JPH0153578B2 - - Google Patents

Description

[Detailed description of the invention] This invention has the ability to form liposomes and is polymerizable.
Lipid-like compounds having sexual functional groups alone or together
mixture with cholesterol or normal phospholipids.
The so-called iron is added to the polymer of liposomes made of
() A composition embedding a porphyrin complex and
The present invention relates to an oxygen adsorbing/desorbing agent comprising the same. Hemoglobin and myoglobin present in the mammalian body
Globin reversible oxygen molecules under physiological conditions
It adsorbs and desorbs oxygen molecules, transporting and storing oxygen molecules.
It is well known that hemoglobin and myo
The binding site for oxygen molecules in globin is iron
() Iron () which is a porphyrin derivative
Although protoporphyrin, this iron ()
When lufilin is separated from the globin protein,
It is immediately oxidized by oxygen, and iron () points are removed.
Lufirin alone may not have the ability to adsorb and desorb oxygen.
well known. Therefore, it has oxygen adsorption/desorption ability.
Let's produce a synthetic iron()porphyrin complex that
Many attempts have been reported to date. example and
Then, J.P. Coleman (J.P.
Collman), Accounts of Chemical Lisa
Accounts of Chemical ResearchTen
265 (1977); F. Basolo, B.
B.M.Hoffman and Jie
J.A. Ibers, ibid.8
384 (1975), etc. However, physiological conditions
Oxygen is reversibly bound under conditions (37℃, water).
Artificial iron ()porphyrin complexes that can be combined are now available.
is not manufactured under slightly non-physiological conditions (non-physiological conditions).
(in protic organic solvents)
Artificial iron ()porphyrin complexes that can be adsorbed and desorbed
It is only now possible to manufacture them. non-physiological condition
A person who can reversibly adsorb and desorb oxygen molecules under certain conditions.
Iron as one of the porphyrin complexes
()-5, 10, 15, 20-tetra (α, α, α, α
-o-pivalamidophenyl)porphyrin complex
(J.P.Collman et al.
Journal of the American Chemical
Society (Journal of the American
(Chemical Society)97 1427 (1975))
Ru. However, this complex in an aprotic organic solvent
Even when a small amount of water coexists, contact with oxygen molecules occurs.
Iron () is immediately oxidized to iron () by
Therefore, reversible binding of oxygen molecules is not possible. this is,
Iron()porphyrin complex with its oxygen bonded
(hereinafter referred to as oxygen complex) is a substance that induces oxidation.
(Water or H₃ ⁺undergoes an irreversible oxidation reaction by
This is because they are placed in an environment where they are easily exposed. like this
water or H₃ ⁺Oxidation of oxygen complexes induced by O
Hemoglobin and myoglobin play a role in suppressing
The globin protein is responsible for the hemoglobin protein.
Robin and myoglobin are effective oxygen molecule adsorbents and desorbers.
It functions well under physiological conditions as
Ru. Reversibly absorbs and desorbs oxygen molecules under physiological conditions
The present inventors used iron()-5,
10, 15, 20-tetra (α, α, α, α-o-piva
lamidophenyl) porphyrin (hereinafter Fe()
(abbreviated as TpivPP) and imidazole derivatives
Lipid-soluble carbonization of liposomes consisting of phospholipid complexes
By embedding the above complex in the hydrogen region,
Irreversible oxidation of oxygen complexes by placing them in a chlorinated environment
We succeeded in suppressing this complex, and developed
Special request for Soum No. 89312 (1982) (Published in 206613 (1982))
The application was filed as That is, it has two long hydrocarbon chains in its molecule.
Liposomes made of glycerophospholipids are shown in Figure 1.
It consists of a phospholipid bilayer membrane as shown schematically in
It is well known that liposomes are formed by
Ru. Such liposomes can contain various compounds.
However, the included part is covered.
determined by the degree of hydrophilicity and hydrophobicity of the inclusion compound.
It will be done. For example, as shown in Figure 1, water-soluble compounds
Lipid-soluble (hydrophobic) compounds are also present in the inner aqueous layer of the posome.
The substance is a lipid-soluble hydrocarbon region within the bilayer membrane of liposomes.
It is known that it is taken into the area. Therefore sparse
Contains aqueous iron ()porphyrin and hydrophobic groups
Hydrophobic complexes consisting of imidazole derivatives are
Embedded in lipid-soluble hydrocarbon regions within the somal bilayer membrane
and immobilized within a hydrophobic microenvironment, the inventors
thought. As is well known, hemoglobin
In bottles and myoglobin, the globin chains form
Iron () protoporph in hydrophobic micro-sites
Water or H by embedding Ilin₃ ⁺O's attack
It suppresses irreversible oxidation of oxygen complexes due to
It is possible to replace this globin chain with liposomes.
Thinking that it might be possible, the inventors filed a patent application in 1983.
This led to the invention of No.-89312. But long
However, without regard to industrial use, the above
Using eel material as a blood substitute with oxygen-carrying function
When considering medical use, phospholipids
It is desirable to use liposomes consisting of only
It cannot be called a thing. That is, such liposo
If a large amount of the drug is administered intravenously to an animal, its
One of its main constituents from red blood cells and tissue cells
The cholesterol that is present in the administered liposol
This can lead to weakening of the animal's red blood cells and
It is said to induce an increase in the amount of free cholesterol in the blood.
There are some drawbacks. Therefore, the above iron () porf
Advantages of phospholipid liposomes containing ilin complexes
The above deficiencies in medical use can be achieved without compromising the
The aim is to provide liposomes that solve these problems.
and iron()-5,10,15,20-tetra(α,
α, α, α-o-pivalamidophenyl) porphyry
A complex consisting of phosphorus and an imidazole derivative is used as phospholipid.
Lipid solubility of liposomes consisting of quality and cholesterol
An application was filed for liposomes embedded in the hydrocarbon region.
(filed on December 28, 1982, patent application No. 1983-234994,
1986-213779 Priority claim United States No.
No. 386060, June 7, 1982). However, in general, phospholipids and phospholipid mixtures
substances or their mixture with cholesterol, etc.
A simple liposome whose constituent components are
Its own stability in water is not that great.
Therefore, it is difficult to maintain the shape for a long period of time, and these materials
When using materials industrially, they are also used as pharmaceuticals.
Even when storing, it is only used for a short period of time or for storage.
The drawback is that it cannot be done. For example, non-physiological conditions
(industrial) use, in some cases
The stability of liposomes is lost due to various additives, etc.
The liposome form is rapidly formed by aggregation and fusion.
Destroyed. Also, when administered into the blood as a medicine,
In some cases, fusion with biological cell membranes or enzymes such as reverse
Degradation of liposomes by chemical substances is also considered. Also,
Due to the osmotic pressure difference between the inner and outer aqueous layers of the liposome, the liposome
Destruction of the membrane membrane occurs. In this way, physical and chemical
Liposomes are always stable due to external chemical factors.
However, when considering various uses, lipo
Increased stability (shape retention ability) of somes
Large is desired. The inventors polymerized liposomes and
The so-called iron within the lipophilic region of the posome polymer
() Oxygen adsorption and desorption by including porphyrin complex
Physically and chemically safer while retaining functionality.
Maintains liposome morphology for long-term use or
Liposomes that can withstand storage - iron () porphyry
The present invention was conceived based on the idea that a complex composition could be obtained.
Ta. According to this invention, the general formula (where n is an integer from 1 to 20)
()-5, 10, 15, 20-tetra (α, α, α, α
-o-alkyl-substituted amidophenyl)porphyry
(Fe()TpivPP, etc.) or general formula (where n is an integer from 10 to 20)
()-5, 10, 15, 20-tetra (α, α, α, α
-o-phosphocholine-substituted amidophenyl)porf
Either one or both of the two axial conformations of Ilin
way of expression (Here, R₁is the imidazo bonded to this
A non-bulky group that does not inhibit the coordination of the metal to the central iron,
and R₂,R₃and R_Fourare hydrogen or sparse, respectively.
at least one of the aqueous substituents
is a hydrophobic substituent)
A complex coordinated with or general formula (where l is 3, 4 or 5, and R_Five,R₆
and R₇are hydrogen atoms or methyl groups, respectively)
Iron ()-2-substituted-5, 10, 15, 20 represented by
-tetra (α, α, α, α-o-pivalamide
) porphyrin complex embedded in the hydrophobic region.
Oxygen absorption and desorption using lipid-based polymerized liposome as an active ingredient
An adhesive is provided. Fe()porphyrin complexes alone contain oxygen.
It cannot be adsorbed or desorbed due to its position. this purpose
Axial position (5th or 6th coordinate) to achieve
It is necessary to coordinate at least one basic ligand to
There is. In this invention, as this axial ligand, the formula
(A) and the iron ()porphyrin shown by formula (B)
in case of The substituted imidazole shown is used. here
So, R₁is the imidazole of formula (A) and (B)
Inhibits the coordination of porphyrins to Fe()
hydrogen, methyl group, ethyl group and
Propyl group (n-propyl group and isopropyl group)
group) is preferred. R₂,R₃and R_FourThat's it
each independently hydrogen, methyl group, or hydrophobic (lipophilic)
at least one hydrophobic (lipophilic) group, and at least one is a hydrophobic (lipophilic)
gender) group. It is commonly known that the group is hydrophobic (lipid-soluble).
Constant R₂or R₃Especially R₂It is. R₁is a methyl group,
In the case of ethyl group or propyl group, two axial coordination
Imidazole of formula (C) at only one of the loci
Derivatives coordinate. Iron ()porphyrin 1 mole
against imidazole induction present in liposomes
The number of moles of the compound (formula (C)) present is R₁is a hydrogen atom field
2 to 30 moles (more preferably 2 to 10 moles)
and R₁is a methyl group, ethyl group, or
In the case of lopyl group, 10 to 100 mol (more preferably
is 30 to 100 moles). Imidazole derivatives as axial ligands (formula (C))
has hydrophobic (lipophilic) substituents. This sparse
Examples of aqueous (lipid-soluble) substituents include C₆~C₃₀of
Alkyl group or trityl group or substituted trityl group
group or carboxylic acid alkyl ester group [Formula] n=1 to 30, or is [Formula] m=3~25, p=0 or 1). Substitution as an axial ligand
Imidazole must have at least one hydrophobic group
is important. According to the research of the present inventors, hydrophobic
Substituted imidazole example without a functional (lipophilic) group
With 1,2-dimethylimidazole as the axial ligand,
In the Fe()TpivPP complex, which has a phospholipid
Forms a stable oxygen complex even when embedded in liposomes
The fact that it oxidizes immediately without oxidation supports the above idea.
There is. Additionally, the hydrophobicity of hydrophobic groups increases (e.g.
In the case of alkyl group, the more the number of carbon atoms increases, the more complex the complex becomes.
Inclusion in liposomes is good, and the production of
The oxygen complex becomes less susceptible to oxidation by water and becomes stable.
become In addition, in the case of a complex represented by formula (D), the molecule
Constituent component contains an imidazole group that coordinates with iron within
Therefore, the imidazole represented by formula (C)
There is no need to use a compound derivative. For the complex represented by formula (A), if n=1, describe
Collman et al., n=2~
20, in the specification of EPC Patent Publication No. 66884
Are listed. The complex represented by formula (B) is disclosed in patent application No. 57-210841 (publicly available).
It can be synthesized according to the method described in
Ru. For example, the expression (where 2 is an integer from 10 to 20)
Acid chloride and J.P. Collman et al., Journal of the
American Chemical Society,97, 1427 (1975)
The expression reported in 5, 10, 15, 20-tetra (α, α,
α,α-o-aminophenyl)porphin (hereinafter referred to as
H₂Abbreviated as TamPP. ) in the presence of a base.
and the obtained formula (Here, n is the same as defined earlier.)
The compound to be treated is treated with anhydrous acetate chloride in the presence of anisole.
By removing the benzyl group with aluminum, the obtained
expression (Here, n is the same as defined earlier.)
Reacting the compound with ferrous bromide in the presence of pyridine
and the obtained formula (Here, n has the same meaning as defined earlier, and
2-chloro-
2-oxo-1,3,2-dioxaphosphorane and
After reacting to form a phosphate ester, excess tor
Phosphocholination is performed by reacting with remethylamine.
The iron porph represented by the general formula (B) by the method of
Ilin derivatives can be produced. The starting material of general formula (I) is synthesized by the following method.
Ta. ω-benzyloxyalkyl halide
George R. Newkome et al., Synthesis,1975, 517.
2-methylpropion produced according to the report of
Dilithium anion of acid and initially low temperature (-70℃~-
20℃), then raise the temperature and react at 30-45℃.
Ta. The reaction mixture was then decomposed with cold dilute hydrochloric acid and the solvent
The extracted crude product is treated with a non-polar solvent such as petroleum acetate.
recrystallized in tel, n-hexane, n-heptane.
ω-benzyloxy-2,2-dimethylalka
phosphoric acid was obtained as colorless crystals. This is a non-polar solvent,
Preferably in benzene or carbon tetrachloride or
After reacting with an excess amount of thionyl chloride without solvent
Concentrate under reduced pressure to obtain ω-benzi represented by general formula (I)
2,2-dimethylalkanoic acid chloride
I got it. raw material formula (Here, n has the same meaning as defined earlier, and X' is
Represents chlorine or bromine. ) denoted by ω-ben
Zyloxyalkyl halide is α,ω-dibromo
1 equivalent of an alkane with sodium benzyl oxide
It can be obtained by refluxing reaction with benzene. Excess amount of carboxylic acid chloride of general formula (I)
H, a known substance₂Anhydrous aprotic solution of TamPP
medium, preferably tetrahydrofuran, dichloromethane
Tan, chloroform, N,N-dimethylforma
Excess triethyl chloride in amide or acetone solution
React at room temperature from 0℃ in the presence of amine or pyridine.
After that, pour it into water, extract it with chloroform, and separate it.
The residue obtained by evaporating the separated extract was evaporated into silica gel.
By purifying by column chromatography
A compound represented by the general formula () is obtained. purification
Recrystallization may be used if necessary. Benzyl ether represented by this general formula ()
dichloromethane to remove the benzyl group of the
Excess amount of anisopropylene in a mixed solvent of nitromethane
in the presence of excess anhydrous aluminum chloride and -5
℃ to 30℃, preferably 15℃ to 25℃ for 2 hours
The reaction was allowed to take place for 1 to 12 hours. Place reaction mixture in ice water
Extract with chloroform and wash the extract with water.
After washing with 4% sodium bicarbonate aqueous solution, separate
The chloroform layer was dried with mirabilite, and the chloroform layer was
The residue obtained by evaporating the
Recrystallized from a mixed solvent of lormethane-benzene.
Ta. Introducing iron into the resulting compound of general formula ()
In J.P. Collman et al., Journal of the
American Chemical Society,97, 1427 (1975),
Reflux under nitrogen gas atmosphere according to the method reported in
in the presence of pyridine in anhydrous tetrahydrofuran
Reacted with excess ferrous bromide. Reduce the pressure of the reaction solution
Dry and add chloroform or chloroform and meth.
Alumina or silica gel using a mixed solvent of alcohol
Purified by column chromatography and purified with hydrobromic acid
The compound of general formula (V) obtained by treatment with
One bromide ion as a counterion in the trivalent state
have At this time, if hydrochloric acid is used instead of hydrobromic acid, the salt
element ions can be converted to iodine ions using hydroiodic acid.
have a function. Phosphocholination of the compound of general formula (V) is N.
