JPH0354671B2 - - Google Patents

Description

[Detailed Description of the Invention] The present invention relates to the general formula (Here, n is an integer from 1 to 20) Iron-5,10,15,20- having one phosphorylcholine group
[α, α, α, α-o-(substituted amido)phenyl]
Concerning porphine complexes. Many iron-porphyrin complexes that can form oxygen complexes in organic solvents at room temperature have been reported. especially
J.P.Collman et al., Journal of the American
Iron()- 5,10,15,20 -tetra[α,
The α,α,α-o-(pivalamido)phenyl]porphine complex is useful in that it forms a very stable oxygen complex in anhydrous aprotic solvents, but it oxidizes quickly in the presence of even small amounts of water. There are drawbacks. As a useful oxygen adsorption/desorption agent, especially when considering biological applications, it is recommended to use water at room temperature or 37℃.
An iron-porphyrin complex that effectively adsorbs and desorbs oxygen is desired. E. Hase-gawa et al., Biochemistry
and Biophysical Research Communications,
105, 1416 (1982), iron()-5,10,15,20-tetra[α,α,α,α-o-(pivalamide)phenyl] was added to liposomes composed of phospholipids.
By embedding porphine complexes, it has become possible to generate oxygen complexes in aqueous media at room temperature. However, although this iron-porphyrin complex has a great stabilizing effect on the oxygen complex due to its four pivalamide groups, it has a drawback in terms of the amount that can be embedded in liposomes due to its structure. The object of the present invention is iron-5,10,15,20-tetra[α,α,α,α-o-(pivalamide)phenyl]
An object of the present invention is to provide an iron-porphin complex that can be more easily embedded in liposomes made of phospholipids while retaining its oxygen adsorption/desorption function. It has the partial structure of a pivalamide group necessary to obtain a stable oxygen complex, and also has a phosphorylcholine group as a hydrophilic moiety at its terminal end, which is similar to the hydrophilic moiety of natural or synthetic phospholipids, especially phosphatidylcholine. The novel compounds presented allow this objective to be achieved. The compound of the present invention has a tetraphenylporphine core portion containing three pivalamide groups as a hydrophobic portion, a terminal phosphorylcholine portion as a hydrophilic portion, and a hydrophobic portion of an alkyl chain of a liposome membrane consisting of a phospholipid such as phosphatidylcholine. The similarity to hydrophilic moieties such as phosphorylcholine moieties facilitates embedding. Moreover, it is a very useful compound in that it has no effect on the stability of oxygen complexes. According to the present invention, novel iron-tetraphenylporphine complexes of the general formula (Here, n is an integer of 1 to 20) 1 equivalent of carboxylic acid chloride is mixed with known 5,10,15,20-tetra[α,α,α,α-o-aminophenyl]porphin (hereinafter referred to as H ₂ TamPP The formula obtained by reacting with 1 equivalent of pivaloyl chloride and 3 equivalents of pivaloyl chloride. (where n is the same as defined above) is reacted with anhydrous aluminum chloride in the presence of anisole to obtain the formula (where n is the same as defined above) is obtained by reacting the compound represented by the formula with ferrous bromide in the presence of pyridine, (Here, n is the same as defined above.) The compound represented by 2-chloro-2-oxo-1,
It can be produced by reacting with 3,2-dioxaphosphorane and further reacting with excess trimethylamine. The starting material for the general formula is a new compound, which was synthesized by the following method. Synthesis of ω-benzyloxyalkyl halide by George R. NewKome et al.
The dilithium anion of 2-methylpropionic acid produced according to the report of 1975, 517.
I reacted with The reaction mixture is then decomposed with cold dilute hydrochloric acid and the crude product extracted with a solvent is recrystallized in a non-polar solvent such as petroleum ether, n-hexane, n-heptane and ω-benzyloxy-2,2-dimethyl The alkanoic acid was obtained as colorless crystals. This is reacted with an excess amount of thionyl chloride in a nonpolar solvent, preferably benzene or carbon tetrachloride, or without a solvent, and then concentrated under reduced pressure to form the ω-
Benzyloxy-2,2-dimethylalkanoic acid chloride was obtained. raw material formula (Here, n has the same meaning as defined above, and X' represents chlorine or bromine.) When n=1X'=Cl, the ω-benzyloxyalkyl halide represented by AJHll et al., Journal of the American
Chemical Society, 48 , 257 (1926), when n = 2 and X' = Br, S. Cremer et al.
