JPH0142926B2 - - Google Patents

Description

[Detailed Description of the Invention] The present invention relates to the general formula () The present invention relates to a therapeutic agent for nephropathy containing an aminobenzoic acid derivative represented by the formula or a pharmaceutically acceptable salt thereof as an active ingredient. Compounds represented by the above general formula () (hereinafter,
Although it has a simple structure, it has extremely low toxicity and has no antibacterial activity, so there is no concern about disturbance of intestinal flora, and it can be administered for a long period of time. Furthermore, it is an extremely safe drug that does not have mutagenicity or affect cellular or humoral immunity, and therefore poses no risk of teratogenicity or allergic reactions to healthy people. In addition, this substance is useful as a therapeutic agent for nephropathy. There are three types of amino group positions in this substance: p-, m-, and o-, and although there may be some differences in activity, all are essentially useful. The substance may be in the form of a salt, in which case any pharmaceutically acceptable salt is included.
These include alkali metal salts, alkaline earth metal salts, and aluminum metal salts. Usually Na,
K, Mg, Ca, Al, etc. are preferred, and Na is particularly preferred. The sugar moiety of the present substance may be any sugar that binds to the amino group of aminobenzoic acid, and is preferably a monosaccharide. Examples of sugars include arabinose, xylose, galactose, glucose, mannose, rhamnose, and the like. The sugar may be in the D or L form or in the form of the α-anomer, β-anomer or a mixture of anomers. Therefore, the present substance can also be an α or β anomer or a mixed anomer thereof. The method for producing this substance is exemplified below. 1.5-5 g of aminobenzoic acid, 2-6.4 g of sugar (L-arabinose, D-xylose, D-glucose, D-galactose, L-rhamnose or D-mannose), and 0.05-0.5 g of ammonium chloride.
94-100% ethanol or pure methanol 10-50
ml under reflux for condensation. If crystals precipitate after being left at room temperature or in a cold place for a while, the reaction solution is filtered, the crystals are thoroughly washed with water, alcohol, ether, etc., and then recrystallized from methanol water or ethanol water. The product can be made into a salt. The physicochemical properties of this substance obtained by the above production method are shown in Table 1 below. Further, infrared absorption spectra are shown in FIGS. 1 to 24. The analysis method in Table 1 is as follows. (1) Melting point Measured using a Yanagimoto micro melting point measuring device. (2) Elemental analysis Measured using Yanagimoto CHN coder MT2 type. (3)Using UV Hitachi EPS-3T self-recording spectrophotometer,
-H was measured using an alcohol-water system, and -Na was measured using water as a solvent. (4) IR Measured using the KBr method using JASCO Model DS-701G. The drawing numbers match the sample numbers in Table 1. [Table] [Table] The toxicological properties of this substance are shown below. (1) Acute toxicity Acute toxicity was investigated by intraperitoneal and forced oral administration using ICR-JCL mice. This substance was dissolved in physiological saline for intraperitoneal administration, and in distilled water for oral administration, and the solution was adjusted to a predetermined amount using a syringe or a stomach tube and administered. After administration, the symptoms of toxicity were continued to be observed, and the LD ₅₀ value was determined from the mortality rate over time up to the 7th day. Survival cases,
In both cases of death, autopsies were performed to obtain findings. _LD50 values are Litchfield-Wilcoxon (Litchfield
−Wilcoxon) calculated using the graphic calculation method. The results are shown in Table 2. Either intraperitoneal or oral
The LD ₅₀ value is 6g/Kg or more, and 6 out of 10 compounds, more than half, have an LD ₅₀ value of 10.
It can be said that it is an extremely safe drug with a value of more than g/Kg. [Table] [Table] (2) Antibacterial activity Dissolve this substance in distilled water to create a 2-fold dilution series, mix this diluted solution with 9 times the amount of heated agar medium, and pour it into a Petri dish. It was made into a flat plate.
The medium is heart infusion agar (bacteria).
