JPS6354386A - Phospholipid and use thereof - Google Patents

Abstract

NEW MATERIAL:A compound expressed by formula I (m represents a positive integer). EXAMPLE:2-Acetoxy-3-(7-carboxyheptyloxy) propyl 2-trimethylammonioethyl phosphate. USE:An antigen useful for preparation of an antibody specific to a platelet activating factor (PAF), which antibody is useful for an assay of PAF. PREPARATION:A compound expressed by formula II (x represents hydroxyl- protecting group; Ph represents phenyl) is reacted with a compound expressed by the formula Z-(CH2)m Y (Z represents Br, I, etc.; Y represents cyano, 2- oxazolinyl, etc.), followed by deprotection of the product and esterification of group Y thereof. Then the produced compound expressed by formula III (R represents 1-4C alkyl) is reacted with a compound expressed by formula IV, and the product is reacted successively with water and triethylamine. The obtained compound expressed by formula V (Me represents methyl) is then successively subjected to hydrolysis, hydrogenolysis and acetylation.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to phospholipids. More specifically, the present invention relates to glycerophospholipids useful in producing antibodies against platelet activating factor (PAF).

PRIOR ART AND PROBLEMS TO BE SOLVED In recent years, PAP expressed by the formula % [wherein n represents 15 or 17] is a phospholipid compound that exists in the living bodies of various animals including humans.
has been discovered, and its effects in living organisms are being studied. This compound has a strong platelet aggregation effect, as well as neutrophil activation and vascular permeability enhancement effects.

It has tissue damaging effects, blood pressure lowering effects, cardiac function suppressing effects, bronchoconstricting effects, etc., and is associated with various effects such as thrombosis, inflammation, nephritis, endotoxin syndrome, phylactic shock, angina pectoris, and myocardial infarction. It is known that it is closely related to diseases. Therefore, it is clinically extremely important to measure the blood PAF concentration of patients with these diseases. However, PAF is an unstable compound that exists only in trace amounts in the blood, and in addition, PAF such as lecithin is present in the blood.
There are a large amount of phospholipid compounds with a structure similar to F, and P
Quantification of AF is extremely difficult, and the fact that PAF has a spectroscopically non-characteristic structure is another reason why it is difficult to measure its concentration.

In order to establish a method for measuring PAF blood concentration by immunoassay, the present inventors developed condensation products of various phospholipid derivatives and proteins for the purpose of producing antigens from which antibodies specific to PAF could be produced. As a result of synthesizing and studying things, we completed the present invention.
-1. q-; knee □1≦), □p-Δ hill (:) ear-C1
! 20-(C1l,)m-COO110-1 where m is not a positive integer] and the compound [1] are administered to a warm-blooded animal other than humans, and the The present invention provides a method for producing antibodies, which comprises collecting antibodies against compound [+].

In the above formula [1], m is preferably a natural number of 30 or less. More preferably, it is a natural number of 20 or less, and most preferably an integer of 7 to 20.

The compound of the present invention [IE] can be synthesized, for example, by the method shown below.

[11] [III]
CIVkoO-〇- [\l]
[■CO[In the formula, Does not represent a lower (C, ,) alkyl group. Me represents medyl, and Ph represents phenyl. m is the same color as above &] Each step in the above reaction formula can all be performed using a known method. For example, in step [a], a compound of general formula [11] and a compound of general formula [■] Z (CHt)i Y [■]
[In the formula, Z represents a leaving group such as bromine, iodine, mesyloxy group, tosyluoquine group, etc. The reaction is carried out by reacting compounds in which m and Y have the same meanings as above. The base used at this time is generally sodium hydroxide, potassium hydroxide, sodium hydride, etc., and the solvent is generally tetrahydrofuran, dimethyl sulfoxide, etc.

Step [b] is a step of removing the protective group X of the alcohol and guiding Y to an ester. Protecting group X of alcohol is acid (e.g., inorganic acid such as hydrochloric acid, sulfuric acid, acetic acid).

It can be easily removed in the presence of organic acids (such as para-toluenesulfonic acid). Y can be easily converted into an ester by acid treatment in the presence of an alcohol (methanol, ethanol, etc.), and into a carboxyl group in the presence of water. The acid used at this time is preferably an inorganic acid such as hydrochloric acid or sulfuric acid. -Once a carboxyl group is obtained, it may be converted into an ester by esterification.