Journal of the American Chemical Society,
86, 4197 (1964). For n=3 to 20 and X'=Br, it can be obtained by refluxing α,ω-dibromoalkane with 1 equivalent of sodium benzyl oxide in benzene. One equivalent of carboxylic acid chloride of the general formula is
H ₂ TamPP (JP Collman et al., Journal of the
American Chemical Society, 97 , 1427 (1975)
) in an anhydrous aprotic solvent containing 1 equivalent of an excess of base, preferably pyridine, triethylamine, 4-(N,N-dimethylamino), preferably tetrahydrofuran, chloroform, dichloromethane, N,N-dimethylformamide or acetone. ) After reacting in the presence of pyridine for 1 to 24 hours at 0°C to room temperature,
Add more than an equivalent amount of pivaloyl chloride and make 1 at room temperature.
The reaction was allowed to occur for up to 24 hours. The reaction solution is poured into water and extracted with ether, chloroform or dichloromethane. After washing with water, the separated organic solvent is dried with Glauber's salt and evaporated to dryness. The resulting residue is purified by silica gel chromatography to obtain the general formula. A compound was obtained. To remove the benzyl protecting group of this compound, excess anhydrous aluminum chloride was added in a mixed solvent of dichloromethane:nitromethane in the presence of an excess amount of anisole to -5°C to 30°C, preferably at 15°C.
The reaction was carried out at a temperature of 2 to 25°C for 2 to 12 hours. The reaction mixture was poured into ice water and extracted with chloroform.
After washing the extract with water and then with a 4% aqueous sodium bicarbonate solution, the separated organic solvent layer is dried with sodium sulfate, and the residue obtained by evaporating chloroform is purified by silica gel chromatography, or with a suitable solvent, e.g. The alcohol represented by the general formula was obtained by recrystallization from benzene, petroleum ether, ether, methanol, or a mixed solvent thereof. To introduce the compound of the general formula obtained, J.
P. Collman et al., Journal of the American
The reaction was carried out with excess ferrous bromide in the presence of pyridine in anhydrous tetrahydrofuran refluxed under a nitrogen gas atmosphere according to the method reported in Chemical Society, 97 , 1427 (1975). The obtained compound of the general formula has one bromide ion as a counter ion in a central iron trivalent state. Phosphorylcholination of compounds of general formula is N.
S.Chandrakumar et al., Tetrahedron Letters,
23, 1043 (1982), we improved the phosphorylcholination reaction of alcohol. A compound of the general formula is prepared from 2-chloro-2-oxo-1,3,2-dioxaphosphoric acid in an anhydrous aprotic solvent such as dichloromethane, chloroform or benzene in the presence of pyridine in an amount of 1 to 2 equivalents based on the alcohol group. 1 to 2 equivalents of the run were added dropwise at 0°C to room temperature, and then allowed to react at room temperature for 6 to 24 hours. The residue obtained by evaporating the reaction solution under reduced pressure was dissolved in acetonitrile or N,N-dimethylformamide, trimethylamine was added, and the mixture was reacted in a sealed stainless steel container at 50°C to 65°C for 12 to 24 hours. The reaction solution was concentrated under reduced pressure, the resulting residue was dissolved in a solvent such as acetone, methanol, acetonitrile, chloroform, or dichloromethane, and the insoluble portion was filtered off. manufactured by Pharmacia) or Toyo Pearl HW-40
The product was purified by column chromatography using an appropriate gel (manufactured by Toyo Soda Co., Ltd.) to obtain the desired compound represented by the general formula (). For one equivalent of this compound, a substituted imidazole ligand such as 1-n-laurylimidazole, 1-
1 such as n-stearylimidazole, 1-phenethylimidazole, 1-tritylimidazole, etc.
~300 equivalents, preferably 5 to 150 equivalents and phospholipids such as egg yolk lecithin, soybean lecithin, egg yolk phosphatidylethanolamine, sphingomyelin,
Single or mixed substances such as dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dimyristoylphosphatidylcholine30
~1000 equivalents of the organic solvent solution was dried under reduced pressure to form a thin film on the wall of the container. Add an aqueous medium such as pure water, buffered solution (PH5-9), physiological saline, etc. to this so that the phospholipid concentration is 25% (w/v) or less, shake to peel off the membrane, and then treat with ultrasound. An aqueous liposome dispersion solution containing an iron()porphin complex represented by the general formula was prepared. To reduce the central iron from trivalent to divalent, add an equivalent to a slight excess of sodium dithionite, sodium borohydride, or ascorbic acid to this liposome dispersion solution by blowing nitrogen gas into the nitrogen atmosphere for a while. Just add it to. Or E for reduction.
