JPS5948811B2 - anti-complement activator - Google Patents

Description

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

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

R^4 represents a lower alkyl group. ] The present invention relates to an anti-complement activator characterized by containing a soya savogenol B derivative represented by the following as an active ingredient. In the above general formula (I), the lower alkyl group represented by R^4 has a carbon number of 1
-6 straight-chain or branched alkyl groups can be mentioned, and specific examples include methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl, and hexyl groups. Compounds related to the compound represented by the above general formula (1) include glycyrrhizin [Yasuhiro Ariga, Hiroyuki Sumi, Yumiko Takada, Meiwa Takada, 17th Plasmin Study Group Program Lecture Abstracts, p. 65 (1977); Motonitsugu, Tomoyuki Mineta, Hiroyuki Sumi, Yumiko Takada, Meiwa Takada, Proceedings of the 14th Complement Symposium, Nos. 79-82
(1977)] and soyasaponin [Isao Kitagawa,
Masayuki Yoshikawa. Part Yoshioka, Chem. Pharm. Bull
5, Vol. 22, p. 1339 (1974); see Vol. 24, p. 121 (1976)].

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 yet on the physiologically active effects of the latter compound. On the other hand, the compound of general formula (1) has a significantly superior anti-complementary activity compared to glycyrrhizin, as is clear from the pharmacological test results described later, and also has an anti-complementary activity that is completely unexpected from soya sabonin. It has an effect. The present invention was completed based on this knowledge. The anti-complement activator of the present invention can be used to treat rheumatoid arthritis, systemic lupus erythematosus, nephritis,
It is used to treat immunological diseases such as autoallergic hemolytic anemia, platelet disorders, and vasculitis, and to improve or prevent pathological reactions that require complement function.

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. Furthermore, it can be used as a therapeutic agent for systemic coagulation syndrome. 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 complex of proteins in body fluids that, in cooperation with antibodies or other factors, plays an important role as a mediator of immune, allergic, immunochemical, and/or immunopathological reactions. It is the body.

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
ullWOrldHealthOrg. ,39935～
938 (1968); Scientific Ameri
can, 229 (Black 5) 54-66 (1973); Me
dicalWOrldNews, lOMon 11th 53~
58, 64-66 (1974); Harvey Lect
ures, 6675-104 (1972);
wEngIandJOwnalOfMedicine
, 287489-495, 545-549, 592-5
96, 642-646 (1972); TheJOhns
HOpkinsMed. J. , l28, 57-74 (1
971); and FederationOnPrOceed
ings, 32l34-137 (1973).

The complement system is thought to consist of the following three subsystems; (1
) Clancy unit (Clq), which has the ability to bind to antibody molecules that detect foreign invaders; (2) activation unit (Clq);
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. In order to minimize damage to the host's own cells π, the duration of its activity must be limited. 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. Immunity is therefore a double-edged sword. Activation of the complement system also promotes blood clotting.

This effect is caused by 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 lupus erythematosus is of this type; 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 lupus erythematosus, this is an autoimmune disease in which the symptoms are caused by pathological effects of the immune system in the host tissues. In summary, the complement system is involved in inflammation,
It is known to be involved in coagulation, fibrinolysis, antibody-antigen reactions 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 causes inflammation and tissue destruction through immunopathological processes. Comments on the nature of certain complement proteins, the manner in which complement binds to biological cell membranes, and the manner in which complement destroys membranes are presented in the Annual Review.
winBlOchemistry, 38, 389 (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 a genetic deficiency or loss of function of the first component inhibitor of complement). It has been reported that it is used in TheNewEnglan.
dJOurnalOfMedicine2868O8~
812 (1972); AllergOlEt. Immu
nOpath, 163-168 (1974): J. A
llergyClln. ImmunOl. K5,298
~302 (1974); and AnnalsOfInt.
enmlMedicine, 84, 58O-593 (1
976).

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

Specifically, the compound of general formula (1) can be prepared by the method of Kitagawa et al. [Isao Kitagawa, Masayuki Yoshikawa, Parti Yoshioka, Chem. Pharm. Bu
ll. , Vol. 22, p. 1339 (1974): Vol. 24, p. 121 (1976)] or according to the method of Kitagawa et al.

For example, soya sabonin B (sawya sabonin I described in the above-mentioned document having soya savogenol B as an aglycone,
The compound of general formula (1) is produced by alcoholysing (meaning a mixture of Reaction Scheme-1) Reaction Scheme-1 υ8i [In the formula, R1 and R4 are the same as above.