Step [Cl] is carried out, for example, by the following method. By reacting compound [■ and compound [IX] in the presence of a base, compound [ XI] [wherein m and R have the same meanings as above] is obtained, and then compound [V] is obtained by reacting with trimethylamine. In this case, compound [XI] can be obtained by using compound [■] as an active derivative and reacting it with compound [TV].

Step [d] is usually carried out by hydrolyzing compound EV] in the presence of a base such as sodium hydroxide or potassium hydroxide.

Step [e] is ethanol, acetic acid, ethyl acetate or
This is carried out by hydrogenolyzing compound [VI] in a mixed solvent thereof using, for example, palladium on carbon as a catalyst.

Step [fl] is carried out by reacting the compound [■] with an acetylating agent (eg, acetic anhydride, acetyl chloride, etc.) in the presence of a base such as pyridine or triethylamine. The reaction temperature is 00-100℃, preferably 0
The temperature is 6-40°C.

Although a typical method for producing compound [1] has been described above, the method for producing compound [I] of the present invention is not limited to only the above method.

The compound thus obtained has two configurations: R-configuration and S-configuration.
Although various optical isomers exist, in the production method of the present invention, R-isomers, mixtures of R-isomers and S-isomers, and racemates are preferably used.

Action The condensation of the compound [I] thus obtained with proteins (e.g., bovine serum albumin, human serum albumin, γ-globulin, hemocyanin, etc.) is carried out using the known acid anhydride method [J,
Immunol, Methods, 10,
61 (1976)] and the carbodiimide method [J.
Cl1n.

Endoct., 33, 171 (1971)]. The usage ratio (weight) of compound [1] and protein is preferably about 1:3. Also, the reaction time is 2
~6 hours is appropriate. The condensation product thus prepared can be stored by dialysis against water at around 4° C. and freeze-drying in a conventional manner.

When the condensation product produced as described above is administered to warm-blooded animals according to a conventional method, it is possible to efficiently produce antibodies that are specific to PAP and have good reactivity.

Examples of warm-blooded animals used include monkeys.

Examples include rabbits, dogs, guinea pigs, rats, sheep, goats, and chickens.

The condensation product is administered to a warm-blooded animal at a site where antibody production is possible, either by itself or together with a carrier or diluent, with subcutaneous injection being preferred. In order to increase antibody production ability during administration, complete Freund's adjuvant or incomplete Freund's adjuvant may be administered.

Antibodies are collected from blood, ascites (preferably blood), etc. of warm-blooded animals. Antibodies can be separated and purified using immunoglobulin separation and purification methods [e.g., salting-out method, alcohol precipitation method, isoelectric focusing method, N pneumophoresis method, adsorption/desorption method using an ion exchanger (e.g., DEAE), ultracentrifugation method, Gel filtration legal.

This is carried out according to a specific purification method in which only a specific antibody is collected using an antigen-antibody conjugate or an active adsorbent, and the binding is dissociated to obtain the antibody.

The obtained antibody can be subjected to a quantitative test for PAP.

For example, antibodies, tritium-labeled PAFC
H-PAF) and blood samples (diluted solutions may be used)
1'AFffi in the sample can be determined by radioimmunoassay or other immunoassay in which the radioactivity of the reacted 'H-PAF is measured.

The present invention will be explained in more detail with reference to production examples below, but the present invention should not be limited thereto.

Production Example 1 2-benzyl-1-(20-cyanoeicosyl)-3-
Tetrahydropyranylglycerin 2-bentrue-3-tetrahydropyranylglycerin 5.32g (20mmol
le), 1,20-dibromoeicosane 17,6
g (40 mmole) and 128 mg (0.4 mmole) of hexadecyltrimethylammonium chloride in 30 J of benzene and 50% sodium hydroxide aqueous solution a9.
6g (120mmole) was added and stirred at 100°C for 20 hours. After cooling, a mixture of n-hexane and ethyl acetate was added to the reaction mixture, the supernatant was removed by decantation, and the aqueous layer was further extracted with n-hexane and ethyl acetate. Combine the organic layers and wash with water.

Dry, concentrate and purify by silica gel chromatography to obtain 2-
6.88 g of benzyl-1(20-bromoeicosyl)-3-tetrahydropyranylglycerin was obtained.

D
The mixture was stirred at 100° C. for 1 hour in MS 040 J. The reaction solution was poured into cold water, extracted with a mixture of ethyl acetate and n-hexane, washed with water, dried, concentrated, and purified using silica gel chromatography. 5.3 g of a colorless waxy target product was obtained.

IR (film, cm-li: 3020, 2925,
285Q, 224Q, *460°1350.1200,
1118.103ONMR (103ON, CDCl,)
δ: 1.15-2.0 (42H, m).