Hasegawa et al., Biochemical and Bio−physical
Research Communications, 104 , 793 (1982)
An enzymatic reduction method reported in 2007 can also be used. When oxygen is blown into the liposome dispersion solution of the iron()-porphine complex obtained in this way at room temperature, it becomes an oxygen complex, and when nitrogen is blown into this, the visible absorption spectrum changes reversibly to the original iron() complex. It was confirmed that it acts as an oxygen adsorption/desorption agent. In particular, a liposome solution can be easily produced even when the molar ratio of the compound represented by the general formula to phospholipid is 1:75 to 1:40. α、α
-o-(pivalamido)phenyl]porphine complex and phospholipid, the ratio is at least 1:100, preferably 1:150 or more, which is a big improvement compared to the point where it is necessary to prepare a liposome dispersion solution. The present invention will be explained in more detail by the following examples, which are not intended to limit the invention. Reference example 1 10-benzyloxydecanyl bromide is 1,10
- 100 g of dibromodecane and an equivalent amount of sodium benzyl oxide were reacted under reflux in tetrahydrofuran, and the precipitate was filtered, concentrated, and then distilled under reduced pressure. Yield 46
g, boiling point 185-189℃/3mmHg. George R. Newkome et al., Synthesis, 1975 ,
517, with lithium diisopropylamide in tetrafuran under nitrogen atmosphere.
- Lithium dianion of methylpropionic acid was generated, 18 g of 10-benzyloxydecanyl bromide was added dropwise at -20°C, and the mixture was reacted at 45°C for 2 hours.
The reaction mixture was added to cold diluted hydrochloric acid and extracted with ether, and the separated ether layer was washed with diluted hydrochloric acid and then with water, separated and dried over Glauber's salt. The crude oil obtained by evaporation to dryness was recrystallized from petroleum ether to obtain 8.4 g of colorless crystals of 12-benzyloxy-2,2-dimethyldodecanoic acid in a yield of 46%. melting point 53
~55℃, Elemental analysis: Calculated value (%) _{as C21H34O3 ;} C75.4, H10.25, Analysis value (%); _C75.64 ,
H10.09. Proton nuclear magnetic resonance spectrum ( _CDCl3 ) δppm: 1.18 (6H, s, _-CH3 ), 1.26
(16H, S, -CH ₂ ), 3.46 (2H, t,
PhCH ₂ OCH ₂ CH ₂ −), 4.51 (2H, s, PhCH ₂ O
-), 7.33 (5H, s, phenyl proton). 3.34 g of this obtained carboxylic acid was dissolved in 5 ml of anhydrous benzene, 1.2 ml of thionyl chloride was added, and the mixture was stirred at room temperature for 12 hours.
After reacting for an hour and drying under reduced pressure, a colorless oil of 12-
Benzyloxy-2,2-dimethyldodecanoic acid chloride was obtained in a yield of 3.53 g. Infrared absorption spectrum (CCl ₄ ) ν1790cm ^-1 [Formula]. Proton nuclear magnetic resonance spectrum (CDCl ₃ ) δppm: 1.28 (22H, S, -
CH ₃ and -CH ₂ -), 3.46 (2H, t, PhCH ₂ O CH ₂
CH ₂ −), 4.50 (2H, s, PhC H ₂ O−), 7.32 (5H,
s, phenyl proton). Example 1 5,10,15,20-tetra [α, α, α, α-o
-(aminophenyl)porphin (hereinafter
Abbreviated as H ₂ TamPP. ) 2.1g in dichloromethane (300ml), added 1ml of pyridine, and then
- 1.09 g of benzyloxy-2,2-dimethyldodecanoic acid chloride was added dropwise and reacted for 3 hours at room temperature, and then 5 ml of pivaloyl chloride (large excess) and 5 ml of pyridine were each added dropwise and reacted for another 2 hours.
