JPS5924967B2 - Antidiabetic agents containing aminobenzoic acid derivatives - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an antidiabetic agent containing a substance represented by general formula (1) or a salt thereof as a main component.

R-NH (□) (R1 represents arabinose, xylose, glucose, galactose, or rhamnose residue) Conventionally, synthetic compounds and antibiotics have been used as anticancer agents, but
Although these drugs have excellent cancer-killing effects, they have the disadvantage of being highly toxic and causing side effects because they also act on normal cells.

Therefore, recently, polysaccharides of various origins have attracted attention because they exhibit anticancer effects by enhancing the immune capacity of the host. The present inventors have already developed and provided to society an anticancer drug made from a polysaccharide derived from basidiomycetes, but during research into the structure and activity of this anticancer drug, we discovered that the antitumor effect of aminobenzoic acid-N-D-mannoside, We found a lowering effect and a blood pressure lowering effect. Subsequently, as a result of further research and improvement, a compound represented by the above general formula (1) with low toxicity and higher medicinal efficacy was discovered, and the present invention was completed. General formula (1)
Although the compound represented by or its salt (hereinafter referred to as 6 substances 1) has a simple structure, it has extremely low toxicity and no antibacterial activity, so there is no need to disrupt the intestinal flora, and it can be used for a long period of time. administration is possible.

Furthermore, it is an extremely safe drug that does not have mutagenicity or affect cell-mediated or humoral immunity, and therefore poses no risk of teratogenicity or allergic reactions in healthy people. In addition, all of these substances have a hypoglycemic effect and are useful as antidiabetic agents. There are three types of positions of the amino group relative to the carboxyl group in this substance: p-, m-, and o-, and although there may be some differences in activity, all are essentially useful.

In addition, the salt of the aminobenzoic acid derivative of the present invention is one in which the hydrogen atom of the -COOH group in the above formula (1) is replaced with an alkali metal, an alkaline earth metal, or an aluminum metal. These metals may be any metals that are acceptable as drugs, and are usually Na, K, Mg,
Ca, Al, etc. are preferred, and Na is particularly preferred. still,
The sugar moiety has a six-membered ring (pyranose) structure. The manufacturing method of this substance is as follows.

Aminobenzoic acid 4,5-59, sugar (L-arabinose,
D-xylose, D-glucose, D-galactose or L-rhamnose) 5-69, ammonium chloride 0.
1 to 0.5 f1 was heated and condensed under reflux of 95 to 1000 t) ethanol or 40 to 907 n1 of pure methanol.

If crystals precipitated after being left at room temperature or in a cold place for a while, the reaction solution was filtered, the crystals were thoroughly washed with water, alcohol, ether, etc., and then recrystallized from methanol water or ethanol water. A well-known method was used to replace the hydrogen of the carboxyl group with a base.

That is, this substance was dissolved in an alcoholic aqueous solvent and an inorganic salt was added for substitution. 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. 1-20. The analysis method in Table 1 is as follows. (1) Melting point Measured using a Yanagimoto micro melting point measuring device.

(2) Optical Rotation Measured using a Yanagimoto direct-reading polarimeter model 0R-50 with an optical path length of 50 mT1L using -H as an alcohol monoaqueous solvent and -Na as a solvent in water.

(3) Elemental analysis Measurement was performed using Yanagimoto CHN coater MT2 type.

(4) Using a UV Hitachi EPS-3T self-recording spectrophotometer, -H was measured using an alcohol monoaqueous system, and -Na was measured using water as a solvent.

(5) IR It was measured by the KBr method using JASCO Model DS-701G.

The drawing numbers correspond to the sample waterfalls in Table 1. Next, the toxicological properties of this substance are shown.

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 and O values were determined from the mortality rate over time up to the 7th day.

The findings were obtained through autopsy in both surviving and dead cases. The LD5O value is determined by the Litchfield-Wilcoxon (Litchfied)
ld-Wil-COxOn) graphic calculation method. The results are shown in Table 2. Both are LD5 regardless of intraperitoneal or oral administration.
With an O value of 69/Kg or higher, it is a low-toxic substance that falls well within the so-called "ordinary drug" category, and 6 out of 10 compounds, more than half, have an LD5O value of 109/Kg or higher, making it extremely safe. It can be said that it is an expensive drug. 2) Antibacterial activity This substance was dissolved in distilled water to prepare a 2-fold dilution series, and this dilution was mixed with 9 times the amount of heated and dissolved agar medium, poured into a Petri dish, and plated.

