JPS5946485B2 - anti-complement activator - Google Patents

Description

[Detailed description of the invention] The present invention relates to anti-complement activators.

More specifically, the present invention shows the general formula [wherein R^1 represents a hydrogen atom or a group H (R^2 represents a hydrogen atom or a hydroxymethyl group, and R^3 represents a hydrogen atom or a rhamnopyranosyl group].

The present invention relates to an anti-complement activator characterized by containing as an active ingredient at least one Soyasapogenol B derivative represented by the following and its salts.

Glycyrrhizin is a compound related to the compound represented by the above general formula (E) [Yasuhiro Ariga, Hiroyuki Sumi, Yumiko Takada-Meiwa Takada, 17th Plasmin Study Group Program Lecture Abstracts, p. 65 (1977); Arimoto Notsugu, Tomoyuki Mineta, Hiroyuki Sumi, Yumiko Takada, Meiwa Takada, Proceedings of the 14th Complement Symposium, pp. 79-82 (1977)] and soyasaponin [Isao Kitagawa, Masayuki Yoshikawa, Parti Yoshioka,
Chem. Pharm. Bull. , Volume 22, No. 13
Page 39 (1974); Volume 24, Page 121 (1974)
1976)] etc. are known.

The former compounds are said to have steroid-like structures and steroid-like actions, such as plasmin,
It has inhibitory effects on urokinase, kallikrein, thrombin and complement. On the other hand, there have been no reports on the physiologically active effects of the latter compound. On the other hand, the compound of general formula () has a significantly superior anti-complementary activity compared to liglycyrrhizin, which is clear from the pharmacological test results described later, and also has an anti-complementary activity that is completely unexpected from soya saponin. It has an active effect. The present invention has been completed based on novel findings. The anti-complement activator of the present invention is used to treat immunological diseases such as rheumatoid arthritis, systemic erythematous lupus, nephritis, autoallergic hemolytic anemia, platelet disorders, and vasculitis, and to improve complement function. It is used to improve or prevent pathological reactions caused by

The anti-complement activating agent of the present invention can also be used to treat non-immunological diseases such as paroxysmal nocturnal hemoglobinuria, hereditary angioneurotic edema, and coronary artery occlusion induced by the action of bacteria or lysosomal enzymes on appropriate complement components. It is used to treat subsequent inflammation, etc. The anti-complement activating agent of the present invention can also be used in the treatment of transplant rejection and blood culture, and can also be used as a blood transport medium. In recent years, theoretical analysis of complement and its effects on the human body or animals has been actively conducted, and it is now known that if a certain compound has an anti-complement activity, it can be used to cause the various symptoms listed above. It is generally accepted that it has therapeutic effects.

For example, Japanese Patent Application Laid-Open No. 53-9325 has the following description. ``Complement'' is a substance in body fluids that, in cooperation with antibodies or other factors, plays an important role in mediating immune, allergic, immunochemical, and/or immunopathological reactions. It is a complex of proteins.

Reactions involving complement occur in serum or other body fluids and are therefore considered humoral reactions. 1 for human blood
One or more proteins are known to be part of the complement system.

These complement proteins are designated by the letter C and a number. They are Cl, C2, C3 to C9. Complement protein C1 actually consists of subunits Clq, Clr and Cl
It is a collection of things called s. The numbers on complement proteins indicate the order in which they are activated, with the exception of C.
Only 4 reacts after C1 and before C2. The proteins in the complement system were given numerical symbols before the order of their reactions was fully understood. A more detailed discussion of the complement system and its role in the body can be found in the following literature examples: B
ullWOrldHeaIthOrg. ,39935～
938 (1968); Scientific Ameri
can, ldan' (ya 5) 54-66 (1973);Me
dicalWOrldNews, lOMon 11th 53~
58, 64-66 (1974); Harvey Lect
ures4” 75-104 (1972); TheNe
wEnglandJournaIOfMedicine
,2j7489~495,545~549,592~5
96, 642-646 (1972); TheJOhns
HOpkinsMed. J. , Ldan'', 57-74 (1
971); and FederationOnPrOceed
ingsl no. 134-137 (1973).