A saturated aqueous sodium bicarbonate solution (200 ml) was added, and the separated organic layer was washed twice with a 4% aqueous sodium bicarbonate solution (200 ml) and water (200 ml), and dried over sodium sulfate. The residue obtained by drying under reduced pressure was purified by silica gel column chromatography using benzene/ether = 7/1 (v/v) as a solvent, and further recrystallized from an ether-methanol mixed solvent to give a reddish brown color. Crystal 5, 10,
15,20-[tri(α,α,α-o-pivalamidophenyl)-α-o-(12-benzyloxy-2,2
-dimethyldodecanamido)phenyl]porphine was obtained in a yield of 1.26 g, 33%. Melting point 87.5~89
℃. Magnetic field desorption mass spectrum M ⁺ 1242
(C ₈₀ H ₉₀ N ₈ O ₅ = 1242). Infrared absorption spectrum (KBr) ν: 3440, 3330, 2960, 2940, 2860,
1690, 1580, 1510, 1450, 1300, 1160, 970,
810, 760, 740 cm ^-1 Proton nuclear magnetic resonance spectrum (CDCl ₃ ) δppm: -2.60 (4H, S, NH in the porphine ring), -0.21 (6H, S, -CH ₂ C ( CH ₃ )
₂ CONH−)0.05, 0.09 (27H, eachs, (C H ₃ ) ₃ C
−CONH−), 3.46 (2H, t, J=6.4Hz,
_{PhCH2OCH2CH2-} ) _{, 4.50} (2H,s, _PhCH2O
-) 7.85 (5H, s, benzyl group phenyl proton). Elemental analysis: Calculated value (%) as C ₈₀ H ₉₀ N ₈ O ₅ ; C77.26, H7.30, N9.01, analytical value (%);
C76.89, H7.31, N8.88 Example 2 0.65 g of benzyl ether obtained in Example 1
dichloromethane (15ml) - nitromethane (15ml)
1 ml of anisole and 2 g of anhydrous aluminum chloride were added to the mixed solvent solution, and the mixture was allowed to react at room temperature for 3 hours.
Pour into ice water (200 ml), extract with 50 ml of chloroform, and mix the separated chloroform layer with water (100 ml), 4%
It was washed with an aqueous sodium bicarbonate solution (twice x 100 ml) and dried with Glauber's salt. The residue obtained by drying under reduced pressure was purified by silica gel column chromatography using a mixed solvent of chloroform/ether = 10/1, and then recrystallized from a benzene-n-hexane mixed solvent to obtain 5 red-purple crystals. ,10,15,20
-[Tri(α,α,α-o-pivalamidophenyl)
-α-o-(12-hydroxy-2,2-dimethyldodecanamido)phenyl]porphin is yielded.
Obtained 0.43 g, yield 72%. Melting point 220-221℃. Infrared absorption spectrum (KBr) ν=3650~3200
(broad), 3440, 3330, 2960, 2930, 2860,
1690, 1580, 1510, 1445, 1300, 1160, 970,
805, 750, 740 cm ^-1 . Proton nuclear magnetic resonance spectrum ( _CDCl3 ) δppm: -2.60 (2H, S, NH in _the porphine ring), -0.19 (6H, s, _-CH2CH2C
( CH ₃ ) ₂ −CONH−) 0.10, 0.06 (27H, eachs,
( CH ₃ ) ₃ CCONH−), 3.79 (2H, t, J=6.5Hz,
HOCH ₂ CH ₂ −), 8.82 (8H, s, porphine ring β
− position proton). Elemental analysis: Calculated value (%) as C ₇₃ H ₈₄ N ₈ O ₅ ; C76.01, H7.34, N9.72, analytical value;
C76.17, H7.46, N9.43. Example 3 0.575 g of porphine obtained in Example 2 was dissolved in 0.3 ml of pyridine and 40 ml of tetrahydrofuran,
Ferrous bromide, tetrahydrate 0.6g under nitrogen gas atmosphere
was added and reacted under reflux conditions for 4 hours. The residue obtained by drying under reduced pressure was mixed with chloroform/methanol = 50/1.
