JPH0573386B2 - - Google Patents

Description

[Detailed description of the invention] Technical field The present invention is a novel fibrin-adsorbing protein.
Concerning the nase complex. Background technology The thrombolytic enzyme urokinase is a thrombosis treatment agent.
Although it is widely used clinically, it does not constitute a blood clot.
Adsorbs to fibrin, a protein component of
urokinase, and the injected urokinase
Because it is rapidly metabolized and excreted,
As expected from in vitro activity.
No therapeutic effect has been obtained. For this reason, Europe, America, etc.
In some cases, large doses of urokinase have been administered.
However, in this case, plasminogen is decreased systemically.
causes activation, decomposition of fibrinogen, etc.
Side effects such as bleeding tendency are also significant. Bloodshed again
is α₂- Plasmin inhibitor (α₂−Plasmin
Inhibitor) and α₂- Macroglobulin (α₂−
Large amounts of plasmin inhibitors such as Macroglobulin)
Contains plasmin activation of urokinase
(formation) of thrombolytic effects. Purpose of invention From this point of view, the present inventors have developed a method for absorbing fibrin.
By binding an adhesive protein to urokinase,
It is possible to impart fibrin-binding properties to rokinase.
Preferably, the urokinase is for therapeutic purposes.
It is well adsorbed to blood clots and becomes difficult to metabolize and excrete.
It is expected that the half-life in blood will be extended, and
is less susceptible to the effects of the above-mentioned plasmin inhibitors.
It is expected that the treatment effect will be increased, and the treatment effect will be further improved.
Because plasminogen is locally activated at
It is expected to reduce side effects such as bleeding tendency.
Based on the idea, the fibrin has adsorbent properties.
We have conducted extensive research with the aim of obtaining kinases.
Ta. As a result, the use of specific protein-binding reagents
various fibrin-adsorbing proteins and urokinase
succeeded in combining and thus obtained
The urokinase complex has a structure that meets the above objectives.
Desired urokinase with bulin adsorption ability
activity and is effective as a new thrombolytic agent.
I discovered that. The present invention is based on this new knowledge.
It was completed based on this. Composition of the invention That is, the present invention provides fibrin-adsorbing proteins and
The general formula is [ka] [In the formula, R is a phenylene group or a cycloalkylene group
, A is a lower alkylene group, B is a substituent
lower alkylthio group or phenyl lower alkylthio group
A lower alkylene group that may have an o group,
l, m and n indicate 0 or 1 at the same time. However,
m and n must not be 0 at the same time. ] is bound through a protein-binding reagent represented by
A fibrin-adsorbing protein characterized by
Concerning the kinase complex. The complex of the present invention can be used as a thrombolytic agent.
Compared to Naze alone, it can be used in a smaller amount to target the thrombus.
Acts selectively (locally), metabolic excretion is delayed,
Moreover, it is less affected by plasmin inhibitors and
It can maintain a long-lasting thrombolytic effect, and also
It is very effective and has few side effects such as sexual bleeding tendency.
be. The present invention provides novel thrombolytic agents useful as such thrombolytic agents.
New complexes and new methods for obtaining them easily and effectively
The pharmaceutical industry and its
This will greatly contribute to other fields of study. The above-mentioned general materials used as protein binding reagents in the present invention
In the compound represented by formula (1), represented by R
As the phenylene group, o-phenylene, m-phenylene
Enylene and p-phenylene groups are replaced by cycloalkyl
As the lene group, cyclopropylene, 1,3-cyclo
Chlobutylene, 1,2-cyclopentylene, 1,
3-cyclopentylene, 1,2-cyclohexylene
1,4-cyclohexylene, 1,3-cyclo
Charcoal such as heptylene, 1,5-cyclooctylene group
Examples of cycloalkylene groups having a prime number of 3 to 8 can be given.
Ru. The lower alkylene group represented by A is methyl
Ren, ethylene, trimethylene, tetramethylene
pentamethylene, hexamethylene, 2-methylene
Alkylene having 1 to 6 carbon atoms such as ru-trimethylene group
lower alkyl group as a substituent represented by B.
Contains thio group or phenyl lower alkylthio group
The lower alkylene group that may be used is the same as above.
Alkylene groups having 1 to 6 carbon atoms and methylthio
Methylene, ethylthiomethylene, butylthiomethi
Ren, hexylthiomethylene, ethylthioethylene
, propylthioethylene, 2-methylthiotri
methylene, 2-ethylthioethylmethylene, 2-
Propylthiotetramethylene, 3-ethylthiome
Thirpentamethylene, 3-ethylthiohexamethylene
Ren, benzylthiomethylene, benzylthiomethylene
lumethylene, benzylthioethylene, 2-bendi
ruthiotrimethylene, 2-benzylthioethylene
Tyrene, 2-{2-(2-phenylethylthio)ethyl
Trimethylene, 3-phenylpropylthio
Methylene, 4-phenylbutylthioethylene, 3
-phenylpentylthiomethylene, 6-phenyl
hexylthioethylene, 3-benzylthiopropyl
ethylene, 2-benzylthiomethylpentamethyl
Charcoal such as lene, 3-benzylthiohexamethylene group, etc.
an alkylthio group or an alkyl moiety with a prime number of 1 to 6;
Substituted with phenylalkylthio group having 1 to 6 carbon atoms
An example is an alkylene group having 1 to 6 carbon atoms as a group.
can be shown. The compound of general formula (1) above may be known or new.
For example, these can be found in the literature [T. Kitagawa et al., J.
Biochem. (Tokyo),79, 233 (1976)] etc.
It can be easily synthesized according to the method. Specifically, an example
For example, it can be produced according to the method shown in the following reaction scheme-1.
It can be done. Reaction formula-1 [ka] [ka] [In the formula, R, A, B, l, m and n are the same as above,
R¹represents a lower alkyl group. ] Aminocarboxylic acid represented by the above general formula (2) and
Reaction with maleimide derivative represented by general formula (3)
is carried out in a suitable solvent in the presence of a basic compound.
be exposed. Basic compounds include potassium oxide,
Sodium bicarbonate, sodium hydride, sodium carbonate
Thorium, sodium hydroxide, potassium hydroxide,
Inorganic bases such as potassium bicarbonate can be used.
As a solvent, use an inert solvent that does not adversely affect the reaction.
You can use everything. Typical solvents and
water, methanol, ethanol, propano
alcohol, butanol, ethylene glycol, etc.
Coles; dimethyl ether, tetrahydrofura
(THF), dioxane, monoglyme, zigra
Ethers such as im; acetone, methyl ethyl
Ketones such as ton; N,N-dimethylformamide
(DMF), dimethyl sulfoxide (DMSO),
Hexamethyl phosphate triamide (HMPA) etc.
Examples include aprotic polar solvents. Aminoca
Usage ratio of rubonic acid (2) and maleimide derivative (3)
are not particularly limited, and can be selected from a wide range as appropriate.
However, the latter is usually 1 to 3 times the former by molar amount,
It is preferable to use about 1 to 1.3 times the molar amount.
The reaction temperature is also not particularly limited, but is usually 0 to
The temperature is preferably about 50℃, preferably about 0 to 30℃,
The reaction time is usually about 1 to 3 hours. Hidden
A compound represented by general formula (4) can be obtained. In the above, instead of maleimide derivative (3)
Using maleic anhydride, convert it to aminocarboxylic acid
Maleic acid derivative obtained by reacting with (2)
can also be expressed by the general formula (4) by subjecting it to a dehydration reaction.
The compound can be obtained. Above
The reaction between nocarboxylic acid (2) and maleic anhydride is
About 1 to 3 times the latter by mole, preferably about 1
Using ~1.3 times the molar amount, the above aminocarboxylic acid (2) and
Used in reaction with maleimide derivative (3)
In a solvent similar to the solvent, usually 20 to 150°C, preferably
At a temperature of about 30 to 100℃, for about 1 to 5 hours.
It takes time. The resulting maleic acid derivative
It is especially important to isolate and purify this substance from the reaction mixture.
It can be subsequently subjected to a dehydration reaction.
The reaction is carried out in the presence of a dehydration condensation agent, without solvent or in an appropriate manner.
It is carried out in a suitable solvent. Dehydration condensation agent used
For example, acetic anhydride, propionic anhydride,
Acids such as butyric anhydride, caproic anhydride, benzoic anhydride, etc.
Examples include anhydrides. Also used as a solvent
dimethyl ether, THF, dioxane,
Ethers such as monoglyme and diglyme; Ben
Aromatic hydrocarbons such as zene, toluene, and xylene;
Halogenated carbonization of methylene chloride, chloroform, etc.
Examples include hydrogens. The amount of dehydration condensation agent used is
is not particularly limited, and is usually a maleic acid derivative.
catalytic amount or more, preferably 10 to 100 times the mole
It is best to use approximately the same amount, and use a large excess as it also serves as a solvent.
It can also be used. The above dehydration reaction is usually about 50~
About 1-5 hours at 150℃, preferably about 90-100℃
It is carried out requiring. The thus obtained compound of general formula (4) is
By reacting droxysuccinimide (5)
The desired maleimide derivative represented by the general formula (1)
The body is acquired. The reaction is preferably carried out in a suitable solvent.
Alternatively, it is carried out in the presence of a dehydration condensation agent. dehydration
As a combination agent, for example, dicyclohexyl carbodi
imide (DCC), trichloroacetonitrile, a
Cetyl ethyl ether, cyanate ethyl ester
Examples of the solvent include diethyl
Ether, THF, dimethoxyethane (DME),
Ethers such as diglyme, DMF, pyridine,
Examples include 2-methylpyrrolidone. General formula (4)
N-hydroxysuccinimide for the compound of
The amount of (5) used is not particularly limited, and is usually equimolar to 3
twice the mole, preferably about the same mole to 1.3 times the mole.
The reaction is generally carried out at a temperature of about 0-50°C, preferably 0-30°C.
The process is completed in about 1 to 3 hours at about ℃. Above
When using a dehydration condensation agent, this is usually
Equimole to 3 times the mole of the compound of general formula (4), preferably
Preferably, use equimolar to 1.3 times the molar amount, and react similarly.
All you have to do is respond. In addition, in the above reaction, the compound of general formula (4)
Alternatively, a compound whose carboxy group is activated
You can also use Examples of such compounds include
For example, an alkylhalocarboxylic acid is added to the compound of general formula (4).
Mixed acid anhydride and acid halogen obtained by reacting
Examples include chloride compounds. Method of using mixed acid anhydrides (mixed acid anhydrides
method), usually first alkyl halide is added to the compound of general formula (4).
rocarboxylic acids, such as methyl chloroformate, bromo
Methyl formate, isobutyl chloroformate, etc.
reaction according to the Bauman reaction, and then the obtained
Isolating the mixed acid anhydride from the reaction system
Instead, add N-hydroxysuccinimide (5) to this.
This is done by reacting. The above shot
The Baumann reaction is carried out using the basic reaction commonly used for the reaction.
compounds such as sodium hydroxide, potassium hydroxide
Mu, sodium carbonate, potassium carbonate, sodium bicarbonate
Inorganic materials such as thorium, potassium bicarbonate, and silver carbonate
Base; sodium methylate, sodium ethylate
Alcoholates such as trimethylamine,
ethylamine, pyridine, N,N-dimethylamine
Niline, N-methylmorpholine, 4-dimethyla
minopyridine, 1,5-diazabicyclo[4,
3,0] nonene-5 (DBN), 1,5-diazabi
cyclo[5,4,0]undecene-5 (DBU),
1,4-diazabicyclo[2,2,2]octane
In the presence of an organic base such as (DABCO), −20 to 100
It is carried out at about ℃, preferably 0 to 50℃,
The reaction time is about 5 minutes to 10 hours. The resulting mixture
Synthetic acid anhydride and N-hydroxysuccinimide (5)
The reaction is generally carried out at -20 to 150℃ in an appropriate solvent.
Preferably carried out at 10-50℃ for about 5 minutes to 10 hours.
It can be done. The solvent used is this type of mixed acid-free
Solvents commonly used in aqueous methods, specifically methylene chloride,
Halogenation of chloroform, dichloroethane, etc.
Carbon; aromatics such as benzene, toluene, xylene, etc.
Aromatic hydrocarbons; diethyl ether, THF,
Ethers such as DME; methyl acetate, ethyl acetate
esters such as alcohol, DMF, DMSO, HMPA, etc.
Any aprotic polar solvent, etc. may be mentioned. the above
The compound of general formula (4) in the mixed acid anhydride method,
Kylhalocarboxylic acid and N-hydroxysuccinic acid
The proportion of imide (5) used is usually about equimolar.
However, for the compound of general formula (4), alkyl halo
Carboxylic acid and N-hydroxysuccinimide (5)
It is preferable to use about 1 to 1.5 times the mole amount. Method using acid halides (acid halides)
method), usually a halogenating agent is added to the compound of general formula (4).
is reacted to form carboxylic acid halide, which is
N-hydroxysuccinimide without isolation
This is done by subjecting it to a reaction with (5). one
The reaction between the compound of general formula (4) and the halogenating agent is
It is carried out either in a medium or in the presence of a solvent. melt
As a medium, you can use something that does not have a negative effect on the reaction, e.g.
Aromatic charcoal such as benzene, toluene, xylene, etc.
Hydrogens; chloroform, methylene chloride, tetrachloride
Halogenated hydrocarbons such as carbon; dioxane,
Ethers such as THF and diethyl ether;
DMF, DMSO, etc. can be used. halogenating agent
, the hydroxyl group of the carboxy group is changed to a halogen.
common halogenating agents such as thionichloride
phosphorus oxychloride, phosphorus oxybromide, phosphorus pentachloride
phosphorus pentabromide, etc. can be used. Representation of general formula (4)
The ratio of the compound and halogenating agent to be used is particularly limited.
The reaction is carried out without a solvent, but the reaction is carried out without a solvent.