] As the acid used for the alcoholysis of Soyasabonin B (), a wide range of acids commonly used for alcoholysis can be used, and specific examples include hydrogen chloride, hydrogen bromide, etc., hydrogen logenide, etc. Strong inorganic acids such as sulfuric acid and nitric acid, trichloroacetic acid,
Examples include strong organic acids such as trifluoroacetic acid, Lewis acids such as aluminum chloride, boron trifluoride, titanium tetrachloride, and titanium tetrabromide.

This alcoholysis is usually carried out at room temperature to 150°C, preferably 50 to 100°C, and the reaction time is usually 1
~6 hours. Representative compounds of general formula α) are as follows.

03-0-(6-0-methyl-β-D-glucuronopyranosyl)-Sawya savogenol BO3-0-(6-0-
Ethyl-β-D-glucuronopyranosyl)-Sawyasapogenol BO3-0-(6-0-hexyl-β-D-glucuronopyranosyl)-Sawyasapogenol BO3-0
-[β-D-galactopyranosyl (1→2)-(6-0
-Methyl-β-D-glucuronopyranosyl)]-Sawyasabogenol BO3-0-[α-L-arabinopyranosyl (1→ no 2)-(6-0-methyl-β-D -glucuronopyranosyl)]-Sawya savogenol BO3-0-
[α-L-rhamnopyranosyl (1→2)-β-D-galactopyranosyl (1→2)-(6-0-methyl-β-D
-Glucuronopyrano 1 syl)] -Sawyasapogenol BO3-0-[α-L-Rhamnopyranosyl (1→2)-
α-L-Arabinopyranosyl (1→2)-(6-0-methyl-β-D-glucuronopyranosyl)]-Sawyasavogenol B1 1 The compound of general formula (1) is:
When used as an anti-complement active agent, it is usually formulated into a pharmaceutical composition together with a pharmaceutical carrier.

The carrier may contain diluents or excipients such as fillers, fillers, binders, wetting agents, disintegrants, surfactants, lubricants, etc. that are commonly used to prepare drugs according to the usage form. include.
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, emulsion suspensions, etc.). When forming tablets, known excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, and silicic acid may be used as carriers; water, ethanol, propanol, etc. Qle, simple syrup,
Binder such as glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone; dried starch, sodium alginate, agar powder, lamina "ria powder", .'sodium bicarbonate, calcium carbonate, Disintegrants such as Twin, sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose; disintegration inhibitors such as white sugar, stearin, cocoa butter, hydrogenated oil; absorption enhancers such as quaternary ammonium bases, sodium lauryl sulfate; glycerin , moisturizing agents such as Denfuso, adsorbents such as starch, lactose, kaolin, bentonite, colloidal silicic acid, etc.; lubricants such as purified talc, stearate, boric acid powder, macrocol, solid polyethylene glycol, etc. can be used. When forming into a pill form, known excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, and talc can be used as carriers; gum arabic powder, tragacanth powder, gelatin, ethanol, etc. Binder: Disintegrants such as laminaria and agar can be used.
Furthermore, the above tablets, etc. may be coated with a conventional coating if necessary, and can be made into sugar tablets, etc.
It can be a gelatin-coated tablet, an enteric-coated tablet, a film-coated tablet, a double tablet, or a multilayer tablet. 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 solutions and suspensions to be used as injections, conventional diluents such as water, ethyl alcoholyl, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and polyoxyethylene are used as diluents. 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. Colorants, preservatives, fragrances, flavors, sweeteners, and other pharmaceutical agents can be included in the anti-complement activator. The active ingredient to be contained in the anti-complement activator, namely the general formula (Iy
The amount of the compound is not particularly limited and can be selected as appropriate, but it is usually 1 to 70% by weight, preferably 5 to 50% by weight of the total composition.

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 2077 cm per day. /Kf
It may be within a certain range, and a sufficiently safe therapeutic effect is 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-(6-0-methyl-β-D-glucuronopyranosyl)-Sawyasapogenol B B. 3-0-[β-D-galactopyranosyl (1→2)
-(6-0-methyl-β-D glucuronopyranosyl)]
-Sawya savogenol B C. 3-0-[α-L-rhamnopyranosyl (1→2)-
β-D-galactopyranosyl (1→2)-(6-0-methyl-β-D-glucuronopyranosyl)]-Sawya sabogenol B D. Glycyrrhizin (comparative compound) (2) Inhibition effect on Forssmann reaction The Forssmann reaction was discovered by Forssmann in 1911.