After inoculation of the pre-cultured test bacteria using Sabouraud agar (fungi), the bacteria were cultured at 37°C for 20-24 hours, and the fungi were cultured at 25°C for 3-7 days, and the presence or absence of growth was examined. The following bacterial species were used as test bacteria. Pseudomonas acruginosa IAM
1514) Escherichia coli IFO 12734) Staphylococcus aureus
209P) Bacillus subtilis (IAM 1069) Baker's yeast ( Saccharomyces cerevisiae)
IAM 4207) Candida albicans ATCC
752) Trichophyton mentagrophytes
IFO 6124) Black mold (Aspergillus niger IAM 3001) As a result, this substance has a 1.
No growth inhibition was observed at a concentration of mg/ml. (3) Mutagenicity First, we conducted an investigation using Rec-assay. That is, a recombinant repair-deficient strain ( Bacillus subtilis
M45) and recombinant repair carrier strain ( B. subtilis
H17) on B-agar medium (meat extract 10
g, polypeptone 10g, NaCl5g, agar 15g,
A line was drawn on 1000 ml of distilled water (PH7.0) so that the starting points did not touch each other. Dissolve this substance in sterile water and absorb 0.05ml onto a circular filter paper with a diameter of 8mm. Immediately leave it standing so as to cover the starting point of the streak, and culture it overnight at 37°C to reach the growth inhibition zone. The length was measured. Kanamycin was used as a negative control, and mitomycin C was used as a positive control. Next, perform a back mutation test on Salmonella
Typhimurium TA98 and TA100 (both require histidine) were used. Soft agar solution (NaCl 6g, agar 6g, distilled water 1000ml) to which 1/10 volume of 0.5mM bithion-0.5mM histidine solution was added
ml) 0.1 ml of bacterial solution and 0.1 ml of drug solution were added to 2 ml, mixed well, and layered on a minimal agar medium. 2 at 37℃
The cells were cultured between eyes and the number of revertant colonies was counted. Furilfuramide (AF2) was used as a positive control. The results of the Rec-assay are shown in Table 3, and the results of the reverse mutation test are shown in Table 4. In Rec-assay, this substance did not exhibit mutagenicity even at high concentrations, but sodium p-aminobenzoate derivatives were particularly excellent. In addition, in a reverse mutation test, there was no change in the mutation incidence rate due to this substance compared to a control without additives, even when a high concentration was applied, proving that it is a highly safe drug. [Table] [Table] [Table] (4) Delayed intradermal reaction To understand the effect of this substance on cellular immunity
A foot pad reaction using sheep red blood cells as an antigen was performed using ICR-JCL mice. Sheep red blood cells were suspended in physiological saline at a volume of 10%, and 0.2 ml of this solution was injected through the tail vein.
Secondary sensitization was performed, and 7 days later, 0.05 ml of a 40% suspension of sheep red blood cells was injected into the footpad for secondary sensitization, and the footpad thickness was measured the next day. This substance was administered intraperitoneally at a dose of 250 mg/Kg five times on consecutive days, mainly on the day of primary sensitization. As a result, no significant difference was observed in the increase in footpad thickness in the group administered with this substance compared to the control (non-administered) group. (5) Antibody production ability In order to understand the effect of this substance on humoral immunity,
10% of sheep red blood cells for ICR-JCL mice
Sensitize by injecting 0.2ml of the suspension through the tail vein.
Blood was collected 7 days after sensitization, and antibody production performance was measured by hemagglutination reaction. The substance was administered intraperitoneally at a dose of 250 mg/Kg five times daily, mainly on the day of sensitization. As a result, no significant difference was observed in the agglutination value between the group administered with this substance and the control group. Next, we will discuss the pharmacological properties of this substance. Puromycin 1.5mg/ for Wistar rats
An animal model of sensitization was created by subcutaneously injecting 100 g body weight/day for 10 consecutive days. This substance was orally administered by force at 100 mg/Kg every day for 10 days, the same as the puromycin administration schedule. As a control, only physiological saline was administered in the same manner instead of this substance. On the 7th day after the end of administration, daily urine was collected using a metabolic gauge, and the daily excreted urine protein amount was calculated from the urine volume and protein concentration. The results are shown in Table 5. All of the compounds tested exhibited inhibitory effects on urinary protein excretion compared to the control. Furthermore, these animals were sacrificed and histopathological examination of the kidneys was performed.