In such cases, the latter is usually used in large excess over the former.
Furthermore, when the reaction is carried out in a solvent, the former is usually used.
On the other hand, the latter should be added in at least an equimolar amount, preferably in an equimolar amount.
is preferably about 2 to 4 times the mole. The reaction is fast
Although not limited to, usually room temperature to about 100℃, preferably
Or at 50-80℃ for 30 minutes to 6 hours.
be exposed. Carboxylic acid halide obtained above
reaction with N-hydroxysuccinimide (5)
is carried out in the presence of a dehydrohalogenating agent.
As a dehydrohalogenation agent, a basic compound is usually used.
is used. A specific example is sodium hydroxide.
potassium hydroxide, sodium carbonate, carbonic acid
Potassium, sodium bicarbonate, potassium bicarbonate
Inorganic bases such as aluminum, silver carbonate; sodium methyl
alcoholates, such as sodium ethylate,
Triethylamine, pyridine, N,N-dimethyl
Aniline, N-methylmorpholine, 4-dimethyl
aminopyridine, DBN, DBU, DABCO etc.
Examples include organic bases. The reaction can be performed without a solvent or with a solvent.
It is also carried out in the presence of Bad for reactions as a solvent
All inert substances that have no effect are used,
For example, chloroform, methylene chloride, carbon tetrachloride
Halogenated hydrocarbons such as diethyl ether
Ethers such as chlorine, THF, and dioxane;
Aromatic hydrocarbons such as zene, toluene, and xylene
esters such as methyl acetate and ethyl acetate;
Aprotic polarity such as DMF, DMSO, HMPA
Examples include solvents. Carboxylic acid halide and N
-The ratio of use with hydroxysuccinimide (5) is
Although not particularly limited, when the reaction is carried out in a solvent.
In this case, the latter is usually at least equivalent to the former.
The amount should be about the same mole to twice the mole amount.
It is better. Although the reaction is not particularly limited,
Normally at about -30 to 100℃, preferably 0 to 50℃,
It takes about 0.5 to 12 hours. In the above reaction scheme-1, represented by general formula (2)
For example, the aminocarboxylic acid that is
It is manufactured according to the method shown in Eq. Reaction formula-2 [ka] [ka] [In the formula, R, A, B, l, m and n are the same as above.
X is a halogen atom, alkanesulfonyloxy,
Arylsulfonyloxy or aralkylsulfo
Indicates a nyloxy group. ] Halogen element represented by X in the above general formula (6)
As children, chlorine, bromine, iodine atoms, etc.
As the methane sulfonyloxy group, methanesulfonyloxy group is
oxy, ethanesulfonyloxy, isopropanol
sulfonyloxy, propanesulfonyloxy
cy, butanesulfonyloxy, tert-butanesulf
Honyloxy, benzanesulfonyloxy, hexa
Sansulfonyloxy groups, etc., are replaced with arylsulfonyloxy groups, etc.
phenylsulfonyloxy,
4-methylphenylsulfonyloxy, 2-methy
Ruphenylsulfonyloxy, 4-nitrophenyl
sulfonyloxy, 4-methoxyphenyl sulfur
Honyloxy, 3-chlorophenylsulfonylo
xy, α- or β-naphthylsulfonyloxy
Substituted or unsubstituted arylsulfonyl groups such as
xy group, or an aralkylsulfonyloxy group
Specifically, benzylsulfonyloxy, 2
-phenylethylsulfonyloxy, 4-phenyl
Rubbutylsulfonyloxy, 4-methylbenzyl
Sulfonyloxy, 2-methylbenzylsulfony
4-nitrobenzylsulfonyloxy, 4-nitrobenzylsulfonyloxy
cy, 4-methoxybenzylsulfonyloxy, 3
-chlorobenzylsulfonyloxy, α- or
is a β-naphthylmethylsulfonyloxy group, etc.
substituted or unsubstituted aralsulfonyloxy groups, respectively
I can give an example. The aminocarboxylic acid represented by general formula (2) is
According to reaction scheme-2, the public represented by general formula (6) is
A known compound is reacted with phthalimide (7), and then
Obtained by hydrolyzing the resulting compound (8)
be done. The reaction between the compound of general formula (6) above and phthalimide (7)
In this case, the aforementioned carboxylic acid halide and N
-Similar to the reaction with hydroxysuccinimide (5)
Reaction conditions can be employed. Compound represented by general formula (8) obtained as above
Hydrolysis of substances can be carried out without solvent or with water, methanol, or ethanol.
Alcohols such as tanol and isopropanol,
Treated with acid or alkali in a suitable solvent such as acetic acid.
This is done by Acids include hydrochloric acid and sulfuric acid.
mineral acids such as acids, and sodium hydroxide as an alkali.
potassium hydroxide, potassium carbonate, sodium carbonate
You can use um etc. Acid or alkali has the general formula
(8), at least equimolar or more,
It is best to use a large excess amount. Reaction temperature is normal room temperature
The temperature should be between 150°C and preferably between 50 and 100°C.
Generally, the reaction is completed in about 0.5 to 24 hours.
In addition, in the above hydrolysis reaction, the phthalimide group
Conversion to an amino group is preferably carried out in a similar solvent,
Under similar reaction conditions, at least equimolar amounts of hydrogen
It can be carried out in the presence of radin. Also, aminocarbon represented by the above general formula (2)
Among acids and compounds of general formula (6) used as raw materials, certain
The species are based on the reaction formulas -3 to -5 shown below.
It can also be manufactured by a method according to the following. Reaction formula-3 [ka] [ka] [ka] [R in the formula¹and X are the same as above. X¹is a halogen source
Child, R²is a lower alkyl group, an aryl group or an alkyl group
M represents a ralkyl group, and M represents a metal atom. ] As a halogen atom defined in each formula above
In other words, chlorine, bromine, iodine atoms, etc. are combined with lower alkyl groups.
as methyl, ethyl, propyl, isopropyl
1 or more carbon atoms such as ru, butyl, pentyl, hexyl, etc.
The linear or branched alkyl group of 6 is replaced with an aryl
Examples of the group include phenyl, 2-methylphenyl, 4
-methylphenyl, 4-nitrophenyl, 2-methylphenyl
Toxiphenyl, 3-chlorophenyl, α-moshi
or β-naphthyl group, etc., as an aralkyl group.
Benzyl, 2-phenylethyl, 4-phenylbu
Chil, 4-methylbenzyl, 4-nitrobenzyl
groups, etc., and as metal atoms, lithium, sodium
In addition to alkali metal atoms such as lium and potassium,
Examples include gnesium and zinc atoms. Below, for each step shown in the reaction scheme-3 above,
Explain in detail. Known compounds represented by general formula (6)' and general
Metal enolate of acetate ester represented by formula (9)
For example, the reaction with THF, dimethoxymethane,
Ether solvents such as diglyme; benzene, tolu
Aromatic hydrocarbon solvents such as ene and xylene;
Aprotic polar solutions such as DMF, DMSO, HMPA, etc.
In a suitable solvent such as
Using 3 times the molar amount, preferably about 1 to 1.5 times the molar amount, -
Around 78℃~50℃, preferably around -78℃~-30℃
It takes about 1 to 5 hours under the temperature conditions of
Ru. In this way, a compound represented by general formula (10) was obtained.
Ru. The reduction reaction of the resulting compound of general formula (10)
Convert the ester moiety of compound (10) to alcohol
However, it is carried out under conditions that do not reduce the nitrile group.
administered. Specifically, for example, hydrolyzing compound (10)
to give the corresponding carboxylic acid, and then add chloro to this.
Methyl formate, ethyl bromoformate, isochloroformate
Basic compound of alkyl halocarboxylic acid such as butyl
to form a mixed acid anhydride, and then
This method of reducing mixed acid anhydrides can be adopted in
Ru. The above hydrolysis reaction is an example of a basic catalyst.
For example, sodium hydroxide, potassium hydroxide, hydroxide
Even if calcium etc. are used, acidic catalysts such as
Acid, trifluoroacetic acid, trichloroacetic acid, water chloride
hydrogen bromide, p-trienesulfonic acid, camphor
It can also be carried out using earthsulfonic acid or the like. basic
When using a catalyst, the reaction can be carried out using water, methanol, or ethanol.
Alcohols such as alcohol, propanol, butanol
solvents, dioxane, THF, diglyme, etc.
Ether solvents, DMF, DMSO, HMPA, etc.
It is carried out in a protic polar solvent. acidic catalyst
When used, the reaction is performed using methylene chloride, chloroform,
halogenated hydrocarbons such as aluminum, trichloroethane, etc.
Preferably it proceeds in a solvent. Use any catalyst
In this case, the catalyst is usually used in excess amount relative to compound (10).
The reaction takes place at a temperature of about 20 to 100℃ for about 1 to 48 hours.
It is completed with. The carboxylic acid and alkali thus obtained
Mixed acid anhydrides by reaction with kylhalocarboxylic acids
is produced by the usual Schotten-Baumann reaction.
The conditions are as described above.
In addition, the mixed acid anhydride obtained by the above method is usually
Reduction reaction that continues without isolation within the reaction system
served. The reduction reaction is carried out using water, methanol,
Alcohol solvents such as tanol and propanol,
Ether-based solutions such as ether, THF, and diglyme
Sodium borohydride in a suitable solvent such as
In the presence of a reducing agent such as lithium borohydride, approximately
At a temperature of 0 to 50°C, preferably about 0 to 20°C,
It usually takes about 1 to 12 hours. The reducing agent is
About 1 to 10 times the mole of mixed acid anhydride, preferably
is used in an amount of about 1 to 3 times the mole. Thus
An alcohol represented by general formula (11) is obtained. Alcohol represented by general formula (11) and general formula (12)
The reaction with the compound represented by is the reaction of a basic compound with
The reaction is carried out in the presence of a suitable solvent. basic compound
As substances, sodium hydroxide, potassium hydroxide
Mu, sodium carbonate, potassium carbonate, sodium bicarbonate
Inorganic materials such as thorium, potassium bicarbonate, and silver carbonate
Base; sodium methylate, sodium ethylate
Alcoholates such as triethylamine,
Lysine, N,N-dimethylaniline, N-methyl
Morpholine, 4-dimethylaminopyridine,
Organic bases such as DBN, DBU, DABCO etc.
It can be done. Solvents include THF, dioxane, DME,
Ether solvents such as diglyme; benzene, tolu
Aromatic hydrocarbons such as ene and xylene; methylene chloride
Halogens such as chloroform, ethylene tetrachloride, etc.
Examples include hydrogenated hydrocarbons. Against alcohol (11)
The amount of compound (12) used is usually about 1 to 3 times the molar amount,
It is preferably about 1 to 1.5 times the molar amount. The reaction is
Usually at a temperature of about 0 to 50℃, preferably about 0 to 20℃
It takes about 1 to 12 hours to complete. Thus the general
A compound represented by formula (6)'' is obtained. In addition, the compound represented by general formula (6)'' is an alcohol
(11) with a suitable halogenating agent, such as thionyl chloride.
phosphorus oxychloride, phosphorus oxybromide, pentoxide
Also by reacting phosphorus, phosphorus pentabromide, etc.
be obtained. The reaction can be carried out using, for example, dioxane,
Ethers such as THF, chloroform, methane chloride
Suitable solvents such as halogenated hydrocarbons such as
In the middle, use less halogenating agent for alcohol (11).
twice the molar amount, usually in excess, around room temperature to approx.
At a temperature of 100℃, preferably around room temperature to 70℃,
This process takes about 1 to 24 hours. Reaction formula-4 [ka] [ka] [ka] [X in the formula¹,R¹,R², M and X are the same as above. ] In reaction scheme-4, the compound of general formula (13) and
The reaction with the compound of general formula (9) is carried out by the reaction scheme -
Compound of general formula (6) and compound of general formula (9) in 3
can be carried out under similar conditions to the reaction with, but the general formula
Amount of compound of general formula (9) used relative to compound (13)
is about 2 to 5 times the molar amount, preferably about 2 to 3 times the molar amount.
It is preferable to consider it as the amount of Reduction reaction of the compound of general formula (14) obtained as above
reaction and the compound of general formula (15) obtained thereby.
The reaction with the compound of general formula (12) or a halogenating agent also
Each step is carried out in the same manner as shown in the above reaction scheme-3.
It can be implemented by Furthermore, from the compound of general formula (16)
The compound of formula (2)' can be produced by following the reaction scheme-2 above.
Compound of general formula (2) from compound of general formula (6) in
It is carried out by the same method as in the production of. In addition, in the above reaction schemes -3 and -4, the output
By selecting the power raw materials accordingly, or each line such as
By repeating the reaction shown in the equation, a.
Obtaining the desired compound with an extended lekylene chain
I can do it. Reaction formula-5 [ka] [ka] [ka] [R in the formula¹is the same as above. ] The oxidation reaction of the compound of general formula (17) can be carried out using an appropriate oxidizing agent.
It is usually carried out in a solvent using as an oxidizing agent
For example, DMSO-acetic anhydride, DMSO-trifluor
Oroacetic acid, DMSO-oxalyl chloride,
DMSO-N,N'-dicyclohexylcarbodiy
mido, pyridium chlorochromate, pyridium
Muzichromate etc. can be used. As a solvent,
Aromatic hydrocarbons such as benzene, toluene, xylene, etc.
Base solvents, methylene chloride, chloroform, etc.
Genated hydrocarbon solvents and the like can be used.
The oxidizing agent is usually about 1 to 5 % of the compound of general formula (17).
twice the molar amount, preferably about 1 to 3 times the molar amount used.
Good. The reaction temperature is usually about 0 to 50°C, preferably
The temperature is about 0 to 20℃, and the reaction takes about 5 to 12 hours according to the general formula.
It ends with. Thus, the compound represented by general formula (18)
things are acquired. The resulting compound of general formula (18) and the compound of general formula (19)
The reaction with the substance is carried out in a suitable solvent, and the reaction with
Examples include ether, THF, ziglyme, and dioxin.