S.Chandrakumar et al., Tetrahedron Letters,
23, 1043 (1982),
The phosphorization reaction was improved. general formula
Compound (V) is mixed with an anhydrous aprotic solvent, e.g.
Dichloromethane, chloroform or benzene
Among them, 1 to 2 equivalents of pi for one hydroxyl group.
2-chloro-2-oxo-1,3, in the presence of lysine
1 to 2 equivalents of 2-dioxaphosphorane at 0°C.
Drop at room temperature, then leave at room temperature for 6 to 24 hours.
It was allowed to react for a while. Obtained by distilling the reaction solution under reduced pressure
Dissolve the residue in acetonitrile or N,N-dimethylphosate.
Dissolve in Lumamide, add trimethylamine and rinse.
12 hours at 50℃ to 65℃ in a stainless steel sealed tube container
The reaction was allowed to occur for up to 24 hours. Strain the reaction mixture
A brown precipitate was obtained. After washing this with acetone,
Cephadex G-25, Cephadex LH-60
(manufactured by Pharmacia), or Toyo Pearl HW
Using an appropriate gel such as -40 (manufactured by Toyo Soda Co., Ltd.),
Column chromatography using methanol or water as solvent
It was purified by Thus, the desired general formula (B)
Iron-5, 10, 15, with phosphocholine groups
20-tetra(α,α,α,α-o-substituted amidoph
(enyl) porphyrin complex is obtained. Complex of formula (B)
The body has a good affinity for liposomes, making them easier to embed.
It's easy. The imidazole derivative represented by formula (C) is a normal
It can be synthesized by applying the method. For example, R₂is hydrophobic
When the compound is a sexual substituent, the compound has the general formula Alternatively, the general formula (R₁,R₃,R_Fouris expressed as (as mentioned above)
Dazole derivatives and general formula Z-R₉ (iii) (However, Z is a chlorine atom, a bromine atom, or an iodine atom.
Represents a child, R₉is (−CH₂)−_tCH₃(t is from 4 to 29
an alkyl group or trityl represented by an integer
group or formula [Formula] (n=1-30) or [formula] (however, m = 3 to 25, p = 0 or 1)
(representing a cyclocarbonyl alkane group)
By desilver halide reaction with halogen hydrogenation reaction
Can be synthesized. Iron-2-substituted-5,10,15,20 represented by formula (D)
-tetra (α, α, α, α-o-pivalamide
) Porphyrin bromide is produced by the following process
It can be synthesized according to Iron-porphyrin imidazole complex as mentioned above
Liposomal polymers (polymeriz) used for embedding the body
The raw material that makes up ``liposomes''
also has one polymerizable functional group and forms liposomes.
as a compound (hereinafter abbreviated as monomer) that can be
For example, 1,2-bis(octadeca-2,4
-dienoyl)glycero-3-phosphocholine (formula
(E)） (B. Hupfer et al., Makromalekular Chemie, 182
Vol. 247-253 (1981)), or dimethyl-
Di(octadeca-2,4-dienoyloxyethyl)
ammonium bromide (formula (F)) (R. Bueschl et al., Federation of European
Biochemical Societies Letters, Volume 150, 38-42
(1982)), or 1-{ω-(p-vinylbenzene)
Nzoyl)alkanoyl}-2-O-alkyl-
rac-glycero-3-phosphocholine (formula (G)) patent
Gansho 57-140963) (Here, q is a positive integer from 1 to 10, and r
is a positive integer between 13 and 21) etc. are available. The monomer represented by formula (E) is described in the literature (H. Hupter
Makromalekular Chemie, vol. 182, 247~
253 (1981)).
was synthesized. That is, the method of Y. Tamura et al.
(Tetrahedron Letters, vol. 22, pp. 1343-1344
(1981), the following process: E,E-2,4-octadecadiene synthesized in
acid with dicyclohexylcarbodiimide and 4-
In the presence of (N,N-dimethylamino)pyridine
Glycerophosphocholine/cadmium chloride complex (C.
M.Gupta et al., Proceedings of the National
Academy of Sciences of the United States
of America, Vol. 74, pp. 4315-4319 (1977)) and reaction.
It was synthesized by The monomer represented by formula (F) is described in the literature (R. Bueschl
et al., Federation of European Biochemistry
Societies Letters, vol. 150, pp. 38-42 (1982))
Therefore, it was synthesized. Produce a phospholipid compound represented by general formula (G)
First, the general formula (however, q is as described above)
general formula for lido (However, r is as described above)
A condensation reaction with a roll derivative gives the general formula get. This condensation is based on the molar ratio of compounds of formula (iv) and formula (v).
Use in a ratio of 0.5:1 to 3:1, and remove an appropriate amount of
A suitable solvent containing a hydrochloric acid agent (such as pyridine) (e.g.
For example, anhydrous tetrahydrofuran, chloroform
temperature of 0°C to 30°C in
Do it in degrees. Reaction time is usually 1 to 8 pm.
It is between. Then, the condensation product represented by formula (vi) was prepared by Von R.
Pharm.Acta Helv., by Hirt et al.33, 349
(1958) as follows:
The desired general formula by reacting in the process
Compound (F) is obtained. (1)Triethylamine ――――――――→ (2)Ag⁺CH₃COO^-Phospholipid compound (G) Note that the acid chloride represented by formula (vi) is first produced by S.
Organic Synthesis, by Swan et al.2, 276
(1950) by improving the method described in (However, as mentioned above, q is a methyl group, R is a methyl group,
methyl, propyl or butyl)
Alkanedicarboxylic acid monoester/monochloride
ethyl bromide in the presence of anhydrous aluminum chloride
When reacted with benzene, the formula (However, q and R are as described above)
A compound is obtained. Then add excess alkali to this
(e.g. sodium hydroxide, potassium hydroxide)
After heating and reacting in alcohol, acidify with hydrochloric acid etc.
sexualized expression The compound shown is obtained. Add this to thionyl chloride or
or by reacting with oxalic acid chloride, etc.
A compound represented by formula (iv) is obtained. The glycerol derivative represented by general formula (v) is D.
by Arnold et al., Lieb.Ann.Chem.703, 234
~239 (1967) by improving the method described in
2-phenyl-5-m-dioxanol and alkyl
After reacting rubromide, sulfuric acid was added in ethanol.
It can be obtained by hydrolysis. Na
Oh, the glycerol derivative represented by general formula (v)
Synthesize those whose chi is 13, 15, 17, 19 or 21
The alkyl bromide, the raw material for this, is a commercially available product.
It is convenient because it can be obtained at a low price. This is how you get it
Compounds of formula (G) require the addition of a polymerization initiator.
Polymerizes rapidly by irradiation with ultraviolet light. Also,
Presence of unsaturated bonds in the obtained polymer compound
Nor. Polymeric liposomes use the above monomers.
(a) Monomeric lipo consisting of monomer alone
some, (b) a mixture of monomer and cholesterol?
Liposomes consisting of (c) monomers and ordinary non-polymerizable
natural or synthetic phospholipids (e.g. lecithyl
N, kephalin, phosphatidylcholine (soybean,
Beef liver, bovine brain, bovine heart muscle and egg yolk phosphatidyl
Natural products such as choline and dipalmylphosphate
Zircholine, distearoylphosphatidylcoli
synthesis of dimyristoylphosphatidylcholine, etc.
), phosphatidylethanolamine, sulphate
Phingomyelin, phosphatidic acid, dialkyl
monomeric resin consisting of a mixture with phosphoric acid, etc.)
Liposomes or (d) monomers and cholesterol
consisting of phospholipids and common natural or synthetic phospholipids.
Polymerization of monomeric liposomes consisting of mixtures
It can be produced by polymerizing a functional group. Li
The molar ratio of each component in the posome is 1 mole of monomer.
In contrast, the total moles of cholesterol and phospholipids are
0.5 or less (more preferably 0.5 to 0.05 mo
). That is, monomeric liposomes (monomeric liposomes)
In this specification,
The main component is filtrate monomer. Iron() or iron()porphyrin complex
For embedding in polymeric liposomes,
General procedure for producing liposomes from natural phospholipids
First, by applying the method, the monomer or monomer and
Mixed with cholesterol or regular phospholipids
Liposomes made of substances (monomeric liposomes)
After taking the complex into and embedding it in the monomer
This is possible by polymerizing the polymerizable functional groups in
be. The method is described below. (Method 1) Iron ()porphyrin complex-containing
Preparation of monomeric liposomes For example, iron()-5,10,15,20-tetra
(α, α, α, α-o-pivalamidophenyl)
Porphyrin bromide (hereinafter referred to as Fe)
Iron () porphyry such as TpivPP・Br) etc.
Substituted imidazole represented by formula (C) and prescribed amount of
(The molar ratio of imidazole/iron porphyrin is
2 to 100) and monomer or monomer and
consisting of cholesterol or normal phospholipids
mixture (however, monomer for iron porphyrin
- and the total weight ratio of cholesterol and phospholipids is 10.
~1000, preferably 10-200) in a suitable solvent,
For example, chloroform, dichloromethane, toluene
benzene or their combination with methanol.
Once dissolved in a mixed solvent, the
using an oil pump or aspirator)
The solvent is distilled off, for example on the inner wall of a glass flask.
Obtain the solid content as a thin film on the surface. Next, the thin
Apply an inert gas (e.g. nitrogen or argon gas) to the membrane solid.
in an aqueous medium (e.g., water,
acid buffered water (PH=5-9), physiological saline,
or Krebs-Ringers solution), then shake.
by stirring in a bowl or mixer
Fe() as an emulsified multilayer liposome membrane
Replacement with iron ()porphyrin such as TpivPP/Br
Liposome dispersion solution containing imidazole
is obtained. further ultrasonication of this
multilamellar liposomes are made into smaller diameter multilayer liposomes.
Multilamellar liposomes or monolamellar liposomes
can be done. (Method 2) Iron ()porphyrin complex-containing
Preparation of monomeric liposomes Iron prepared according to the method described in (Method 1)
() Monomerits embedded with porphyrin complexes
In an inert gas atmosphere,
A suitable reducing agent, e.g. ascorbic acid, under ambient conditions.
Equivalent mole or more of iron()porphyrin etc.
By adding iron () porphyrin to iron
() reduce to porphyrin, or
Oxygen mixtures with suitable reducing action, e.g.
β- as described in Etsuo Hasegawa et al., patent application No. 107462-1983
Nicotinamide adenine dinucleotide phosphate
(NADP⁺), D-glucose-6-phosphate
(G6P), glucose-6-phosphate dehydrogena
-ase, ferredoxin and ferredoxin-
A enzyme consisting of NADP reductase (FNADPR)
elemental system (add appropriate amount of catalase to this enzyme system)
It is preferable to leave it. ) iron () porphyrin complex
1 D-glucose-6-phosphate per mole of body
Used in a molar or higher ratio, with a pH of 5 to 9.
Reduction is carried out below to produce iron ()porphyrin.
By reducing iron() to porphyrin
Fe() from porphyrin and substituted imidazoles
Monomeric liposomes embedding complexes consisting of
can be prepared. (Method 3) Iron ()porphyrin complex-containing
Preparation of monomeric liposomes Inert gas atmosphere (e.g. nitrogen gas, alkaline gas)
A predetermined amount of Fe()TpivPP・
With iron ()porphyrin and formula (C) such as Br
The indicated substituted imidazole (imidazole/iron
Porphyrin molar ratio = 2 to 100) in a suitable solvent
For example, chloroform, dichloromethane, benzene
toluene, or a mixture of these and methanol.
Dissolve in a mixed solvent and add Fe()TpivPP・Br to this.
Sodium dithionite, etc. in an amount equal to or more than the equivalent mole of
Add the reducing agent aqueous solution, shake and stir, and add the water
- Fe()TpivPP in organic solvent heterogeneous layer system
Reduces central iron to divalent. Next, this organic layer
After collecting and dehydrating the monomer or
Monomer and cholesterol or regular phospholipid
(However, for iron porphyrins,
total monomers, cholesterol and phospholipids
Add a weight ratio of 10 to 1000, preferably 10 to 200).
I got it. Then, the solvent was distilled off from this system under reduced pressure.
For example, it can be solidified as a thin film on the inner wall of a flask.
Get the form part. These operations are under inert gas
Alternatively, it can be performed by blowing in CO gas.
(Operation under inert gas is more preferred). C.O.
If gas is used, heat deaeration is performed at the end.
This can be easily removed. Next, the above thin film
solid in an aqueous medium (e.g.
For example, water, phosphate buffered water (PH = 5-9), physiological saline
Add salt water or Krebs-Ringers solution)
After that, shake or mix with a mixer.
As an emulsified multilayer liposome membrane by
Fe()porphyrins such as Fe()TpivPP
and a complex consisting of a substituted imidazole.
An aqueous posome dispersion solution is obtained. further this
Multilamellar liposomes by sonication
into smaller-diameter multilamellar liposomes or single-layered liposomes.
It can be a stratified liposome. In addition, before
Reduction with sodium dithionite aqueous solution
instead of palladium-carbon/hydrogen gas
Rebates are also available. For example, Fe()
Iron ()porphyrin and iron such as TpivPP and Br
Dry dichloromethane with midazole derivative
or dissolved in benzene/methanol mixed solvent,
Add a small amount of palladium carbon and add water to this.
Fe is reduced at room temperature by sufficiently blowing elementary gas into Fe.
Iron ()porph such as ()TpivPP (formula (A)))
Prepare the ilin solution and filter the palladium carbon.
A filtrate obtained separately may be used. (Method 1), (Method 2) and (Method
3) in which the complexes of formula (A) and formula (B) are embedded.
Nomeric liposomes are obtained. Note that the iron-porphyrin complex represented by formula (D) is modeled as
When embedding in Nomeric liposomes
is the operation of (Method 1), (Method 2), and (Method 3) above.
in production without using substituted imidazoles.
Then, iron ()pol such as Fe()TpivPP and Br
The formula except that the central iron is the valence instead of the fillin.
Corresponds to (D) and bromide ion as counter ion
In exactly the same way using a compound with
More equivalent iron() or iron()porphy
Monomeric liposome dispersion water containing phosphorus
Solutions can be prepared. Iron () or iron thus prepared
() Monomerits embedded with porphyrin complexes
The average particle size of liposomes is determined by the above preparation method.
By controlling the power and time of the ultrasonic treatment operation.
It can be adjusted in the range of several μm to 200 Å. The iron-porphyrin thus prepared was
The morphology of the embedded monomeric liposomes
To polymerize and microcapsule while maintaining
is an inert gas (e.g. nitrogen, argon, monoxide)
A suitable water-soluble polymerization initiator under an atmosphere (carbon, etc.)
(e.g. potassium persulfate, ammonium persulfate,
2,2-Azobis(2-amidinopropane)・2
hydrochloride, etc.) in monomeric liposome dispersion in water.
After adding it to the liquid, for example, perform a polymerization reaction by heating, or
Alternatively, use a light source such as an ordinary low-pressure or high-pressure mercury lamp.
Use ultraviolet light total light or ultraviolet monochromatic light (e.g. 240nm,
Rapid polymerization reaction by irradiation (310nm)
progresses to the desired iron()porphyrin complex
Polymeric liposome dispersion aqueous solution embedding
can be prepared. In addition, the above-mentioned (Method 1), (Method
2) In (method 3), a fat-soluble polymerization initiator
(e.g. azobisisobutyronitrile)
Azo initiators such as cyclohexanitrile, peracids
benzoyl, t-butyl hydroperoxide, etc.