The complement system is thought to consist of the following three subsystems: (1
) Sensing unit (Clq), which has the ability to bind to antibody molecules that detect foreign invaders; (2) Activating unit (
Clr, Cls, C2, C4, C3) which provides positions on adjacent membranes; and (3) attack units (C5, C6
, C7, C8 and C9) This creates holes in the membrane. Units that attack membranes do not choose a specific opponent, destroying intruders simply because they spawn near them. The duration of its activity must be limited to minimize damage to the host's own cells. This limitation is achieved partly by naturally occurring attenuation of activated complement and partly by interference with inhibitors and destructive enzymes. However, limiting complement is not complete, and therefore damage to host cells often occurs. Damage to host cells can therefore often occur. Immunity is therefore a double-edged sword. Activation of the complement system also promotes blood clotting.

This effect occurs through complement-mediated release of coagulation factors from platelets. Biologically active complement fragments and complexes are engaged in reactions that damage host cells, and these pathogenic reactions result in immune complex diseases. For example, in some types of nephritis, complement destroys the basal membrane of the kidney and proteins in the blood escape into the urine. Disseminated erythematous lupus is of this kind; its symptoms are nephritis, visceral lesions and skin rash. Injecting large amounts of antitoxin to treat diphtheria and tetanus often results in serum sickness, an immune complex disease. Rheumatoid arthritis is also due to immune complexes. Like disseminated erythematous lupus, this is an autoimmune disease in which symptoms are produced by pathological effects of the immune system in the host tissues. In summary, the complement system is known to be involved in inflammation, coagulation, fibrinolysis, antibody-antigen responses, and other metabolic reactions. In the presence of antibody-antigen complexes, complement proteins participate in a series of reactions that irreversibly destroy biological cell membranes when they come near them.

Complement thus forms part of the body's defense mechanism against infection, but on the other hand it causes inflammation and tissue destruction through immunopathological processes. Comments on the nature of some complement proteins, the manner in which complement binds to biological cell membranes, and the manner in which complement destroys membranes can be found in the Annual Review.
ewinBiOchemistry, 38389 (19
69). The known complement inhibitors epsilon-aminocaproic acid, suramin sodium and tranexamic acid are effective in treating hereditary angioneurotic edema (a condition resulting from genetic deficiency or loss of function of the first component inhibitor of complement). It has been reported that T
heNewEnglandJournalOfMedi
808-812 (1972): Al
lergOlEt. ImmunOpath, Hl63~
168 (1974); J. AllergyClln. I
mmunOl. Jf6.5, 298-302 (1974
); and AnnalsOfInternalMedi
cine, Encounter 580-593 (1976).

” The compound of general formula (1) is a known or new compound.

These compounds can be prepared, for example, by the method of Kitagawa et al. [Isao Kitagawa, Masayuki Yoshikawa, Parti Yoshioka, Chem. Pharm・Bull. , Vol. 22, p. 1339 (1974); see Vol. 24, p. 121 (1976)] or by the method of Kitagawa et al. Manufactured by 〜、、(In the formula, R1 is the same as above.) Conventionally known reaction conditions for ester hydrolysis can be widely applied to the hydrolysis of the compound of general formula (). Usually done.

Any conventional catalyst can be used, specifically mineral acids such as hydrochloric acid, sulfuric acid, nitric acid, sodium hydroxide, potassium hydroxide, sodium carbonate, sodium hydrogen carbonate, potassium carbonate, potassium hydrogen carbonate, etc. Examples include inorganic basic compounds. Preferably, an inorganic salt-rising compound is used as a catalyst. Any commonly used inert solvent can be used, specifically water, lower alcohols such as methanol, ethanol, propanol, etc.
Examples include ethers such as dioxane and tetrahydrofuran, dimethyl sulfoxide, dimethyl formamide, and mixed solvents thereof. This hydrolysis usually occurs at room temperature to 1
The reaction is preferably carried out at 50°C, preferably from 50 to 110°C, and the reaction time is usually about 1 to 6 hours. General formula (1)
Representative compounds among the compounds are as follows.