Ethers such as San, benzene, toluene, xylene
Aromatic hydrocarbons such as ethylene, methylene chloride, chloro
Halogenated hydrocarbons such as roform, DMF,
Aprotic polar solvents such as DMSO, HMPA, etc.
I can give an example. General formula (19) for the compound of general formula (18)
The amount of the compound used is usually about 1 to 5 times the molar amount, preferably about 1 to 5 times the molar amount.
Preferably, the amount is about 1 to 3 times the molar amount, and the reaction is about 20 to 3 times the molar amount.
Under temperature conditions of 100℃, preferably about 20-50℃,
It is completed in about 5 to 12 hours, and as a result, the formula expressed by the general formula (20) is obtained.
A compound is obtained. The reduction reaction of the compound of general formula (20) is generally carried out using hydrogen.
In the atmosphere, methanol, ethanol, propanol
Contact in an alcoholic solvent such as alcohol, butanol, etc.
Reduction catalyst e.g. 5% palladium-carbon, 10% palladium
This is carried out using dium-carbon or the like. The catalyst is
Usually 50 mg to 1 g per 1 g of the compound of general formula (20), preferably
Approximately 50 to 200 mg of alcohol is used, and the reaction is carried out at approximately 0 to 50°C.
Complete in about 12 to 72 hours, preferably at 0 to 30℃
do. Addition of the compound of general formula (21) obtained above
The water splitting reaction is a general reaction shown in the reaction scheme-3 above.
It can be carried out under the same conditions as the hydrolysis reaction of formula (10).
can be expressed by the desired general formula (2)″.
can be obtained. In each of the above reaction schemes, each target compound
The substance can be easily isolated and purified by conventional separation means.
Ru. Such separation methods include precipitation, extraction, and recycling.
Crystal method, column chromatography, preparation
Examples include thin layer chromatography. The fibrin-adsorbing protein used in the present invention is
A protein that has the property of binding to ebulin.
A typical example is plasmin heavy chain (plasmin
HC), the active center site is inhibited with an irreversible inhibitor.
Protein component enzymes are added to plasmin and plasminogen.
Kringle obtained by reacting elements
1 to 3, the same kringle 4, protein to fibronectin
Fibronectic tissue obtained by the action of white degrading enzyme
The N-terminal domain of the protein, the domain adjacent to the C-terminal domain of the same
For example, main etc. Currently, one of the representative fibrin-adsorbing proteins mentioned above is
About plasmin, which gives plasmin heavy chain,
Detailed research is being conducted on the subject [B.Wiman
and P. Wall′en, Thrombosis Research,1,
213 (1977)]. In other words, human plasmin adsorbs fibrin.
The heavy chain (molecular weight approximately 60,000) and active center involved in
Containing light chains (molecular weight approximately 30,000) are linked by two S-S
It is a protein with a molecular weight of about 90,000, and the heavy chain has 5
Two highly homologous unit structures (called “kringles”)
Kringle 1~
3 has a strong fibrin adsorption site, and kringle
4 is also known to have adsorption properties [L.
Sottrup-Jensen et al., Progress in Chemical
Fibrinolysis and Thrombolysis,3, 191
(1978)]. In addition, fibronectin has a molecular weight of approximately
400,000 sticky polymeric glycoproteins that form on the cell surface.
present in the surface, extracellular matrix and blood; cell-matrix
between cells, cell-cell adhesion, or normal cell shape.
state maintenance, cell migration, wound healing, phagocytosis, blood
It is thought that it functions in coagulation, etc.
The bioactivity of fibronectin collagen,
Parin, glycosaminoglycan, fibrin, fine
This is due to specific binding to cell membrane components, etc. Hui
The bronectin molecule consists of at least 6-7 proteins.
It is composed of a domesic structure that is resistant to tearase.
The fibronectin exhibits such diverse ligands.
The binding activity for each domain is determined by the binding activity of each domain to each ligand.
This is said to be due to the fact that it shows specific binding activity to
[R.O.Hynes and K.M.Yamada, J.Clll
Biol.95, 369 (1982)]. Analysis of domain structure?
Since fibronectin in human plasma has an N-terminal
and fibrin binding with a molecular weight of approximately 30,000 near the C-terminus.
It is known that there are two types of domains with activity.
In the present invention, these fibrin bonds are
Filter active fibronectin fragments
Used as ebulin-adsorbing protein. Fibrin-adsorbing protein is used in the production of the complex of the present invention.
The plasmin heavy chain used for this purpose is described, for example, in the literature [Eur.
J.Biochem.57, 441 (1975)] etc.
which partially reduces plasmin
It has a mercapto group obtained by The plasmin
The heavy chain, for example, converts plasmin into 2-mercaptoeta
The heavy chain and light chain are partially reduced in the presence of alcohol, etc.
Cut the S-S bond between the chains and convert the reaction solution into heavy chains.
Lysine-sepharose, etc., which has an affinity for
It can be separated and obtained by deposition and elution.
Wear. Plasma in which the active center site is inhibited with an irreversible inhibitor
Sumin, for example, uses the literature [Methods in Ensymol.
45, 256 (1976)]. That is, it can be obtained by reacting urokinase-activated plasmin with tosyl-arginine-chloromethyl ketone and purifying the reaction solution by gel filtration. Kringles 1 to 3 and 4, which are obtained by the action of protein component enzymes on plasminogen, are described, for example, in the literature [Progress in Chemical Fibrinolysis and
Thrombolysis, 3 , 191 (1978)] or the literature [Thromb.Res, 10 , 213 (1977)]. That is, plasminogen can be isolated and obtained by limited decomposition with a proteolytic enzyme such as elastase or alphaarchymotryplysin, adsorption and elution with lysine-sepharose, etc., and then gel filtration. Obtained kringle 1~
The molecular weights of Kringle 3 and Kringle 4 are about 30,000 to 36,000 and about 10,000. Plasminogen is one of the precursors of proteolytic enzymes present in blood and can be easily purified using lysine-sepharose or the like [Methods in Enzymol., 34 , 424 (1974)]. In addition, the N-terminal domain and the domain adjacent to the C-terminal domain of fibronectin obtained by treating fibronectin with a protease can be obtained from human plasma, for example, in the literature [J. Biol. Chem., 257 , (9), 5263].
(1982)] using gelatin-cepharose, heparin-cepharose, etc., is subjected to limited digestion with a protease such as trypsin or plasmin, and the target domain is isolated from the target domain as described in the literature [J.Biol.Chem. , 258 , (5), 3332 (1983)]
It can be isolated and obtained by purification according to . The molecular weights of the obtained N-terminal domain and the domain adjacent to the C-terminal domain are approximately 31,000 and 34,000, respectively.
It is. Further, in the present invention, the urokinase may be various known ones without particular limitation, such as low molecular weight urokinase and high molecular weight urokinase.
Weight−Urokinase, LMW−UK, average molecular weight
32000; High Molecular Weight-Urokinase,
HMW-UK, average molecular weight 54000, Biochemistry,
5, 2160 (1966)] and the heavy chain of the above-mentioned urokinase. Here HMF−UK
For example, the heavy chain of HMW-UK is partially reduced in the presence of thiols such as 2-mercaptoethanol, dithiothreitol, dithioerythritol, etc. to form an S-S bond between the heavy chain and the light chain. It can be separated and obtained by reductively cleaving the reaction mixture, and then adsorbing and eluting the reaction solution on a resin that has an affinity ligand for the active center of the HWF-UK heavy chain [H. Sumi and KC Robbins,
J. Biol. Chem., 258 (13), 8014 (1983)]. Among the above fibrin-adsorbing proteins, plasmin has a free mercapto group (SH) in its molecule.
For compounds other than HC, a mercapto group or a mercapto group protected with a mercapto protecting group is introduced into the molecule, and then a binding reaction is performed with urokinase. The reaction for introducing the mercapto group or protected mercapto group can be carried out using various known reagents, such as acetylmercaptosuccinic anhydride, 2-iminothiolane (2-
iminothiolane), N-succinimidyl-3-(2
-pyridyldithio)propionate, etc. These reagents are usually used in an amount of about 1 to 50 times, preferably about 3 to 20 times, per mole of fibrin-adsorbable protein, and about 1 to 5 moles, preferably about 1 to 5 times, per mole of fibrin-adsorbable protein. 1-2
A molar amount is introduced. In the binding reaction between the fibrin-adsorbing protein and urokinase, preferably, the urokinase and the protein-binding reagent of general formula (1) are reacted in advance, and then the fibrin-adsorbing protein or a mercapto group or a protected mercapto group is added to the reaction. This is done by reacting with the introduced fibrin-adsorbing protein. In the above, the ratio of the protein binding reagent of general formula (1) to urokinase is usually about 1 to 50 times the molar amount, preferably about 3 to 20 times the molar amount, and about 1 to 10 times the molar amount, preferably about 1 to 10 times the molar amount per 1 mole of urokinase. About 1-2 moles of protein binding reagent are bound. In addition, the binding reaction between urokinase into which the above protein binding reagent has been introduced and fibrin-adsorbing protein (or one into which a mercapto group or a protected mercapto group has been introduced) can be carried out, for example, in an aqueous solution, physiological saline, or PH4.
The reaction is carried out in a conventional buffer solution having a pH of 10 to 10, preferably a buffer solution having a pH of 6 to 8, at a temperature of 0 to 40 DEG C., preferably around room temperature, preferably in a nitrogen stream. The reaction is usually completed within several minutes to about 24 hours. As the buffer solution used above, any of various known buffer solutions that do not contain ammonia or amino groups can be used. In the above binding reaction, the ratio of both raw material proteins to be used is determined as appropriate, but is usually about 1:5 to 5:1 (molar ratio), preferably about 1:2 to
The molar ratio is preferably 2:1. The complex produced in this way mainly contains 1 urokinase per 1 mole of fibrin adsorbent protein.
~5 mol, preferably approximately 1 mol, is bound, and after the binding reaction is completed, it can be easily isolated and purified by conventional methods such as dialysis, gel filtration, fractional precipitation, affinity chromatography, etc. can. Moreover, it can be preserved by the usual freeze-drying method. The complex obtained as described above has fibrin adsorption ability and urokinase activity, and is effective in treating thrombosis. When the complex of the present invention is used as a thrombolytic agent, it is prepared in the form of a crude drug together with a conventional pharmaceutical carrier. The dosage unit form of the thrombolytic agent can be selected from various forms depending on the therapeutic purpose, but it is usually used as an injection. The injection is sterilized by conventional methods and preferably isotonic with blood. Examples of the diluent used in forming the injection into the form of an injection include various commonly used diluents in this field, such as water and physiological saline. In this case, the thrombolytic agent may contain a sufficient amount of salt, glucose, or glycerin to prepare an isotonic solution, and may also contain conventional solubilizing agents, buffers, soothing agents, preservatives, etc. In addition, coloring agents, fragrances, flavoring agents, sweeteners, etc., and other pharmaceuticals may be added to the thrombolytic agent as necessary. The amount of the complex of the present invention to be contained in the thrombolytic agent is not particularly limited and is appropriately selected from a wide range, but is usually about 0.01 to 30% by weight in the total pharmaceutical composition.
It is preferable to set the amount to be contained. The above-mentioned thrombolytic agents are administered by various methods depending on the form, but are usually administered intravenously as an injection alone or mixed with a replacement fluid such as glucose or amino acid infusion. The dosage is appropriately selected depending on the purpose of use, symptoms, etc., but it is usually about 1,000 to 500,000 units per day as the active ingredient, fibrin-adsorbing protein-urokinase complex.
Kg, which may be administered in 2 to 4 doses. Examples Hereinafter, examples of the production and preparation of fibrin-adsorbing protein, urokinase, and protein binding reagent used in the production of the complex of the present invention will be listed as reference examples, and then examples of the production of the complex of the present invention will be listed as examples. An example of a pharmacological test of the obtained complex is given below. The methods for measuring various activities and testing for other physical properties in each example are as follows. Activity measurement of Γ plasminogen A plasminogen sample was diluted with 50% glycerol, 2mM hydrochloric acid and 5g/polyethylene glycol (PEG6000, manufactured by Wako Pure Chemical Industries, Ltd.) solution, and 100μ of the diluted solution was added to 0.05M Tris-HCl. 0.1M-NaCl buffer (PH7.4) 600μ
and 600 units of streptokinase (1.5
Add 100 μg of Validase (manufactured by Nippon Redary Co., Ltd.)/ml of the same buffer solution, stir, and store at 37℃ for 10 minutes.
incubate for 1 minute, then substrate S-2251
(HD-Val-Leu-Lys-p-nitroaniline, manufactured by Daiichi Chemical Co., Ltd.) 200μ (0.7μmol) is added and incubated at 37°C for 2 minutes. 50%
Add 100μ of acetic acid aqueous solution to stop the reaction.
The absorbance is measured at 405 nm, and the activity of the sample is calculated from the same absorbance of standard plasminogen. Γ Urokinase activity measurement Synthetic substrate method; Urokinase sample
Dilute to an appropriate concentration with an aqueous solution containing 0.15M NaCl and 5 g/polyethylene glycol (PEG 6000), and add 0.05M Tris-HCl buffer (PH=
8.4) Add 800μ and heat at 37°C for 1 minute, then add substrate S-2444 (PyroGlu-Gly-Arg-p
-Nitroanilide, manufactured by Daiichi Kagaku Yakuhin Co., Ltd.)
Add 100 μM of the above buffer solution at 0.3 μM, and incubate at 37° C. for 2 minutes. Stop the reaction by adding 100μ of 50% acetic acid aqueous solution.