(organic peroxide initiators, etc.) in advance.
Lipid-soluble region within the bilayer membrane of Merritsk liposomes
The resulting monomeric
For example, by heating the posome-dispersed aqueous solution.
By performing a polymerization reaction, the desired purpose can be achieved in the same way.
Polymerite containing iron()porphyrin complex
An aqueous solution of Tsuku liposome dispersion is obtained. When using carbon monoxide gas as an inert gas
After polymerization, add nitrogen, argon, etc. to the solution.
Operation to remove carbon monoxide gas by blowing in gas
It is a good idea to do this. Monomeric embedding of iron()porphyrin complex
When polymerizing Tsuku liposomes by the above method,
Iron is removed by various radical species generated during the polymerization reaction.
() Porphyrin becomes iron () Porphyrin
Iron is easily reduced in this case as it is automatically reduced.
() Polymerites embedded with porphyrin complexes
Aqueous liposome dispersion solutions can be easily prepared.
If the iron()porphyrin complex is formed after the polymerization reaction,
If any residue remains, use an inert gas (e.g. nitrogen or
a suitable reducing agent, e.g. glucose
s, cysteine, ascorbic acid, glutathione
Cysteamine, 2-Mercaptopropion
acid, ethanethiol, 1,2-ethanediol, etc.
The reduction can be carried out by adding equal moles or more of the same mole or more. After the polymerization reaction, the obtained polymeric liposol
For example, dextran, hydroxyl, etc.
Blood expanders such as ethyl starch and albumin
An aqueous solution containing amino acids, sugars, vitamins, etc.
After addition, it may be used for medical purposes, for example.
stomach. Iron (porf) manufactured in this way
Polymeric liposomes embedding ilin complexes
Dispersed aqueous solution has reversible oxygen adsorption and desorption due to oxygen partial pressure difference
The iron()porphyrin can stably exhibit its function.
The complex is stable even under quasi-physiological conditions at room temperature and in water.
Forms a stable oxygen complex and functions as an oxygen adsorption/desorption agent
You can. Therefore, industrially, oxygen
Artificial blood is used as a concentrate, etc., and for medical purposes.
The water can be used as a perfusion preservation solution for organs. Iron-Porph constituting the oxygen adsorption/desorption of this invention
Polymeric liposomes embedding ilin complexes
Since the liposome membrane is polymerized, the membrane structure is
is stable and can itself be stably dispersed over long periods of time
At the same time, even if administered into the blood as a medicine, it will not affect the living body.
Fusion with cell membranes and decomposition by enzymes such as lipase
even more so than in monomeric liposomes.
There are very few. As a result, in living organisms, decomposition is
It progresses gradually, so its components are released rapidly.
Less fear (reduced toxicity). Also, liposomes
liposomes due to the osmotic pressure difference between the inner and outer aqueous layers of
Destruction of the membrane is also less likely to occur. Or for industrial use
in contact with an organic solvent (e.g. ethanol)
Even if it comes to this, it is not easily destroyed. Furthermore, lipids,
Cholesterol or iron ()porphyrin complex
Motility in the liposome membrane of the body is suppressed
Therefore, a homogeneous film composition can be achieved and the hydrophobic region is also stable.
The oxygen of the iron()-porphyrin complex is secured in
In addition to stably exhibiting adsorption/desorption ability, oxygenation complex
It also improves the lifespan of the body. In addition, iron embedded in polymeric liposomes
() Publication of EP patent for porphyrin complexes
As stated in the specification of Publication No. 66843,
Alkylamidophenyl group in the compound of formula (A)
terminal hydrogen is carboxyl group, carboxylic acid ester
Those substituted with ru groups, hydroxyl groups, etc. can also be used.
It will be done. Examples will be described below. Synthesis example 1,2-bis(octadeca-2,4-dienoy)
) Synthesis of glycero-3-phosphocholine 78.8g of metallic sodium to 1.5g of absolute ethanol
Add sodium ethoxide solution and methyl chloride under ice cooling.
After blowing in mercaptan gas to saturate it,
Add 520g (3.11 mole) of ethyl bromoacetate and stir at room temperature.
After one day of reaction, the precipitate was separated and the liquid was dried under reduced pressure to obtain
Distill the oily substance under reduced pressure to boiling point 64-65℃ (13mmHg)
Ethyl 2-methylthioacetate as a colorless oil of
The ester was obtained in a yield of 371.3 g with a yield of 90%. 2-methylthioacetic acid ethyl ester 350g
(2.61 mole) was made into a 0.5 methanol solution and
Sodium iodine 558.5g (2.61mole) in 4.5 water
After adding it to the solution in an ice bath, it was reacted at room temperature for 2 days to cause precipitation.
After washing the pores with another chloroform, remove the solution with chloroform.
The obtained chloroform layer was extracted with sulfuric anhydride.
After drying with sodium, drying under reduced pressure yields a colorless oil.
2-Methylsulfinyl acetic acid ethyl ester
Quantity: 256 g, yield: 65%. 2-Methylsulfinyl acetic acid ethyl ester
280g (1.87mole) of trifluoroacetic acid (1.0) dissolved in
Cool the liquid on ice and add 391 g of trifluoroacetic anhydride dropwise.
Afterwards, 418g of 1-hexadecene dichloromethane
Add chlorine (0.5) solution, return to room temperature, and react for 3 hours.
After that, it is dried under reduced pressure, further purified by reduced pressure distillation, and boiled.
As a colorless oil at a temperature of 178-182℃ (0.006mmHg)
Ethyl 2-methylthio-E-4-octadecenate
The ester was obtained in a yield of 476.6 g with a yield of 72%. Ethyl 2-methylthio-E-4-octadecenoate
460g (1.29mole) of ester as a 0.5 solution of acetic acid.
After dropping 180ml of 30% hydrogen peroxide solution over 2 hours in an ice bath.
After reacting for another 2 hours at room temperature, 1 part of water was added.
Extracted with chloroform (2 x 1.5) and combined
After washing the chloroform layer with water, dry it with Glauber's salt and reduce the pressure.
The corresponding sulfoxide is dried under pale yellow oil.
Approximately 490g of this was obtained without purification.
After refluxing for 5 hours, the
The crude oil obtained by distilling off the toluene under pressure is
Gel 7.0Kg, benzene-petroleum ether as solvent
Column chromatography using a mixed solvent (1:2 volume ratio)
Matogram purification and silica gel thin layer chromatography
(Rf value 0.35: solvent benzene)
The solvent was distilled off, and then dried using a vacuum pump.
By drying, E, E-2, 4 becomes a colorless oil.
-Yield of octadecadienoic acid ethyl ester
Obtained 224 g, yield 56%. 223g (0.72mole) of this ethyl ester is
(2.0) solution in 2N sodium hydroxide solution
By adding 0.6 of the solution and stirring at room temperature for 36 hours.
Precipitate generated by hydrolysis and neutralization to PH3.0 with dilute hydrochloric acid
, washed with dilute hydrochloric acid and water, and then dried.
The obtained solid was mixed with dichloromethane-n-hexane.
Recrystallize from solvent to obtain colorless crystals of E,E-2,4-
Obtained octadecadienoic acid in a yield of 153.2g and 76%.
Ta. Melting point 68-70℃. Elemental analysis value (%): C77.18 (77.09); H11.64
(11.50) (However, in parentheses
C₁₈H₃₂O₂The value is . ) E,E-2,4-octadecadienoic acid 41g
(0.146 mol) and L-α-glycerophosphocholine.
Cadmium chloride complex (manufactured by Sigma) 19.3g
(0.044 mol) and 4-dimethylaminopyridine 8.0 g
(0.071 mol) dissolved in anhydrous dichloromethane (0.6)
Distill the solution with 200ml of glass beads while stirring.
Cyclohexylcarbodiimide 33.3g (0.162mol)
Add dichloromethane (0.2) solution and protect from light.
After reacting at room temperature for 80 hours, separate the precipitate and remove the solvent under reduced pressure.
The yellow solid obtained by distillation was dissolved in chloroform/methanol.
Mixed solvent (300ml) of water/water (volume ratio 4/5/1)
Dissolve in ion exchange resin AG501-X8 (D)
Pass through a column (manufactured by Iorad) and concentrate again.
A silica gel column chromatogram of the obtained solid
Purified. Solvent: chloroform, chloroform/
Methanol = 9/1 (v/v), chloroform/metal
Tanol = 5/5 (v/v), chloroform/meth
Kol = 1/9 (v/v) and gradually change chloro
Form/methanol = 1/9 (v/v) elution fraction
19.0 g of almost pure product was obtained. even more refined
A neutral alumina column is used to produce chloro
Form/methanol = 10/1 (v/v) solvent
Elute the target fraction, collect it, concentrate it, and add benzene to it.
The title compound is obtained as a white powder by freeze-drying.
The product was obtained in a yield of 13.7 g and a yield of 39%. Infrared absorption spectrum; ν (KBr) 3400, 2960,
2860, 1715, 1640, 1620, 1470, 1250, 1110,
1090, 1070, 1000, 970cm^-1. Proton nuclear magnetic resonance spectrum: δ (CDCl₃) 0.88 (6H, t: 18'-methyl group), 3.34 (9H,
s:N(CH ₃)₃), 5.73 (2H, d, J = 15Hz; 2′ position
-proton), 6.16 (4H, n; proton at 4' and 5' positions)
), 6.9 (2H, m; 3' proton) ppm. ¹³C nuclear magnetic resonance spectrum: δ (CDCl₃) 166.8, 166.4 (1′), 146.0, 145.8, 145.5, 145.2
(3′ and 5′), 128.2 (4′), 118.4 (2′), 70.7 (
2),
66.2 (5), 63.5, 63.2 (1 and 3), 59.5 (4), 54
.3
(6), 33.1 (6′), 31.9 (16′), 29.7, 29.5, 29.
Four,
28.7 (7′-15′), 22.7 (17′), 14.1 (18′) ppm. Synthesis example 2 1-{9-(p-vinylbenzoyl)nonanoy
2-O-stearyl-rac-glycero-3
-Synthesis of phosphocholine (A) 1-(p-vinylbenzoylnonanoyl)-g
Synthesis of lycerol-stearyl ether-2 (A-1) J.Amer.Chem by H.Hibbert et al.
Soc.,62, 1601-1613 (1929)
2-phenyl-5-m-dioki obtained according to
Sanor [Formula] to D.Arnold et al. Jus.Lieb.Ann.Chem.,709, 234-239
(1967).
2-stearyloxy-1,
3-propanediol was synthesized. Elemental analysis value: H12.84 (12.8) (weight%, same below) C73.75 (73.3) However, the values in parentheses are C_{twenty one}H₄₄O₃calculated value of ¹³C-NMR spectrum δ (ppm) value: {CDCl₃, tetramethylsilane (same as below)} 【table】 IR spectrum (KBr pellet) (cm^-1):3350,
2940, 2870, 1470, 1120,
1080, 980 (B) p-(ethoxycarbonylnonanoyl)phene
Synthesis of nylethyl bromide Organic Synthesis by S. Swan et al.2,
276 (1950)
sebacic acid monoethyl ester, monochrome
Lido 10.0 g (40.2 mmol), Bromoethyl
6.2 grams (33.5 mmol) of rubenzene and
A mixture consisting of 60 ml of dry nitrobenzene was cooled on ice.
Stir it down and add anhydrous aluminum trichloride to it.
11.2 grams (83.8 mmol) was added. this
The mixture was stirred in an ice bath for 4 hours and then brought to room temperature.
Stirred overnight. After cooling again in an ice bath, this mixture
Add ice and water to the mixture, then add 5.7 ml of concentrated hydrochloric acid.
I added it slowly. Add methylene chloride to this
After adding appropriate amount of water and shaking, methylene chloride
Separate the water layer and add methylene chloride to the aqueous layer.
In addition, further extraction was performed. methylene chloride layer
After dehydration with combined sodium sulfate, methylene chloride and
and nitrobenzene were evaporated off under reduced pressure, and the silica
Fractionation using a Kagel column (diameter 4cm, length 35cm)
The desired product was obtained. Yield 6.6 grams (yield
rate 49%). Mass spectrum: M⁺=396(C₂₀H₂₉O₃Br₁minutes
molecular weight 396, 398) Silica gel TLC: Rf = approx. 0.47 (Benzene/ether = 20/1) (ultraviolet absorption,
(Colored with bromthymol blue) IR spectrum (cm^-1) (sodium chloride pellets
): 2940, 2860, 1730, 1680, 1610, 1440,
1410, 1370, 1260, 1220, 1180, 1130, 1095,
1030 Elemental analysis value: C60.20 (60.5) H 7.61 (7.4) However, the values in parentheses are
C₂₀H₂₉O₃Br₁calculated value of ¹³C-NMR spectrum δ value: 【table】 (C) p-(chlorocarbonylnonanoyl)styrene
synthesis of (C-1) p-(ethoxycarbonyl obtained in (B) above)
nonanoyl) phenylethyl bromide 3.3g
Rum (8.3 mmol) in 40 ml of primary ethanol
and 6 grams of powdered potassium hydroxide.
This was heated under reflux for 2.5 hours, then cooled on ice.
did. The formed precipitate was collected by filtration and dried.
After that, it was dissolved in 100ml of water. Add concentrated hydrochloric acid to this solution.
Add 5 ml, collect the formed precipitate, and dry under reduced pressure.
It was dry. Heat this product with 50 ml of toluene and petroleum
Recrystallized from a mixture of 150 ml of ether.
The desired product (p-(hydroxycarbonyl)
(nanoyl)styrene) was obtained. Yield 1.1 grams
(yield 46%). ¹³C-NMR spectrum (δ value): 【table】 ¹H-NMR spectrum δ (ppm): (CDCl₃, trimethylsilane) 1.34(s, 8H, (-CH₂)−_Four) 2.31(d,4H,-C(O)CCH₂− 2.35(t, 2H, -CH₂C(O)-O-) 2.95(t, 2H,
【formula】) 5.49(q, 2H, -C=CH₂) 5.86 (q, 1H, -CH=C) 7.70(q, 4H, [formula] IR spectrum (cm^-1) (KBr pellets):
3430, 3160, 2945, 2940, 2860, 1725, 1685,
1610, 1470, 1440, 1410, 1380, 1310, 1245,
1190, 1120, 995, 980, 910, 840 Mass spectrum: M⁺=288(C₁₈H_{twenty four}O₃molecule of
Amount 288) (C-2) p-(hydroxycarbonate obtained in (C-1) above)
rubonylnonanoyl) styrene 1.0 g (3.5
25 ml of dry benzene and dimethyl
One drop of formamide was added and stirred. This
Add 1 ml (11.8 mmol) of oxalic acid dichloride dropwise.
After stirring at room temperature for 2 hours, the solvent was removed under reduced pressure.
I left. Dry the residue with an oil pump to form the desired
The acid chloride product p-(chlorocarboni
(lunonanoyl) styrene was obtained. IR spectrum (KBr pellet): 1805cm^-1
(νCO(COCl)) (D) 1-(p-vinylbenzoylnonanoyl)-g
Synthesis of lycerol-stearyl ether-2 2-stearyloxy-1,3-propanedi
0.995 g (2.89 mmol) of dry
30 ml ethylene chloride and 0.5 ml dry pyridine
was added and stirred. In addition, in (B-2) above,
The entire amount (3.5 mmol) of the acid chloride obtained was
Add a solution dissolved in 10ml of ethylene chloride for 10 minutes.