An inhibitory effect on glomerular basement membrane thickening was observed in cases where this substance was administered. [Table] As is clear from the table above, this substance had a significant effect on suppressing the excretion of proteinuria. Furthermore, when a search was conducted using 5/6 nephrectomized rats, a similar inhibitory effect on urinary protein excretion was observed. Next, we will discuss the formulation of this substance. When this substance is used as a therapeutic agent for nephropathy, it can be used in a form convenient for obtaining the medicinal effect depending on the type and symptoms of the disease, and can be used alone or in combination with a pharmaceutically acceptable diluent and other drugs. Can be used as a mixture. The substance is applied orally or parenterally.
Therefore, it may take any form for oral or parenteral administration. The substances can be provided in dosage unit form.
It contains an effective amount of the active ingredient, and its forms include powders, granules, tablets, sugar-coated tablets, capsules, suppositories,
It can take the form of a suspension, solution, emulsion, ampoule, injection solution, etc. The diluent may be solid, liquid, semi-solid, or in an ingestible capsule, such as the following: i.e. excipients, fillers, binders, wetting agents, disintegrants, surfactants,
These include lubricants, dispersants, buffers, fragrances, preservatives, solubilizing agents, and solvents. Furthermore, one type or a mixture of one or more types of these may be used. The therapeutic agent for nephropathy of the present invention can be produced by any known method. The active ingredient in the compositions used in the present invention generally comprises from 0.01 to 100 wt%. The therapeutic agent for nephropathy of the present invention can be administered orally or parenterally to humans and animals, but oral administration is preferred. Oral administration includes sublingual administration. Parenteral administration also includes injections, such as subcutaneous, intramuscular, intravenous, infusion, and the like. The therapeutic agent for nephropathy of the present invention is used for renal diseases. For example, it is effective for renal failure, glomerulonephritis, diabetic nephropathy, nephrotic syndrome, etc. The dosage of the therapeutic agent for nephropathy of the present invention depends on whether it is an animal or a human, and is influenced by age, individual differences, medical conditions, etc. In some cases, doses outside the range shown below may be administered, but in general, humans When targeted, the oral dosage of this substance is 1 kg body weight, 0.1 to 1000 per day.
mg, preferably 1 to 500 mg, the same parenteral dosage is 0.01 to 200 mg, preferably 0.1 to 100 mg.
Administer in 4 to 4 divided doses. Hereinafter, the present invention will be explained in more detail by showing formulation examples and manufacturing examples of the substance of the present invention. Formulation Example 1 This substance (sodium p-aminobenzoate-N-
L-arabinoside) 10 parts heavy magnesium oxide 15 parts lactose 75 parts are uniformly mixed to make a powder or fine granules. Further, this powder was put into a capsule container to form a capsule. Formulation Example 2 This substance (sodium o-aminobenzoate-N-D
-xyloside) 45 parts starch 15 parts lactose 16 parts crystalline cellulose 21 parts polyvinyl alcohol 3 parts water 30 parts are mixed uniformly, crushed, granulated, dried, and sieved to form granules. Formulation Example 3 Granules were made in the same manner as in Formulation Example 2 using sodium o-aminobenzoate-N-D-glucoside instead of sodium o-aminobenzoate-N-D-xyloside. 4 parts of calcium stearate is added to 96 parts of the drug and compressed to form tablets with a diameter of 10 mm. Formulation Example 4 This substance (sodium p-aminobenzoate-N-L
-Rhamnoside) Granules are prepared in the same manner as in Formulation Example 2 using 94 parts polyvinyl alcohol 6 parts water 30 parts. 10 parts of crystalline cellulose is added to 90 parts of the obtained granules and compressed to form tablets with a diameter of 8 mm, and syrup gelatin and precipitated calcium carbonate are added to form sugar-coated tablets. Formulation Example 5 This substance (sodium p-aminobenzoate-N-
D-galactoside) 0.6 parts nonionic surfactant 2.4 parts physiological saline 97 parts are mixed under heating and sterilized to prepare an injection. Production example 1 p-aminobenzoic acid-N-L-arabinoside-
Method for producing Na salt: 4.6 g of p-aminobenzoic acid, 5 g of L-arabinose, and 0.5 g of ammonium chloride are heated and condensed in 40 ml of 94% ethyl alcohol under reflux. If the reaction solution is left in the refrigerator, crystals will precipitate. The reaction solution was filtered, the crystals were washed with ether, and recrystallized several times from 50% methyl alcohol to obtain colorless needle-like crystals. The yield was 45.8%. The thus obtained p-aminobenzoic acid-N-L-arabinoside was gradually dissolved in a 1% aqueous solution containing a calculated amount of NaOH, and the insoluble matter was filtered.