2, 30 (2H, t), 3.35-4, 0 (9H
, +a), 4.61(Il, br), 4.70(
2H,s), 7.35(5B,m) Production Example 2 2-benzyloxy-3-(20-methoxycarbonyleicosyloxy)propertool 2.8g of the nitrile obtained in Production Example 1 was added to 20ml of methanol. While stirring, hydrochloric acid gas was blown into the solution to saturate it. Furthermore, 1 at room temperature
After stirring for an hour, the reaction solution was concentrated and 50 d of ether was added to the residue.

Concentrated hydrochloric acid was added and stirred at room temperature. The ether layer was washed with water, 2 parts of sodium bicarbonate solution, dried, concentrated, and purified by silica gel chromatography. Colorless waxy object 1
．． 80g was obtained.

I R (KBr, cm-'): 3450, 2920.2
850.1735, 1460°137G, 1240.1
110.104ON MR (90MHZ, CD
C13) δ: 1.15-1.75 (3611, m).

2.29 (21+, t), 3.41 (211, t
), 3.63(3H,s), 3.5-3.8(5
H, m), 4.65 (2tl), 7.31 (5H, m)
Production Example 3 2-benzyloxy-3-(20-methoxycarbonyleicosyloxy)propyl 2-trimethylammonioethyl phosphate 1.7 g of the alcohol obtained in Production Example 2 was dissolved in 20% toluene, and bromoethyl was dissolved under water cooling. 1.20 g of phosphodichloridate and 0.69 g of triethylamine were added and stirred at room temperature for 2.5 hours. The reaction solution was concentrated, 4 liters of INN hydrochloric acid was added and extracted with ether.
After washing with water and layer width, the residue was dissolved in 25 toluene containing 5 g of trimedelamine and stirred at room temperature for 3 days. The reaction solution was concentrated to dryness, and the residue was purified by silica gel chromatography (eluent: methanol). 1.70 g of the target product was obtained.

I R (KBr, am-'): 2925, 2850,
1740,1465,1235°1090.105O NMR (90M) (Z, CD C13) δ:
1.15-1.8 (36H, m).

2.30 (2H, t), 3.17 (9H, s), 3.4
1 (211,t), 3.85 (3H.

s), 3.5-4.08 (7H, m), 4.20 (21
! , m), 4.65 (2H, s).

7.31 (5H, m) Production Example 4 2-benzyloxy-3-(20-hydroxyeicosyloxy)propyl 2-trimethylammonioethyl phosphate 1.7 g of the ester obtained in Production Example 3 was dissolved in methanol. 2
5d was added with 25ml of dissolved IN sodium hydroxide and stirred at room temperature for 3 hours. Add acetic acid 2- to the reaction solution and concentrate.
The residue was purified by silica gel chromatography (eluent: methanol). 1.51 g of the target product was obtained. Colorless solid.

I R (K B r, cm-ri: 2925, 2850
, 1710, 1470, 1240° 1050.97O N M R (90 MHZ, CD Cl s +
CD s OD ) δ: 1.11.8 (36H, m)
, 2.27 (2H, t), 3.10 (911, s), 3
．． 2-4.3 (IIH, m), 4.7 (28, s), 7
．． 37(5H,+n) Production Example 5 3-(20-carboxyeicosyloxy)-2-hydroxypropyl 2-trimedelammonioethyl phosphate 990+ng of the benzylic compound obtained in Production Example 4 was mixed with 25 mL of ethanol, 20 d of acetic acid, Dissolve in a mixture of 5 d of water and lO%
Hydrogenolysis was carried out using 200 mg of palladium on carbon. The catalyst was filtered off, the reaction solution was concentrated, and acetone was added to perform reprecipitation to obtain 796 mg of the desired product as a colorless powder.

I R (K B r, cm-t: 2910, 2g45
．． 17QO, 1465, 1210° 1075.105O NMR (105ON, CDCl3+CD30D) δ:
1.1-1.75 (3611, m), 2.27 (2
1+, t), 3.20(911,s), 3
．． 35-3.7 (6H, m), 3.8-4.05 (3H
, m), 4.25 (2H, m) Production Example 6 2-acetoxy-3-(20-carboxyeicosyloxy)propyl 2-trimethylammonioethyl phosphate 770 mg of the compound obtained in Production Example 5 was added to pyridine 6. ,
The mixture was stirred at 80°C for 12 hours in a diluted acetic anhydride mixture. The reaction solution was concentrated to dryness, and the residue was purified by silica gel chromatography. Re-precipitate from chloroform-methanol-acetone to obtain the desired product 497 as a colorless powder.
mg was obtained.

