JPS635010B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a phospholipid liposome containing a so-called polyphyllin metal complex and an oxygen adsorbing/desorbing agent comprising the liposome. Iron ()porphyrin complexes in hemoglobin and myoglobin reversibly adsorb and desorb oxygen molecules. In order to synthesize a complex with an oxygen adsorption/desorption function similar to such a natural porphyrin iron() complex,
Many studies have been published so far. Examples include J.P. Collman, Accounts of Chemical
Research 10 265 (1977); F. Basolo, B.M.
Hoffman and JA, Ibers, ibid., 8 384
(1975); Hidetoshi Tsuchida, “Biological systems and their models from the perspective of complex chemistry” (Gakkai Publishing Center) (1978). However, if even a small amount of water coexists with these complexes, they are immediately oxidized, making it impossible to generate oxygen complexes. For this reason, the development of iron()porphyrin complexes that provide oxygen complexes even in the coexistence of water at room temperature is being promoted. By the way, when considering the medical and medicinal purposes of oxygen carriers, safe metabolism of porphyrin complexes in vivo is essential. It is said that the structure of the iron()-porphyrin complex that can be metabolized in vivo requires hydrogen to be present at the meso position of the porphyrin ring (KM Smith, ed., Porphyrins and
Metalloporpyrins, Elsevier Pue.1975, etc.). An example of an iron()porphyrin complex that satisfies this condition and is reported to be able to form an oxygen complex under room temperature conditions although in an organic solvent is cofacial (face-to-face) diporphyrin (CKChang).
et al., J. Am. Chem. Soc. 1981, 103 , 5236-5238). However, this face-to-face diporphyrin also does not form a stable complex in an aqueous system. Therefore, the object of the present invention is to form a stable oxygen complex in an aqueous phase or aqueous medium at room temperature, and to create a metabolizable porphyrin iron () complex that can reversibly adsorb and desorb oxygen depending on the oxygen partial pressure difference. The goal is to provide a system. According to this invention, the above object is achieved by the formula (Here, M is copper or zinc, R is hydrogen or a substituent, R ₁ is hydrogen or a substituent, R ₂ is a hydrophobic substituent, R ₃ and R ₄ are each hydrogen or a substituent)
This is achieved by including a facing-type diporphyrin metal complex having an axial ligand in a phospholipid liposome containing an axial ligand. The present inventors thought that by placing the face-to-face diporphyrin metal complex represented by formula (1) in a specially designed hydrophobic field, a stable oxygen complex could be formed even in a system where water coexists. As a method of placing an iron complex with a poorly water-soluble porphyrin complex as a ligand in a hydrophobic field in water, it is also possible to use various micelle-forming agents such as synthetic surfactants. However, when considering the medical purpose of oxygen carriers, the toxicity of commercially available synthetic surfactants becomes a problem, so the present inventors created liposomes using phospholipids, which have almost no toxicity. The present invention was completed by conducting research on inclusion of the complex of formula (1). Phospholipids include soybean phosphatidylcholine,
Natural products such as bovine liver phosphatidylcholine, bovine brain phosphatidylcholine, bovine heart muscle phosphatidylcholine, egg yolk phosphatidylcholine, phosphatidic acid, kephalin, phosphatidylethanolamine, and sphingomyelin can be used. , synthetic ones such as dipalmitoylphosphatidylcholine. In the facing-type diporphyrin metal complex represented by formula (1) used in this invention, copper and zinc are suitable as the central metal M of the porphyrin dicarboxyl body facing the iron ()porphyrin (porphyrin diamino body). . Other metals, such as cobalt and iron, react with the oxygen bonded to the opposing iron ()porphyrin, making it impossible to achieve reversible oxygen adsorption and desorption. When the central metal M is copper or zinc, reversible oxygen adsorption and desorption is possible. When M is zinc, although the life of the oxygen complex is relatively short, there are no problems with metabolism or toxicity;