Heart infusion agar (bacteria) and Sabouraud agar (fungi) were used as the culture medium, and after smearing and inoculating the pre-cultured test bacteria, the bacteria were incubated at 37°C for 20-24 hours. The fungi were cultured at 25° C. for 3 to 7 days, and the presence or absence of growth was examined. The following bacterial species were used as test bacteria. Pseudomonas aeruginosa
asaeruginOsaIAMl5l4) Escherichia coli (E
scherichiacOliIFOl2734) Staphylococcus aureus
s2O9P) Bacillus subtili
sIAMlO69) Baker's Yeast (SaccharOmyc)
Candida albicans ATCC752
TrichOphytOnmentagrOp
hytesFO6l24) Black mold (Aspergill)
usnigerIAM3OOl) As a result, this substance has 1T against all bacteria! ! g/d showed no growth inhibition.

3) Mutagenicity First, we conducted a study using Rec-Assay.

That is, a recombinant repair-deficient strain (Bacillus subt.
ilis M45) and the recombinant repair carrier strain (B. subtil
isHl7) on B-1 agar medium (10f
11 Polypeptone 109, NaC259, Agar 15f!
, distilled water 1000a1 PH7.0) were streaked so that the starting points did not touch each other. Dissolve this substance in sterile water,
After absorbing the 0.04a onto a circular piece of paper with a diameter of 8m1, it was immediately left to stand so as to cover the starting point of the drawing line, and was placed at 37°C.
The cells were cultured late and the length of the growth inhibition zone was measured. Kanamycin was used as a negative control, and mitomycin C was used as a positive control. Next, the reverse mutation test is carried out using SalmOnellatype.
This was carried out using hj-MuriumTA98 and TAlOO (both require histidine).

0.5n1M pithione-0.5mM histidine solution ν1
Add 0 volume of soft agar solution (NaC269, agar 69, distilled water 1000ml) to 2m' of bacterial solution 0.1a1 chemical solution 0.1a
was added, mixed well, and layered on a minimal agar medium.

The cells were cultured at 37°C for 2 days 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 the Rec-Assay, this substance did not show mutagenicity even at high concentrations, but the sodium P-aminobenzoate derivative was particularly excellent. In addition, in the reverse mutation test, no change was observed in the mutation incidence rate due to this substance compared to the non-additive control even when a high concentration was applied, proving that it is a highly safe drug. 4) Delayed intradermal reaction ICR-JC to understand the effect of this substance on cell-mediated immunity
Pole reaction using sheep red blood cells as an antigen using L mice (
FOOtpadreactiOn) was performed.

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 to perform primary sensitization.
After a further 7 days, a 40% volume suspension of sheep erythrocytes 0.05
Secondary sensitization was performed by injecting m1 into the feet, and foot thickness was measured the next day. This substance is 250η/K around the day of primary sensitization.
g was administered intraperitoneally 5 times daily. As a result, no significant difference was observed in the increase in foot 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, ICR-J
CL mice were given 0.0% suspension of sheep red blood cells.
2mj was injected into the tail vein for sensitization, and on the 7th day after sensitization, blood was collected and antibody production ability was measured by hemagglutination reaction.

The substance was intraperitoneally administered at a dose of 250 η/Kg five times on consecutive days, 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.

1) Hypoglycemic effect Streptomycin 60η/Kg was administered intraperitoneally to 10-week-old Wistar rats, and urinary glucose was confirmed to be positive one week later. Furthermore, regular insulin was administered to reduce urinary sugar and blood sugar. Only animals in which high urinary sugar and hyperglycemia were confirmed again after a few days, although a decrease was observed, were used as diabetic model animals.

Using 5 animals per group, dissolve this substance in distilled water and add 3
It was orally administered at 0T19/Kg or 300T/Kg.