The mixture was slowly added dropwise and stirred overnight. This anti
Methylene chloride was distilled off from the reaction mixture, and the residue was
was dissolved in chloroform. Add this solution to 0.5N
Hydrochloric acid, water, 2% sodium carbonate aqueous solution and water
After sequentially washing with water, the mixture was dehydrated with sodium sulfate.
This was subjected to evaporation treatment under reduced pressure, and the residue was dried.
After that, benzene/ether = 9/1 was added as the effluent solvent.
Using a silica gel column (diameter 2 cm, length 30
The desired ether product was obtained which was purified by cm). Silica gel TLC (monospot): Rf=0.13
(Benzene/ether = 9/1) Rf=0.28 (cyclohexane/ethyl acetate=
3/1) (Ultraviolet absorption, presentation by bromothymol blue)
(Color available) Mass spectrum: M⁺=614 (molecular weight 614) Elemental analysis value: C76.38 (76.2) H10.82 (10.7) However, the value in ( ) is C₃₉H₆₆O_Five
calculated value of ¹³C-NMR spectrum (δ value): 【table】 IR spectrum (cm^-1) (KBr pellets): 3450,
2930, 2870, 1750, 1690, 1610, 1475, 1400,
1370, 1295, 1230, 1185, 1135, 995, 980, 910,
840, 730 (E) 1-{9-(p-vinylbenzoyl)nonanoy
}-2-O-stearyl. rac.glycero-3
-Synthesis of phosphocholine Cl₂P(O)OCH₂CH₂Dry Br3.0g
It was dissolved in 10 ml of loform and cooled on ice. On top of this
1.0 g (29 mm) of the ether product obtained in (C)
mol) dissolved in 10 ml of dry chloroform.
Pour the solution and 4 ml of dry triethylamine into a dry container.
Separately dissolve each solution in 10ml of loloform.
were simultaneously dripped from the dripping funnel. This reaction mixture
After stirring the mixture at room temperature for 20 hours, 0.5M potassium chloride was added.
Slowly drop 10 ml of the lithium aqueous solution into the chamber.
Stir at room temperature for 1 hour. Add 10ml of methanol to this
After addition, collect the chloroform layer and wash once with water.
Cleaned and evaporated to dryness. The residue is further diluted with pentoxide.
After vacuum drying over phosphorus, add to 15 ml of dry butanone.
Dissolved. This solution is made of stainless steel pressure-resistant
After pouring into a tube (for 200ml) and cooling to -25℃,
15 ml of dry trimethylamine was added and the container was tightly stoppered.
This reaction mixture was mixed with polyethylene glycol at 60°C.
After reacting for 9 hours in a water bath, the precipitate formed was
It was collected by filtration and washed with dry butanone. The product obtained in this way was directly mixed with methanol 70
ml, add 2.5 g of silver acetate and store in the dark.
The mixture was stirred at room temperature for 1.5 hours. After filtration, filtrate
After evaporating and drying the residue under reduced pressure, silica
Gel column (chloroform/methanol/water =
65/25/4). In this way, the desired host
A phatidylcholine type phospholipid compound was obtained. collection
Amount: 0.60 grams (yield 28%). Silica gel TLC (chloroform/methanol
/ water = 65/25/4): Rf = 0.29 (ultraviolet absorption,
(Colored with bromthymol blue)¹³C
−NMR spectrum (δ value): 【table】 【table】 IR spectrum (cm^-1): 3450, 2940, 2870,
1735, 1685, 1615, 1470, 1410, 1360, 1240,
1210, 1095, 1080, 975, 925, 845 Nitrogen analysis value: N1.80 (1.79) However, the numbers in parentheses are
C₄₄H₇₈N₁O₈P₁calculated value. (F) 1-{9-(p-vinylbenzoyl)nonanoy
)-2-O-stearyl-rac-glycero-3
-Synthesis of phosphocholine (alternative method) 2-chloro-2-oxy-1,3,2-dio
Ether obtained from 4.56 g of Kisaphosphorane in above (C)
16.9 g of product, 5.7 ml of triethylamine, and
4-(N,N-dimethylamino)pyridine 05g
into a solution dissolved in 300 ml of anhydrous benzene,
Thereafter, the mixture was stirred at room temperature for 12 hours. The resulting trifecta
After separating the amine hydrochloride, it was concentrated under reduced pressure to form a pale red oil.
I got something like that. Add this to 80ml of anhydrous acetonitrile.
Dissolve and add 20ml of trimethylamine to stainless steel
Pour into a steel pressure-resistant reaction tube (for 200ml) and heat at 60℃.
After reacting for 12 hours, the mixture was concentrated under reduced pressure to obtain a light brown solid.
Silica gel column (silica gel 800g; solvent column)
loloform/methanol/water = 65/25/4)
The title is obtained by purifying and freeze-drying from benzene.
of phospholipid compounds were obtained. Yield 14.6g (yield 68
%) TLC,¹³C-NMR and IR spectra and
The nitrogen analysis value is the same as that of the compound obtained in (E) above.
I did it. Synthesis example 3 dimethyl-di(octadeca-2,4-dienoy)
Synthesis of ammonium bromide Literature (R. Bueachl et al., Federation of
European Biochemical Societies' Letters, 150
Synthesized according to the method described in Vol., pp. 38-42 (1982).
did. ¹³C-NMR spectrum (δ value, CDCl₃): 【table】 ¹H-NMR spectrum (δ value, CDCl₃): 0.88
(t, 6H, -CH₂CH₃), 1.3(s(broad), 22H,
(−CH₂)−₁₁), 2.17 (m, 4H, −CH=CH−CH₂
-), 3.61(s, 6H, N-CH₃), 4.25 (m, 4H, −
CH₂-O-), 4.69(m, 4H, N-CH₂−),
5.75 (d, 2H, [formula]), 6.2 (m, 4H, −CH=CH−CH₂−), 7.3(m, 2H,
【formula】), Synthesis example 4 Iron()-2{(1′-imidazolyl)propyla
Minocarbonyloxymethyl}-5,10,15,
20-tetra(α,α,α,α-o-pivalamide
Synthesis of phenyl)porphyrin bromide (i) J.P.Collman et al., J.Am.Chem.Soc., 97,
1427 (1975).
5, 10, 15, 20-tetra (α, α, α, α
-o-pivalamidophenyl)porphyrin (pivaramidophenyl)
Phettofuensporphyrin (abbreviated as PFP (H))
)) 20.2g (20mmol) in chloroform 1.5
Dissolve Cu(CH) under reflux at boiling point₃C.O.₂)₂・H₂O 6.0g
(30 mmol) in methanol saturated solution
added. After refluxing for 30 minutes, concentrate under reduced pressure and
The mixture was crystallized by adding mol. Chloroform-mer
When recrystallized from tanol, Picketteffens
Copper divalent complex of porphyrin (Cu()−PFP
(H)) was obtained. Yield 20.1g (yield 93.8%), melting point (mp) > 300℃ TLC RF=0.49 (silica gel plate, Ben
Zen/Ether (1/1 (v/v)) IR spectrum (KBr) 1690 (ν_C=O, amide)
cm^-1other Visible spectrum (CHCl₃) λmax411, 534,
568 (shoulder absorption) nm FDMS spectrum m/e1071 (M⁺) Elemental analysis (C₆₄H₆₄N₈O_Fouras Cu) Analysis value (calculated value) H; 5.87 (6.01), C; 71.40
(71.65), N; 10.29 (10.44)% (ii) Add 19.0g (17.7mmol) of Cu()-PFP(H)
Dissolved in 1.5 ml of lormethane. Separately, DMF68.5
ml (0.885mol) and POCl₃82.5ml (0.885mol)
Prepared by mixing below room temperature under ice cooling
Vilsmeier complex in solution
was added dropwise over 30 minutes at room temperature. 8 hours boiling point reflux
After that, it was returned to room temperature. The resulting dark green potato
After injecting the sodium salt solution into ice water, concentrate it at room temperature.
Add 500ml of ammonia water and react for 1 hour.
Using benzene/ether (2/1 (v/v)) solvent
Purification using a silica gel column (7cmφ x 35cm)
After recrystallization from acetone-methanol, 2
- formyl picketfuensporphyrin copper
A divalent complex (Cu()-PFP(CHO)) is obtained.
Ta. Yield 12.8g (yield 65.7%), mp260~262℃ TLC
Rf=0.26 (silica gel plate, benzene/ethyl
-tel (1/1 (v/v)) IR spectrum (KBr) 1690 (ν_C=O, amide),
1675 (ν_C=O, aldehyde) cm^-1other Visible spectrum (CHCl₃) λmax423, 545,
586nm FDMS spectrum m/e1099 (M⁺) Elemental analysis (C₆₅H₆₄N₈O_FiveCu₁as) Actual value (calculated value) H: 5.88 (5.86), C: 70.66
(70.92), N; 9.96 (10.18)% (iii) Cu()PFP(CHO) 6.0g (5.6mmol) with concentrated sulfur
Dissolve homogeneously in 120ml of acid and react at room temperature for 2 hours.
I let it happen. Under ice cooling, 7.5N-ammonia water/jiku
Lormethane (1/1 (v/v)) injected into 1.2
Ta. The chloroform layer was washed with water, dried, and then dried under reduced pressure.
and the residue was dissolved in benzene/ether (1/1 (v/
v)) Silica gel column using solvent (5.5cmφ
×35cm). Acetone-methanol?
Recrystallize from
Porphyrin (2H-PFP (CHO)) was obtained. Yield 3.54g (yield 60.9%), mp258~260℃ TLC
Rf=0.44 (silica gel plate, benzene/ethyl
ether/acetone (8/8/1 (v/v/v))) IR spectrum (KBr) 1690 (ν_C=O, amide),
1675 (ν_C=O, aldehyde) cm^-1other Visible spectrum (CHCl₃) λmax429, 521,
559, 599, 655nm FDMS spectrum m/e1038 (M⁺) Elemental analysis (C₆₅H₆₆N₈O_Fiveas) Actual value (calculated value) H: 6.16 (6.40), C: 74.83
(75.12), N; 10.65 (10.78)% PMR spectrum (CDCl₃)δ(ppm)−2.41
(singlet, 2H, pyrrole N-H), 0.068,
0.120, 0.125 (respectively singlet, 36H, −CH₃), 7.09
~8.87 (multiplet, 26H, phenyl ring, porphyry
Phosphorus ring hydrogen, -CONH-), 9.41 (singlet, H,
[Formula]), 9.47 (singlet, H, - CHO). CMR spectrum (CDCl₃) δ (ppm) 26.45
(−CH₃), 38.95, 38.84(-C(CH₃)₃, 115.07
~149.94 (phenyl ring porphyrin ring carbon),
175.39, 175.51, 175.60 (−CONH−), 188.69
(-CHO). (iv) 2.29g (2.20mmol) of 2H-PFP(CHO) was chlorinated.
Dissolved in loform/methanol (5/1 (v/v))
and NaBH_FourAfter adding 330mg (8.72mmol)
The reaction was allowed to take place at room temperature for 10 minutes. To refine this
2-Hydroxymethylpiquetfuens
Porphyrin (2H−PFP(CH₂OH)) is obtained
Ta. Yield 2.22g (yield 96.8%) mp＞300℃TLC Rf
= 0.51 (chloroform/methanol (10/1
(v/v))) IR spectrum (KBr) 1690 (ν_C=O, amide),
1060(ν_C-OH)cm^-1et al., 1675 (ν_C=O,aldehyde)
cm^-1Disappearance Visible spectrum (CHCl₃) λmax417, 511,
542 (shoulder breathing), 585, 640nm FDMS spectrum m/e1040 (M⁺) Elemental analysis (C₆₅H₆₈N₈O_Fiveas) Actual value (calculated value) H: 6.80 (6.58), C: 74.78
(74.97), 10.65 (10.76)% PMR spectrum (CDCl₃)δ(ppm)−2.62
(singlet, 2H, pyrrole N-H), 0.036,
0.051, 0.058, 0.083 (singlet, 36H, −
CH₃), 2.79 (triple line, H, -OH), 4.95 (double line
line, 2H, −CH₂OH), 7.15-8.83 (multiplet,
26H, phenyl ring, porphyrin ring hydrogen, −
CONH−), 8.99 (single line, H,
【formula】). CMR spectrum (CDCl₃) δ (ppm) 26.42
(−CH₃), 38.87(-C(CH₃)₃), 60.15(−
CH₂OH), 114.02-148.20 (phenyl ring, pol
firin ring carbon), 175.51, 175.77, 175.90 (−
CONH−). (v) 2H−PFP(CH₂OH) 104mg (0.10mmol)
Dissolve in 20ml of dichloromethane and cool to 0°C.
Ta. Carbon tetrachloride solution containing 1.0 mmol of phosgene
After adding 0.23 ml, it was allowed to react for 1 hour. to room temperature
After returning and reacting for another hour, remove the solvent and filtrate.
When excess phosgene is distilled off under reduced pressure and dried, 2-
Chlorocarbonyloxymethyl picketophene
Sporphyrin (2H−PFP(CH₂OCOCl))
It was obtained quantitatively in the form of hydrochloride. TLC Rf=0.68 (silica gel plate, Ben
Zen/Ether/Acetone (5/5/1 (v/
v/v)), 2H-PFP(CH₂OH) is Rf under the same conditions
=0.44 FDMSm/e1102(M⁺) (vi) 2H-PFP (CH₂OCOCl) 110mg
(0.10 mmol) in 20 ml of dichloromethane,
1-(3-aminopropyl)imidazole 125mg
(1mmol), triethylamine 0.14ml (1mmol)
was added and reacted at room temperature for 16 hours. Remove solvent under vacuum
After distillation, wash with water, dry, and chloroform/methanol
Silica gel using 10/1 (v/v) solvent
By purifying with a column (2cmφ x 20cm)
the law of nature, was gotten. Yield 75mg (yield 59.5%) TLC Rf=0.39 (silica gel plate, black
Roform/methanol (10/1 (v/v)) IR spectrum (KBr) 1720 (ν_C=O, ureta
), 1690 (ν_C=O, amido) cm^-1other Visible spectrum (CHCl₃) λmax418, 512,
544 (shoulder breathing), 585, 640nm Elemental analysis (C₇₂H₇₇N₁₁O₆as) Actual value (calculated value) H: 6.37 (6.51), C: 72.23
(72.52), N; 12.78 (12.92)% PMR spectrum (CDCl₃)δ(ppm)−2.60
(singlet, 2H, pyrrole N-H), 0.007,
0.051, 0.075, 0.117 (respectively singlet, 36H, −
CH₃), 2.03 (quintet, 2H, OCONHCH₂CH
₂CH₂), 3.19 (quartet, 2H, OCONH
CH₂CH₂CH₂), 4.09 (triple line, 2H,
OCONHCH₂CH₂CH ₂), 5.04 (double line, H,
CH₂OCONH), 5.73 (multiplet, 2H, CH
₂OCONH), 7.03-8.83 (multiplet, 30H, Fueni
ring, porphyrin ring, and imidazole ring
element, −CONH−). CMR spectrum (CDCl₃) δ (ppm) 26.45
(−CH₃), 31.43, 37.78, 44.21
(CH₂CH₂CH₂), 38.89(-C(CH₃)₃), 61.32
(CH₂OCONH), 114.37-151.22 (phenyl ring,
porphyrin, and imidazole ring carbon),
156.37 (−OCONH−), 175.46, 175.72, 176.16
(−CONH−). Dissolve 38 mg (0.032 mmol) in 10 ml of THF and add nitrogen
FeBr under boiling point reflux in air flow₂・4H₂O92.1mg
(0.32mmol) and pyridine 0.026ml (0.32mmol)
added. After reacting at the same temperature for 2.5 hours, dry under reduced pressure.
and chloroform/methanol (10/1 (v/
v) Silica gel column (2 cmφ×
20cm), trivalent iron ions are used as counterions.