When copper is used, its oxygen transport ability is extremely good. R may be hydrogen or any other substitution in view of the performance of the complex, but from the viewpoint of synthesis, an alkyl group such as ethyl or pentyl is convenient. The face-to-face diporphyrin metal complex alone has almost no effect of adsorbing and desorbing oxygen through coordination, and in order to achieve this purpose, it is necessary to coordinate one basic ligand at the axial position. In this invention, as shown in the above formula (1), as this axial ligand, the formula The substituted imidazole shown is used. Here, R ₁ is a group that does not inhibit the coordination of the imidazole to the Fe()porphyrin complex, and is a substituent such as hydrogen or a methyl group, an ethyl group, and a propyl group (including n-propyl group and isopropyl group). It is. R ₂ is a hydrophobic group. Examples of such hydrophobic groups include _C5 - _C30 alkyl groups or trityl groups or substituted trityl groups or carboxylic acid alkyl ester groups (

[Formula]n=1 to 30). It is important for the substituted imidazole as an axial ligand to have a hydrophobic group as R ₂ for ease of inclusion in liposomes. The more hydrophobic the hydrophobic group becomes (for example, the number of carbon atoms increases in the case of an alkyl group),
The complex is better included in the liposome, and the generated oxygen complex is stabilized as it becomes less susceptible to oxidation by water. R ₃ and R ₄ are hydrogen or an optional substituent (eg, an alkyl group). Note that the facing-type diporphyrin metal complex can be synthesized, for example, through the following process. In order to include the complex of formula (1) in phospholipid liposomes, in an inert atmosphere (e.g. nitrogen gas),
The face-to-face diporphyrin metal complex and an excess amount of substituted imidazole are dissolved in a suitable solvent such as dichloromethane, and the central iron of the face-to-face diporphyrin metal complex is reduced to a divalent state using a reducing agent such as sodium niotinate. Next, an excess amount (for example, 200 times the mole or more) of phospholipid relative to the face-to-face diporphyrin metal complex is added, and the solvent is distilled off. These operations
It is best to do this by blowing in CO gas. Finally, CO gas can be easily removed by thermal degassing. Next, this is added to an aqueous medium (e.g., water, phosphate buffered water, physiological saline) under an inert gas atmosphere, and subjected to ultrathermal treatment to form liposomes embedding the complex of formula (1). is obtained. The thus obtained liposome according to the present invention stably exhibits the reversible oxygen adsorption/extraction function of the complex of formula (1) clathrated therein, and the complex of formula (1) can coexist with water at room temperature. Forms a stable oxygen complex even at low temperatures. Also,
It is biocompatible because it uses phospholipids. Therefore, the liposome of the present invention has the characteristics of a biologically applicable oxygen adsorption/desorption agent. Synthesis examples and examples of the present invention will be shown below. Synthesis Example 1 Face-to-face iron()porphyrin complex (M= in formula (1)
Synthesis of copper, R=ethyl) (A) Synthesis of porphyrin dihydrazide Mesoporphyrin-dimethyl ester (This synthesis was performed by J.P.Collman et al., J.Am.Chem.Soc.1980,
102, 6024-6036) in distilled pyridine 4, add 400 ml of anhydrous hydrazine,
Boiling point reflux was carried out for 36 hours in the dark under nitrogen. This solution was left standing in a refrigerator overnight, and the precipitated crystals were collected, washed with methanol until the pyridine odor disappeared, and then vacuum-dried at room temperature to obtain reddish-brown fine crystals. Yield 17g. Yield 85%. Infrared spectrum: ν _c=0
1650cm ^-1 (B) Synthesis of porphyrin diamino compound 17 g of porphyrin dihydrazide obtained in (A) above
was dissolved in acetic acid 9, added with 900 ml of 3N hydrochloric acid, added dropwise with 300 ml of saturated aqueous sodium nitrite solution under ice cooling, stirred for 10 minutes, and then dissolved in saturated aqueous sodium acetate solution.