Measure the absorbance at 405 nm. The activity of the sample is calculated from the absorbance obtained by performing the same procedure using standard urokinase. Fibrin plate method; Method A: Add 0.3% bovine serum fibrinogen (manufactured by Povite) to 6 ml of 0.05M veronal buffer (PH = 8.0) containing 0.1M-NaCl and add a final 0.02M
of CaCl ₂ and a final concentration of 3 NIH units/ml bovine thrombin (manufactured by Mochida Pharmaceutical Co., Ltd.), stirred, and spread on a shear plate (inner diameter 8.5 cm) to prepare a fibrin plate. 10μ of the urokinase sample dissolved in the above buffer of 0.1% rabbit serum albumin (manufactured by Sigma) is spotted on the above fibrin plate, and after incubation at 37°C for 16 hours, the diameter of the lysis circle is measured. Method B: Dissolve 2 g of fibrinogen (Fraction I; Sigma) in 200 ml of 0.1M phosphate buffer (PH=7.2). One vial (1000 units) of bovine thrombin (manufactured by Mochida Pharmaceutical Co., Ltd.) is dissolved in 200 ml of the same buffer, and 21 ml of this is added to 21 ml of the above fibrinogen solution and stirred to prepare a fibrin plate. Thereafter, the fibrinolytic activity of the sample was determined in the same manner as method A above, except that bovine serum albumin (BSA) was used instead of rabbit serum albumin.
Measured as the diameter of the melting circle. Measurement of Γ protein amount Lowry method [OHLowry et al, J.Biol.
Chem., 193 , 265 (1951)]. Quantification of ΓSH group After degassing 100μ of the sample, add 0.2M Tris-HCl buffer (PH = 8.2) 1 which was replaced with degassed _N2 .
ml, then add 100μ of 0.01M-5,5'-dithiobis(2-nitrobenzoic acid) dissolved in degassed methanol, stir, and leave at room temperature for about 30 minutes, then measure the absorbance at 412 nm. do.
The SH group amount of the sample is calculated using 2-mercaptoethanol as a standard. Quantification of ΓS-acetyl mercapto group After degassing a 100μ sample, degassed 0.5M-
After deacetylation, 10μ of a hydroxyamine solution (adjusted to pH 7.3 with 5N-NaOH) was added, stirred, and incubated at 37°C for 20 minutes, and the generated SH groups were measured according to the method for quantifying SH groups described above. Quantitative determination of Γm-maleimidobenzoyl group (MB group) After degassing 100μ of a sample, 25μ of an aqueous solution of 2-mercaptoethanol (35 nanomoles) is added and incubated at 37°C for 20 minutes. Above above
The amount of residual 2-mercaptoethanol is measured in the same manner as the determination of SH groups, and the amount of MB groups in the sample is converted. ΓSDS polyacrylamide gel (SDS-PAGE)
Electrophoresis Samples were transferred to 0.02M Tris acid buffer (PH=8.0) containing 40% glycerol and 2% SDS.
After heating at 100°C for 2 minutes, the method of UK Laemli et al. was performed using a 12.5% separation gel and a 4% concentration gel [Nature
(London), 227 , 680 (1970)] and gradient gel PAA 4/30 manufactured by Pharmacia. After electrophoresis, stain with mercury bright blue (CBB), and calculate the molecular weight using a standard protein (Molecular Weight Kit, Pharmacia). Reference Example 1 Production of protein binding reagent Production of (2S)-3-benzylthio-2-maleimidopropionic acid succinimide ester (Reagent 1) Method of O. Keller and J. Rudinger [Helv.Chim.Acta, 58 , 531 (1975)], 8.44 g of S-benzyl-L-cysteine (40 m
200 ml of saturated sodium bicarbonate solution (mol)
-6.2 g (400 mmol) of N-methoxycarbonylmaleinimide in 100 ml of THF mixture at 0°C
added. After stirring for 10 minutes, add 300ml of water and 150 THF.
diluted with ml. The reaction mixture was incubated at room temperature for 30 min.
The mixture was subsequently stirred at 40°C for 30 minutes. After neutralizing with concentrated sulfuric acid, the solution was concentrated under reduced pressure until the volume of the solution was about 200 ml. After extraction with ethyl acetate, it was dried over anhydrous sodium sulfate (Na ₂ SO ₄ ) and concentrated to obtain a white solid. This was subjected to silica gel column chromatography (from 90:10:5 chloroform-ethyl acetate-acetic acid to 40:60:5 chloroform-ethyl acetate-acetic acid) to give (2S)-3-benzylthio-2-maleimidopropionic acid 2.29 g
I got it. mp86-87℃ Next, 2.29g (7.87mmol) of the (2S)-3-benzylthio-2-maleimidopropionic acid
995mg of N-hydroxysuccinimide
(8.67mmol) and dimethoxyethane (DME)
40 ml were mixed, and 1.79 g (8.67 mmol) of dicyclohexylcarbodiimide (DCC) was added to the mixture at 0° C. under an argon atmosphere. The reaction mixture was stirred at 0° C. for 1 hour and at room temperature for 3 hours.
After cooling to 0°C, add a few drops of acetic acid and add 1
Stir for hours. After removing insoluble matter, the liquid was concentrated under reduced pressure, and then subjected to silica gel column chromatography (95:5 methylene chloride-ethyl acetate).
80:20 methylene chloride-ethyl acetate) was carried out to obtain 2.75 g (yield 90%) of the title compound (Reagent 1). mp; 133-134°C (methylene chloride-ether) IR (KBr); 1832, 1800, 1755, 1724 cm ^{-1 1} H-NMR (CDCl ₃ , 90MHs); δppm = 2.70 (s, 4H), 3.14 (m, 2H), 3.60 (s, 2H), 4.97 (m, 1H), 6.63 (s, 2H), 7.17 (s, 5H) MS (m/s) = 388 (M ⁺ ) Analysis value (%); C55. 90 H4.03 N7.39 Calculated value (%) (as C ₁₈ H ₁₆ O ₆ N ₂ S); C55.66 H4.15 N7.21 (±)-4-ethylthio-2-maleimidobutyric acid succinimide ester ( Production of Reagent 2) The title compound was obtained in the same manner as above by converting (±)-ethionine to (±)-4-ethylthio-2-maleimidobutyric acid. mp; 57-59°C (methylene chloride-ether) IR (KBr); 1838, 1800, 1752, 1725 cm ^{-1 1} H-NMR (CDCl ₃ , 90MHs) δppm = 1.20 (t, J = 6.6Hz, 3H), 2.26-2.66 (m, 6H), 2.74 (s, 4H), 5.21 (m, 1H), 6.68 (s, 2H) MS (m/z) = 340 (M ⁺ ) Analysis value; z = 340.0736 Calculation value ( (as C ₁₄ H ₁₆ O ₆ N ₂ ); M=340.07290 Production of (4-maleinimidophenyl)acetic acid succinimide (Reagent 3) Kava et al. (MPCava, AA Deana, K. Mutt
and MJMitchell) method [Org.Synth.,
Coll. Vol. 5, 944 (1973)], add 40 ml of a DMF solution of 10 g (66.2 mmol) of 4-aminophenyl acetic acid to 15 ml of a DMF solution of 7.14 g (72.8 mmol) of maleic anhydride at room temperature under an argon atmosphere. added. The reaction mixture was stirred at room temperature for 3 hours and then diluted with 250 ml of ether-benzene (1:1).
The precipitate was separated. The resulting precipitate was mixed with 2.87 g (35 mmol) of sodium acetate and 70 ml of acetic anhydride, and the mixture was stirred at 100°C for 1 hour under an argon atmosphere. After concentration under reduced pressure, the residue was dissolved in ethyl acetate.
Diluted with 300ml. The ethyl acetate layer was washed with saturated brine, dried over Na ₂ SO ₄ , concentrated, and subjected to silica gel column chromatography (90:10:5
Chloroform-ethyl acetate-acetic acid 50:50:
5-chloroform-ethyl acetate-acetic acid) to obtain 8.72 g (yield 57%) of (4-maleinimidophenyl)acetic acid as a white solid. 2.31 g (10 mmol) of (4maleinimidophenyl)acetic acid obtained above was mixed with 1.15 g (10 mmol) of N-hydroxysuccinimide and 30 ml of DME, and the mixture was placed at 0°C under an argon atmosphere.
After adding 2.27 g (11 mmol) of DCC and stirring the reaction mixture at 0°C for 4 hours, insoluble matter was removed.
After concentrating the liquid, silica gel column chromatography (80:20 chloroform-ethyl acetate)
2.85 g (yield: 87%) of the title compound (Reagent 3) was obtained. mp: 153-154°C (methylene chloride-ether) Production of (2-maleinimidophenyl)acetic acid succinimide ester (Reagent 4) The title compound (Reagent 4) was obtained in the same manner as above. mp; 158-159°C (methylene chloride-ether) (3-maleinimidophenyl) Production of acetic acid succinimide ester (Reagent 5) The title compound (Reagent 5) was obtained in the same manner as above. mp; 150-151℃ (methylene chloride-ether) Production of 4-maleimidoethylbenzoic acid succinimide ester (Reagent 6) Potassium phthalimide 37g (0.2mol)
α- at room temperature under argon in a 200ml suspension in DMF.
Bromo-p-tolnitrile 19.6g (0.1mol)
50ml of DMF solution was added. The reaction mixture was stirred at 90°C for 3 hours and then cooled to room temperature. The mixture was poured into 400 ml of ice-water and extracted with benzene-ether (1:1). The organic layer was washed with saturated brine, dried (Na ₂ SO ₄ ), and concentrated.
4-phthalimidomethylbenzonitrile 24.9g
(yield 95%). Next, 26.0 g (99 mmol) of 4-phthalimidomethylbenzonitrile obtained in the same manner as above.
was mixed with 6.32 ml (0.129 mol) of hydrazine-hydrate and 300 ml of methanol and the mixture was refluxed for 1 hour. After cooling to room temperature, it was concentrated. residue
1 moldm ^-3 was dissolved in 300 ml of NaOH and extracted with ether. The ether layer was washed with saturated brine, dried (MgSO ₄ ), and concentrated to obtain 11.8 g (yield 90%) of 4-aminomethylbenzonitrile. Furthermore, 11.2 g (86.2 mmol) of the obtained 4-aminomethylbenzonitrile was mixed with 86.2 ml of 3 moldm ^-3 KOH aqueous solution and 100 ml of THF, and the mixture was refluxed under argon for 24 hours. After cooling to room temperature,
It was concentrated to about 100ml. The resulting solution was neutralized with concentrated hydrochloric acid. The precipitated white crystals were separated. 11.2 g of 4-aminomethylbenzoic acid (yield
86%). 3.02g of 4-aminomethylbenzoic acid obtained
(20 mmol) was mixed with 100 ml of saturated aqueous NaHCO ₃ solution, and 3.1 g (20 mmol) of methoxycarbonylmaleimide was added into the mixture at 0°C. at 0℃
After stirring for 10 minutes, 200 ml of water was added. After stirring at 30°C for 1 hour, the pH was adjusted to 2 with concentrated sulfuric acid. After extraction with ethyl acetate, the organic layer was washed with saturated brine. Drying (Na ₂ SO ₄ ) and concentration gave 3.18 g (87% yield) of crude 4-maleimidomethylbenzoic acid. 1.61 g (14.0 mmol) of N-hydroxysuccinimide and 50 ml of DME were added to 2.49 g (10.8 mmol) of the obtained crude 4-maleimidomethylbenzoic acid.
was added to the mixture, and 2.88 g of DCC (14.0
mmol) was added. The reaction mixture was stirred at 0° C. for 1 hour and at room temperature for 3 hours. After cooling to 0°C, a few drops of acetic acid were added. After stirring at 0°C for 1 hour, insoluble matter was removed. After concentrating the liquid, the residue was subjected to silica gel column chromatography (70:30 chloroform-ethyl acetate) to obtain the title compound (reagent 6).
3.19g (yield 90%) of mp; 191-193°C (methylene chloride-ether) Production of 3-maleimidoethylbenzoic acid succinimide ester (Reagent 7) The title compound (Reagent 7) was obtained in the same manner as above. mp; 132-134℃ (methylene chloride-ether) Production of 4-(3-maleimidopropyl)benzoic acid succinimide ester (Reagent 8) Diisopropylamine 10.2ml (73mmol)
A hexane solution of butyllithium (2.4 moldm ^-3
Solution) 27.5 ml (66 mmol) was added and stirred for 15 minutes. After cooling to -78°C, 20 ml of a THF solution containing 8.07 ml (60 mmol) of t-butyl acetate was added thereto. After stirring for 30 minutes, 30 ml of a THF solution containing 9.8 g (50 mmol) of α-bromo-p-tolnitrile was added. After stirring at −78 °C for 1 h, saturated NH ₄ Cl water was added.
Added 20ml. After raising the temperature to room temperature, it was diluted with 100 ml of water. After extraction with ether, drying (MgSO ₄ ),
Concentration gave an oil. This oil was subjected to silica gel column chromatography (90:10 hexane-ethyl acetate to 60:40 hexane-ethyl acetate) to obtain 9.8 g of 4-(2-t-butoxycarbonylethyl)benzonitrile (yield 85
%) was obtained as an oil. 4-(2-t-butoxycarbonylethyl)
2.31g of benzonitrile and 3ml of trifluoroacetic acid
and 30 ml of methylene chloride, and the mixture was stirred at room temperature for 13 hours. After concentration, the resulting residue was azeotroped with toluene. 1.75 g of crude 4-(2-t-butoxycarbonylethyl)benzonitrile
(yield 100%). This compound was used in the next reaction without purification. Crude 4-(2-t-butoxycarbonylethyl)
3.27g (18.7mmol) of benzonitrile, 2.86ml (20.6mmol) of triethylamine and 40ml of THF
Mix with ethyl chloride formate at 0 °C in the mixture 1.96
ml (20.5 mmol) was added. After stirring the reaction mixture at room temperature for 3 hours, insoluble materials were removed. The liquid was concentrated to give a yellow oil. This oil was diluted with 100 ml of benzene. saturates the benzene layer
Washed with aqueous NaHCO ₃ , dried (Na ₂ SO ₄ ), and concentrated to give a yellow oil. Add this oil to THF−
Dissolved in 40 ml of H ₂ O (8:2). 0℃ in this
Then 2.13 g (56.1 mmol) of NaBH ₄ was added. After stirring the reaction mixture at 0°C for 2 hours, 3moldm ^-3 HCl
Pour into 100ml of water. After extraction with ethyl acetate, it was dried (Na ₂ SO ₄ ) and concentrated to obtain 2.71 g (yield 90%) of 4-(3-hydroxypropyl)benzonitrile as an oil. 16.1 g (0.1 mol) of 4-(3-hydroxypropyl)benzonitrile was added to triethylamine.