The filtrate was concentrated under reduced pressure, a large excess of acetone was added, dehydrated, and dried to obtain colorless crystals. Yield 100%,
The overall yield was 45.8%. Production example 2 o-aminobenzoic acid-N-L-arabinoside-
Production method of Na salt: 2.3 g of o-aminobenzoic acid, L-arabinose
2.5 g of ammonium chloride and 0.2 g of ammonium chloride were heated and condensed in 30 ml of methyl alcohol under reflux. After the reaction, if left at room temperature, crystals will precipitate.
The reaction solution was filtered and the crystals obtained were washed with water, methyl alcohol, and ether to obtain colorless needle-like or disk-like crystals. The yield was 61.3%. The thus obtained o-aminobenzoic acid-N-L-arabinoside was gradually dissolved in a 1% aqueous solution containing a calculated amount of NaOH, and the insoluble matter was filtered.
The filtrate was concentrated under reduced pressure, a large excess of acetone was added, dehydrated, and then dried to obtain colorless crystals. The yield was 100%, and the total yield was 61.3%. Production example 3 p-aminobenzoic acid-N-D-xyloside-
Production method of Na salt: p-aminobenzoic acid 2.3g, D-xylose 2.5g
g, ammonium chloride 0.05g in ethyl alcohol
Condensate by heating in 25 ml under reflux. There is precipitation of crystals during the reaction, but by adding solvent,
Continue heating to complete the reaction. After standing in a cold place, the reaction product was filtered, and the crystals were washed with water, dilute methyl alcohol and a small amount of ether, and recrystallized from 94% ethyl alcohol to obtain colorless needle-shaped crystals. The yield was 73.7%. p-Aminobenzoic acid thus obtained
N-D-xyloside with calculated amount of NaOH 1
% aqueous solution, insoluble matter was filtered, the filtrate was concentrated under reduced pressure, a large excess of acetone was added, dehydrated, and dried to obtain colorless crystals. Yield 100%,
The total yield was 73.7%. Production example 4 o-aminobenzoic acid-N-D-xyloside Na
Salt manufacturing method: o-aminobenzoic acid 2.3g, D-xylose 2.5g
g, 0.2 g of ammonium chloride in ethyl alcohol
Condensate by heating in 35 ml under reflux. After the reaction, concentrate to about 1/2 under reduced pressure and leave to stand at room temperature to observe crystal precipitation. After filtering the reaction solution,
Wash the crystals with water, methyl alcohol, and ether,
Recrystallization from ethyl alcohol gave colorless needle-like crystals. The yield was 74.6%. O-aminobenzoic acid thus obtained
N-D-xyloside with calculated amount of NaOH 1
% aqueous solution, filtered out insoluble matter, concentrated the filtrate under reduced pressure, added a large excess of acetone, dehydrated and dried to obtain colorless crystals. Yield 100%, Total
The yield was 74.6%. Production example 5 p-aminobenzoic acid-N-D-glucoside-
Production method of Na salt: p-aminobenzoic acid 5g, D-glucose 6.4
0.5 g of ammonium chloride was condensed by heating under reflux in 500 ml of 94% ethyl alcohol. After the reaction, the mixture was concentrated to about 1/3 under reduced pressure and left in a cool place, whereupon the entire liquid became gel-like. Add a small amount of water, warm again to dissolve, and then leave it in the refrigerator to see crystals precipitate. The reaction solution was passed through the mouth, the crystals were washed with water, dilute methyl alcohol, and a small amount of ether, and recrystallized from 50% methyl alcohol to obtain colorless needle-shaped crystals. The yield was 33.7%. p-Aminobenzoic acid thus obtained
1 containing calculated amount of NaOH containing N-D-glucoside
% aqueous solution, insoluble matter was filtered, the filtrate was concentrated under reduced pressure, a large excess of acetone was added, dehydrated, and dried to obtain colorless crystals. Yield 100%,
The total yield was 33.7%. Production example 6 o-aminobenzoic acid-N-D-glucoside-
Production method of Na salt: o-aminobenzoic acid 4.6g, D-glucose 6.0
g, 0.5 g of ammonium chloride is heated and condensed in 40 ml of 95% ethyl alcohol under reflux. After the reaction, concentrate to about 1/3 under reduced pressure and store in the refrigerator.