400 ml was added and the azide was extracted with dichloromethane. The oil layer was washed with ice water, then with an ice-cooled saturated aqueous sodium bicarbonate solution, dried over sodium sulfate, and then dried under reduced pressure at room temperature. This azide was dissolved in 10 dehydrated toluene and refluxed at boiling point for 2 hours in the dark under nitrogen. Add 10% of 3N hydrochloric acid to this
was added and refluxed at boiling point for an additional 3 hours in the dark under nitrogen with vigorous stirring. After cooling to room temperature, the green aqueous layer was separated, washed with toluene, and dried under reduced pressure. This amine hydrochloride was dissolved in methanol, aqueous ammonia was added to make the solution basic, and the diaminated product was extracted with dichloromethane. After drying and concentrating this dichloromethane solution with potassium carbonate,
Chloroform: Methanol: Triethylamine =
Silica gel column purification was performed using 95:5:0.5 as a developing solvent. The second stream was collected and recrystallized with a chloroform-methanol solvent containing ammonia, washed with water, and dried under vacuum at room temperature for 2 days to obtain brown fine crystals. Yield: 10g. Yield 64%. Thin layer chromatography: R _f =0.1 (CHCl ₃ /MeOH = 96/4). λ _nax
403nm (in _CHCl3 ). NMR( _CDCl3 )−3.73(2H,
NH), 1.87 (6H, CH ₂ CH ₃ ), 3.65 (12H, CH ₃ ),
4.10 (4H, CH ₂ CH ₃ ), 5.25 (4H, CH ₂ NH ₂ ),
10.12ppm (4H, mesoH) (C) Synthesis of copper porphyrin di(p-nitrophenyl ester) 20g of mesoporphyrin dimethyl ester was dissolved in dimethylformamide 5, added with 20g of cupric chloride, and dissolved at 100℃ for 5 After stirring for a minute, the mixture was dried under reduced pressure. This was dissolved in dichloromethane and washed with water three times. Concentrate the dichloromethane solution and
The mixture was recrystallized by adding methanol, collected, washed with methanol, and dried under vacuum at 50°C overnight to obtain reddish-purple fine crystals. This was dissolved in 25% of pyridine, 11% of a 2N potassium hydroxide aqueous solution and 8% of isopropyl alcohol were added, and the mixture was refluxed at boiling point for 20 hours with vigorous stirring.
This was dried under reduced pressure to remove the solvent, dissolved in water 5, added with hydrochloric acid to collect the precipitate, thoroughly washed with water, and then vacuum-dried at 50°C overnight. The obtained hydrolysis product was dissolved in pyridine 10 which had been dehydrated and distilled, 100 g of para-nitrophenyl trifluoroacetate was added, and the mixture was stirred at room temperature in the dark under nitrogen for 2 days. To this was added n-hexane 20, and the mixture was allowed to stand in a freezer for several hours, and the precipitated crystals were collected, washed with hexane until the pyridine odor disappeared, and vacuum-dried overnight at room temperature to obtain red-purple fine crystals. Yield: 12g. Yield 40%. Mass spectrum M ⁺・
843. λ _nax 400 nm (in _CHCl3 ). Thin layer chromatography: R _f ] 0.9 ((CHCl ₃ /MeOH=96/4) (D) Synthesis of face-to-face porphyrin Copper porphyrin di(p-nitrophenyl ester) obtained in (C) above 1.7 g (2 mmol) ) was heated and dissolved in dehydrated distilled pyridine 10, and 1.0 g (2 mmol) of the porphyrin diamino compound obtained in (B) above was added to the solution.
The solution dissolved in dehydrated distilled pyridine was added all at once, and the mixture was stirred and reacted at 70°C in the dark under nitrogen for 8 hours. After the reaction was completed, pyridine was removed by drying under reduced pressure, dissolved in dichloromethane, 300 ml of 0.5M aqueous sodium hydroxide solution was added, and after vigorous stirring, the oil layer was washed with water.
Dry with potassium carbonate. After concentrating this dichloromethane solution, chloroform:methanol=96:4
A silica gel column purification was performed using this as a developing solvent to remove unreacted substances. After concentrating the desired fraction, it was recrystallized using chloroform-toluene as a solvent, and the collected crystals were washed with toluene and vacuum-dried at room temperature overnight to obtain dark purple fine crystals. Yield: 1.13g. Yield 54%. Thin layer chromatography: R _f =0.3 (CHCl ₃ /MeOH = 96/4). λ _nax 383 nm (in _CHCl3 ). Mass spectrum M ⁺・
1045. (E) Synthesis of face-to-face iron ()porphyrin complex After heating and dissolving 1.0 g of face-to-face porphyrin obtained in (D) above in distilled dimethylformamide, 10 g of ferrous chloride was added under nitrogen, and the mixture was kept at 80°C in the dark for 6 hours. Stirred.
After confirming the completion of iron introduction using a visible spectrum, the mixture was dried under reduced pressure, dissolved in dichloromethane, thoroughly washed with water, concentrated, and recrystallized by adding petroleum ether. The precipitated crystals were collected and dried under vacuum at room temperature to obtain black-purple fine crystals. Yield 0.9g. Yield 86%. λ _nax 382 nm (in _CHCl3 ).