3 hours after administration. and 6hr. Blood was collected from the eyes, and glucose was measured using a RaBA kit (manufactured by Chugai Enzyme Method). The results of the average values are shown in Table 5. The blood sugar level actually lowered by administration of this substance was greater than that of the control for all compounds, but sodium O-aminobenzoate-N-L
-arabinoside, sodium O-aminobenzoate-N-
D-glucoside, sodium P-aminobenzoate-N-
L-rhamnoside and sodium O-aminobenzoate-N
-L-rhamnoside had a significant hypoglycemic effect. In general, even if drugs are highly effective, many have strong side effects and cannot be administered over a long period of time. As mentioned above, this substance not only has low acute toxicity, but also exhibits no mutagenicity, allergenicity, or adverse effects on intestinal flora, and its LD5O value is at least 250 times higher than the 30 mg/Kg dose that shows medicinal efficacy. dosage, and the safety factor is extremely high. From this point of view as well, this substance can be said to be a drug that can be safely administered over a long period of time. Furthermore, diabetes is a long-term disease, and once the administration of hypoglycemic agents has been started, it is impossible to stop taking them, and in this sense, the present substance can be said to be useful as an anti-diabetic agent. Next, we will discuss the formulation of this substance.

When used as an anti-diabetic agent, this substance can be used in any form convenient for obtaining medicinal efficacy depending on the type and symptoms of the disease, and may be used alone or in a mixture with pharmaceutically acceptable diluents and other drugs. Can be used.

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.

They contain an effective amount of the active ingredient and can take the form of powders, granules, tablets, dragees, capsules, suppositories, suspensions, solutions, emulsions, ampoules, injections, and the like. 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,
Lubricants, dispersants, buffers, fragrances, preservatives, solubilizing agents,
Such as solvents. Furthermore, one type or a mixture of one or more types of these may be used. The medicament of the present invention can be produced by any known method.

The active ingredients in the compositions used in the present invention generally range from 0.01% to 100% by weight. %included. The pharmaceutical agent 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 includes injections such as subcutaneous, intramuscular, intravenous, infusion, and the like. The dosage of the medicine of the present invention depends on whether it is an animal or a human being, and is influenced by age, individual differences, medical conditions, etc., and in some cases, doses outside the range shown below may be administered.
Generally, when targeting humans, the oral dosage of this substance is 0.1 to 500 Tf1fi1 per body weight 1K9 per day, preferably 1 to 250 Tf1fi1, and the parenteral dosage is also 0.0
1 to 200η, preferably 0.1 to 100TIJ? 1
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 Mix them uniformly to make a powder or fine granules.

Further, this powder was put into a capsule container to form a capsule. After uniformly mixing and blending Formulation Example 2, the mixture is crushed, granulated, dried, and sieved to form granules.

Formulation Example 3 Sodium O-aminobenzoate in Example 2
Sodium O-aminobenzoate instead of xyloside
Granules are prepared in the same manner using N-D-glucoside, 4 parts of calcium stearate are added to 96 parts of the granules, and the mixture is compression-molded to form tablets with a diameter of 10 m11 L.

Formulation example 4 Granules are prepared in the same manner as in Example 2 using

10 parts of crystalline cellulose is added to 90 parts of the obtained granules and compressed to form tablets with a diameter of 8 um, and syrup gelatin and precipitated calcium carbonate are added to form sugar-coated tablets.
Formulation Example 5 is heated and mixed and then sterilized to prepare an injection.

Production Example 1 Production method of P-aminobenzoic acid-NL-arabinoside monosodium salt 4.69% of P-aminobenzoic acid, 59% of L-arabinose, and 0.59% of ammonium chloride were heated under reflux in 40a9401) ethyl alcohol. Condense.

If the reaction solution is left in the refrigerator, crystals will precipitate. The reaction solution was passed through the mouth, the crystals were washed with ether, and recrystallization was repeated several times from 50% methyl alcohol to obtain colorless needle-shaped crystals. The yield was 45.8%.

Thus obtained, P-aminobenzoic acid-N-L
- Gradually dissolve the arabinoside in a 1% aqueous solution containing the calculated amount of NaOH, filter out the insoluble material, concentrate the oral fluid under reduced pressure, add a large excess of acetone, dehydrate, and dry to obtain colorless crystals. Ta.

The yield was 100%, and the total yield was 45.8%.