If you leave it overnight, you will see the precipitation of crystals. The reaction solution was passed through the mouth, the crystals were washed with water, methyl alcohol, and ether, and recrystallized twice from methyl alcohol to obtain colorless needle-like crystals. The yield was 4.6%. O-aminobenzoic acid thus obtained
1 containing calculated amount of NaOH containing N-D-glucoside
% aqueous solution, filtered out insoluble matter, concentrated the filtrate under reduced pressure, added a large excess of acetone, dehydrated and dried to obtain colorless crystals. 100% yield, ToTal
The yield was 4.6%. Production example 7 p-aminobenzoic acid-N-D-galactoside-
Method for producing Na salt: 1.5 g of p-aminobenzoic acid, 2 g of D-galactose, and 0.1 g of ammonium chloride are heated and condensed in 30 ml of 94% ethyl alcohol under reflux. After the reaction, concentrate under reduced pressure and leave in a cool place to observe crystal precipitation. The reaction solution was passed through the mouth, the crystals were washed with water, dilute methyl alcohol and a small amount of ether, and recrystallized from methyl alcohol to give colorless needles. The yield was 18.1%. p-Aminobenzoic acid thus obtained
N-D-galactoside was gradually dissolved in a 1% aqueous solution containing the calculated amount of NaOH, the insoluble matter was filtered, the filtrate was concentrated under reduced pressure, a large excess of acetone was added, and after dehydration, it was dried to form a colorless product. Obtained crystals. Yield 100%,
The total yield was 18.1%. Production example 8 o-aminobenzoic acid-N-D-galactoside-
Production method of Na salt: 2.4 g of o-aminobenzoic acid, D-galactose
3.0 g of ammonium chloride and 0.2 g of ammonium chloride were heated and condensed in 30 ml of 95% ethyl alcohol under reflux. After the reaction, concentrate to about 1/2 under reduced pressure and leave to stand at room temperature to observe crystal precipitation. Pass the reaction solution through the mouth, wash the crystals with water, methyl alcohol, and ether, and reduce to 95%.
Recrystallization from ethyl alcohol gave colorless needle-like crystals. The yield was 16.4%. O-aminobenzoic acid thus obtained
Gradually dissolve N-D-galactoside in a 1% aqueous solution containing the calculated amount of NaOH, filter out insoluble matter, concentrate the filtrate under reduced pressure, add a large excess of acetone, dehydrate, and dry to obtain colorless crystals. Obtained. Yield 100%, Total
The yield was 16.4%. Production example 9 p-aminobenzoic acid-N-L-rhamnoside-
Production method of Na salt: 3 g of p-aminobenzoic acid, 4 g of L-rhamnose
g, and 0.1 g of ammonium chloride are heated and condensed in 94% ethyl alcohol under reflux cooling. After the reaction, if left at room temperature, crystals will precipitate. The reaction solution is filtered, the crystals are washed with water and diluted methyl alcohol, and then recrystallized from 50% methyl alcohol to obtain colorless needle-shaped crystals. The yield was 30.9%. The p-aminobenzoic acid-N-L-rhamnoside thus obtained was gradually dissolved in a 1% aqueous solution containing a calculated amount of NaOH, the insoluble materials were filtered, and the filtrate was concentrated under reduced pressure. Excess acetone was added and the mixture was dehydrated and dried to obtain colorless crystals. Yield 100%,
The total yield was 30.9%. Production example 10 o-aminobenzoic acid-N-L-rhamnoside-
Production method of Na salt: 2.3 g of o-aminobenzoic acid, 2.8 g of L-rhamnose
g, 0.2 g of ammonium chloride in methyl alcohol
Heat condensation in 25 ml under reflux. After the reaction, if left at room temperature, crystals will precipitate.