27.8 ml (0.2 mol) and 300 ml of methylene chloride were mixed, and 15.5 ml (0.2 mol) of methanesulfonyl chloride was added to the mixture at 0°C. The reaction mixture was stirred at room temperature for 12 hours and then concentrated. The residue was diluted with 500 ml of benzene and then washed with saturated brine. Dry (Na ₂ SO ₄ ) and concentrate to give 24.0 g of crude 4-(3-mesyloxypropyl)benzonitrile (yield
100%) was obtained as an oil. Potassium phthalimide 21.1g (0.114mol)
13.6 g of crude 4-(3-mesyloxypropyl)benzonitrile (57 m
mol) of DMF solution was added. The reaction mixture was stirred at 90°C for 3 hours and then cooled to room temperature. After pouring into 200 ml of ice-water, the mixture was extracted with ether-benzene (1:1). The organic layer was dried (Na ₂ SO ₄ ) and concentrated to give 14.0 g of 4-(3-phthalimidopropyl)benzonitrile (yield: 85
%) was obtained. mp; 138-140℃ 10 g (30 mmol) of 4-(3-phthalimidopropyl)benzonitrile and hydrazine hydrate
1.89 ml (39 mmol) and 130 ml of methanol were added and the mixture was refluxed for 3 hours. After cooling to room temperature, it was concentrated. Residue 1moldm ^-3 NaOH200ml
The mixture was dissolved in water and then extracted with ether. It was dried (K ₂ CO ₃ ) and concentrated to obtain 4.32 g (yield 90%) of 4-(3-aminopropyl)benzonitrile. mp; 72-74°C 5.34 g of the obtained 4-(3-aminopropyl)benzonitrile was mixed with 30 ml of 3moldm ^-3 KOH and 30 ml of ethanol, and the mixture was refluxed for 24 hours. After cooling to room temperature, it was concentrated to remove most of the ethanol. The aqueous layer was neutralized with concentrated hydrochloric acid. The deposited precipitate was separated. 4.36 g (yield 73%) of 4-(3-aminopropyl)benzoic acid was obtained. This compound was used in the next reaction without purification. 4-(3-aminopropyl)benzoic acid 900mg
750 mg (5 mmol) of methoxycarbonylmaleimide was added at 0° C. to 25 ml of a solution of (5 mmol) dissolved in saturated aqueous NaHCO ₃ solution. at 0℃
After stirring for 10 minutes, it was diluted with 50 ml of water. After stirring at 30°C for 1 hour, post-treatment was carried out according to the usual method, and a crude 4
-(3-maleimidopropyl)benzoic acid 907
g (yield 70%) was obtained. 3.86 g (14.9 mmol) of crude 4-(3-maleimidopropyl)benzoic acid was mixed with 2.23 g (19.4 mmol) of N-hydroxysuccinimide and 3.97 g of DCC.
(19.4 mmol) and 100 ml of DME, and the mixture was stirred at 0° C. for 1 hour and then at room temperature for 3 hours. A white solid was obtained after normal work-up. Silica gel column chromatography (90:10 chloroform-ethyl acetate) was performed to obtain 4.77% of 4-(3-maleimidopropyl)benzoic acid succinimide ester (Reagent 8).
g (yield 90%) was obtained. mp; 122-124°C (methylene chloride-ether) Production of 3-(3-maleimidopropyl)benzoic acid succinimide ester (Reagent 9) The title compound (Reagent 9) was obtained in the same manner as above. mp; 148-149℃ (methylene chloride-ether) Production of 3-[4-maleimidopropyl)phenyl]propionic acid succinimide ester (Reagent 10) 44 ml (0.315 mol) of diisopropylamine
Butyllithium in 200ml of THF solution
168ml (286m) of 1.70moldm ^-3 hexane solution
mol) was added at 0°C. After stirring for 10 minutes at 0°C,
Cooled to -78°C. In this, 35 t-butyl acetate
ml (260 mmol) was added and stirred for 1 hour. α、
α′-dibromo-p-xylene 26.4g (0.1mol)
300 ml of THF solution was added into the lithium enolate. After stirring the reaction mixture for 12 hours at -78°C to room temperature, 100ml of saturated oxalic acid solution was added. After separating the organic layer, it was extracted with ether. The organic layer was dried (MgSO ₄ ) and concentrated to give an oil. This was subjected to silica gel column chromatography (90:10
3-[4-(2
-t-butoxycarbonylethyl)phenyl]
25.0g of t-butyl propionate (yield 75%)
was obtained as an oil. t-Butyl 3-[4-(2-t-butoxycarbonylethyl)phenyl]propioate 18.0g
(54 mmol), LiAlH ₄ 2.05 g (54 mmol) and
It was mixed with 100 ml of THF and the mixture was stirred at 0° C. for 1 hour. 1 moldm ^-3 after dilution with 200 ml of ether
of NaOH was added. After removing insoluble matter,
The liquid was concentrated to obtain an oil. This was subjected to silica gel column chromatography (80:20 hexane-ethyl acetate) to obtain 7.70 g (yield 54%) of t-butyl 3-[4-(3-hydroxypropyl)phenyl]propionate as an oil. . 4.82 of the obtained tert-butyl 3-[4-(3-hydroxypropyl)phenyl]propionate.
g (18.3 mmol) to triethylamine 5.09 ml
(36.6 mmol), methanesulfonyl chloride 2.84
ml (36.6 mmol) and 50 ml of methylene chloride were mixed and this was stirred at room temperature for 12 hours. After concentration, it was diluted with benzene. The benzene layer was washed with saturated NaCl water, dried (Na ₂ SO ₄ ), and concentrated to give 3-[4-
6.29 g (yield 100%) of t-butyl (3-mesyloxypropyl)phenyl]propionate was obtained as an oil. It was used in the next reaction without purification. To the t-butyl 3-[4-(3-mesyloxypropyl)phenyl]propionate obtained above, 6.77 g (36.6 mmol) of potassium phthalimide and
100ml of DMF was mixed and the mixture was stirred at 90°C for 3 hours. Pour the reaction mixture into ice water,
It was then extracted with benzene-ether (1:1), dried (Na ₂ SO ₄ ) and concentrated to give a yellow oil. This was subjected to silica gel column chromatography (97:3 benzene-ethyl acetate) to obtain 5.75 g (80% yield) of t-butyl 3-[4-(3-phthalimidopropyl)phenyl]propionate as an oil. . The above 3-[4-(3-phthalimidopropyl)
6.88g of t-butyl phenylpropionate
(17.5 mmol) to hydrazine hydrate 1.10 ml (22.8
mmol) and 50 ml of methanol were mixed and the mixture was refluxed for 1 hour. After cooling this to room temperature, it was concentrated. The residue was dissolved in 200 ml of 1 moldm ^-3 NaOH, then extracted with ether and dried (K ₂ CO ₃ ).
and concentrated to give 3-[4-(3-aminopropyl)
4.14g of t-butyl phenylpropionate
(yield 90%) was obtained as an oil. The above t-butyl 3-[4-(3-aminopropyl)phenyl]propionate was mixed with 6 ml of trifluoroacetic acid and 50 ml of methylene chloride, and the mixture was stirred at room temperature for 48 hours. After concentration, the residue was azeotroped with toluene. Add 30 ml of ethanol and 18 ml of 3moldm ^-3 KOH to the residue obtained here,
The mixture was refluxed for 24 hours. After cooling to room temperature, it was concentrated to remove ethanol. The aqueous layer was neutralized with concentrated hydrochloric acid and concentrated to obtain 3.42 g of crude 3-[4-(3-aminopropyl)phenyl]propionic acid. This compound was used in the next reaction without purification. The above crude 3-[4-(3-aminopropyl)phenyl]propionic acid was dissolved in saturated _NaHCO3 ,
2.64 g (17.5 mmol) of methoxycarbonylmaleimide was added to 90 ml of this solution at 0°C.
After stirring at 0°C for 10 minutes, it was diluted with 180ml of water. After stirring the reaction mixture at 30℃ for 1 hour, the pH was adjusted to 2 with concentrated sulfuric acid.
I made it. Extracted with ethyl acetate, washed with saturated NaCl aqueous solution, dried (Na ₂ SO ₄ ) and concentrated to give 3-
3.16 g (yield 70%) of [4-(3-maleimidopropyl)phenyl]propionic acid was obtained. 2.48 g (8.64 m
mol) of N-hydroxysuccinimide 1.29g
(11.2 mmol), DCC2.31g (11.2 mmol) and
Mix 50ml of DME and keep the mixture at 0℃ for 1 hour.
The mixture was then stirred at room temperature for 3 hours. Reaction mixture to 0
℃, add a few drops of acetic acid, and add 1
After stirring for an hour, insoluble matter was removed. The liquid was concentrated to obtain a white solid. This was subjected to silica gel column chromatography (70:30 methylene chloride-
3-[4-(3-maleimidopropyl)phenyl]propionic acid succinimide ester (Reagent 10) 3.0 g (yield 90
%) was obtained. mp; 159-160°C (methylene chloride-ether) Production of 3-[2-(3-maleimidopropyl)phenyl]propionic acid succinimide ester (Reagent 11) The title compound (Reagent 11) was obtained in the same manner as above. . IR (neat); 1822, 1792, 1740, 1710 cm ^{-1 1} H-NMR (CDCl ₃ ); δ = 1.85 (m, 2H), 2.69 (s, 4H), 2.69 (m, 6H), 3.46 (t, J = 6.2Hz, 2H), 6.49 (s, 2H), 7.03 (s, 4H) MS (m/z) = 384 (M ⁺ ) Analysis value; m/z = 384.1325 as C ₂₀ H ₂₀ O ₆ N ₂ Calculated value; M=384.1321 Preparation of 3-[3-3-maleimidopropyl)phenyl]propionic acid succinimide ester (Reagent 12) The title compound was obtained in the same manner as above. mp; 121-122℃ (methylene chloride-ether) Production of 4-(2-maleinimidophenyl)butyric acid succinimide ester (Reagent 13) 2-(2-nitrophenyl)ethanol 5.51
g (33.3 mmol), DMSO50 ml, benzene 50 ml
and 1.31 ml (17 mmol) of trifluoroacetic acid and 2.7 ml of pyridine in a mixture of 21 g (0.1 mol) of DCC.
(33.3 mmol) was added. After stirring the reaction mixture at room temperature for 4 hours, 300 ml of an aqueous solution of 8.82 g (70 mmol) of oxalic acid dihydrate was added thereto.
After stirring at room temperature for 30 minutes, it was diluted with 500 ml of ethyl acetate. After separating the organic layer, wash with saturated NaCl,
Dry (Na ₂ SO ₄ ) and concentrate to give a yellow oil. Silica gel column chromatography (95:5 benzene-ethyl acetate) gave 2-nitrophenylacetaldehyde as an unstable oil. The yield is 4.50g (yield 81
%). 4.50 g (27 mmol) of the above aldehyde, 18.0 g (53 mmol) of methoxycarbonylmethylenetriphenylphosphorane and 100 g of methylene chloride
ml was mixed and this was refluxed for 12 hours. After cooling to room temperature, it was concentrated. The residue was subjected to column chromatography (97:3 benzene-ethyl acetate) and a 60:40 mixture of methyl 4-(2-nitrophenyl)-3-butenicate and methyl 4-(2-nitrophenyl)crotonate was used as an oil at 2.58 g.
g (yield 43%) was obtained. Mixture of nitroesters obtained above 1.71
g (7.74 mmol) were mixed with 200 mg of 10% PD-C and 30 ml of methanol, and the mixture was stirred at room temperature under a hydrogen atmosphere for 3 days. After removing insoluble matter, the liquid was concentrated to obtain 1.0 g (yield 67%) of methyl 4-(2-aminophenyl)butyrate as an oil. 1.0g (5.18mmol) of the obtained oil
10 ml of 1 moldm ^-3 KOH and 20 ml of ethanol were mixed, and this was refluxed for 12 hours. After cooling to room temperature, it was concentrated to remove ethanol. water layer
After neutralizing with 1moldm ^-3 HCl, the crude 4-
1.02 g of (2-aminophenyl)butyric acid was obtained. This compound was used in the next reaction without purification. 1.02 g of crude 4-(2-aminophenyl)butyric acid obtained above was dissolved in saturated NaHCO ₃ , and 782 mg (5.18 mmol) of methoxycarbonylmaleimide was added to 26 ml of NaHCO ₃ aqueous solution at 0°C. 10 at 0℃
After stirring for a minute, it was diluted with 50 ml of water. After stirring at 30°C for 1 hour, the mixture was cooled to 0°C, a few drops of acetic acid was added thereto, and the mixture was further stirred at 0°C for 1 hour to remove insoluble materials. The liquid was concentrated to give a yellow oil. This was dispersed and subjected to TLC (70:30:0.5 ethyl acetate-chloroform-acetic acid) to yield 367 mg of 4-(2-maleinimidophenyl)butyric acid (based on methyl 4-(2-aminophenyl)butyrate). rate 27
%) was obtained as an oil. Obtained 4-(2-maleinimidophenyl)
Butyric acid 367mg (1.42mmol), N-hydrocysuccinimide 212mg (1.85mmol), DCC 381mg
(1.85 mmol) and 20 ml of DME were mixed, and the mixture was stirred at 0° C. for 1 hour and at room temperature for 3 hours. Then, the reaction mixture was cooled to 0°C, a few drops of acetic acid was added thereto, and the mixture was stirred at 0°C for 1 hour. After removing insoluble matter, the liquid was concentrated to obtain a yellow oil. This was subjected to preparative TLC (70:30 chloroform-ethyl acetate) to yield 379 mg of the title target compound (yield:
75%) was obtained as an oil. IR (CHCl ₃ ); 1808, 1780, 1745, 1708 cm ^{-1 1} H-NMR (CDCl ₃ ); δ = 2.94 (m, 2H), 2.46 (m, 4H), 2.69 (s, 4H), 6.69 (s , 2H), 7.23 (m, 4H) MS (m/z) = 356 (M ⁺ ) Analysis value; m/z = 356.1006 Calculated value as C ₁₈ H ₁₆ O ₆ N ₂ ; M = 356.1008 4-(3 - Maleimidophenyl)acetic acid succinimide ester (Reagent 14) Production of the title target compound (Reagent 14) in the same manner as above.