I R (KBr, cm""): 2910, 2g40,
1730,1462.1372゜1232.1080.
1050.965NMR (90MHZ, CD
Cl s + CD s OD ) δ: 1.2-1
．． 8 (36H, n+), 2.07 (3H, s), 2.2
7(2H,t), :(,21(9H.

s), 3.35-3.70 (6H, m), 3.99 (2
H), 4.27 (2H, m).

5.13 (IH, quint) Production Example 7 2-benzyl-1-[7-(4,4-dimethyl-2-oxacylyl)hebutyl-3-tritylglycerin 2-benzyl-1-)lythylglycerin 10.5 g, 2
-(7-bromoheptyl')-4,4-dimethyl-2-
8.3 g of xacillin was dissolved in a mixed solution of 50 dDMSO and 30 dTHF, and 8.4 g of KOH powder was added at once. After stirring vigorously at room temperature for 3 hours, the mixture was poured into cold water, extracted with a mixture of n-hexane and ethyl acetate, washed with water, dried, concentrated, and purified by silica gel chromatography to obtain 11.5 g of the desired product. Colorless liquid.

I R (film, c+++-'): 3050.30
20.2925, 2855.1665°1492, 14
50.1365, 1120.1092N M R (90
MHz, CD C13) δ: 1.23 (6H,
s), 1.2-1.8 (IOH, m), 2.23 (21
1, t), 3.2-3.8 (7H, m), 3.86 (2
H, s), 4.64 (2H, s) Production Example 8 2-benzyl-1-(7-medoxycarbonylheptyl)glycerin 10 g of the trityl compound obtained in Production Example 7 was mixed with 60% acetic acid 10
It was combed at 0 am and stirred at 85°C for 1.5 hours. The crystals precipitated by cold soaking were filtered off, and the filtrate was concentrated to dryness. The residue was made into a chloroform solution, neutralized with aqueous sodium bicarbonate, dried, condensed, and purified by silica gel chromatography to obtain colorless oily 2-
5.0 g of benzyl-3-[7-(2-methyl-2-aminopropyloxycarbonyl)hebutyl]glycerin was obtained.

700 mg of this compound was diluted with methanol and
Add 5.1 d of sodium methoxide and heat under reflux for 3 hours. Neutralized with cold soaked acetic acid, concentrated, and purified by silica gel chromatography to obtain colorless oily product 1-1.
Obtained 63 mg j-0I R (Jiln, em-'):
3440.3015.2920, 2850, 1730°1492, 1450.1435.1360.1250.
1200.111ONMR (90MHz, CD C13
) δ 2 1.30-t, 8(to)(,+τ1).

2.28 (2B, t), 2.50 (IH, br)
, 3.41(211,tl 3.5-3.Pi(
5H, m), 3.63 (3+!, s), 4.65 (2H
, d), 7.32 (5H) Production Example 9 2-benzyloxy-3-(7-medoxycarbonylhebutyloxy)propyl 2-trimethylammonioethyl phosphate 2501?1 g of the alcohol obtained in Production Example 8 was The same reaction and purification as in Production Example 3 were carried out to obtain the target product 365.
I got a B. Colorless waxy.

I R (KBr, cm-'): 3370, 3020.
2925.2850, 1732°1480.1230.
108g, 1Q50,97ON M R (90M HZ
, CD CI3) δ: 1.29-1.8 (IOH,
m).

2, 28 (21+, t), 3.27 (9H, s)
, 3.4-3.77 (7B, m), 3.95 (
211, m), 4.28 (2H, m), 4.66 (2H
, s), 7.32 (5H, 111) Production Example 10 2-acetoxy-3-(7-carboxyheptyloxy)propyl ll-limethylammonioethyl Posfu s, -h The compound obtained in Production Example 9 , Hydrolysis, debenzylation, and acedelation were performed in the same manner as in Production Examples 4 and 5.6,
177 mg of a colorless wax-like target product was obtained.

I R (K B r1cm''''): 2915.28
50,172+3.1552.147g.

1370, 1238.1085.1060,970,7
2ONMR[90MH2, CDC15+CD30D(1
:3)] δ: 1.35-1.8 (1011, m),
2.07 (311, S), 2.25 (21i, t
), 3.23 (9H, s), 3.47 (2H, t
), 3.5-3.6 (411, m), 4.01 (
211, m).