03-0-(β-D-glucuronopyranosyl)-Sawyasapogenol BO3-0-[β-D-galactopyranosyl (1→2)-β-D-glucuronopyranosyl]- Soyasapogenol BO3-0-[α-L-arabinopyranosyl (1→2)-β-D-glucuronopyranosyl]-Sawyasapogenol BO3-0-[α-L-rhamnopyranosyl ( 1→2) -β-D-galactopyranosyl (1→2
)-β-D-glucuronopyranosyl]-Sawyasapogenol B03-0-[α-L-rhamnopyranosyl (1→2
)-α-L-arabinopyranosyl (1→2)-β-D-
Glucuronopyranosyl]-Sawyasapogenol B The compound of general formula (1) can form salts with various pharmaceutically acceptable basic compounds.

Examples of basic compounds that can be used for the above salt formation include inorganic basic compounds such as sodium hydroxide, potassium hydroxide, aluminum hydroxide, sodium carbonate, potassium carbonate, and sodium hydrogen carbonate, as well as piperazine, morpholine, piperidine, ethylamine, dimethyl Examples include organic basic compounds such as amine and triethylamine. General formula ()
When the compound and its salt are used as an anti-complement active agent, they are usually put into the form of a pharmaceutical composition together with a pharmaceutical carrier.

The carrier includes diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, and lubricants that are commonly used to prepare drugs according to the usage form. do. The dosage unit form of the anti-complement active agent of the present invention can be appropriately selected depending on the therapeutic purpose. Typical examples include tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, suppositories, and injections (solutions, emulsions, suspensions, etc.).
When forming tablets, known carriers such as lactose, sucrose, sodium chloride, glucose, urea,
Excipients such as starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid; water, ethanol, propanol,
Simple syrup, glucose solution, starch solution, gelatin solution,
Binders such as carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone; dried starch, sodium alginate, agar powder, laminaria powder, sodium bicarbonate, calcium carbonate, twine, sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose Disintegrants such as white sugar, stearin, cocoa butter, hydrogenated oils, etc. Absorption enhancers such as quaternary ammonium bases, sodium lauryl sulfate; Moisturizing agents such as glycerin, starch, starch, lactose, kaolin, etc. Adsorbents such as bentonite and colloidal silicic acid; lubricants such as purified talc, stearate, boric acid powder, macrocol, and solid polyethylene glycol can be used. When forming into a pill form, known excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, and talc are used as carriers; combinations of gum arabic powder, tragacanth powder, gelatin, ethanol, etc. Agent: Disintegrants such as laminaria and agar can be used. Furthermore, the above-mentioned tablets can be coated with a conventional coating, if necessary, to form sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, film-coated tablets, double tablets, or multilayer tablets. When forming a suppository, known carriers such as polyethylene glycol, cacao butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerade, etc. can be used. When forming liquids and suspensions used as injections, as a diluent,
Conventional substances such as water, ethyl alcohol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene solpit, zorbitan ester, etc. can be used. Further, it is preferable that the liquid preparation is sterilized and isotonic with blood. In this case, a sufficient amount of salt, glucose, or glycerin to prepare an isotonic solution is included in the solution, or conventional solubilizing agents, buffers, soothing agents, preservatives, etc. are added as necessary. Coloring agents, preservatives, fragrances, flavoring agents, sweetening agents, and other pharmaceutical agents can be incorporated into the therapeutic agent. Active ingredient to be contained in the anti-complement activator, i.e. general formula (1)
The amount of the compound or its salt is not particularly limited and can be selected as appropriate, but is usually 1 to 70% by weight of the entire composition. , preferably 5-5
Preferably, it is 0% by weight.

The above-mentioned anti-complement activators are administered in various ways depending on the form.

For example, oral administration can be adopted in the case of tablets, pills, solutions, suspensions, emulsions, granules, and capsules. In the case of an injection, it may be administered intramuscularly, intradermally, subcutaneously, or intraperitoneally. In the case of suppositories, it is administered rectally. The dosage of the anti-complement activator is appropriately selected depending on the purpose of use, symptoms, etc., but the amount of the active ingredient is usually 0.5 to 20 WII per day! /I
The amount may be within the range of about <g, and sufficiently safe therapeutic effects are shown when used within this range.