It began with the discovery that immunizing rabbits with tissue from guinea pigs and other animals produced antibodies that lysed sheep red blood cells. This meant that the same antigen as sheep red blood cells was present in the tissues of guinea pigs and other animals, and this was called the Forssmann antigen, and the antibody came to be called the Forssmann antibody. Forssmann antigen exists in human tissues, guinea pigs, sheep, horses, cats, chickens, turtles, bacteria, etc.
It does not exist in snails, pigeons, and frogs [J. Buch
binder:HeterOphilephenOme
nainimmunOlOO-Review. Arch
．． Pathcl. , l9, pp. 841-880 (193
See 5)]. Generally, the Forssman reaction uses sheep red blood cells as an antigen, immunizes an animal such as a rabbit that does not have this antigen to obtain an antiserum (FOrssman antibody), and injects this antiserum intravenously into a guinea pig. Guinea pigs injected intravenously with this antiserum die within minutes. In other words, the Forssmann reaction involves the production of antibodies against Forssmann antigens (glycolipids) on cell surfaces and connective tissue, and the cytolysis (Coombs &Gell's allergy type) caused by injecting the antibody into an animal that has the antigen. ) can be said to be a consistent fatal reaction [Clinical As
pectsOfImmunOlOgyThirdedi
tiOnEditedbyP. G. H. Gell, R.
R. A. COOmbsandP. J. Lachmann
Blackwell ScientificPubllc
atiOnsOsneYMead,OxfOrd,85
MarylebOne High Street, LOnd
OnWIM3DE, AlicjaB. Pelczars
kaandAdOlphP. ROs7kOwskiJO
urnalOfPharmacOlOgyAndExp
erimentalTherapeutics, vOl
l85, Black 1, pp. 116-126, InhibitO
rsOfFOrssmanGuineaPig゛Ana
phylaxis""]. In the cytolytic type of allergy, complement in the blood is activated along with the antigen-antibody reaction, and C
3a and C5a anaphylatoxins (Anaphyla
tOxin) is released, increasing the permeability of blood vessels, and especially in the Forssmann reaction of guinea pigs, blood components leak into the alveoli, leading to airway obstruction and death. Therefore, if the formation of anaphrytoxins such as C3a and C5a is suppressed by anti-complement activators, death can be avoided even by administration of Forssmann antibody (hemolysin). Hemolysin [Anti-Sheep Erythrocyte Rabbit Antibody (Anti-Sheeph)
eatenstrOmataRabbitSerun
)] is prepared according to the method described in Immunochemistry, pp. 830-834 (1973), written by Yuichi Yamamura, published by Asakura Shoten. Hemolysin 1me/from one side of the hind limb vein of a guinea pig
Immediately after administering Kf, a test compound dissolved in a 5% ethanol aqueous solution is administered through the vein of the other hind limb. Table 1 shows the survival status of the guinea pigs 24 hours after administration. 5
(: Group administered with F6 ethanol only (control)
will all die within 5 minutes.

Compounds (A, B and C) of general formula (1) are Comparative Compound 1
) shows a remarkable inhibitory effect on the Forssmann reaction. That is, the compound of general formula (1) exhibits significantly superior anti-complement activity compared to comparative compounds. (3) Nephrotoxin (Ne
Rat nephrotoxin (hereinafter abbreviated as "NT") can be obtained as follows.

Mouse kidney cortex
x) with an equal volume of physiological saline, and the homogenate was mixed with Freund Complate adjuvant (F
reund9scOmpleteadjuvant;D
ifcO) at a ratio of 1:1, and 2 m2 of the resulting mixture was intramuscularly injected into rabbits (body weight 31007) to immunize them. After 1.5 months, blood was collected from the rabbit's heart to obtain serum.

The obtained serum was inactivated at 56°C for 30 minutes and then incubated at 40°C (
fl) Fractionation by salting out with saturated ammonium sulfate. A fraction of imino γ-globulin (IgG) is collected to obtain NT. In the test, a Wista strain (150 to 160 tons) was tested.
A male mouse (Wister) is used.

