- (2-methylimidazolyl) octanoic acid / hydrochloride in Example 3, 12-imidazolyl decanoic acid was used. -2- (N- (8- (imidazolyldodecanoyl)) succinimidyl-L-glutamate) methyl-5,10,15,20-tetrakis according to exactly the same procedure as in Examples 1-6 except that the hydrochloride salt was used. (Α, α, α, α-o- (1-methylcyclopentanoyl) aminophenyl) porphyrin iron (III) complex / bromide was synthesized quantitatively.

<Analysis results>
Thin layer chromatography (chloroform / methanol: 10/1 (volume / volume): Rf: 0.37 (monospot))
Infrared absorption spectrum (cm ^-1 ): 1739 (νC = O (ester)), 1683 (νC = O (amide))
UV-visible absorption spectrum (CHCl ₃ , λmax: 420, 503, 578, 647, 685 nm)
ESI-MS spectrum (m / z): 1674 [M-Br] +.

Example 9
Instead of 2-hydroxymethyl-5,10,15,20-tetrakis (α, α, α, α-o-pivalamidophenyl) porphyrin in Example 1, 2-hydroxymethyl-5,10,15,20- Tetrakis (α, α, α, α-o- (adamantanoyl) aminophenyl) porphyrin, Fmoc-L-aspartic acid (t-butyl ester) instead of Fmoc-L-glutamic acid (t-butyl ester) Except for the use of 2- (N- (8- (2-methylimidazolyloctanoyl)) succinimidyl-L-glutamate) methyl-5,10,15,20-tetrakis (following the same procedure as in Examples 1-6) α, α, α, α-o- (adamantanoyl) aminophenyl) porphyrin iron (III) complex / bromide was quantitatively synthesized.

<Analysis results>
Thin layer chromatography (chloroform / methanol: 10/1 (volume / volume): Rf: 0.54 (monospot))
Infrared absorption spectrum (cm ^-1 ): 1740 (νC = O (ester)), 1685 (νC = O (amide))
UV-visible absorption spectrum (CHCl ₃ , λmax: 420, 503, 582, 649, 679 nm)
ESI-MS spectrum (m / z): 1826 [M-Br] +.

Example 10
The porphyrin iron (III) complex / bromide 48.2 μg (0.03 μmol) synthesized in Example 6 was changed to 10 mL of anhydrous toluene solution, and after nitrogen substitution, the mixture was stirred and mixed with a dithionite aqueous solution in a heterogeneous system for about 2 hours. Reduced to iron (II). Under a nitrogen atmosphere, only the toluene layer was extracted, dehydrated and dried over anhydrous sodium sulfate, filtered, and the resulting toluene solution was transferred to a measurement cell and sealed. Thus, a toluene solution of a porphyrin iron (II) complex was obtained. The visible absorption spectrum of this solution is λmax: 440, 538, and 557 nm, and the complex corresponds to a 5-coordinate deoxy form in which one imidazole is coordinated.

  When oxygen gas was blown into this solution, the spectrum immediately changed and a spectrum of λmax: 425, 551 nm was obtained. This clearly shows that it is an oxygenated complex. When nitrogen gas was blown into this oxygenated complex solution for 1 minute, the visible absorption spectrum reversibly changed from the oxygenated spectrum to the deoxy spectrum, and it was confirmed that the adsorption / desorption of oxygen occurred reversibly. The operation of blowing oxygen and then blowing nitrogen was repeated, and oxygen adsorption / desorption could be continuously performed.

Example 11
To a solution of porphyrin iron (III) complex / bromide 482 μg (0.3 μmol) synthesized in Example 6 in ethanol (2.0 mL) was added a dithionite aqueous solution in a carbon monoxide atmosphere, and the mixture was stirred for 5 minutes. Upon reduction, a carbon monoxide complex was formed, and the color of the solution changed to orange. After confirming the formation of a carbonyl complex by absorption spectrum measurement, phosphoric acid of recombinant human serum albumin (rHSA, 0.075 μmol, 4.98 mg) is slowly added through a Teflon tube at a carbon monoxide pressure. The solution was dropped into a buffered aqueous solution (pH 7.3, 30 mM) (FepivP-SI / rHSA = 4/1 (mol / mol)). The solution was slowly stirred at room temperature for 1 hour, and the resulting solution was dialyzed against an aqueous phosphate buffer solution (pH 7.3, 30 mM) for 15 hours to remove ethanol. The obtained porphyrin iron (II) complex-bound albumin was stored refrigerated (4 ° C.) under light shielding as a carbon monoxide complex.