In order to clarify the present invention more clearly, the results of pharmacological tests on the compounds of the present invention are listed below.

1) Test compound A. 3-0-(β-D-glucuronopyranosyl)-Sawyasapogenol BB. 3-0-[β-D-galactopyranosyl (1→2)-β-D-glucuronopyranosyl]-Sawyasapogenol BC. 3-0-[α-L-rhamnopyranosyl (1→2)-β-D-galactopyranosyl (1→2)-β-D-glucuronopyranosyl]-Sawyasapogenol BD. 3-0-[α-L-arabinopyranosyl (1→2)-β-D-glucuronopyranosyl]1-
Soya Sapogenol BE. 3-0-[α-L-rhamnopyranosyl (1→2)-α-L-arabinopyranosyl (1
→2) β-D-glucuronopyranosyl]-Sawyasapogenol BIF. Glycyrrhizin (comparative compound) 2) Anti-complement activity The anti-complement activity was measured and confirmed according to the test method described in "Immunochemistry", pages 830-834 (published by Asakura Shoten, edited by Yuichi Yamamura et al., 1973). recognized.

That is, 0.5 ml of an aqueous dispersion of the test compound in a test tube, 1×1
08 cells/WLI of sensitized blood cells (EA) 0.5 ml of 5-fold dilution of veronal-buffered saline containing isotonic gelatin (G
1 ml of VB++) and 0.57 n1 of complement serum (g-P・2c) diluted 150 times with GVB+8 diluent were collected and incubated at 37°C for 60 minutes, then diluted with 5 ml of ice-cold physiological saline.
1 and centrifugation, and then measuring the absorbance of the separated supernatant using an absorbance meter 0D413 to determine how much hemolysis of sensitized blood cells is inhibited by the test compound. be done. Table 1 shows the anti-complement activity (50% hemolysis inhibition activity value, γ/d) measured according to the above method.
3) Toxicity test Test compounds A to E were administered intraperitoneally, and three test compounds were administered intravenously to measure acute toxicity (LD5O value, Wlf7/I<g) to mice.

The results are shown in Table 1 below. As is clear from Table 1 above, the compounds A to E of the present invention exhibit significantly superior anti-complement activity compared to Compound P (comparative compound).

Therapeutic effect on O nephrotoxin (NephrOtOxin) type nephritis Rat nephrotoxin (hereinafter abbreviated as "NT") can be obtained as follows.

Mouse kidney cortex (KidneycOrt)
ex) with an equal volume of physiological saline, and the homogenate was blended.
ifcO) at a ratio of 1:1, and 2 m2 of the resulting mixture was intramuscularly injected into rabbits (body weight 3100 fl) to immunize them.

After 1.5 months, blood was collected from the rabbit's heart to obtain serum.

After inactivating the obtained serum at 56°C for 30 minutes, 40%
Fractionate by salting out with saturated ammonium sulfate. A fraction of imino γ-globulin (1fG) is collected to obtain NT. In the test, Wlst breeds weighing 150 to 160 years were tested.
er) male mice are used. The compound of the present invention was administered 1 hour before NT administration, and every 24 hours,
The dose is intraperitoneally administered once per day from 3 days before to 10 days after the administration. In addition, 1 ml of NT was intravenously injected into the tail vein of the mouse. Physiological saline is used as a control. PrOteinuria (total amount excreted in the urine in 24 hours) was purified by BOvineSerumAlbumin (BOvineSerumAlbum) with sulfosalicylic acid.
Measured by turbidity method using in) as a control. The results obtained are shown in Table 2. Note that the number of days is the number of days that have passed since administration was administered 1 hour before NT administration. Also, the amount of protein uria is ~
/day. Protein uria in a mouse under normal conditions is 0.5
~5η/day, but if it exceeds this value, especially 10W
If it is 9 days or more, it can be said that nephritis has developed. As is clear from Table 2, nephritis developed in the control group, and in the case of the compound of the present invention, the amount of proteinuria for 10 days from the time of administration was almost the same as in mice under normal conditions, and the primary It can be seen that the reaction and secondary reactions are suppressed.