The compound of general formula (1) is administered intraperitoneally every 24 hours at a dose of 10 doses per animal once a day from 3 days before to 10 days after administration, starting from the time when the compound of general formula (1) is administered 1 hour before NT administration. In addition, 1me of NT was intravenously injected into the tail vein of the mouse. Physiological saline is used as a control. Protein uria (PrOte)
inuria) (total amount excreted in the urine in 24 hours) was treated with bovine serum albumin (total amount excreted in urine in 24 hours) by sulfosalicylic acid.
It is measured by the turbidity method using BOvineSerumAlbunlln) 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 W! /day. Proteinuria in mice under normal conditions is 0.5 to 5 mf/day, but if it exceeds this value, especially if it exceeds 10711 f1/day, 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 general formula α), the amount of proteinuria for 10 days from the time of administration was almost the same as in mice under normal conditions. It can be seen that the primary reaction and secondary reaction are suppressed. (4) Heyman
ymann) type nephritis, body weight 18
Male mice of the Wista strain, ages 0 to 2007, are used.

Remove the mouse kidney cortex and homogenize it with an equal volume of physiological saline. 150% of the homogenate
Centrifugation was performed at 0 G for 1 hour, and the supernatant was collected using the method of Edginton et al. [T. S. EdgingtOnetal, Jour
nalOfExperimentalMedicinl
, 127, 555] and ``Freund Combrate Ajuband 37Ra'' (Di
(manufactured by fcO) at a weight ratio of 0.4:1 and 0.5 ml of the mixture was injected into the abdominal cavity of a mouse. Thereafter, the same amount of this is administered every two weeks until the proteinuria level reaches 100 or more per day (6 to 8 weeks). The compound of general formula (1) was administered to mice (weighing about 300 to 350?) that had developed Heymann's nephritis every 24 hours at a dose of 107 nf per mouse! The amount of proteinuria (Mf/day) was measured in the same manner as above.

A similar test was conducted 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 400 to 500 tons.
Therefore, the normal amount of protein uria is considered to be 5-15~/day. As is clear from Table 3, Compound A has the effect of curing Heymann's nephritis. (5) Toxicity test Test compounds A, B, and C are administered intraperitoneally to determine acute toxicity (LD5O value) to mice.

The results are shown in Table 4. Formulation examples of the compound of general formula (1) are listed below as reference examples, and then production examples are listed.

Reference Example 10 soybeans were crushed, defatted with hot hexane, and then heated and extracted with methanol under reflux.

The methanol extract was concentrated to dryness to obtain a residue of 1.4 kf, which was partitioned with butanol and water (1:1). After concentrating the butanol layer, it was dissolved in a small amount of methanol, and poured into a large amount of ether to give a coarse residue. Precipitate sabonin. This crude saponin is taken out in a furnace, dissolved again in a small amount of methanol, and then passed through a 200f activated carbon column and eluted with methanol to obtain 30f of crude saponin. After washing this crude saponin with butanol, it was further washed with VC. Suspended in 5% caustic soda aqueous solution and centrifuged to remove impurities, colorless Soya sabonin B8. lt is obtained. 3. Dissolve the obtained Soya sabonin B5OO7V in 30 ml of methanol and add 1.5 m2 of 15N monosulfuric acid.
Reflux for 5 hours.

Cold water is added to the reaction mixture, and the precipitate formed is collected and thoroughly washed with water. The precipitate was adsorbed onto a silica gel column and chloroform-ethanol (20:1, 200ml, 10:1, 1
50me, 5:1,200m2, 2:1,200m2 volume ratio), and the melting point was 249-25 from the 20:1 fraction.
3-0-(6-0-methyl-β-D-glucuro・
Nopyranosyl)-Sawya Sabogenol B7lT! f,
From the 10:1 fraction, 30-[β
-D-galactopyranosyl (1→2) (6-0-methyl-β-D-glucuronopyranosyl)]-Sawya savogenol B and 3-0-[α- L-
Arabinopyranosyl (1→2)-(6-0-methyl-β
-D-glucuronopyranosyl)]-Sawya savogenol B was obtained from a 5:1 fraction of 3-0 with a melting point of 270-274°C.
-[α-L-rhamnopyranosyl (1→2)-β-D-galactopyranosyl (1→2)-(6-0-methyl-β-
D-glucuronopyranosyl)]-soyasabogenol B are obtained, respectively. General formula (1) for semi-synthetic glycerade base
The above compound is added, mixed and suspended at 50°C, poured into a mold, allowed to cool naturally, and taken out to obtain a suppository.

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). R^4 represents a lower alkyl group. An anti-complement activator characterized by containing a soyasapogenol B derivative represented by the following as an active ingredient.