Purify by alumina column chromatography using a mixed solvent of
was added and stirred, followed by drying with Glauber's salt. After drying under reduced pressure, the residue was recrystallized from a methanol/dichloromethane mixed solvent to give black-purple crystals of bromo{5,10,
Yield: 0.56 g of 15,20-[tri(α,α,α-o-pivalamidophenyl)-α-o-(12-hydroxy-2,2-dimethyldodecanamido)phenyl]porphynato}iron () , with a yield of 87%. Melting point 235-237℃. Magnetic field desorption mass spectrum (M+
1) ⁺ 1287, [(M+1)-Br] ⁺ 1207 (C ₇₃ H ₈₂ O ₅ ,
(as FeBr=1286, Fe=57, Br=79). Infrared absorption spectrum (KBr) ν: 3440, 2970, 2940,
2860, 1690, 1585, 1510, 1445, 1300, 1260,
1000, 810, 760 cm ^-1 . _{Elemental analysis} : _{C73H82N8O5 ,}
Calculated value as FeBr (%); C68.11, H6.42,
N8.70, analysis value (%); C67.84, H6.46, N8.58. Example 4 To a solution of 0.345 g of the compound obtained in Example 3 and 0.15 g of triethylamine in dichloromethane (20 ml), 0.2 g of 2-chloro-2-oxo-1,3,2-dioxasphorane was added and reacted at room temperature for 12 hours. Afterwards, it was dried under reduced pressure. The residue was dissolved in acetonitrile (20 ml), 10 ml of trimethylamine was added, the tube was sealed and reacted at 60°C for 15 hours, and then the solvent was distilled off under reduced pressure. The resulting residual solid was dissolved in chloroform (100 ml) and washed with water. The chloroform layer was separated, dried over Glauber's salt, and evaporated to dryness. Then, it was dissolved in methanol and purified by column chromatography using Toyo Pearl Gel HW-40s (manufactured by Toyoso Co., Ltd.) using methanol as a solvent. Thus iron()-5,10,15,20-{tri(α,α,α-o
-pivalamidophenyl)-α-o-[12-(2'-trimethylaminoethyl)phosphoryloxy-2,
0.086 g of 2-dimethyldecanamide]phenyl}porphine complex (hereinafter abbreviated as Fe(piv3.dodop)) was obtained in a yield of 23%. Melting point 178-180℃.
Infrared absorption spectrum (KBr) ν: 3600-3200
(broad), 3440, 2960, 2940, 2860, 1690,
1582, 1510, 1445, 1300, 1070, 1000, 810, 760
cm ^-1 . Elemental analysis: Calculated value (%) as C ₇₈ H ₉₄ N ₉ O ₈ P・Fe・3H ₂ O; C65.67, H7.07, N8.83, analytical value (%); C65.34, H7.48 , N8.80 Oxygen adsorption test Iron()-porphine complex synthesized in Example 4
A chloroform-methanol mixed solution containing 1.24 mg of N-laurylimidazole, 1.06 mg (5 times equivalent to iron () complex) and 66 mg of egg yolk phosphatidylcholine (about 50 times equivalent to iron () complex) was placed in a container under reduced pressure. It dried as a thin film on the wall. Add 10 ml of 0.1M phosphate buffer solution and shake to obtain an emulsified solution, and then undergo ultrasonic stirring treatment (30 ml) under nitrogen atmosphere.
(min) to obtain a homogeneous aqueous solution of liposome dispersion containing the iron() complex. After blowing nitrogen gas into this solution for 20 minutes to deoxygenate it,
NADP ⁺ 1mg, glucose-6-phosphate 10mg, ferredoxin 0.02mg, ferredoxin-NADP ⁺
Add 0.1 mg of reductase, 0.05 mg of catalase, and 0.08 mg of glucose-6-phosphate dehydrogenase, and leave to stand at room temperature for 6 hours to reduce iron()-
A porphine complex lipolyme solution was obtained. Measure the visible absorption spectrum and find out the maximum absorption wavelength of this solution.
After confirming that it was a reduced type showing wavelengths of 427, 533, and 560 nm, oxygen gas was then blown in for 30 seconds. The spectrum changes immediately, and the absorption maximum wavelengths are 424 and 538n.
It was confirmed that an oxygen complex exhibiting m was formed. When nitrogen gas was again blown in for 2 minutes to create a nitrogen atmosphere, the spectrum returned to the original reduced type (427, 533, 560 nm), so it was concluded that adsorption and desorption of oxygen could occur reversibly. Further, the half-life of the produced oxygen complex was determined to be about 2 hours from the results of tracking changes over time.

Claims (1)

[Claims] 1 formula (Here, n is an integer of 1 to 20) Iron-5,10,15,20- having one phosphorylcholine group
[α, α, α, α-o-(substituted amido)phenyl]
Porphin complex.