The reaction solution is filtered, the crystals are washed with water and methyl alcohol, and then recrystallized from 50% methyl alcohol to obtain colorless needle-shaped crystals. The yield was 9.8%. O-aminobenzoic acid thus obtained
N-L-rhamnoside with calculated amount of NaOH 1
% aqueous solution, filtered out insoluble matter, concentrated the filtrate under reduced pressure, added a large excess of acetone, dehydrated and dried to obtain colorless crystals. Yield 100%, Total
The yield was 9.8%. Production example 11 p-aminobenzoic acid-N-D-mannoside-
Manufacturing method of Na salt: 2 g of p-aminobenzoic acid, 3 g of D-mannose
and 0.2 g of ammonium chloride are heated under reflux for about 1 hour in 10 ml of ethyl alcohol to carry out a condensation reaction. After the reaction, the product is left at room temperature to precipitate crystals. Next, the crystals were separated, washed with water, dilute ethyl alcohol, and a small amount of ether, and then recrystallized using 50% methyl alcohol to obtain colorless needle-like crystals of p-aminobenzoic acid-N-D-mannoside. I got it. The yield was 56.1%. The hydrate of p-aminobenzoic acid N-D-mannoside obtained as described above was mixed with a calculated amount of NaOH.
The mixture is gradually added to a 1% aqueous solution containing the following ingredients to form a precipitate. When this precipitate is collected, dehydrated, and dried, colorless crystals are obtained.
These crystals were further recrystallized using an aqueous solution consisting of 5 parts of water and 1 part of acetone to obtain colorless crystals of sodium p-aminobenzoate-N-D-mannoside. The yield was 95%, and the total yield was 53.3%. Production example 12 m-aminobenzoic acid-N-D-mannoside-
Production method of Na salt: 2 g of m-aminobenzoic acid, 3 g of D-mannose
g, 0.2 g of ammonium chloride in ethyl alcohol
Heat condensation in 10 ml under reflux in a water bath at 95-96°C. After a while of heating the reaction product, a thick crystal mass is precipitated. The crystals obtained by passing the reaction solution through the mouth were thoroughly washed with water and methyl alcohol, and then recrystallized from methyl alcohol to obtain colorless needle-like crystals. Yield 33.0%
It was hot. The m-aminobenzoic acid thus obtained
1 containing calculated amount of NaOH containing N-D-mannoside
% aqueous solution, and if any insoluble matter was present, it was filtered, the oral liquid was concentrated under reduced pressure, a large excess of ethanol was added, and after dehydration, it was dried to obtain colorless crystals. Yield 100
%, and the total yield was 33%.

[Brief explanation of drawings]

Figures 1 to 24 of the attached drawings are shown in Table 1.
Infrared absorption spectra of each compound are shown in Nos. 1 to 24, respectively.

Claims (1)

[Claims] 1 General formula () A therapeutic agent for nephropathy containing as an active ingredient at least one aminobenzoic acid derivative or a pharmaceutically acceptable salt thereof. 2. The therapeutic agent for nephropathy according to claim 1, wherein R is a monosaccharide. 3. The therapeutic agent for nephropathy according to claim 2, wherein the monosaccharide is selected from the group consisting of arabinoside, xyloside, glucoside, galactoside, rhamnoside, and mannoside. 4. The therapeutic agent for nephropathy according to claim 1, wherein the pharmaceutically acceptable salt thereof is a sodium salt.