Example 12
The molecular weight of the porphyrin iron (II) complex-bound albumin obtained in Example 11 was measured by Matrix-Assisted Laser Desorption Time of Flight Mass Spectra (MALDI-TOFMS) AXIMA-CFR (KRATOS). A molecular ion peak appeared at m / z: 70,643, suggesting that the porphyrin iron (II) complex is covalently bonded to albumin by an amide bond. Usually, when the molecular weight of a complex in which a porphyrin metal complex is included in albumin is measured by MALDI-TOFMS or ESI-TOFMS measurement, the porphyrin metal complex is eliminated during ionization, so the mass of albumin itself (m / z : 66,500) only. This result shows that the molecular ion peak was clearly observed as a result of the covalently binding of the porphyrin metal complex to albumin. It was revealed that about 3 molecules of porphyrin iron (II) complex were bound per albumin molecule.

Example 13
A porphyrin iron (II) complex-bound albumin (carbon monoxide complex) solution (4 mL) prepared in Example 11 was placed in a 1 cm quartz spectroscopic cell, and cooled with an ice-water bath, while oxygen was vented (flowed), a halogen lamp (500 W) was used to irradiate the porphyrin iron (II) complex-bound albumin aqueous solution (10 minutes). Then, the ultraviolet visible absorption spectrum measurement of the obtained aqueous solution was performed. After confirming the formation of the oxygen complex, deoxygen was prepared by aeration with nitrogen and deoxygenation. The absorption spectrum of porphyrin iron (II) complex-bound albumin in a nitrogen atmosphere shows a Fe (II) 5-coordinated high spin complex type, and in the same way as in toluene solution, deoxy having an axial base coordinated to central iron in the molecule. It became clear that it was a body. When oxygen was bubbled there, it immediately shifted to an oxygen complex type spectrum, and the oxygen bond reversibly changed in response to the oxygen partial pressure. The half-life of the oxygen complex was 5 hours at 37 ° C. Moreover, when carbon monoxide was aerated, a stable carbon monoxide complex was obtained.

Claims (10)

Formula [I]:

(Here, R ₁ is a linear or alicyclic hydrocarbon group which may have a substituent, R ₂ is an alkylene group, and R ₃ is a transition metal ion M in the 4th to 5th periods of the periodic table.) Porphyrin compounds represented by a group that allows coordination of an imidazolyl group to the central transition metal ion M when coordinated, R is a methylene group or an ethylene group), or transitions in the 4th to 5th periods of the periodic table The metal complex in which the metal ion M is coordinated. R ₁ is a linear or alicyclic hydrocarbon group having a substituent at the 1-position, R ₂ is a C ₁ -C ₁₀ alkylene group, and R ₃ is a hydrogen atom or a methyl group, an ethyl group or The porphyrin compound or metal complex thereof according to claim 1, which is a propyl group. R ₁ is a 1,1-disubstituted C ₁ -C ₁₈ alkane group, 1-substituted cyclopropyl group, 1-substituted cyclopentyl group, 1-substituted cyclohexyl group, 2-substituted norbornyl group (substituents in these groups are The porphyrin compound or metal complex thereof according to claim 2, which is a methyl group, an alkylamide group, an alkyl ester group or an alkyl ether group), a 1-methyl-2-cyclohexenyl group, or a 1-adamantyl group.   The metal complex according to any one of claims 1 to 3, wherein M is Fe or Co.   The metal complex according to claim 4, wherein the valence of Fe is +2 or +3.   The metal complex according to claim 4, wherein Co has a valence of +2.   A porphyrin metal complex-bound albumin obtained by reacting the succinimidyl group of the metal complex according to any one of claims 1 to 6 with albumin.   The porphyrin metal complex bound albumin according to claim 7, wherein the succinimidyl group reacts with a lysine amino group of the albumin to form an amide bond.   9. The porphyrin metal complex-bound albumin according to claim 8, wherein the albumin is human serum albumin, bovine serum albumin, recombinant human serum albumin, or albumin multimer.   An oxygen infusion containing the porphyrin metal complex-bound albumin according to claim 9.