4.29 (211, +n), 5.15 (ill, Qu
int) Production Example 11 2-acetoxy-3-(7-carboxyheptyloxy)propyl 2-acetoxy-3-(7-carboxyheptyloxy)propyl 2-Trimedelammonioethyl phosphate condensate and bovine serum albumin 2-acetoxy-3-(7-carboxyheptyloxy)propyl 2 - Trimethylammonioethyl phosphate (72 mg) and bovine serum albumin (200 m
After dissolving each of g) in 100 ml of water and mixing, hydrochloric acid/l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (188 mg) was added, and the mixture was stirred at room temperature for 4 hours. Next, 190+ng of the target product was obtained by dialysis (water, 2Q, X5) at 4°C, followed by lyophilization.

Production Example 12 2-acetoxy: 3-(20-carboxyeicosyloxy)propyl 2-trimethylammonioethyl
Condensate of phosphate and bovine serum albumin 2-acetoxy-3-(20-carboxyeicosyloxy)propyl 2-trimethylammonioethyl phosphate (50 mg) was dissolved in ethanol (4 ml), and bovine serum albumin (100 n+g) was dissolved. Aqueous solution (water: 4
5d), hydrochloric acid/1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (91D) was added, and the mixture was stirred at room temperature for 2 hours.

Next, dialysis (water, 2 (2×5) l) and lyophilization at 4° C. yielded the desired product 95B.

Production Example 13 Production of anti-PAP antibody 2 mg of the condensate obtained in Production Example 11 was dissolved in 1 ηJ of physiological saline and added to complete Freund's adjuvant (Preundco
Complete adjuvant) [Paraffinura containing lanolic acid and killed tuberculosis bacteria] was added and mixed well to prepare an emulsion, which was injected subcutaneously into several places in rabbits. One month later, an emulsion was prepared in the same manner using incomplete Freund's adjuvant [paraffin oil containing lanolic acid] and injected subcutaneously into the rabbit. This operation was repeated 5 times at monthly intervals, and 10 days after the final injection, heparinized blood was collected, plasma was obtained by a conventional method, and then the method of Steinbuch et al. [Aeh, Biochem.

Biophys, 134. 279 (1969)]
Anti-PAF antibody (IgG) was purified using That is, 10 volumes of plasma and 20 volumes of acetate buffered RCI (60mM
, l'184.0) and 750 μQ of caprylic acid were mixed and stirred for 30 minutes. The supernatant obtained by centrifugation at 3000 rpm for 15 minutes at 4°C was diluted with 15 mM sodium acetate.
was adjusted to 5.7, and then dialyzed against 15mM acetate buffer (pH 5,7) 6σ at 4°C. After repeating this operation 5 times, 1.2 g of diethylaminoethyl cellulose was added to the solution containing the dialyzed IgG and stirred for 15 minutes.
Filtered through a glass filter.

The obtained filtrate was concentrated using a membrane (Centriflow) (protein 5%: 2, 9 mg/d).

Radioimmunoassay was performed using the IgG fraction purified above. That is, Buffer 1 (100mM
Phosphate buffer, pH 7.4; 0.1% gelatin, 0.9
IgG fraction (protein content: Q, 01 mg/d) diluted with 5% NaCl and 0.01% NaNs)
After mixing 0 μg and various amounts of PAF, lysoPAF, and Bu[er I containing L-α-lecithin, 'H-PAF(
Specific activity: Buffer I (40,000 dpm) containing 90 Ci/ +++++++ole)
Q was added and left at room temperature for 1 hour. Further, leave at 4°C for 1 hour, then cool with water using 100 μQ of dextran charcoal.
was added and 10 minutes later, the mixture was centrifuged at 3,000rpo+ for 10 minutes. Supernatant 18 using a liquid scintillation counter
0 μg of 'H radioactivity was measured.

PAF of 0.11-1O0n has 3H radioactivity (3H-P
AP and anti-1'AFii'i1. lyso-PAF and L-α-lecithin had little effect on 3H radioactivity up to 11000n. In other words, this antibody was shown to specifically react with PAF.

Effects of the Invention The compound [1] of the present invention is useful for producing antibodies that specifically react with PAF, and the produced antibodies are useful for quantifying PAF.

Claims (2)

[Claims] (1) Formula ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ A compound represented by the formula [wherein m represents a positive integer]. (2) A compound represented by the formula ▲ There are mathematical formulas, chemical formulas, tables, etc. A method for producing an antibody, which comprises collecting the antibody.