Complexes introduced with Br I got it. Yield 30mg (yield 71.0%) TLC Rf=0.27 (silica gel plate, chloro
Form/methanol (10/1 (v/v)) IR spectrum (KBr) 172.5 (ν_C=O, urethane),
1690 (ν_C=O, amido) cm^-1other Visible spectrum (CHCl₃) λmax417, 505,
575, 640, 657 (shoulder absorption) nm Elemental analysis (C₇₂H₇₅N₁₁O₆Fe₁Br₁as) Actual value (calculated value) H: 5.60 (5.70), C: 64.80
(65.21), N; 11.38 (11.62)% Synthesis example 5 1-n-lauryl-2-methylimidazole
synthesis 2-Methylimidazole 19g (0.23mole), bromide
Mix 25g (0.10mole) of lauryl and heat to 200℃ for 10 hours
A heating reaction occurred. 10% K after cooling₂C.O.₃500ml of aqueous solution
After addition and stirring, ether extraction (2 x 300ml)
did. Combine the organic layers and add saturated NaHCO₃aqueous solution
(2 x 200ml), washed with saturated NaCl aqueous solution, and separated.
and dry Na₂S.O._FourIt was dried. Brown after being subjected to evaporation
An oily substance was obtained, which was transferred to a silica gel column (silica gel column).
Gel 500g; CHCl₃:MeOH=10:1)
Ta. Yield 20gr, yield 79%. This is distilled under reduced pressure and boiled.
The point 156-158°C/4 mmHg fraction was used. Mass spectrum: M⁺=250, ¹H-NMR spectrum (CDCl₃, TMS): δ (ppm) 6.88 (d, J = 1Hz), 6.76 (d, J =
1Hz): 1m 4th place, 5th place protons 3.89 (2H, t, J=7Hz): 1st place N-CH ₂ −CH₂ 2.36 (3H, s): 1m2CH ₃ IR spectrum (NaCl plate) cm^-1:3400, 2930,
2860, 1525, 1500, 1465, 1425,
1375, 1280, 1145, 995, 720, 680 Synthesis example 6 Synthesis of 1-trityl-2-methylimidazole 2-methylimidazole-Ag salt 4.3g (0.023mol)
Suspend and stir trityl chloride in 50 ml of toluene.
7.0 g (0.025 mol) was added and heated under reflux for 10 hours. room temperature
The insoluble part (AgBr) was separated and the liquid was concentrated.
A tan solid was obtained. Silica gel column (silica gel column)
200g, CHCl₃/MeOH=20/1),
Recrystallized from benzene-heptane system. yield
3.34g, yield 45%. Mass spectrum: M⁺324.
¹H-NMR spectrum (CDCl₃, TMS) δ
(ppm): 7.00-7.40 (15H, m): Phenylproto
6.86, 6.76 (each 1H, d, J = 1Hz): Imida
Sol 4th and 5th positions - proton 1.67 (3H, s):
Imidazole 2-CH₃. IR (KBr disk): 3350, 3070, 3030, 1600,
1520, 1490, 1450, 1395, 1305,
1240, 1180, 1140, 1070, 1035,
1000, 990, 910, 880, 865, 760,
750, 700cm^-1 Synthesis example 7 Methyl 11-[1-(2-methyl)imidazoly
[L] Synthesis of undecanoate 25g of 11-bromoundecanoic acid and 100ml of benzene
Stir at room temperature for 4 hours in the presence of 13 ml of oxalyl chloride.
Ta. Next, add 50ml of methanol and leave it overnight, then use the usual method.
Treatment with 11-bromoundecanoic acid methyl ester
23g (yield 87.5%) was obtained. 8.2g of 2-methylimidazole to 2.4g of NaH/
It was slowly added to a suspension solution of 200 ml of DMF. addition
After heating at 90℃ for 1 hour, the previously obtained 11-bromoun
Decanoic acid methyl ester was added dropwise. Then at 2 o'clock
After heating and stirring at 90° C. for a while, the mixture was treated in a conventional manner. Silica gel column (CHCl₃/CH₃OH＝9/1
(v/v)) and purified using methyl 11-{1-
(2-methyl)imidazolyl}undecanoate
9.5g (yield 41%) was obtained. IR (liquid film): 1740cm^-1(ν_C=O,ester) ¹H-NMR spectrum (TMS, CDCl₃)δ
(ppm) 1.28 (singlet, 12H, NCH₂CH₂(CH ₂)
₆CH₂CH₂C.O.₂−), 1.68 (multiline, wide, 4H,
NCH₂CH ₂(CH₂)₆CH ₂CH₂C.O.₂−), 2.31 (Mie
line, 2H, -CH ₂C.O.₂−), 2.37 (single line, 3H, i
midazole ring-CH ₃), 3.66 (singlet, 3H, −
COOCH ₃), 3.80 (triple line, 2H, NCH ₂−),
6.80 (double line, imidazole ring 5th proton),
6.90 (double line, proton at position 4 of imidazole ring) MS spectrum: M⁺280 Synthesis example 8 Synthesis of 1-laurylimidazole 20g imidazole and 25g lauryl bromide
(0.10mole) was mixed and heated to 200℃ for 10 hours.
Add 500ml of 10% potassium carbonate water and extract with ether.
(2 x 300ml), washed with water, separated and dried under reduced pressure.
The obtained brown oil was transferred to a silica gel column (solvent chloro
Form/methanol = 10/1 (v/v)), then
It was obtained by distillation under reduced pressure in a yield of 75%. Boiling point 145-150℃/
5mmHg IR spectrum (NaCl plate) ν3400, 3120, 2930,
2860, 1505, 1465, 1380, 1285, 1230, 1110,
1085, 1030, 910, 810, 730, 670cm^-1 PMR spectrum (CDCl₃) δ (ppm) 0.88 (3H,
t), 1.26 (18H, s), 1.8 (2H, t), 3.92 (2H,
t), 6.90 (1H, s), 7.50 (1H, s), 7.46 (1H,
s). Synthesis example 9 (A) 10-benzyloxydecanyl bromide is 1,
Sodium equivalent to 100 g of 10-dibromodecane
Reducing benzyl oxide in tetrahydrofuran
Flow reaction is carried out, the precipitate is filtered, concentrated and then distilled under reduced pressure.
Ta. Yield 46g, boiling point 185-189℃/3mmHg. George R. Newkome et al., Synthesis,1975,
517. under a nitrogen atmosphere.
lithium diisopropylamide during the run.
Lithium dianio of 2-methylpropionic acid
10-benzyl oxide at -20°C.
After dropping 18g of canyl bromide, incubate at 45℃ for 2 hours.
I responded. Add the reaction mixture to cold dilute hydrochloric acid and evaporate.
The separated ether layer was extracted with dilute salt.
Washed with acid and then water, separated and dried with Glauber's salt.
Ta. The crude oil obtained by evaporation to dryness is converted into petroleum ether.
Recrystallized from 12-benzyl oxychloride as colorless crystals
Yield: 8.4g of ci-2,2-dimethyldodecanoic acid,
Obtained with a yield of 46%. Melting point 53-55℃. Elemental analysis:
C_{twenty one}H₃₄O₃Calculated value (%); C75.4, H10.25,
Analysis value (%); C75.64, H10.09, proton nuclear magnetism
Gas Resonance Spectrum (CDCl₃) δppm: 1.18 (6H,
s, -C(CH ₃)₂COOH), 1.26 (16H, s, -
OCH₂(CH ₂)₈CH₂−), 3.46 (2H, t,
PhCH₂O.C.H ₂CH₂−), 4.51 (2H, s, PhCH
₂O−), 7.33 (5H, s, phenyl proton). 3.34 g of this obtained carboxylic acid was added to anhydrous benzene.
Dissolve the solution in 5 ml of water, add 1.2 ml of thionyl chloride, and bring to room temperature.
React for 12 hours and dry under reduced pressure to form a colorless oil.
12-benzyloxy-2,2-dimethyldode
Kanoyl chloride was obtained in a yield of 3.53 g. infrared absorption
Spectrum (CCl_Four)ν1790cm^-1,
[Formula] Proton nuclear magnetic resonance spectrum (CDCl₃) δppm: 1.28 (22H, s, -CH ₃and-
CH ₂−), 3.46 (2H, t, PhCH₂O.C.H ₂CH₂
−), 4.50 (2H, s, PhCH ₂O−), 7.32 (5H,
s, phenyl proton). (B) 5, 10, 15, 20-tetra (α, α, α, α-
o-aminophenyl)porphin (hereinafter
H₂(abbreviated as TamPP). J.P. Collman et al.
Journal of the American Chemical
Society,97, 1427 (1975)
did. H₂TamPP1.0g anhydrous tetrahydrofuran
(40 ml) solution, add 0.81 ml of pyridine and leave at room temperature.
12-benzyloxy-2,2- obtained in (A) above
Drop 3.53g of dimethyldodecanoyl chloride,
The reaction was allowed to proceed for 3 hours. Extract with ether and wash with water.
After drying the separated ether layer with Glauber's salt, the pressure was reduced.
The crude product obtained by drying was mixed with benzene:ether.
Silica gel column with mixed solvent (volume ratio 15:1)
Chromatographically purified as a brown oil 5,10,
15,20-tetra [α, α, α, α, -o-(12-
Benzyloxy-2,2-dimethyldodecana
Yield 1.69g of mido)phenyl]porphyrin,
Obtained with a yield of 60%. Infrared absorption spectrum (CHCl₃) ν3440, 3330,
3000, 2930, 2860, 1680, 1580, 1510, 1450,
1300, 1100, 970, 910, 770cm^-1. Proton nuclear magnetic resonance spectrum (CDCl)₃)
δppm: −2.6 (2H, s, N in the porphyrin ring
H) _, -0.23 (24H, s, -C( CH3 ) _2CONH- ),
3.46 (8H, t _{, PhCH2OCH2CH2-} ) , _4.50
(8H, s, PhC H ₂ O−), 7.12 (4H, s,
[Formula]), 7.32 (20H, s), 8.82 (8H, s). (C) 5, 10, 15, 20-tetra [α, α, α, α,
-o-(12-benzyloxy-2,2-dimethyldodecanamido)phenyl]porphyrin
Make 1.68g into a mixed solvent solution of 25ml of anhydrous dichloromethane and 25ml of nitromethane, and add 2ml of anisole.
After that, 2 g of anhydrous aluminum chloride was added and reacted at room temperature for 4 hours. Pour into 100ml of ice water,
Excess aluminum chloride was decomposed and extracted with dichloromethane. The separated dichloromethane layer was washed with water and then with a 10% aqueous sodium bicarbonate solution, separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was recrystallized from benzene to give purple plate-like crystals of 5,10,15,20-tetra [α, α, α,
Yield α, -o-(12-hydroxy-2,2-dimethyldodecanamido)phenyl]porphine
Obtained 1.10 g, yield 80%. Melting point 127-129.5℃ Magnetic field desorption mass spectrum: 1579 (M+1) ⁺ Infrared absorption spectrum (KBr) ν3600-3350
(wide range), 3440, 3330, 2940, 2860, 1690,
1585, 1515, 1450, 1302, 1060, 970, 810,
770, 740 cm ^-1 proton nuclear magnetic resonance spectrum (CDCl ₃ ).
δppm: −2.59 (2H, s, pyrrole NH ), −
0.22 (24H, s, -C( CH3 ) ₂ -CONH-) _, 3.64
(8H, t, HOC H ₂ CH ₂ −), 7.15 (4H, s)
7.36-8.73 (16H, m), 8.82 (8H, s). In addition, 4.50 (8H, s) and 7.32 derived from benzyl group
The absorption of (20H, s) disappeared. Elemental analysis: Calculated value (%) as C ₁₀₀ H ₁₃₈ N ₈ O ₈ ; C76.00, H8.80, N7.09, analytical value (%);
C75.62, H8.90, N7.09. (D) 5, 10, 15, 20-tetra [α, α, α, α,
A solution of 0.65 g of -o-(12-hydroxy-2,2-dimethyldodecanamido)phenyl]porphine in anhydrous tetrahydrofuran (50 ml) was added with 0.3 ml of pyridine, and after purging with nitrogen, ferrous bromide and tetrahydrate were added. 2.0 g of the product was added and the mixture was refluxed for 3 hours under nitrogen. After extraction with chloroform and washing with water, the separated chloroform layer was dried with Glauber's salt and the solvent was distilled off under reduced pressure. The resulting residue was purified by alumina column chromatography using a mixed solvent of chloroform/methanol (volume ratio 9/1). . The eluate solution was stirred with 2 ml of 48% hydrobromic acid, dried with Glauber's salt, and evaporated to dryness to give a black-purple solid of bromo{5,10,
15,20-tetra [α, α, α, α-o-(12-
Yield of hydroxy-2,2-dimethyldodecanamido)phenyl]porphynato}iron ()
Obtained 0.38g, yield 54%. Melting point 76-79°C Magnetic field desorption mass spectrum: 1713 (M+1) ⁺ However, molecular formula C ₁₀₀ H ₁₃₆ N ₈ O ₈ FeBr = 1712. Infrared absorption spectrum (KBr): ν3600~3150
(broad), 3440, 2930, 2860, 1690, 1580,
1510, 1440, 1300, 1075, 1000, 805, 760,
715cm ^-1 Elemental analysis Calculated value (%) as C ₁₀₀ H ₁₅₆ N ₈ O ₈・FeBr; C70.13, H8.00, N6.54, analytical value (%);
C70.37, H8.40, N6.63. (E) Phosphocholination of the tetraalcohol obtained in (D) above was carried out by NSChandrakumar et al.
It can be carried out as reported in Tetrahedron Letters, 23 , 1043 (1982). Bromo {5, 10, 15,
0.15 g of 20-tetra[α, α, α, α, -o-(12-hydroxy-2,2-dimethyldodecanamido)phenyl]porphynato}iron () was dissolved in anhydrous dichloromethane (10 ml), and 0.07 ml of triethylamine and 2-chloro-2-oxo-
0.08 g of 1,3,2-dioxaphosphorane was added and reacted at room temperature for 12 hours. Completion of the reaction can be confirmed by silica gel thin layer chromatography (solvent chloroform/methanol = 10/1) from the disappearance of a spot with an Rf value of 0.30 and the formation of a spot with an Rf value of 0.40. The solvent was distilled off under reduced pressure, the residue was dissolved in 25 ml of anhydrous acetonitrile, and 5 ml of trimethylamine was added at -60 to -40°C, the mixture was sealed in a pressure-resistant stainless steel container, heated to 60 to 65°C, and reacted for 16 hours. The black precipitate obtained by cooling to room temperature was dissolved in methanol and added to Cephadex LH-
The eluate was evaporated to dryness and then vacuum-dried in the presence of phosphorus pentoxide. As a black solid, iron()-5,10,15,20-tetra{α,α,α,α, -o-(12(2'-trimethylammonioethoxy)phosphinatoxy-
2,2-dimethyldodecanamide phenyl}
A porphyrin complex was obtained in a yield of 0.18 and 90%. Melting point 145~150℃ Infrared absorption spectrum (KBr): ν3650~3150
(broad), 3430, 2940, 2860, 1690, 1580,
1510, 1440, 1220, 1080, 1000, 950, 800,
760, 710cm ^-1 Visible absorption spectrum (H ₂ O): λmax412,
564nm Synthesis Example 10 (A) 1,18-dibromooctadecane is e.g.