Production Example 2 Production method of 0-aminobenzoic acid-N-L-arabinoside mono-Na salt 2.39% of anthranilic acid, 2.59% of L-arabinose, and 0.29% of ammonium chloride are heated and condensed in 30a methyl alcohol under reflux.

After the reaction, if left at room temperature, crystals will precipitate.

The reaction solution was passed through the mouth, and the crystals were washed with water, methyl alcohol, and ether to obtain colorless needle-like or disk-like crystals. Yield 61
．． It was 3%.

The thus obtained anthranilic acid-N-L-arabinoside was gradually dissolved in a 1% aqueous solution containing a calculated amount of NaOH, the insoluble material was filtered off, the oral solution was concentrated under reduced pressure, and a large excess of acetone was added. was added, dehydrated and dried to obtain colorless crystals.

The yield was 100 (:Ff), and the total yield was 61.3%.

Production example 3P-aminobenzoic acid-N-D-xyloside monosodium salt production method P-aminobenzoic acid 2.3f1sD-xylose 2.59, ammonium chloride 0.05g 25
Heat condensation in m2 ethyl alcohol under reflux.

Although crystals precipitate during the reaction, add more solvent and continue heating to complete the reaction.

After standing in a cold place, the reaction solution was sifted, 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 Cf). The thus obtained P-aminobenzoic acid mono-N-D-xyloside was gradually dissolved in a 1% aqueous solution containing a calculated amount of NaOH, the insoluble matter was sifted through the mouth, the oral liquid was concentrated under reduced pressure, and a large amount of Add excess acetone,
After dehydration and drying, colorless crystals were obtained.

The yield was 100%, and the TOtal yield was 73.7%.

Production Example 4 Production method of 0-aminobenzoic acid-N-D-xyloside mono-Na salt 2.39% of anthranilic acid, 2.511% of D-xylose, and 0.29% of ammonium chloride are heated and condensed in 35Tnt ethyl alcohol under reflux.

After the reaction, about ? When concentrated and left at room temperature, crystals begin to precipitate.

The reaction solution was passed through the mouth, the crystals were washed with water, methyl alcohol, and ether, and recrystallized from ethyl alcohol to obtain colorless needle-like crystals. The yield was 74.6%.

The thus obtained anthranilic acid-N-D-xyloside was gradually dissolved in a 1% aqueous solution containing a calculated amount of NaOH, the insoluble material was filtered off, the oral solution was concentrated under reduced pressure, and a large excess of acetone was added. was added, dehydrated and dried to obtain colorless crystals.

The yield was 1000 t), and the TOtal yield was 76.4 (fl).

Production example 5P-aminobenzoic acid-N-D-glucoside-
Production method of Na salt 50ml 194 of P-aminobenzoic acid 59, D-glucose 6.49, ammonium chloride 0.59
% ethyl alcohol under reflux.

After the reaction, about ? When concentrated and left in a cool place, 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. Pass the reaction solution and remove the crystals.
Wash with water, dilute methyl alcohol and a small amount of ether;
Recrystallization from 50% methyl alcohol gave colorless needle-like crystals.

The yield was 33.701).

Thus obtained, P-aminobenzoic acid-N-D
- The glucoside was gradually dissolved in a 1% aqueous solution containing the calculated amount of NaOH, the insoluble matter was filtered through the mouth, the oral fluid was concentrated under reduced pressure, a large excess of acetone was added, and after dehydration, it was dried to obtain colorless crystals. .

The yield was 100%, and the TOtaI yield was 33.7%.

Production example 60-aminobenzoic acid-N-D-glucoside-
Production method of Na salt: 4.6 g of anthranilic acid, 6.09 g of D-glucose, 0.5 f of ammonium chloride! 40a95
% ethyl alcohol under reflux.

Approximately under reduced pressure after the reaction? Concentrate the solution and leave it in the refrigerator overnight to 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%.

The thus obtained anthranilic acid-N-D-glucoside was gradually dissolved in 1 (fl) aqueous solution containing a calculated amount of NaOH, the insoluble matter was filtered through the mouth, the oral fluid was concentrated under reduced pressure, and a large excess was removed. Acetone was added and the mixture was dehydrated and dried to obtain colorless crystals.

Yield 100%, TOtal yield 4.60! ).