was obtained as an oil. IR (CHCl ₃ ); 1808, 1780, 1743, 1718 cm ^{-1 1} H-NMR (CDCl ₃ ); δ = 2.02 (m, 2H), 2.63 (m, 4H), 2.73 (s, 4H), 6.69 (s , 2H), 7.06 (m, 4H) MS (m/z) = 356 (M ⁺ ) Analytical value; m/z = 356.1006 Calculated value as C ₁₈ H ₁₆ O ₆ N ₂ ; M = 356.1008 12-maleimide Production of dodecanoic acid succinimide ester (Reagent 15) 12-amidonododecanoic acid 4.30 g (20 mmol),
A mixture of 2.0 g (20 mmol) of maleic anhydride and 200 ml of chloroform was refluxed at room temperature for 2 hours and then for 5 hours. After cooling the reaction mixture to room temperature, it was concentrated to about 50 ml. The mixture was cooled to 0° C., and the precipitate deposited was separated. This product was used as it was in the next reaction. To the solid obtained above, 820 mg of sodium acetate (10 m
mol) and 20 ml of acetic anhydride, and the mixture is
The mixture was stirred at 90°C for 1 hour. After concentration, the residue was diluted with ethyl acetate. Washing with saturated brine, drying (Na ₂ SO ₄ ) and concentration gave a yellow semi-solid.
This was dissolved in THF (30 ml) − H ₂ O (50 ml) − CH ₃ COOH
(10 ml), the mixture was stirred at room temperature for 1 hour. After concentration, the mixture was extracted with ethyl acetate, dried (Na ₂ SO ₄ ), concentrated, and azeotroped with toluene to obtain a yellow solid. This was subjected to silica gel column chromatography (90:10:0.5 benzene-ethyl acetate-acetic acid to 70:30:0.5 benzene-ethyl acetate-acetic acid) to obtain 1.23 g of a white solid. 1.23 g (4.32 mmol) of this solid was mixed with 595 mg (5.18 mmol) of N-hydroxysuccinimide,
Mixed with 1.07 g (5.18 mmol) of DCC and 30 ml of DME, the mixture was heated at 0 °C for 1 h and then at room temperature for 3 h.
After stirring for an hour, the mixture was cooled to 0°C, a few drops of acetic acid was added thereto, and after further stirring at 0°C for 1 hour, insoluble materials were removed and the liquid was concentrated to obtain a white solid. This was subjected to silica gel column chromatography (2:1 ethyl acetate-hexane), and 12
-Maleimidododecanoic acid succinimide ester 1.52g (yield 90%) was obtained. mp; 67-68°C (methylene chloride-ether-hexane) The structural formulas of the reagents obtained in each of the above examples and other protein binding reagents are shown in Table 1 below. For known compounds in the table, citations or manufacturing company names are also listed in the notes section. [Table] [Table] [Table] [Table] [Table] [Table] Production example 2 Production of fibrin-adsorbing protein Plasminogen kringles 1 to 3 and 4
(1) Plasminogen human plasma (Japanese Red Cross Society) 5 and trasylol (Bayer) 50000 KIU
After adding (Kallidinogenase Inactivator Unit), lysine-sepharose (BAChibber, Method in Enzymology,
34, 424 (1974)]) 300ml
(packed volume) and stir at room temperature for 3 hours. The resin was then collected in a glass filter and diluted with 0.3M sodium phosphate buffer (PH
7.4, containing 10 KIU/ml Trasylol) 4, packed into a column (5 cmφ), continued washing overnight with the same buffer 5, and washed with 0.2 M sodium phosphate buffer (PH 7.4, 0.05 M ε-aminocaproic acid). (EACA)), eluted at 280n
Measure the absorbance (OD) at
Gradually add ammonium sulfate at a rate of 40g/100ml and after stirring for an additional 15 minutes,
Centrifuge at 10,000 rpm for 10 minutes to obtain a precipitate. This precipitate was dissolved in a small amount of 0.1M-NH ₄ HCO ₃ and equilibrated with the same solution.
150 (Falmacia) or Cefacryl S
−300 Super Fine (Falmacia)
Apply to a column (5 cm φ x 95 cm) packed with and develop with the same solution. Discarding the protein near the void volume, the next main peak fraction with an O.D of 280 nm is mixed and lyophilized to obtain about 600 mg of pure human plasminogen. Its specific activity averaged 20 casein units/mg protein. (2) Gringle 1 to 3 This production was carried out by Strappjjansen (L.
Sottrup-Jensen et al.'s method [Progress in
Chemical Fibrinolysis and Thrombolysis
3191 (1978)] with some modifications. That is, 500 mg of human praminogen and 3.6 mg of trypsin inhibitor (derived from bovine pancreas, manufactured by Sigma) were added.
0.1M− _{NH4HCO3 − NH4OH} buffer (PH8.3)
Dissolve in 32.6ml and then stir while stirring.
Add 0.53g of NH ₄ HCO ₃ to give a concentration of 0.3M.
After dropping 0.8 ml of elastase (Sigma, type) solution dissolved in 0.1M-NH ₄ HCO ₃ to a concentration of 2 mg/ml, the mixture was stirred at room temperature for 3.5 hours, and then elastatinal (protein serum) was added. Add 8 mg of NH 4 HCO 3 and stir for 20 minutes, add 0.67 g of NH ₄ HCO ₃ and stir for an additional 20 minutes. Then at 16000rpm
Centrifuge for 20 minutes, collect the supernatant, and collect 0.5M−
Cephadex G- equilibrated with NH ₄ HCO ₃
75 Superfine (Fuymacia) or Cefa Acryl S-200 Superfine (2.6 cmφ x 98 cm) column at 4°C. Discard the protein near the void volume and collect the next main peak at O.D280nm and freeze-dry it. Dissolve this in approximately 40 ml of 0.1M-NH ₄ HCO ₃ , centrifuge at 8000 rpm for 10 minutes, collect the supernatant,
Apply to a lysine-Sepharose column (1.6cmφ x 30cm) equilibrated with the same solution in advance,
Contains 0-10 mmol of ε-aminocaproic acid (EACA) after washing with 850 ml of 0.1M-NH ₄ HCO ₃
Gradient elution with 0.1M NH ₄ HCO ₃ solution (approximately 25 ml/h). Approximately 120 mg of neoplasminogen (kringle 5 + light chain) is obtained from the pass-through wash. The O.D280 peak part of the eluate was mixed and transferred to a Sephadex G-25 column (5 cm
After desalting with φ x 23 cm), freeze-dry to obtain about 150 mg of kringles 1-3 (1+2+3). (3) Kringle 4 The second peak fraction following the gel-prone main peak of the elastase-decomposed product of plasminogen in (2) above was lyophilized and dissolved in a small amount of about 80 mg of 0.1M-NH ₄ HCO ₃ . , Lysine-Sepharose column (3 cm) equilibrated with the same solution.
After washing with about 500 ml of the same solution, elute with 0.1M-N ₄ HCO ₃ ^- 50mM-EACA, collect the peak at 280 nm O.D. ) and lyophilized to obtain about 50 mg of kringle 4. (4) Plasminogen kringles 1 to 3 and 4 were obtained in the same manner as in (2) and (3) above, except that alpha-chymotrypsin (manufactured by Sigma) was used instead of elastase. Production of the N-terminal domain of fibronectin (1) Fibronectin Purification of fibronectin from human plasma was performed by M. Hayashi and K.M.
Yamada) method [J.Biol.Chem, 257 (9),
5263 (1982)]. i.e. human plasma 2.5
Add 62.5 ml of 0.2M EDTA and 12.5 ml of an ethanol solution of 0.2M phenylmethanesulfonyl fluoride to the solution, centrifuge at 8500 x g for 20 minutes at 4°C, and incubate the resulting supernatant at 37°C for 10 minutes. After warming and degassing for 5 minutes, add 0.15M NaCl and 5
Apply to a Sepharose CL-4B precolumn (25 cmφ x 20 cm) equilibrated with 10 mM Tris-HCl buffer (PH7.4) containing mM-EDTA. The flow-through was adsorbed on a gelatin-sepharose column (2.5 cmφ x 20 cm, equilibrated with the same solution as above), and then 10
Wash with mM-Tris-HCl (PH7.4), and then
Wash with 10mM Tris-HCl buffer (PH7.4) containing 0.15M NaCl and 5mM EDTA, and elute with 10mM Tris-HCl buffer (PH7.4) containing 4M urea. The obtained protein peak fraction was applied to a heparin-sepharose column (2.5 cmφ x 20 cm, 0.1 M NaCl and
Adsorb to 10mM-Tris-HCl buffer (PH7.4) containing 0.5mM-EDTA),
After washing with the same solution used for equilibration, 0.5M
Elute with 10 mM Tris-HCl buffer (PH 7.4) containing -NaCl. Ammonium sulfate is added to the obtained fibronectin solution to achieve 40% saturation and concentrated to obtain about 450 mg of fibronectin concentrate. (2) Fibronectin N-terminal domain M. Hayashi and KM
Yamada) method [J.Biol.Chem, 258 , (5),
3332 (1983)], the fibronectin concentrate obtained in (1) above was diluted with 1mM-CaCl ₂ and
Dialyze against 50mM Tris-HCl buffer (PH7.0) containing 30mM NaCl, and 115ml of this solution
(containing 344 mg of fibronectin) was heated at 30°C, then treated with trypsin (Sigma) 34.4μ
g (0.688 ml aqueous solution) and digested at 30°C for 5 minutes. Then make a final 1mM.
After adding 0.56 ml of an ethanol solution of 0.2M phenylmethanesulfonyl fluoride to inactivate trypsin, add 10M ethanol solution containing 0.1mM phenylmethanesulfonyl fluoride.
Dialyze against M-Tris-HCl buffer (PH7.0) for 4 hours at room temperature. Dialysis fluid
Apply to DEAE-cellulose column (manufactured by Watmann, DE-52, 2.5cmφ x 8cm, equilibrated with 10mM Tris-HCl buffer (PH7.0)), wash with the same solution as the equilibration solution, and then 0.5M-
Elute with 10 mM tolyl-HCl buffer (PH7.0) containing NaCl. Combine the protein peaks of the flow-through and wash fractions and analyze with Amicon PM-10.
Concentration is performed by ultrafiltration using an ultrafiltration membrane (manufactured by Amicon) to obtain about 51 mg of fibronectin N-terminal domain with a molecular weight of about 30,000. (3) The N-terminal domain of fibronectin was obtained in the same manner as in (2) above using plasmin (manufactured by Kabi Co., Ltd.) instead of trypsin. Production of a domain adjacent to the C-terminus of fibronectin M. Hayashi and KMYamada
method [J.Biol.Chem, 258 , (5), 3332 (1983)]
DEAE of the tryptic digest of fibronectin obtained in Reference Example 2-(2) above with reference to
- Cellulose adsorption, purification from liquid fraction. That is, the same fraction was added to a 10mM Tris-HCl buffer (PH7.0) containing _1mM MgCl2.
After dialysis with 4, gelatin-Sepharose column (1.5cmφ×
30cm). Collect the O.D280nm peak fraction of the passthrough, add EDTA to a final concentration of 2mM, and add 10mM Tris-HCl buffer (PH7.0) containing 1mM EDTA in advance.
Pass through a heparin-Sepharose column (1.5 cmφ x 30 cm) equilibrated with O of the passing liquid.
D280nm peak fractions were collected and dialyzed against 10mM Tris-HCl buffer (PH7.0)4, followed by DEAE-cellulose (1.5cm) equilibrated with the same solution.
φ×6cm). After washing with the same solution,
0.06M, 0.08M, 0.1M, 0.12M, 0.14M,
Elute stepwise with 10mM Tris-HCl buffer (PH7.0) containing 0.16M and 0.3M NaCl. Fractions eluted with 0.1-0.12M NaCl are collected and concentrated using an Amicon PM-10 ultrafiltration membrane to obtain a domain adjacent to the C-terminus of fibronectin with a molecular weight of approximately 34,000. Production of plasmin heavy chain (HC) (1) EERickli et al. method [Eur.J.
Biochem., 59 , 411 (1975)]. That is, 150 mg of plasminogen purified in (1) of Reference Example 2 was mixed with 0.1M-NaCl and
Urokinase (manufactured by Nippon Chemical Research Co., Ltd.) was dissolved in 30 ml of 0.05 M Tris-HCl buffer (PH = 7.8) containing 25% glycerol.
Add units and incubate for 8 hours at room temperature. Further, 1500 units of the same urokinase are added and incubated at room temperature for 16 hours.
This solution was purged with nitrogen, and 2-mercaptoethanol was added to a final concentration of 0.1M.
Leave at 20℃ for 20 minutes. After that, it was cooled on ice, and 0.15M−NaCl, 0.01M− was replaced with nitrogen in advance.