Lester Friedman and Arnon Shani;
Journal of the American Chemical
Society, 96 , 7101-7103 (1974). In the same manner as in Synthesis Example 9(A), 18.3 g (yield: 41%) of 18-benzyloxyoctadecanyl bromide was obtained from 42 g of 1,18-dibromooctadecane instead of 1,10-dibromodecane. 18 g of this was reacted with lithium dianion (equivalent mole) of 2-methylpropionic acid, and the crude oil obtained after extraction and post-treatment in the same manner as in Reference Example 1 was obtained using a mixed solvent of benzene:ether (volume ratio 15:1).
Purification by silica gel column chromatography using 20-benzyloxy-
2,2-dimethyleicosanoic acid was obtained in a yield of 5.7 g and a yield of 31%. Melting point 72-73℃ _Elemental analysis: Calculated value _{as C29H50O3} (%);
C77.97, H11.28, analysis value (%); C78.25,
H11.21. Infrared absorption spectrum (KBr) ν: 2930,
2860, 1705, 1470, 1130, 1120, 950, 740,
700 cm ^-1 . Proton nuclear magnetic resonance spectrum ( _CDCl3 )
δppm: 1.18 (6H, s _, -C( CH3 ) _2COOH ),
1.25 (32H, brs, PhCH ₂ OCH ₂ ( CH ₂ ) ₁₆ CH ₂
−), 3.46 (2H, t, J=6.5Hz, PhCH ₂ OC H
₂ CH ₂ −), 4.50 (2H, s, PhC H ₂ O−), 7.33
(5H, m, benzene ring proton). 5.2 g of the obtained carboxylic acid was reacted with 4 ml of thionyl chloride at room temperature for 4 hours and dried under reduced pressure to obtain 5.6 g of 20-benzyloxy-2,2-dimethyleicosanoic acid chloride as a colorless solid. Infrared absorption spectrum (CCl ₄ ) ν: 2930,
2860, 1790, 1460, 1365, 1100, 905, 700cm
^-1 . (B) 5.6 g of the obtained 20-benzyloxy-2,2-dimethyleicosanoic acid chloride
_H2 TamPP1.3g anhydrous tetrahydrofuran (50
ml) and pyridine (1.5 ml) solution and Synthesis Example 9 (B)
React and post-process and purify under the same conditions as 5.
10, 15, 20-tetra (α, α, α, α, -o-
Yield of (20-benzyloxy-2,2-dimethyl-eicosanamido)phenyl]porphine
Obtained 3.70 g, yield 80%. Melting point 33~35℃ Elemental analysis: Calculated value (%) as C ₁₆₀ H ₂₃₀ N ₈ O ₈ ; C80.29, H9.68, N4.68, analytical value (%);
C80.11, H9.88, N4.77. Infrared absorption spectrum (KBr) ν: 3440,
2920, 2850, 1690, 1580, 1510, 1450, 1360,
1300, 1100, 965, 800, ^{750cm -1} . Proton nuclear magnetic resonance spectrum ( _CDCl3 )
δppm: −2.60 (2H, s, N in the porphine ring
−H ), −0.22(24H, s, −C( CH ₃ ) ₂ −CONH
−), 3.45 (8H, t, J=6.4Hz, PhCH ₂ OC H
₂ CH ₂ −), 4.49 (8H, s, PhC H ₂ O−), 7.13 )7.31 (20H, s, [formula]), 8.76 (4H, d, J=7Hz,
[Formula]), 8.82 (8H, s, proton at β-position of porphine ring) (C) 5,10,15,20-tetra [α, α, α, α,
-o-(20-benzyloxy-2,2-dimethyleicosanamido)phenyl]porphine
3.5 g was reacted with 6 g of anhydrous aluminum chloride in a mixed solvent of dichloromethane (30 ml), nitromethane (15 ml), and anisole (3 ml) in the same manner as in Synthesis Example 9 (C), and then subjected to post-treatment and extraction operations to dryness under reduced pressure. The resulting residue was purified by silica gel column chromatography (solvent chloroform/methanol = 15/1) and recrystallized from a mixed solvent of dichloromethane and methanol. 5, 10, 15, 20-tetra [α, α, α,
1.89 g of α,-o-(20-hydroxy-2,2-dimethyleicosanamido)phenyl]porphine was obtained in a yield of 64%. Infrared absorption spectrum (KBr) ν: 3430,
3320, 2920, 2860, 1675, 1580, 1510, 1470,
1450, 1300, 970, 805, 760, 720 cm ^-1 Proton Nuclear Magnetic Resonance Spectrum ( _CDCl3 )
δppm: −2.60 (2H, s, N− H in the porphyrin ring), −0.21 (24H, s, −C( CH ₃ ) ₂ −
CONH−), 3.69 (8H, t, J=6.4Hz, HOC H
₂ CH ₂ −), 7.14 (4H, s, amide group −CON H
-), 8.73 (4H, d, J = 7Hz,
[Formula]), 8.82 (8H, s, proton at β position of porphine ring). Elemental analysis: Calculated value (%) as C ₁₃₂ H ₂₀₂ N ₈ O ₈ ; C78.14, H9.98, N5.52, analytical value (%);
C78.42, H10.20, N5.39. (D) 5, 10, 15, 20-tetra [α, α, α, α,
-o-(20-hydroxy-2,2-dimethyleicosanamido)phenyl]porphine 1.06g
was purified by reaction, post-treatment, and silica gel column chromatography (chloroform/methanol = 25/1 mixed solvent) in the same manner as in Synthesis Example 9(D), and further recrystallized from a dichloromethane/methanol mixed solvent. . Black-purple crystals of bromo {5, 10, 15, 20-tetra [α, α, α,
α, -o-(20-hydroxy-2,2-dimethyleicosanamido)phenyl]porphynato}
Iron () was obtained in a yield of 0.82 g and a yield of 73%. melting point
49-50℃ Infrared absorption spectrum (KBr) ν: 3430,
2930, 2860, 1690, 1580, 1510, 1460, 1440,
1300, 1000, 800, 760, 720cm ^-1 Elemental analysis: C ₁₃₂ H ₂₀₀ N ₈ O ₈ FeBr・1/2CH ₂ Cl ₂
Calculated value (%); C72.16, H9.19, N5.08, analytical value (%); C72.41, H9.09, N4.92. (E) Bromo {5, 10, 15, 20-tetra [α, α,
0.55 g of α,α, -o-(20-hydroxy-2,2-dimethyleicosanamido)phenyl]porphynato}iron () was added to 2-chloro in the presence of 0.3 ml of triethylamine in the same manner as in Synthesis Example 9(E). After reacting with 0.30 g of -2-oxo-1,3,2-dioxaphosphorane, the residue was dried under reduced pressure and dissolved in N,N-dimethylformamide (15 ml).
and acetonitrile (15 ml), reacted with 10 ml of trimethylamine, and after similar post-treatment and purification, iron()-5,10,15,20-tetra {α, α, α,
0.45 g of α, -o-(20-(2'-trimethylammonioethoxy)phosphinatoxy-2,2-dimethyleicosanamido)phenyl}porphyrin complex was obtained in a yield of 61%. Melting point 235~
237℃ Infrared absorption spectrum (KBr) ν: 3430 (wide range), 2940, 2860, 1690, 1590, 1515, 1470,
1440, 1300, 1230, 1090, 1000, 970, 810,
760, 730cm ^-1 Elemental analysis: Calculated value (%) as C ₁₅₂ H ₂₄₈ N ₁₂ O ₂₀ P ₄ Fe・9H ₂ O; C62.83, H9.23, N5.78, Analysis value (%); C62. 55, H9.42, N5.99. Synthesis Example 11 1-{9-(p-vinylbenzoyl)nonanoyl}-2-O-myristyl-rac-glycero-3
- Synthesis of phosphocholine (A) Synthesis of 1-(p-vinylbenzoylnonanoyl)glycerol-myristyl ether-2 1.0 g of 2-myristyloxy-1,3-propanediol and p synthesized in (C) of Synthesis Example 2 1-(p-vinylbenzoylnonanoyl)glycerol myristyl ether-2 was synthesized by reacting with -(chlorocarbonylnonanoyl)styrene according to the method of Synthesis Example 2(D). Yield 36%. Silica gel TLC: Rf = 0.17 (benzene/ether = 9/1) (ultraviolet absorption, coloring with bromothymol blue) Mass spectrum: M ⁺ = 558 (molecular weight 558) ¹ H-NMR (deuterochloroform, TMS): δ
(ppm) 0.88 (t, 3H, -CH ₃ ); 1.26 (s wide,
(−CH ₂ ) − _o ); 1.6 (t, 2H, [formula]); 2.08 (t, 1H, −OH); 2.32 (t, 2H, −
CH ₂ COO−); 2.92 (t, 2H,
[Formula]); 3.5-3.7 (m, 5H, -CH ₂ -, [Formula]); 4.15 (d, 2H, [Formula]); 5.35, 5.82, 6.7 (d, d, q, 1H, 1H, 1H, -CH= _CH2 ); 7.69(d,
d, 2H, 2H, [Formula]) ¹³ C-NMR (deuterochloroform, TMS): δ
(ppm) [Table] [Table] (B) 1-{9-(p-vinylbenzoyl)nonanoyl}-2-O-myristyl-rac-glycero-3
-Synthesis of phosphocholine According to the method (E) of Synthesis Example 2, the above 1-(p-
It was synthesized by phosphocholination of vinylbenzoylnonanoyl)glycero-myristyl ether-2. Yield 30%. Silica gel TLC: Rf=0.27 (chloroform/methanol/water=65/25/
4) (Ultraviolet absorption, coloration due to bromothymol blue) Elemental analysis value (weight%): N1.90 (1.93) (Calculated value of C ₄₀ H ₇₀ N ₁ O ₈ P ₁ in parentheses) ¹³ C-NMR (Deuterated chloroform, TMS): δ
(ppm) [Table] IR spectrum (chloroform solution) cm ^-1 :
2940, 2870, 1730, 1680, 1610, 1470, 1410,
1360, 1220, 1180, 1090, 975, 920 Example 1 In a nitrogen gas atmosphere, Fe()
TpivPP・Br1mg (8.7×10 ^-4 mmol), 1-n-
Lauryl-2-methylimidazole 11mg (4.3×
10 ^-2 mmol) in 5 ml of toluene and add
5 ml of an aqueous solution in which 10 mg of sodium dithionite was dissolved was added, and the mixture was shaken. After standing still, the toluene layer was collected, and a small amount of 4 Å of molecular sieves was added thereto for dehydration treatment. To this toluene solution, 15 mg (0.02 mmol) of dipalmitoylphosphatidylcholine and 1,2-di(octadeca-2,4-dienoyl) were added.
Glycero-3-phosphocholine 150mg (0.19mmol)
A solution prepared by dissolving this in 2 ml of toluene and saturated with nitrogen gas was added thereto. Toluene was distilled off under reduced pressure. The thin film-like solid material remaining on the inner wall surface of the flask was further subjected to sufficient vacuum treatment to completely remove toluene. Then, after adding 10 ml of phosphate buffered water (PH7.0) from which oxygen gas had been removed, ultrasonic stirring (20 kHz, 60 W) was performed for 45 minutes under nitrogen gas without ice cooling. 5, 10,
15,20-tetra(α,α,,α,α,-o-pivalamidophenyl)porphine iron()-mono(1-
An aqueous monomeric liposome dispersion solution of the n-lauryl-2-methylimidazole complex was obtained. The maximum absorption wavelength λmax of the visible absorption spectrum of the solution under nitrogen gas is 532 and 562 nm,
It corresponded to a deoxy type complex. The shape of the absorption spectrum and the absorption maximum wavelength are also consistent with literature values for the same type of complex in anhydrous benzene solution. The monomeric liposome dispersion aqueous solution prepared in this way was transferred to a quartz glass cell container under a nitrogen gas atmosphere, and the container was tightly stoppered. This was irradiated with light in a 50℃ water bath using a 30-160W low-pressure mercury lamp (Riko Kagaku Sangyo Co., Ltd. UVL-30LA or UVL-160LA type) at a location approximately 5 cm from the light source for 30 minutes. A photopolymerization reaction was carried out for 5 hours to obtain the desired aqueous polymeric liposome dispersion solution. When oxygen gas was blown into this polymeric liposome dispersion aqueous solution (in a nitrogen gas atmosphere) at 25°C, a single peak with a wavelength of λmax 546 nm was newly observed in place of the two deoxy-type peaks. This is consistent with literature values for the oxygenated complex of Fe()TpivPP complex. By bubbling nitrogen gas into this oxygenated complex solution for a short time, a reversible change in the deoxy spectrum was observed, confirming reversible oxygen adsorption and desorption. Example 2 Fe()TpivPP・Br 1 mg (8.7×10 ^-4 mmol), 1-n-laurylimidazole 1.0 mg (4.3
×10 ^-3 mmol) and 1,2-di(octadeca-
2,4-dienoyl)glycero-3-phosphocholine 150 mg (0.19 mmol) Cholesterol 11.6 mg
(0.03 mmol) and 10 mg (0.014 mmol) of dipalmitoylphosphatidylcholine were dissolved in 5 ml of toluene, and the toluene was distilled off under reduced pressure to thoroughly remove the thin film-like solid substance remaining on the inner wall of the flask. The solvent was completely removed by vacuum treatment. to this,
After adding 10 ml of physiological saline (PH7.0) from which oxygen gas had been removed under a nitrogen gas atmosphere, ultrasonic stirring (20 kHz, 50 W) was performed for 60 minutes under nitrogen gas. −tetra(α, α, α, α−
(o-pivalamidophenyl)porphine iron ()-
An aqueous monomeric liposome dispersion solution of di(1-n-laurylimidazole) complex was obtained. The obtained solution was irradiated with light in the same manner as in Example 1 to obtain the desired aqueous polymeric liposome dispersion solution. After adding ascorbic acid in a molar amount 5 times that of iron porphyrin, incubation was carried out at 37°C for 3 hours. The absorption maximum wavelength λmax of the visible absorption spectrum of the solution under nitrogen gas was 533 nm and λshoulder 560 nm, which corresponded to the deoxy type.