]) Male mice of the Wista strain weighing 180 to 200 y are used for testing the therapeutic effect on Heymann type nephritis.

Remove the mouse kidney cortex and homogenize it with an equal volume of physiological saline. 1500 ml of the homogenate
G for 1 hour, and the supernatant was collected using the method of Edginton et al. [T. S. EdgingtOnetal, Journ
alOfExperimentalMedicinl4
A mixture of ``Blend Comptoadjuvant 37Ra'' (manufactured by DifcO) at a weight ratio of 0.4:1 was used as an adjuvant, and 0.5111 was added to the intraperitoneal cavity of a mouse. Inject inside. Thereafter, administer the same amount of this adjuvant every 2 weeks until protein uria is 100 or more per day (6 to 8 weeks).
. Two doses of the compound of the present invention were administered to mice (weighing approximately 300 to 3,500 kg) that had been decapitated to develop Heymann's nephritis.
A dose of 3 w/mouse was administered intraperitoneally every 4 hours for 7 days, and the amount of proteinuria (WIV' days) was measured in the same manner as above. One test was conducted in the same manner using physiological saline as a control group. Table 3 shows the results obtained by repeating the same test three times. Two to three weeks after the start of the experiment, the weight of the mouse was 40.
0 to 500r, and the normal amount of proteinuria is 5 to 15
It is thought that Tf9/day.

As is clear from Table 3, it can be seen that the compounds of the present invention have the effect of curing Heymann type nephritis. Reference Examples and Examples will be given below to explain the present invention in more detail.

Reference Example 3-71Wlg of 0-(6-0-methyl-β-D-glucuronopyranosyl)-Sawyasapogenol B was dissolved in 2d of methanol, and 7l of 1N-NaOIi<. Add solution 2a and reflux for 2 hours.

Add cold water to the reaction mixture and adjust the pH to about 1 with 1N-HCI aqueous solution.
and extract with n-butanol. The n-butanol layer was concentrated to dryness under reduced pressure, and the residue was dissolved in chloroform-acetone (
1:1 volume ratio) to give 50η of 3-0-(β-D-glucuronopyranosyl)-Sawyasapogenol B.
get. o Melting point 231-232°C (decomposition) O elemental analysis value Calculated value as C36H58O9 (to) C68. ll, H9.2l actual measurement value (to)
C67.85, H9. O8 O silica gel thin layer chromatography (using "Kiesel Gel F254", manufactured by Talc) 1. Chloroform-methanol monohydrate (65:35:8 volume ratio) Rf=0.382
．． Isopropanol-2N ammonia water (100:15
Volume ratio) Rf = 0.21 0 Solubility Easily soluble in methanol, ethanol, n-propanol, n-butanol, alkaline aqueous solution, pyridine, dimethyl sulfoxide, dimethyl formamide, soluble in acetone, ethyl acetate, methyl ethyl ketone, It is sparingly soluble in benzene, chloroform, diethyl ether, n-hexane, and petroleum ether.

The compound of the present invention and glucose are dissolved in distilled water for injection and then injected into a 5TI11 ampoule.

After purging with nitrogen, autoclaving is performed at 121° C. for 15 minutes to obtain an injection. The compound of the present invention is added to a semi-synthetic glycerade base, mixed and suspended at 50°C, poured into a mold, allowed to cool naturally, and taken out to obtain a suppository.

The compound of the present invention, Abysiel, cornstarch, and magnesium stearate are mixed, polished, and then tableted using a sugar-coated RlOmlL kine.

Claims (1)

[Claims] 1 General formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [In the formula, R^1 is a hydrogen atom or a group ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (R^2 is a hydrogen atom or hydroxymethyl (R^3 represents a hydrogen atom or a rhamnopyranosyl group). ] An anti-complement activator characterized by containing at least one Soyasapogenol B derivative represented by the following and its salt as an active ingredient.