Lysine-Sepharose column (manufactured by Daiichi Chemical Co., Ltd., 5 cmφ x 10 cm) sufficiently equilibrated with 0.01 M sodium phosphate buffer (PH = 7.2) containing EDTA and 0.001 M 2-mercaptoethanol. After washing with the same buffer solution, elute with the same buffer solution containing 0.05M-6-aminohexanoic acid, collect the peak fraction of absorbance at 280 nm, and use Amicon PM-10 ultrafiltration membrane. Concentrate under nitrogen pressure. The above buffer solution excluding 2-mercaptoethanol is added to this and concentrated again. Repeat this operation several times to obtain about 60 mg of plasmin HC. This was replaced with nitrogen and stored at -80°C. Approximately 3 moles of SH groups were detected per mole of this product. (2) Plasminogen purified from human blood
300 mg of 0.05 M Tris-HCl buffer containing 0.1 M NaCl and 25% glycerol (PH =
7.8) Dissolve in 60 ml, add 18,000 units of urokinase, and incubate at 25°C for 8 hours. Additionally, 9000 units of urokinase were added.
Incubate for 16 hours at 25°C. A solution of 34 mg of p-nitrophenyl-p-guanidinobenzoate hydrochloride dissolved in 0.4 ml of DMF was added to this solution, and after stirring at 37°C for 10 minutes, the atmosphere was replaced with nitrogen, and 2-mercaptoethanol was added to the final solution.
Add to a concentration of 0.1M and stir at 20°C for 20 minutes. After that, it was cooled on ice, and 0.2M-NaCl, 0.01M-EDTA and nitrogen-substituted
0.01M sodium phosphate buffer containing 0.001M 2-mercaptoethanol (PH=
7.2) was applied to a lysensepharose column (5 cmφ x 15 cm) that had been sufficiently equilibrated, washed with the same buffer, and then 0.001M-6-aminohexanoic acid, 0.15M-NaCl, 0.01M-EDTA and 0.01M sodium phosphate buffer containing 0.001M 2-mercaptoethanol (PH=
After washing with 7.2), elute with the same buffer containing 0.003M-6-aminohexanoic acid, and analyze at 280 nm.
Collect the absorbance peak fractions at Amicon
With PM-10 ultrafiltration membrane (manufactured by Amicon),
Concentrate to 50 ml under nitrogen pressure. The concentrated solution was replaced with 0.15M−CaCl and
The column was applied to a Sephadex G-25 column (5 cmφ x 20 cm) equilibrated with 0.01M sodium phosphate buffer (PH=7.2) containing 0.01M EDTA, and eluted with the same buffer. The absorbance peak fraction at 280 nm was collected and concentrated under nitrogen pressure using an Amicon PM-10 ultrafiltration membrane to give approximately 100 mg.
Obtain plasmin HC. Approximately 5 moles of SH groups were detected per mole of this product. Production of plasmin with irreversibly inhibited active center (TLCK-treated plasmin) (1) 200 mg of human plasminogen was added to 10 ml of 0.05 M Tris-HCl buffer (PH9.0) containing 0.02 M lysine and 0.1 M NaCl. Dissolve, add 3.3 ml of glycerol and stir. Add approximately 4,500 units of urokinase solution to this solution and heat at 25°C.
Incubate for 20 hours. Then 1N−
Adjust the pH to approximately 7.4 with HCl, add 0.18 ml of a 0.5 M DMF solution of tosyl lysine-chloromethyl ketone (TLCK) (to a final concentration of 6.6 mM),
After incubating for 2.5 hours at 25℃, 1N-HCl
to pH 3, and dialyzed overnight with 10 ^-3 N-HCl (2
×2) and freeze-dry the internal solution to obtain the desired
Approximately 150 mg of TLCK-treated plasmin was obtained. (2) In the same manner as in (1) above, instead of TLCK, a low-molecular serine protease inhibitor such as diisopropylfluorophosphate and phenylmethylsulfonyl fluoride is used to obtain plasmin whose active center is irreversibly inhibited. . Reference Example 3 Preparation of urokinase LMW-UK LMW-UK (molecular weight 32000, manufactured by Nippon Chemical Research) was concentrated to approximately 10 mg/ml using an Amicon PM-10 ultrafiltration membrane, and then concentrated with 0.01M sodium phosphate buffer ( Dialyze overnight at 4°C (PH=7.0). The supernatant obtained by centrifuging the internal solution (8000 rpm x 10 minutes) is used as the LMW-UK concentrate. HMW-UK Using HMW-UK (molecular weight 54000, manufactured by Nippon Chemical Research Co., Ltd.), the same procedure as above was carried out.
Obtain HMW-UK concentrate. HMW-UK heavy chain HMW-UK 1.3 million units (by synthetic substrate method,
The same applies below) (containing 0.05M Tris-HCl buffer, PH8.0, 0.15M NaCl and 0.02MEDTA) was added to 2ml of a solution containing nitrogen, and the final concentration was
Add 2-mercaptoethanol to a concentration of 0.01M, and stir at room temperature for about 10 hours under a nitrogen stream. After cooling, add 2 ml of 0.05M aqueous sodium iodoacetate solution and stir in the dark for 20 minutes. The reaction solution was then dialyzed against 0.1M sodium phosphate buffer (PH7.5) containing 0.4M NaCl, and then benzamidine-CH-Sepharose (method of L. Holmberg et al.) equilibrated with the same solution was used. [Biochemica et Biophysica Acta,
455, 215 (1976)] column (1.5 cmφ x 4 cm). The same column,
After washing with the same solution, elute with 0.1M acetic acid containing 0.4M NaCl. The protein peak fraction after elution was filtered using Diaflow ultrafiltration membrane PM-10 (manufactured by Amicon).
After concentration, the solution is dialyzed at 4°C against a 0.01M sodium phosphate buffer (PH7.0) substituted with nitrogen. By the above operations, about 1 million units of urokinase (active heavy chain) with a molecular weight of about 32,000 are obtained. Example 1 6.7 mmol of m-maleimidobenzoyl N-hydroxysuccinimide ester (compound code number
NS2002, Pierce, hereinafter referred to as "MBS")
Add the DMF solution dropwise with stirring and stir at room temperature for 30 minutes. This was equilibrated with a degassed 0.1 M sodium phosphate buffer (PH = 6.0) and a Sephadex G-25 fine column (1.5 cmφ x
20cm) and develop with the same buffer. 280n
The peak fraction of absorbance at m is collected to obtain 0.9 mmol of LMW-UK to which the MB group is attached. In this product, an average of 2.7 moles of MB groups were bonded to 1 mole of LMW-UK. 123 mg of plasmin HC obtained by the same operation as in Reference Example 2 (1) and MB group-containing obtained above
Mix 29.5 mg of LMW-UK, replace with nitrogen thoroughly,
Stir at room temperature for 3 hours. 2-mercaptoethanol was added to the mixture to give a final concentration of 1 mM, and after stirring for 10 minutes, N-ethylmaleimide was added to the mixture to give a final concentration of 2 mM, and the mixture was stirred for 20 minutes at room temperature. This was equilibrated with a 0.1M sodium phosphate buffer containing 0.4M NaCl to a Sephadex G-25 fine column (2.6cmφ x 30mm).
Benzamidine-CH-Sepharose (method of L. Holmberg et al. [Biochemica et Biophysica Acta, 445 , 215
(1976)]) column (2.5 cm
After washing with 900ml of the same buffer,
0.1M acetic acid aqueous solution containing 0.1M-NaCl and
Elute with a 6M urea aqueous solution, combine the absorbance peak fractions at 280 nm, and gel-filter through a Sephadex G-25 column (5cmφ x 25cm) equilibrated with a 0.1M ammonium bicarbonate aqueous solution.The absorbance peak obtained The fraction was applied to a lysine-Sepharose column (1.6 cmφ x 14 cm) equilibrated with a 0.1 M aqueous ammonium hydrogen carbonate solution, and washed with 400 ml of the same solution and then with 100 ml of a 0.2-M aqueous 6-aminohexanoic acid solution. Elute with 2M-KSCN aqueous solution. The absorbance peak fraction of the eluate was dehydrochlorinated and concentrated using Amicon PM-10.
22.3 mg of the plasmin HC-LMW-UK complex of the present invention (hereinafter referred to as "complex A") is obtained. The obtained complex A was confirmed as a band with a molecular weight of 80,000 to 90,000 on SDS-PAGE, and was confirmed as a band with a molecular weight of 80,000 to 90,000.
Approximately 1 mole of plasmin HC is bound to 1 mole of UK. Examples 2 to 4 As a protein binding reagent in Example 1 above
In place of MBS, each of the following compounds shown in Table 1 above was used, and plasmin shown in Table 2 below was used in the same manner.
Obtain each of the HC-LMW-UK complexes. [Table] Each of the obtained complexes (complexes B to D) was
Both were confirmed as bands of 80,000 to 90,000 by PAGE, and the molar ratio of LMW-UK and plasmin HC was about 1:1. Example 5 A DMF solution of 4 mmol MBS was added dropwise to 1 ml of the LMW-UK concentrate obtained in Reference Example 3 (LMW-UK amount 500,000 units/ml) with stirring.
Stir for 30 minutes at room temperature. This was applied to a Sephadex G-25 fine column (1.5 cmφ x 20 cm) equilibrated with a degassed 0.1M sodium phosphate buffer (PH=7.0) and developed with the same buffer. The absorbance peak fraction at 280 nm is fractionated to obtain 400,000 units of LMW-UK to which the MB group is bonded. This is for 1 mole of LMW−UK
An average of 2 moles of MB groups were bonded. Plasmin HC 8 mg obtained by the same operation as in Reference Example 2 (2) and MB group-containing obtained above
400,000 units (2.05 mg) of LMW-UK were mixed, the atmosphere was sufficiently replaced with nitrogen, and the mixture was stirred at room temperature for 3 hours. 2-mercaptoethanol was added to the mixture to give a final concentration of 1 mM, and after stirring for 10 minutes, N-ethylmaleimide was added to the mixture to give a final concentration of 2 mM, and the mixture was stirred for 20 minutes at room temperature. This was gel-filtered using a Sephadex G-25 fine column (2.6cmφ x 30cm) equilibrated with 0.1M sodium phosphate buffer containing 0.4M NaCl, and the peak fraction with absorbance at 280 nm was collected. This was applied to benzamidine-CH-Sepharose equilibrated with the above buffer, and after washing with 900 ml of the same buffer, 0.1M-
Sephadex G was eluted with a 0.1M aqueous acetic acid solution containing NaCl and a 6M aqueous urea solution, the absorbance peak fractions at 280 nm were combined, and this was equilibrated with a 0.1M aqueous ammonium bicarbonate solution.
After gel filtration with a 25 column (5 cmφ x 25 cm), the obtained absorbance peak fraction was applied to a lysine-Sepharose column (1.6 cmφ x 14 cm) equilibrated with a 0.1 M ammonium bicarbonate aqueous solution, and 400 ml of the same solution was applied. , and then eluted with 0.2M 6-aminohexanoic acid. The absorbance peak fraction of the eluate was dehydrochlorinated and concentrated using Amicon PM-10 to obtain 200,000 units (4.5 mg) of the plasmin HC-LMW-UK complex of the present invention (hereinafter referred to as "complex E"). get. The obtained complex E was confirmed as a band with a molecular weight of approximately 90,000 on SDS-PAGE, and was identified as LWM-
Approximately 1 mole of plasmin HC is bound to 1 mole of UK. Examples 6-27 In Example 5 above, as a protein binding reagent
Using each of the following compounds shown in the table above in place of MBS, each plasmin HC-LMW-UK complex shown in Table 3 below is obtained in the same manner. [Table] Each of these complexes was confirmed as approximately 90,000 bands on SDS-PAGE, and the molar ratio of LMW-UK and plasmin HC was approximately 1:1. Example 28 (1) Preparation of acetylmercapto-kringles 1 to 3 Kringles 1 to 3 obtained in Reference Example 2-(2)
329.07 mg (855 nanomoles) was dissolved in 3 ml of 0.125 M sodium phosphate buffer (PH 7.2), and 78 µ of a 20 mg/ml DMF solution of S-acetylmercaptosuccinic anhydride (Kringle 1
About 10 times the molar amount of ~3) was added dropwise, and stirring was continued for 30 minutes at room temperature.
Cephadex G equilibrated with EDTA (containing EDTA)
Apply to a -25 fine column (1.5 cmφ x 20 cm), develop with the same buffer, and collect about 8 ml of the peak at 280 nm O.D. Thus 1.75 acetylmercapto groups per mole of kringles 1-3.
Obtain the object in which the moles are combined. Recovery rate is approximately 93
It was %. (2) Preparation of maleimidobenzoyl-LMW-UK 3.05 LMW-UK concentrate obtained in Reference Example 3
ml (930 nanomoles, approximately 6 million units) of MBS
Add 112μ of 24.8mg/ml DMF solution dropwise with stirring, and stir at room temperature for 30 minutes. I degassed this
It was applied to a Sephadex G-25 Phite column (1.5 cmφ x 20 cm) equilibrated with 0.1M sodium phosphate buffer (PH=6.0) and developed with the same buffer. The absorbance peak fraction (8 ml) at 280 nm was collected, and LMW-
Get UK. This is per mole of LMW−UK
It contained 3.5 moles of MB groups, and the protein recovery rate was 90% and the activity recovery rate was approximately 75%. (3) Manufacture of complex Mix 7.8 ml of acetylmercapto-kringles 1 to 3 obtained in (1) above and 7.8 ml of maleimidobenzoyl-LMW-UK obtained in (2), and after thoroughly purging with nitrogen, Add 1.6 ml of a 0.5M aqueous hydroxylamine solution (PH7.3) purged with nitrogen, and stir at room temperature for 3 hours. Add 2-mercaptoethanol to this to a final concentration of 1mM,
After stirring for 5-10 minutes, N-ethylmaleimide is added to a final concentration of 2mM and stirred at room temperature for 20 minutes. This reaction solution was gel-filtered through a Sephadex G-25 fine column (2.6cmφ x 30cm) equilibrated with 0.1M sodium phosphate buffer (PH7.4) containing 0.4M NaCl, and 280n
The peak fraction with the absorbance of
After washing with 900ml of the same buffer, 0.1M acetic acid aqueous solution containing 0.1M NaCl and 6M urea aqueous solution
Elute with 100 ml, combine the absorbance peak fractions at 280 nm, and add this to a Sephadex G- _{25 column} (5 cmφ x
After gel filtration with 25cm), apply to a lysine-Sepharose column (1.6cmφ x 14cm) equilibrated with the same solution, wash with 400ml of the same solution, and then apply 2M-KSCN.