Mass spectrum M ⁺・1099. Synthesis Example 2 In the same manner as in Synthesis Example 1, a face-to-face iron()porphyrin complex (M=copper, R=hexyl in formula (1)) was synthesized. λ _nax 385 nm (in _CHCl3 ). Mass spectrum M ⁺・
1211) Synthesis Example 3 A face-to-face iron ( ) porphyrin complex (M in formula (1) = zinc , R=ethyl) was synthesized. λ _nax 384 nm (in _CHCl3 ). Mass spectrum M ⁺・
1101 Example 1 The complex obtained in Synthesis Example 1 in a nitrogen atmosphere
0.6mg and 1-n-laurylimidazole 7mg
was dissolved in 5 ml of dichloromethane, 5 ml of an aqueous solution containing excess sodium dithionite was added thereto, and after shaking and standing, the dichloromethane layer was collected. After dissolving 80 mg of egg yolk phosphatidylcholine in 2 ml of dichloromethane and saturated with nitrogen gas,
It was added to the dichloromethane solution above. After removing dichloromethane under reduced pressure, add 10% of phosphate buffered water (PH7.0)
ml and subjected to ultrasonic stirring (20kHz, 100W) for 10 minutes in a nitrogen atmosphere to obtain Synthesis Example 1 Complex -
A liposome-dispersed aqueous solution of a mono(1-n-laurylimidazole) complex was obtained. The maximum absorption wavelength of the visible absorption spectrum of the solution under nitrogen is
The wavelengths were 391 nm, 533 nm (shoulder), and 570 nm, which corresponded to the deoxy type. By blowing oxygen, the maximum absorption wavelengths became 392 nm, 536 nm, and 573 nm. Moreover, the half-life of this oxygen complex was 30 minutes. Note that when carbon monoxide gas was blown into the aqueous solution of the oxygen complex, the spectrum of the carbon monoxide complex shifted to 396, 530, and 573 nm, confirming that the complex of Synthesis Example 1 was not degraded. In this example, the visible absorption spectrum before the reduction operation in the process of preparing the oxygen adsorbent/desorbent is shown as line a, that after preparation as line b, that of the oxygen complex as line c, and that of the carbon monoxide complex as line d. Shown in Figure 1. Example 2 Synthesis example 1 complex-mono(1-n-stearylimidazole ) A liposome-dispersed aqueous solution of the complex was obtained. By blowing oxygen, the maximum absorption wavelength is 392, 537,
An oxygen complex with a wavelength of 574 nm was produced. The half-life of this oxygen complex was 25 minutes. Example 3 In Example 1, dipalmitoylphosphatidylcholine was used instead of egg yolk phosphatidylcholine.
An aqueous liposome dispersion solution of Synthesis Example 1 complex-mono(1-n-laurylimidazole) complex was obtained using exactly the same conditions and method except that 0.1 g was used. The half-life of the oxygen complex formed by oxygen injection was 45 minutes. Example 4 Synthesis Example 3 complex-mono(1-n-
A liposome-dispersed aqueous solution of the laurylimidazole complex was obtained. Maximum absorption wavelength achieved by oxygen injection
Oxygen complexes with wavelengths of 395, 536, and 573 nm were produced, and their half-lives were 20 minutes.

[Brief explanation of the drawing]

FIG. 1 is a spectrum diagram of an oxygen complex and a carbon monoxide complex before and after reduction during the preparation of the oxygen adsorbing/desorbing agent in Example 1.

Claims (1)

[Claims] 1 formula (Here, M is copper or zinc, R is hydrogen or a substituent, R ₁ is hydrogen or a substituent, R ₂ is a hydrophobic substituent, R ₃ and R ₄ are each hydrogen or a substituent)
A phospholipid liposome characterized by including a face-to-face diporphyrin metal complex represented by: 2. The phospholipid liposome according to claim 1, wherein R is an ethyl group or a pentyl group. 3. The phospholipid liposome according to claim 1 or 2, wherein _R2 is a _C5 - _C30 alkyl group, a trityl group or a substituted trityl group, or a carboxylic acid alkyl ester group. 4 formula (Here, M is copper or zinc, R is hydrogen or a substituent, R ₁ is hydrogen or a substituent, R ₂ is a hydrophobic substituent, R ₃ and R ₄ are each hydrogen or a substituent)
An oxygen adsorbing/desorbing agent comprising a phospholipid liposome that includes a facing-type porphyrin metal complex represented by: 5. The oxygen adsorbing/desorbing agent according to claim 4, wherein R is an ethyl group or a pentyl group. _6. The oxygen adsorbing/desorbing agent according to claim 4 or 5, wherein _R2 is a C5 to _C30 alkyl group, a trityl group, a substituted trityl group, or a carboxylic acid alkyl ester group.