Production Example 7 Production method of P-aminobenzoic acid-N-D-galactoside-Na salt 30 ml of P-aminobenzoic acid 1.59, D-galactose 29, and ammonium chloride 0.19
Heat condensation in 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, and the crystals were washed with water, diluted methyl alcohol and a small amount of ether, and recrystallized from methyl alcohol to obtain colorless needle-like crystals.

Yield 18.10! ). The thus obtained P-aminobenzoic acid mono-N-D-galactoside was gradually dissolved in a 1% aqueous solution containing a calculated amount of NaOH, the insoluble material was filtered through the mouth, the oral liquid was concentrated under reduced pressure, and a large excess of of acetone was added, and the mixture was dehydrated and dried to obtain colorless crystals.

The yield was 100%, and the TOtal yield was 18.10 t).

Production example 80-Aminobenzoic acid-N-D-galactoside-Na salt production method Anthranilic acid 2.49, D-galactose 3.09, ammonium chloride 0.29 were added to 30a9
Heat condensation in 5% ethyl alcohol under reflux.

After the reaction, about ? When concentrated and left at room temperature, crystals begin to precipitate.

The reaction solution was passed through the mouth, the crystals were washed with water, methyl alcohol, and ether, and recrystallized from 95% ethyl alcohol to obtain colorless needle-like crystals.

The yield was 16.4%.

The thus obtained anthranilic acid-N-D-galactoside was gradually dissolved in an aqueous solution of 1(Ff) containing a calculated amount of NaOH, the insoluble material was filtered through the mouth, and the oral liquid was concentrated under reduced pressure.
A large excess of acetone was added and the mixture was dehydrated and dried to obtain colorless crystals.

The yield was 100(f) and the TOtal yield was 16.4%.

Production Example 9 Production method of P-aminobenzoic acid-N-L-rhamnoside mono-Na salt P-aminobenzoic acid 3f! , L-rhamnose (49%), and ammonium chloride (0.19%) are condensed under heating under reflux cooling in 94% ethyl alcohol.

After the reaction, if left at room temperature, crystals will precipitate.

The reaction solution was passed through the mouth, the crystals were washed with water and diluted methyl alcohol, and then recrystallized from 50 (fl) methyl alcohol to obtain colorless needle-shaped crystals.

The yield was 30.9%.

Thus obtained, P-aminobenzoic acid-N-L
- 101 containing a calculated amount of NaOH containing 0.29 rhamnosides
) It was gradually dissolved in an aqueous solution, the insoluble material was passed through the mouth, the oral liquid was concentrated under reduced pressure, a large excess of acetone was added, and after dehydration, it was dried to obtain colorless crystals.

The yield was 100(f), and the TOtal yield was 30.901).

Production Example 100-Aminobenzoic acid-N-L-rhamnoside monosodium salt production method Anthranilic acid 2.39, L-rhamnose 2.89, and ammonium chloride 0.29 are condensed under reflux in 25 ml of methyl alcohol.

After the reaction, if left at room temperature, crystals will precipitate.

The reaction solution was passed through the mouth, the crystals were washed with water and methyl alcohol, and then recrystallized from 50% methyl alcohol to obtain colorless needle-shaped crystals. The yield was 9.8%.

0.21 of the thus obtained anthranilic acid-N-L-rhamnoside was gradually dissolved in a 1% aqueous solution containing a calculated amount of NaOH, the insoluble matter was sifted through the mouth, and the oral liquid was concentrated under reduced pressure. A large excess of acetone was added and the mixture was dehydrated and dried to obtain colorless crystals.

The yield was 100%, and the TOtal yield was 9.8%.

[Brief explanation of the drawing]

1 to 20 are infrared absorption spectrum diagrams of the substances of the present invention.

Claims (1)

[Claims] 1 At least one aminobenzoic acid or its salt represented by the general formula ▲ Numerical formula, chemical formula, table, etc. ▼ (where R_1 represents an arabinose, xylose, glucose, galactose, rhamnose residue) An antidiabetic agent containing. 2 General formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (However, R is arabinose, xylose, glucose,
The antidiabetic agent according to claim 1, which is represented by galactose and rhamnose residues. 3. The antidiabetic agent according to claim 1 or 2, which is in an oral administration form. 4. The antidiabetic agent according to claim 1 or 2, which is in a parenteral administration form.