Elutes in aqueous solution. The eluate is desalted and then lyophilized to obtain a complex of the present invention in which LMW-UK and kringles 1 to 3 are bound. The activity recovery rate is approx.
It was 34%. Hereinafter, this will be referred to as "complex AB." This was confirmed as approximately 60,000 bands on SDS-PAGE, and the binding ratio of both raw materials was 1:1 (molar ratio). Example 29 LMW-UK concentrate prepared in Reference Example 3,
4.53ml (882 nanomoles, 6 million units) and Reference Example 2
31.44 mg (925
A composite of the present invention was obtained in the same manner as in Example 28 using nanomol). The activity recovery rate was about 24%. This is called "complex AC." Example 30 (1) Preparation of acetylmercaptokringel 4 18.1 of Kringel 4 obtained in Reference Example 2-(3)
mg (1.87 micromol), 10 times the molar amount of S-acetylmercaptosuccinic anhydride (18.7
micromole) in the same manner as in Example 28 (1) to obtain the target product in which 2.0 moles of acetyl meltcapto group were bound per mole of Kringel 4. (2) Preparation of maleimidobenzoyl-LMW-UK 2.7 ml of LMW-UK concentrate obtained in Reference Example 3
Example 28 using (823 nanomoles, 4 million units)
Similarly to (2), the MB group per mole of LMW-UK is
3.34 mol of maleimidobenzoyl-
Obtain LMW−UK. (3) Production of complex Acetyl mercapto-Kringel 4 obtained in (1) above and maleimidobenzoyl-obtained in (2)
A composite of the present invention is obtained in the same manner as in Example 28 (3) using LMW-UK. This is called “complex AD”
shall be. Example 31 (1) The TLCK-treated plasmin obtained in Reference Example 2-(1) was mixed with 5 times the molar amount of S-acetylmercaptosuccinic anhydride (10 mg/ml DMF solution),
The reaction was carried out in the same manner as in Example 28 (1), and the acetylmercapto group was added per mole of the above plasmin.
A compound with 1.86 moles bound is obtained. Recovery rate 88
%. (2) LMW-UK concentrate obtained in Reference Example 3 (approx.
LMW
Maleimidobenzoyl-LMW-UK with 3.86 mol of MB groups bound per mol of -UK is obtained. (3) Using the compound obtained in (1) above and the compound obtained in (2), the complex of the present invention is obtained in the same manner as in (3) of Example 28. However, 2-mercaptoethanol was not added after the reaction, and the affinity chromatography procedure for urokinase during complex purification was omitted, and the gel filtrate of the reaction solution was directly applied to a lysine-sepharose column (1.6 cmφ x 5 cm). ),
Thereafter, wash, elute and desalt, and lyophilize in the same manner. The obtained complex is referred to as "complex AE." Example 32 (1) Fibronectin N obtained in Reference Example 2-(2)
25 mg of terminal domain (approximately 30 mg of solution previously dialyzed against 0.125 M sodium phosphate buffer (PH7.2))
ml) with its 20 parts molar amount of S-acetylmercaptosuccinic anhydride (20 mg/ml DMF solution),
The reaction was carried out in the same manner as in Example 28 (1), and per mole of the above fibronectin N-terminal domain,
A compound to which 2.5 moles of acetylmercapto groups are bonded is obtained. The recovery rate was 95%. (2) 3 ml of LMW-UK concentrate obtained in Reference Example 3
Example using (780 nanomoles, approximately 5 million units)
Similarly to (2) of 28, per mol of LMW−UK,
Maleimidobenzoyl-LMW-UK to which 2.3 moles of maleimidobenzoyl groups are bonded is obtained. (3) 8 ml of the fragment solution obtained in (1) above and 8 ml of the maleimidobenzoyl-LMW-UK solution obtained in (2)
After deaerating and purging with nitrogen, add 0.5M-hydroxylamine solution (5N-NaCl to pH
Add 1.6 ml of 7.3) and stir at room temperature for 2.5 hours under a nitrogen stream to react. Next, 2-mercaptoethanol is added to a final concentration of 1 mM, and the mixture is stirred for 10 minutes. N-ethylmaleimide is then added to a final concentration of 2 mM, and the mixture is stirred for about 10 minutes. The reaction solution was transferred to a Sephadex G-25 fine column (3 cmφ x 30 cm, 0.05M-NaCl and
10mM Tris-HCl buffer (PH) containing 100KIU/ml Trasylol (Bayer)
7.5)) and develop with the same buffer. The obtained protein peak fraction was applied to a fibrin-monomer-Sepharose 6B column (DL
The method of Heene and FRMatthas [Thromb.
Adjusted by [Research, 2 , 137 (1973)]
2.5cmφ×10cm, 0.05M−NaCl and 100KIU/ml
Equilibrated with 10mM Tris-HCl buffer (PH7.5) containing tradiol), washed with the same buffer, and eluted with 10mM-Tris-HCl buffer (PH7.5) containing 8M-urea. . The obtained protein peak fraction was concentrated with ammonium sulfate, and then the concentrated solution was treated with Cephacryl S-
200 super fine column (2.5cmφ×97cm,
The fractions exhibiting urokinase activity with a molecular weight of around 60,000 were collected, concentrated with ammonium sulfate, and then purified with phosphate buffered saline. Dialyzed against saline, the complex of the present invention is about 20
Obtain a solution containing mg. The obtained complex is referred to as "complex AF."<Pharmacologicaltests> (1) Fibrinolytic activity test (a) The dissolving activity of Complex A obtained in Example 1 was measured by the fibrin plate method (Method A). The results are shown in Figure 1. In FIG. 1, the horizontal axis shows the urokinase activity (unit/ml) measured by the synthetic substrate method, and the vertical axis shows the diameter (mm) of the lysis circle measured by this method.
Further, in FIG. 1, line 1 indicates the composite A obtained in Example 1 of the present invention, and line 2 indicates LMW-UK (control). From the figure, the complex A of the present invention has a concentration of 10 units/ml.
It is clear that the fibrinolytic activity of LMW-UK is comparable to that of the control LMW-UK, and that it exhibits a higher lytic activity than the control at lower concentrations. (b) The dissolving activity of several of the complexes obtained in Examples 5 to 27 was measured by the buibbrin plate method (Method B). A similar lytic activity test was conducted in the presence of a plasmin inhibitor. The results are shown in Figure 2. In FIG. 2, the horizontal axis shows the urokinase activity (unit/ml) measured by the synthetic substrate method, and the vertical axis shows the diameter (mm) of the lysis circle measured by this method. Also, in Figure 2, lines 3 to 8
respectively indicate the following. 3; LMW-UK (control) 4; Complex E 5; Complex G 6; Complex N 7; LMW-UK + plasmin inhibitor 8; Complex E + plasmin inhibitor In addition, in the plasmin inhibitor addition group, M .Moroi et al.'s method [J.Biol.Chem.
251, 5956 (1976)] was added to 0.5 ml of the sample and measured in the same manner. From FIG. 2, it can be seen that the complexes E, G and N of the present invention
retains urokinase lytic activity and 1
Control LMW-UK at ~100 units/ml
Furthermore, complex E exhibits higher lytic activity than 4, even in the presence of plasmin inhibitor.
It is clear that at ~20 units/ml it exhibits higher lytic activity than LMW-UK. Incidentally, as a result of performing the same test on other complexes of the present invention obtained in each of the above-mentioned Examples, it was confirmed that all the complexes had substantially the same urokinase activity. In addition, the results of measuring the dissolution activity of the composites obtained in Examples 28 to 32 by the fibrin plate method (Method A) are shown in Figures 3 to 3.
It is shown in FIG. The vertical and horizontal axes in each figure are the same as in FIG. 1, and lines 9 to 17 in the figures indicate the following, respectively. 9; Complex AB 10; Complex AC 11; LMW-UK (control) 12; HMW-UK (control) 13; Complex AD 14; Complex AE 15; LMW-UK (control) 16; Complex AF 17 ;LMW-UK (control) From Figures 3 to 5, all complexes had stronger fibrinolytic activity than the control LWM-UK, and the difference was as the sample concentration decreased as shown on the horizontal axis. It can be seen that this becomes more noticeable. (2) Fibrin adsorption ability test (a) The fibrin binding ability of Complex A obtained in Example 1 was tested by the following method. i.e. fibrin-monomer-sepharose 6B (Hahne (D.
L.Heene) et al.'s method [Thrombosis
Research, 2 , 137 (1973)] filled with 3 ml of 0.135M NaCl.
After equilibration with 0.005M sodium phosphate buffer (PH = 7.4) containing , and wash with 30 ml of the above buffer.
Elution was performed with the above buffer containing 10mM-6-aminohexanoic acid, and plasminogen and plasmin HC were determined from the absorption at 280 nm of the eluate fraction, as well as complex A and HMW-UK.
The urokinase activity of the eluate fractions was measured by the synthetic substrate method, and the recovery rate for each elution fraction was determined, and this was taken as the fibrin adsorption capacity. As a result of the above test, Complex A of the present invention retains approximately 1/2 of the fibrin adsorption capacity of plasminogen (24%) and approximately 1/4 of plasmin HC (39%), and retains approximately 1/4 of the fibrin adsorption capacity of urokinase (approximately 1%). ) showed about 10 times stronger fibrin adsorption ability (10%). In addition, the complex AE is approximately 15% further complex
AF showed a strong fibrin adsorption capacity of about 80%. (b) The fibrin binding ability of the complex of the present invention is also
It was tested by the following method. i.e. fibrin-
Monomer - Sepharose 6B (same as above)
A column packed with 20 ml of 0.005 M sodium phosphate salt buffer (PH
= 7.4), each complex of the present invention, HMW-UK or plasmin HC (0.5 mg each) dissolved in the above buffer is applied, and washed with 200 ml of the above buffer. The above buffer containing 10mM-6-aminohexanoic acid and 2M-
Plasmin HC was eluted with the same buffer containing KSCN, and plasmin HC was determined by absorbance at 280 nm, and each complex and HMW-UK were determined by urokinase activity measured using a synthetic substrate method.
The percentage of the adsorbed content (eluted fraction) to the total recovery rate of the fibrin adsorbed content and non-adsorbed content is determined, and this is taken as the fibrin adsorption capacity. The results are shown in Table 4 below. [Table] Furthermore, when the same test as above was repeated for the composites obtained in each of the above Examples other than the composites shown in Table 4, all the composites showed approximately the same adsorption capacity (%). These results confirm that the complex of the present invention retains a strong fibrin adsorption ability derived from the fibrin adsorption protein. (3) Therapeutic effect on rabbit experimental pulmonary embolism This test was conducted according to the method of O. Matsuo et al. [Thrombosis Research, 24 , 347 (1981)]. Specifically, an artificial thrombus was prepared from fresh human blood mixed with ¹²⁵ I-fibrinogen, and this was injected into the rabbit jugular vein under anesthesia.
Create a pulmonary embolism model. Complex A obtained in Example 1 or HMW-UK as a control
Alternatively, physiological saline is administered through the ear vein, blood is collected over time, its radioactivity is measured, and its percentage is calculated from the total radioactivity of the administered thrombus. After 6 hours, the lungs are removed, the remaining thrombus is collected, and the lysis rate of the administered thrombus is determined from its radioactivity. Furthermore, the recovery rate of radioactivity in the lungs and urine is calculated. The results are shown in Table 5 and Figure 6 below. Lines 1 to 4 in the figure indicate groups to which the following substances were administered, respectively. 1; Physiological saline (control) 2; HMW-UK (30,000 units/Kg body weight, control) 3; Complex A (10,000 units/Kg body weight) 4; Complex A (30,000 units/Kg body weight) [ Table] As is clear from Table 5 above, HMW−
When administering UK30,000 units/Kg body weight, there was no difference at all from the case of physiological saline alone, but in the groups administered with Complex A of 30,000 units/Kg body weight and 10,000 units/Kg body weight,
The radioactivity in the blood was significantly high throughout the period in a dose-dependent manner, and the thrombolytic rate calculated from the radioactivity of the collected thrombi also increased in a dose-dependent manner, indicating a significantly high thrombolytic effect. I understand. In addition, no bleeding tendency was observed in the group administered with complex A. Furthermore, it was confirmed that the above test results were substantially the same when any of the complexes B to AF were used instead of the complex A.

[Brief explanation of the drawing]

Figures 1 to 5 are diagrams showing the fibrinolytic activity of the complex of the present invention. In FIG. 6, the therapeutic effect of the complex on experimental pulmonary embolism in rabbits was investigated by dissolving isotope-labeled thrombus, and the vertical axis shows the amount of isotope in the blood that appeared after dissolution.

Claims (1)

[Claims] 1. Fibrin-adsorbing protein and urokinase are
General formula: [In the formula, R is a phenyl group or a cycloalkylene group, A is a lower alkylene group, and B is a lower alkylene group that may have a lower alkylthio group or a phenyl lower alkylthio group as a substituent,
l, m and n each represent 0 or 1. However, 1, m
and n must not be 0 at the same time. ] A fibrin-adsorbing protein-urokinase complex, characterized in that the complex is bonded via a protein-adsorbing reagent represented by the following.