The shape of the absorption spectrum and the absorption maximum wavelength are also consistent with literature values for the same type of complex in anhydrous benzene solution. Using this solution, we checked the reversible adsorption and desorption of O ₂ complexes at room temperature. As in Example 1, a visible absorption spectrum (single peak absorption band at λmax 543 nm) corresponding to the oxygenated complex was obtained by blowing nitrogen gas into the solution. or,
By blowing nitrogen gas into this solution, the original deoxy substance spectrum was obtained, confirming the reversible adsorption and desorption of oxygen. Example 3 Dipalmitoylphosphatidylcholine and 1,
Instead of adding 2-di(octadeca-2,4-dienoyl)glycero-3-phosphocholine, dimethyl-di(octadeca-2,4-dienoyl) synthesized in Synthesis Example 3 was used.
5,10,15,20-tetra(α,α,α,α-o-pi varamide phenyl)porphine iron()-mono(1-n-lauryl-2
An aqueous monomeric liposome dispersion solution of the (-methylimidazole) complex was obtained. The visible absorption spectrum of the solution under nitrogen gas is λmax 532 nm,
It was 562nm. After preparing an aqueous polymeric liposome dispersion solution according to the method of Example 1, the formation of an oxygen complex was confirmed in the same manner. Example 4 In Example 1, dipalmitoylphosphatidylcholine and 1,2-di(octadeca-2,4
-dienoyl)glycero-3-phosphocholine, 1-{9-(p
-vinylbenzoyl)nonanoyl}-2-O-stearyl-rac-glycero-3-phosphocholine 170
Using exactly the same method except that 5,10,15,20-tetra (α, α, α, α
-o-pivalamidophenyl)porphine iron ()
An aqueous monomeric liposome dispersion solution of -mono(1-n-lauryl-2-methylimidazole) complex was obtained. Polymerik according to the method of Example 1.
After preparing the liposome dispersion aqueous solution, the generation of oxygen complexes was similarly confirmed. Example 5 In Example 1, instead of Fe()TpivPP.Br1-lauryl-2-methylimidazole, 1.3 mg (8.7×10 ^-4 mmol) of the iron Fe()porphyrin bromide synthesized in Synthesis Example 4 was used. A monomeric liposome dispersion aqueous solution of the corresponding iron()porphyrin complex was obtained by the same method except that the above method was used. The solution visible absorption spectrum under nitrogen gas was λmax of 535 nm and 563 nm. After preparing polymeric liposomes according to the method of Example 1, the production of oxygen complexes was confirmed in the same manner. The λmax of the oxygen complex was 544 nm. By bubbling nitrogen gas into this oxygenated complex solution for a short time, a reversible change in the deoxy spectrum was observed, confirming reversible oxygen adsorption and desorption. Example 6 Fe()TpivPP・Br1 mg (8.7×10 ^-4 mmol), 1-lauryl-2-methyl-imidazole
11 mg (4.3×10 ^-2 mmol), dipalmitoylphosphatidylcholine 30 mg (0.04 mmol), 1-{9-
(p-vinylbenzoyl)nonanoyl}-2-O-
Stearyl-rac-glycero-3-phosphocholine
170 mg (0.22 mmol) and 2 mg (8.2×10 ^-3 mmol) of azobiscyclohexanitrile were dissolved in toluene 10
ml, toluene was distilled off under reduced pressure. The thin film-like solid substance remaining on the inner wall surface of the flask was further subjected to sufficient vacuum treatment to completely remove the solvent. After adding 10ml of physiological saline (PH7.0) from which oxygen gas has been removed under nitrogen gas atmosphere, ultrasonic stirring (20k
Hz, 50W) operation for 60 minutes, 5, 10, 15, 20−
An aqueous monomeric liposome dispersion solution of tetra(α,α,α,α-o-pivalamidophenyl)porphine iron()-mono(1-n-lauryl-2-methylimidazole) complex was obtained. The obtained solution was shaken in a bath at 60°C to perform polymerization, thereby obtaining the desired aqueous polymeric liposome dispersion solution. A visible absorption spectrum corresponding to the oxygenated complex was obtained by bubbling oxygen gas into the solution. In addition, by blowing nitrogen gas into this solution, the original deoxy substance spectrum was obtained, confirming the reversible adsorption and desorption of oxygen. Example 7 Fe()TpivPP・Br0.55mg (4.6×10 ^-4 mmol), 1-n-laurylimidazole 1.09mg (4.6
x 10 ^-3 mmol) and 74 mg (9.22 x 10 ^-2 mmol) of 1,2-bis(octadeca-2,4-dienoyl)glycero-3-phosphocholine were dissolved in benzene, and the benzene was distilled off under reduced pressure. Further drying was performed using a vacuum pump to obtain a thin film-like solid on the inner wall surface of the flask. After adding 25ml of pure water to this, ultrasonic stirring (20kHz,
60W) treatment for 30 minutes to obtain a monomeric liposome dispersion aqueous solution containing 5,10,15,20-tetra(α,α,α,α-o-pivalamidophenyl)porphyrin iron complex. This solution was transferred to a quartz glass cell container and argon gas was blown into it to create an argon gas atmosphere.The solution was sealed and polymerized by irradiating it with light using a low-pressure mercury lamp (30W) in a water bath at 50℃ at a distance of approximately 5cm from the light source. I did this. To confirm polymerization, collect 0.2 ml of this solution with a syringe, dilute it to 100 ml with pure water, measure the ultraviolet absorption spectrum, and confirm that 1,2-bis(octadeca-2,4
The absorption maximum at 255 nm based on the dienoyl structure of -dienoyl)glycero-3-phosphocholine disappeared over time with light irradiation. Approximately 4
After some time, the absorption at 255 nm completely disappeared, confirming the completion of polymerization. Note that the concentration measured in this ultraviolet absorption spectrum is 5, 10, 15, 20-tetra (α, α,
No absorption by the α,α-o-pivalamidophenyl)porphyrin iron complex was observed, and the polymerization could be confirmed without any influence. While the monomers used in this experiment were soluble in chloroform, ether, etc., the polymeric liposomes obtained as described above were insoluble in these organic solvents. This also supports the formation of polymers. Example 8 In Example 7, 1,2-bis(octadeca-
Except that 72 mg of 1-{9-(p-vinylbenzoyl)nonanoyl}-2-O-stearyl-rac-glycero-3-phosphocholine was used instead of 74 mg of 2,4-dienoyl)glycero-3-phosphocholine. The same method was used to obtain the ultraviolet absorption spectra.
Polymerization was confirmed by the fact that the absorption at 254 nm completely disappeared after 4 hours. The same method was repeated using 67 mg of dimethyl-di(octadeca-2,4-dienoyloxyethyl) ammonium bromide in place of 74 mg of 1,2-bis(octadeca-2,4-dienoyl)glycero-3-phosphocholine. Completion of polymerization was confirmed by the complete disappearance of absorption at 255 nm in the ultraviolet absorption spectrum after about 4 hours. This polymeric liposome was also insoluble in organic solvents such as chloroform and ethanol. Example 9 Bromo{5,10,
15,20-tetra [α, α, α, α-o-(2,2
-dimethylhexadecaneamide)phenyl]porphynato}iron () 1.62 mg (8.7 × 10 ^-4 mmol),
1-n-laurylimidazole 2.05mg (8.7×10 ^-3
mmol) was made into a 5 ml toluene solution and reduced with an aqueous sodium dithionite solution in the same manner as in Example 1, and further 12.5 mg (approximately 0.02 mmol) of dimyristoylphosphatidylcholine and 1,2-bis(octadeca-2, By adding 150 mg (0.19 mmol) of 4-dienoyl)glycero-3-phosphocholine and operating according to Example 1,
15,20-tetra [α, α, α, α-o-(2,2
-dimethylhexadecanamido)phenyl]porphine iron()-bis(1-n-laurylimidazole) complex monomeric liposome dispersion aqueous solution was obtained. The absorption maximum wavelength λmax of the visible absorption spectrum of the solution in a nitrogen gas atmosphere is 533, 560
(Shoulder absorption) nm, and it was confirmed to be a deoxy hexacoordination type complex. The monomeric liposome dispersion aqueous solution prepared in this way was subjected to a photopolymerization reaction under the same conditions as in Example 1 to obtain a desired polymeric liposome dispersion aqueous solution. When oxygen gas is blown into this at 37℃,
The absorption maximum wavelength of the visible absorption spectrum immediately changes from 533 and 560 nm (shoulder absorption) in the deoxy type.
A new observation was made from the absorption of a single peak at 537 nm, confirming the formation of an oxygenated complex. Furthermore, by blowing nitrogen gas into this oxygenated complex solution for several minutes, the visible absorption spectrum returned to that of the deoxy type, confirming that reversible oxygen adsorption and desorption occurred. Example 10 Iron ()-5, 10, 15, 20-tetra [α, α,
α,α-o-[20-(2′-trimethylammonioethoxy)phosphinatoxy-2,2-dimethyleicosanamide]phenyl}porphyrin complex
2.46 mg (8.7 x 10 ^-4 mmol), 1-n-laurylimidazole 1.0 mg (4.3 x 10 ^-3 mmol), dipalmitoylphosphatidylcholine 10 mg (0.014 mmol) and 1,2-bis(octadeca-2, 4-
Dienoyl)glycero-3-phosphocholine 150mg
(0.19 mmol) was dissolved in 5 ml of toluene and operated in exactly the same manner as in Example 2 to obtain an aqueous monomeric liposome dispersion solution containing the deoxy type complex. The absorption wavelengths of the visible absorption spectrum were 533 nm and 560 nm (shoulder absorption), confirming that it was a deoxy type. This solution was photopolymerized in exactly the same manner as in Example 1 to obtain an aqueous polymeric liposome solution, and after confirming the deoxy type by visible absorption spectrum, oxygen gas was blown into the polymeric liposome to obtain a visible absorption corresponding to an oxygenated complex. A spectrum (single peak absorption band at λmax 541 nm) was obtained. Furthermore, by blowing nitrogen gas into this aqueous oxygenated complex solution, the spectrum returned to the original aqueous deoxy complex solution, confirming reversible adsorption and desorption of oxygen. Example 11 Following the method of Example 2, Fe()TpivPP・
Br0.55 mg (4.6×10 ^-4 mmol), 1-n-laurylimidazole 1.09 mg (4.6×10 ^-3 mmol) and 1,
2-di(octadeca-2,4-dienoyl)glycero-3-phosphocholine 74 mg (9.2×10 ^-2 mmol)
A 20 ml aqueous solution was prepared containing polymeric liposomes containing Fe()porphyrin complexes consisting of: Even when this solution was left at room temperature for about 3 weeks, no phase separation or precipitation occurred, confirming that this polymeric lipome structure was extremely stable. For comparison, in a monomeric liposome dispersion aqueous solution having the same composition as above that was not subjected to the polymerization operation, a considerable amount of phase separation, that is, precipitation due to dislocation of liposomes, was observed after about 3 days. Example 12 In Example 11, in place of 1,2-bis(octadeca-2,4-dienoyl)glycero-3-phosphocholine, the same mole of 1-{8-(p-vinylbenzoyl)nonanoyl}-2- stearyl-rac-
Glycero-3-phosphocholine or dimethyl-
Corresponding using di(octadeca-2,4-dienoyloxyethyl)ammonium bromide
Polymeric compounds containing Fe()porphyrin complexes
An aqueous liposome dispersion solution was obtained, and it was similarly confirmed that the polymeric liposome structure was extremely stable. Example 13 Iron ()-5, 10, 15, 20-tetra [α, α,
α,α-o-[20(2′-trimethylammonioethoxy)phosphinatoxy-2,2-dimethyleicosanamide]phenyl}porphine complex 0.86 mg
(3.03×10 ^-4 mmol), 1-n-laurylimidazole 0.36 mg (1.53×10 ^-3 mmol) and 1,
24.3 mg (3.04 x 10 ^-2 mmol) of 2-bis(octadeca-2,4-dienoyl)glycero-3-phosphocholine was dissolved in 5 ml of methanol, the solvent was distilled off under reduced pressure, and the flask was dried using a vacuum pump. A thin film was applied to the vessel wall. Add to this 0.05M-phosphate buffered water (PH
7.0) Add 25 ml and ultrasonic stirring (20kHz,
60W) for 20 minutes to obtain an almost homogeneous and transparent monomeric liposome dispersion aqueous solution. For polymerization, 5 ml of this aqueous solution was transferred to a quartz glass cell container, carbon monoxide gas was blown into it to create a carbon monoxide gas atmosphere, and after the cap was tightly capped, the light source was placed in a water bath at 50°C using a low-pressure mercury lamp (30W). Light was irradiated at a distance of about 2 cm from the surface, and the reaction was allowed to occur for 4 hours. Polymerization was confirmed by collecting 0.2 ml of the solution after the reaction, diluting it with 10 ml of ionized water, and confirming that the absorption at λmax 257 nm in the ultraviolet absorption spectrum completely disappeared. When the visible absorption spectrum of the solution polymerized for 4 hours was measured, λmax423,
Iron()-5,10,15,20-tetra {α,
α,α,α-o-[20-(2'-trimethylammonioethoxy)phosphinatoxy-2,2-dimethyleicosanamide]phenyl}porphin-
Absorption based on the CO (carbon monoxide) addition complex of the (1-n-laurylimidazole) complex was observed. When argon gas was bubbled into this solution for 20 minutes to remove the added CO, an aqueous polymeric liposome dispersion solution containing the deoxy type complex was obtained. The maximum wavelength of the visible absorption spectrum was 426 and 535 nm, and it was confirmed that it was a deoxy type. When oxygen gas was blown into this deoxy type complex solution, the spectrum immediately changed to give a visible absorption spectrum (λmax 424, 542 nm) corresponding to an oxygenated complex. Furthermore, by blowing argon gas into this aqueous oxygenated complex solution, the spectrum returned to the original aqueous deoxy complex solution, confirming reversible adsorption and desorption of oxygen. Example 14 In Example 13, instead of 24.3 mmol of 1,2-bis(octadeca-2,4-dienoyl)glycero-3-phosphocholine, 1-{9-(p-vinylbenzoyl)nonanoyl}-2-O -stearyl-rac-glycero-3-phosphocholine 23.7 mg
Iron()- ^5,10,15,20 -tetra(α,α,α,α-o-[20-(2′-trimethyl ammonioethoxy)phosphinatoxy-2,2
-dimethyleicosanamide]phenyl}porphine-(1-n-laurylimidazole) complex
An aqueous polymeric liposome dispersion solution containing a CO addition complex (λmax 540 nm) was obtained. When argon gas was blown into this for 20 minutes, the visible absorption spectrum gave a λmax of 535 nm, confirming that it was a deoxy type aqueous solution. Furthermore, when oxygen gas is injected,
When an oxygen complex with a wavelength of λmax of 542 nm was generated and argon gas was again blown into the complex, the visible absorption spectrum returned to that of the original deoxy complex, confirming reversible adsorption and desorption of oxygen. Example 15 Iron()-5,10,15,20-tetra {α, α,
α,α-o-[20-(2′-trimethylammonioethoxy)phosphinatoxy-2,2-dimethyleicosanamide]phenyl}porphine complex 1.72
mg (6.07 x 10 ^-4 mmol), 1-n-laurylimidazole 0.42 mg (1.78 x 10 ^-3 mmol) and 1,2-bis(octadeca-2,4-dienoyl)glycero-3-phosphocholine 48.6 mg (6.07 ×
10 ^-2 mmol) was dissolved in 5 ml of ethanol, the solvent was distilled off under reduced pressure, and the solution was dried using a vacuum pump to form a thin film on the wall of the flask. To this was added 15 ml of 0.05M phosphate buffered water (PH7.0), and ultrasonic stirring (20 kHz, 60 W) was performed for 20 minutes in an ice bath to obtain an almost uniform and transparent monomeric liposome dispersion aqueous solution. This aqueous solution was transferred to a quartz glass cell container, argon gas was blown into it to create an argon gas atmosphere, and after the solution was tightly capped, it was irradiated with light using a low-pressure mercury lamp (30W) in a water bath at 50°C and reacted for 8 hours to complete the reaction. A polymerized liposome dispersion aqueous solution was obtained. Most of the original iron() complex has been reduced to the deoxy type complex of iron() during the polymerization reaction, but in order to still completely reduce it to the deoxy type, ascorbic acid 0.7 mg (4.0
×10 ^-3 mmol) and allowed to stand for 1 hour on a water bath at 37°C to obtain a polymeric liposome dispersion aqueous solution in which a complete deoxy type complex (visible absorption spectrum absorption maximum wavelength 535 nm) was embedded. When oxygen gas is blown into the solution, an oxygenated complex (542 nm) is obtained, and when argon gas is further blown into the solution, it returns to the original deoxy complex solution (535 nm), confirming that oxygen can be adsorbed and desorbed reversibly. Example 16 1-{9-(p-vinylbenzoyl)nonanoyl}-2-O-stearyl-rac-glycero-3-
Phosphocholine 48.2 mg (6.18 × 10 ^-2 mmol), iron () 5,10,15,20-tetra (α, α, α, α
-o-[20-(2'-trimethylammonioethoxy)phosphinatoxy-2,2-dimethyleicosanamide]phenyl}porphyrin complex 3.5 mg
(1.24 x 10 ^-3 mmol) and 0.87 mg (3.71 x 10 ^-3 mmol) of 1-n-laurylimidazole were prepared in methanol solution (10 ml), and the solvent was distilled off under reduced pressure to obtain a thin film of solid on the wall of the flask. Add 15 ml of 0.05 M phosphate buffered water (PH7.0) to this and perform ultrasonic stirring (20 kHz, 40 W) at 30 to 40°C for 20 minutes to determine the pore diameter.
The mixture was filtered through a 0.45 μm membrane filter to obtain an almost transparent and uniform aqueous monomeric liposome dispersion solution. Transfer 5 ml of this aqueous solution to a quartz glass cell container, blow in argon gas for 30 minutes to create an argon gas atmosphere, seal it tightly, and irradiate it with a low-pressure mercury lamp (30 W) in a 50°C water bath at a distance of about 2 cm from the light source. 2 hours) to polymerize. To confirm polymerization, take a portion of the solution after light irradiation, dilute it with 0.05M phosphorus buffer, measure the ultraviolet absorption spectrum,
This was carried out by the disappearance of absorption at λmax 267 nm due to the p-vinylbenzoyl group of the raw material. The maximum wavelength of the visible absorption spectrum after polymerization is 536 nm, and it is a deoxy hexacoordination type, and it is clear that the iron ( ) complex is completely reduced to the iron ( ) complex during polymerization. When oxygen gas was blown into this deoxy hexacoordination complex solution, the spectrum immediately changed and the solution became an oxygenated complex aqueous solution with a maximum absorption wavelength of 543 nm. When argon gas is passed through again for 1 to 2 minutes, the maximum absorption wavelength is
Reversible adsorption and desorption of oxygen was confirmed by returning to the deoxy hexacoordination form at 536 nm. Example 17 In Example 16, additionally 5 mg of cholesterol
(1.29 × 10 ^-2 mmol) was added to make a methanol solution, prepare a monomeric liposome dispersion aqueous solution in the same manner, and further photopolymerize it. After polymerization, a deoxy hexacoordination complex aqueous solution (visible absorption spectrum maximum wavelength 536 nm) It was confirmed that when oxygen gas was blown into this, an oxygenated complex aqueous solution (maximum wavelength 543 nm) was formed. Example 18 Polymeric liposome aqueous solutions containing iron()porphyrin complexes prepared by various compositions and methods gradually oxidize and change to iron()complexes when left in the atmosphere for a long time. The iron() complex does not have the ability to adsorb and desorb oxygen, so it is reduced to iron() by reducing agent again.
By reducing it to a complex, this solution can be reused as an oxygen adsorption/desorption agent. We investigated usable re-reduction reagents using the following method. The polymeric liposome aqueous solution containing the iron() porphyrin complex prepared in Example 16 was left in a dark place under the atmosphere for 5 days to oxidize and form an iron() complex aqueous solution. This iron () complex aqueous solution (iron () complex concentration approximately 8.2×
After substituting 10 ^-5 M) with nitrogen gas and adding about 5 times the equivalent amount of reducing agent, it was confirmed that re-reduction could be achieved by generating a visible absorption spectrum maximum wavelength of 536 nm based on iron (). Reagents that could be used for the reduction include sodium dithionite, sodium borohydride, ascorbic acid, glutathione, cysteine, cysteamine, 2-mercaptopropionic acid, ethanethiol, and 1,2-ethanedithiol. Ta. Although glucose could not be reduced at about 5 times the equivalent, it had the ability to reduce at about 1% (w/v) concentration or higher. Example 19 In Example 16, 1-{9-(p-vinylbenzoyl)nonanoyl}-2-O-myristyl-rac-glycero-3-phosphocholine (6.18×
We obtained an aqueous polymeric liposome solution containing the corresponding iron()porphyrin using the same method except that 10 ^-2 mmol), and further confirmed that this reversibly adsorbed and desorbed oxygen. Example 20 In Example 16, iron()-5,10,15, which was synthesized in Synthesis Example 9, was used as iron()porphyrin.
20-tetra(α,α,α,α-o-[12-(2′-trimethylammonioethoxy)phosphinatoxy-2,2-dimethyldodecanamide]phenyl}
We obtained a polymeric liposome aqueous solution containing the corresponding iron ()porphyrin in exactly the same manner except that porphyrin (1.24 × 10 ^-3 mmol) was used, and furthermore, we confirmed that this reversibly adsorbed and desorbed oxygen. confirmed. Example 21 Iron ()-5,10,15,20-tetra {α, α,
α,α-O-[20-(2′-trimethylammonioethoxy)phosphinatoxy-2,2-dimethyleicosanamide]phenyl}porphine complex
3.72 g (1.28 mmol) 1-n-laurylimidazole 0.90 g (3.84 mmol) and 1-{9-(p-vinylbenzoyl)nonanoyl}-2-0-stearyl-rac-glycero-3-phosphocholine 50 g ( 64 mmol) was dissolved in a mixed solvent of ethanol (50 ml) and benzene (800 ml), the pressure was reduced using a vacuum pump, and the mixture was lyophilized to powder. Add physiological saline (1) to this and use a probe-type ultrasonic stirring device under a water bath.
Ultrasonic dispersion treatment at 20KHz, 160~200W output
By performing this for 40 minutes, a monomeric liposome dispersion aqueous solution was obtained. After neutralizing the pH to 8.0 with a 0.1N aqueous sodium hydroxide solution, the solution was filtered through a filter with a pore size of 0.45 μm (manufactured by Millipore), and the resulting liquid was replaced by argon gas being blown at a rate of 500 ml/min for 1 hour. Thereafter, the mixture was transferred to a photoreactor container designated for No. 1, and a photopolymerization reaction was carried out at 50° C. for 3 hours in an argon gas atmosphere using a 160W low-pressure mercury lamp (manufactured by Riko Kagaku Sangyo). It was passed through a 0.45 μm filter under an argon gas atmosphere to obtain a deoxy solution (about 1.0) having a polymerizable phospholipid concentration of 5 wt/vol% and a heme concentration of about 1.28 mM. The pH of the solution was 7.5. When this solution was stored in a container filled with argon gas or nitrogen gas, it showed stability for more than one year without any change in the solution state in the temperature range from 4°C to 35°C. Further, the half life of the oxygenated complex obtained by blowing oxygen gas into the obtained deoxy compound solution was about 1 day. Example 22 Iron ()-5,10,15,20-tetra {α, α,
α,α-O-[20-(2′-trimethylammonioethoxy)phosphinatoxy-2,2-dimethyleicosanamide]phenyl}porphine complex
0.262 g (0.09 mmol), 0.064 g (0.27 mmol) of 1-n-laurylimidazole, and 3.6 g (4.5 mmol) of 1,2-bis(octadeca-2,4-dienoyl)glycero-3-phosphocholine in ethanol. (10ml)
- It was dissolved in a mixed solvent of benzene (90 ml) and pulverized by reducing the pressure with a vacuum pump and freeze-drying. 360 ml of physiological saline was added thereto, and ultrasonic dispersion was performed for 40 minutes at 20 KHz and an output of 160 to 200 W using a probe-type ultrasonic stirrer under a water bath to obtain a monomeric liposome-dispersed aqueous solution. After neutralizing the pH of this solution to 7.5 with a 0.1N aqueous sodium hydroxide solution, it was passed through a filter (manufactured by Millipore) with a pore size of 0.22 μm. Oxygen gas was removed by blowing argon gas into the liquid at a rate of 500 ml/min for 1 hour, and the mixture was transferred to a 500 ml reaction vessel and placed in an argon gas atmosphere using a low-pressure mercury lamp (160 W) in a photoreactor (Riko Kagaku Sangyo Co., Ltd.). (manufactured by)
Light irradiation was carried out at 30-50°C for 5 hours. Completion of polymerization was confirmed by diluting the reaction solution 250 times with water and measuring the ultraviolet absorption spectrum by the disappearance of absorption by diene groups at λmax 255 nm. Furthermore, the fact that a deoxy type complex solution was obtained was confirmed by transferring a portion of the solution to a cell with an optical path length of 1 mm under argon gas and measuring the visible absorption spectrum, and by the absorption at λmax 536 and 560 nm (shoulder absorption). When oxygen gas was blown into this deoxy type complex solution, the absorption position in the visible absorption spectrum immediately changed, giving an absorption of λmax 545 nm, which corresponds to an oxygenated complex. The change over time of the obtained oxygenated complex into an oxidized form was followed by visible absorption spectroscopy, and it was found that the half-life of the oxygenated complex was about 5 days. Furthermore, the solution of the deoxy type complex obtained after the photoreaction could be stored stably for more than one year without any change in the solution state in the temperature range from 4°C to 35°C by storing it in a sealed container filled with nitrogen or argon gas. At this time, the pH of the solution is 7.0 to 7.5, the heme concentration is about 250 μM, and the concentration of polymerizable phospholipid is about 1 wt/
It was vol%. An oxygen-nitrogen mixed gas with a known oxygen partial pressure is blown into the obtained deoxy type solution, and when gas equilibrium is reached, the visible absorption spectrum is measured, and the oxygen bonding rate is determined from the amount of change in absorption at λmax 536 nm. An equilibrium curve was plotted (Figure 2, curve a). The oxygen partial pressure (P _1/2 O ₂ ) when half the amount of oxygen is combined is approximately 30 mmHg from curve a in Figure 2,
The value is close to the blood value (curve b, 20 mmHg) and is different from the myoglobin value (curve c (0.37 to 1.0 mmHg). This value indicates that the compound of the present invention has sufficient ability to transport oxygen. Example 23 At the polymerized phospholipid concentrations obtained in Examples 21 and 22,
A wt/vol% deoxy type solution can be concentrated to an arbitrary concentration using an ultra-superconcentrator. For example, when using UF Membrane NMWL10000 as the ultrafiltration membrane and Pellicon Lab Cassette (both manufactured by Millipore) as the device, add 1 wt/vol% solution of 1 to 20 wt/vol%.
It took 1-2 hours to concentrate to vol%. All concentration operations were performed under a nitrogen or argon gas atmosphere, and about 200 ml of the desired deoxy type solution with a concentration of 20 wt/vol% was obtained from the solution of Example 16, and about 18 ml of the solution of Example 17. The physical constants of the 20 wt/vol% solution were in the range of specific gravity 1.01-1.02, viscosity 3.5-4.2 cp, and immersion pressure 320-350 mOsm/. solution 100
van Slyke's device used to measure the oxygen binding capacity per ml of blood (the measurement method is provided by Kentaro Takagi,
Edited by Akira Okamoto, Physiology System, Physiology of Blood and Breathing,
Pages 876-885 (1968), based on Igaku Shoin literature. ), it was found that approximately 87% of the oxygen was bound to the theoretical value at a heme concentration of 10.0mM, and as shown in the table, it was found to be almost comparable to blood. [Table] Example 24 In Example 22, 0.9 mmol of the non-polymer additive was added together with 1,2-bis(octadeca-2,4-dienoyl)glycero-3-phosphocholine, and then polymerization was carried out in the same manner. A deoxy heme solution embedded in liposomes was obtained. The stability of the oxygenated complex obtained by blowing oxygen gas into this was compared by determining the half-life. Additives include egg yolk phosphatidylcholine, cholesterol,
Using phosphatidylserine and egg yolk phosphatidylethanolamine (both manufactured by Sigma),
The table below shows the results of a comparison using solutions at pH 7.0. The additives either did not affect the lifetime of the oxygenated complex or had the effect of extending its lifetime. [Table] No additives 5

Claims (1)

[Claims] 1. General formula (n is an integer from 1 to 20) iron ()-
5, 10, 15, 20-tetra (α, α, α, α-o-
Alkyl-substituted amidophenyl)porphyrin or general formula (Here, n is an integer from 10 to 20) iron()-5,10,15,20-tetra(α,α,α,α
-o-phosphocholine-substituted amidophenyl) porphyrin has the formula (Here, R ₁ is a non-bulky group that does not inhibit the coordination of the imidazole to the central iron to which it is attached,
and R ₂ , R ₃ and R ₄ are each hydrogen or a hydrophobic substituent, and at least one of them is a hydrophobic substituent), or a complex coordinated with an imidazole compound represented by the general formula (where l is 3, 4 or 5, and R ₅ , R ₆
and R ₇ are hydrogen atoms or methyl groups, respectively)
Iron ()-2-substituted-5, 10, 15, 20 represented by
- An oxygen adsorption/desorption agent containing as an active ingredient a lipid-based polymerized liposome in which a tetra(α,α,α,α-o-pivalamidophenyl)porphyrin complex is embedded in a hydrophobic region. 2. Claim 1, wherein the polymerized liposome is derived by polymerization of a monomeric liposome containing 1,2-bis(octadeca-2,4-dienoyl)glycero-3-phosphocholine as a main component.
Oxygen adsorption/desorption agent described in Section 1. 3. Claim 1, wherein the polymerized liposome is derived by polymerizing a monomeric liposome containing dimethyl-di(octadeca-2,4-dienoyloxyethyl)ammonium bromide as a main component. Oxygen adsorption/desorption agent. 4 General formula for polymerized liposomes (Here, q is an integer from 1 to 10, and r is 13
Induced by polymerization of monomeric liposomes mainly composed of 1-{ω-(p-vinylbenzoyl)alkanoyl}-2-O-alkyl-rac-glycero-3-phosphocholine (integer from 1 to 21) The oxygen adsorbing/desorbing agent according to claim 1, which is an oxygen adsorbing/desorbing agent. 5. The oxygen adsorbing/desorbing agent according to any one of claims 2 to 4, wherein the monomeric liposome contains a non-polymerizable phospholipid. 6. The oxygen adsorbing/desorbing agent according to any one of claims 2 to 5, wherein the monomeric liposome contains cholesterol.