JPS591686B2 - Antitumor agent containing aminobenzoic acid derivatives - Google Patents

Description

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

R-M 卜〈２;＞(1)（However, R1 represents arabinose, xylose, glucose, galactose, rhamnose residue） Conventionally, synthetic compounds and antibiotics have been used as anticancer agents, but these are not carcinogenic. Although they were highly effective, they had the disadvantage of being highly toxic and causing side effects because they also affected 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, the antitumor effect of aminobenzoic acid-N-D-mannoside, We found a lowering effect and a blood pressure lowering effect. After that, as a result of further research and improvement, the above-mentioned general medicines were found to be less toxic and more effective. The present invention was completed by discovering the compound shown in 1). Although the compound represented by the general formula (1) (hereinafter abbreviated as "this substance") has a simple structure, it has extremely low toxicity and has no antibacterial activity, so there is no need to worry about disturbing the intestinal flora. Long-term 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 anti-intestinal ulcer effects and are useful as anticancer 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.

The salt of the aminobenzoic acid derivative of the present invention is one in which the hydrogen atom of the -COOH group in formula (1) is replaced with an alkali metal, an alkaline earth metal, or an aluminum metal. These metals may be any metal as long as it is acceptable as a drug, and usually Na. K. Mg,C
a. Al is preferable, and Na is particularly preferable. The sugar moiety has a 6-membered ring (pyranose) structure. The manufacturing method of this substance is as follows.

Aminobenzoic acid 4.5-57, sugar (L-arabinose,
D-xylose, D-glucose, D-galactose or L-rhamnose) 5-6f7, ammonium chloride 0
．． 1 to 0.5y was heated and condensed under reflux of 40 to 90 ml of 95 to 100% ethanol or pure methanol.

If crystals precipitated after being left at room temperature or in a cold place for a while, the reaction solution was thoroughly 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: −
H was measured using an alcohol-aqueous system, and -Na was measured using water as a solvent with an optical path length of 50 mm.

(3) Elemental analysis Measured 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) Measured by the KBr method using IR Japan Spectroscopy DS-701G model.

Note that the drawing numbers match the sample waste 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 LD5O value was 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 (Litchfie)
ld-WillcOxOn) graphic calculation method.

The results are shown in Table 2. Both are LD regardless of intraperitoneal or oral administration.
With a 5O value of 67/K9 or higher, it is a low-toxic substance that falls well within the category of so-called "ordinary drugs," and 6 out of 10 compounds, more than half, have an LD5O value of 107/K9.
Based on the above, it can be said that this drug is extremely safe. (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 volume of heated and dissolved agar medium, poured into a Petri dish, and plated.

Heart infusion agar (bacteria) and Saburoichi agar (fungi) were used as the culture medium, and after inoculation of 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
scherichiacOlllFOl2734) Staphylococcus aureus
us2O9P) Bacillus subtilis
lsIAMlO69) Baker's Yeast (SaccharOmy
cescerevisiae IAM42O7) Candida albicans ATCC752
) TrichOphytOnmentagI'
0Ph37teSF06124) Black mold (Asperg
illunigerIAM3OOl) As a result, this substance did not inhibit the growth of any bacteria at a concentration of 1/ml.

(3) Mutagenicity First, an investigation using Rec-Assay was conducted.

That is, a recombinant repair-deficient strain (Bacillus subt.
ilis M45) and the recombinant repair carrier strain (B. subtil
lsHl7) on B-agar medium (Nikukozu 107,
Polypeptone 107, NaCl 5tl, agar 15t1, distilled water 1000ml, pH 7.0) were streaked so that the starting points did not touch each other.

Dissolve this substance in sterile water and add 0.04a to 8mm in diameter.
After absorbing the mixture onto a round paper, the mixture was immediately left to stand so as to cover the starting point of the streak, and cultured overnight at 37°C to measure the length of the growth inhibition zone. Kanamycin was used as a negative control, and mitomycin C was used as a positive control. Next, perform the reverse mutation test with S
almOnellatyPhimUrillmTA98
and TAlOO (both require histidine).

0.5mM biotin-0.5mM histidine solution 1/1
Add 0 volume of soft agar solution (NaCl6y, agar 6y1 1000ml of distilled water) to 2d of bacterial solution 0.1ml11 chemical solution 0.1ml
1 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 In order to understand the effect of this substance on cell-mediated immunity, ICR-JCL mice were used to perform a foot-hop reaction (FOOt) using sheep red blood cells as an antigen.
padreactiOn) was performed.

Hedge's red blood cells were suspended in 10% physiological saline, and 0.2 mL of this solution was injected through the tail vein for primary sensitization, and after 7 days, 0.05 mL of a 40% suspension of sheep red blood cells was added.
1 was injected into the foot to perform secondary sensitization, and the foot thickness was measured the next day. This substance is 250mg mainly on the day of primary sensitization.
/K9 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-administration) group.

(5) Antibody production ability In order to understand the effect of this substance on humoral immunity, ICR-J
For CL mice, a 10% volume suspension of Higgi's red blood cells was administered at 0.
2ml 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.

This substance is 250Tf19/Kg mainly on the day of sensitization.
was administered intraperitoneally 5 times daily. 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) Anti-tumor effect 1 x 106 SareOrlla-180 cells were transplanted subcutaneously in the axillary region of 6-week-old ICR-JCL female mice (purchased from Nippon Clea Co., Ltd.), and 10 cells were administered every other day for 24 hours after transplantation.
100 m9/time of this substance dissolved in sterile physiological saline.
K9, 500 w/K9 and 1000 W/Kg were orally administered.

Tumor nodules were excised on the 25th day after metastasis, and the growth inhibition rate (1.R.%) was calculated using the following formula. Note that 10 mice were used in each group.

T: Administration group average tumor weight C: Control group average tumor weight The results are shown in Table 5.

All of the compounds tested showed antitumor activity;
Generally, aminobenzoic acids with an amino group in the para position to the carboxyl group showed higher activity than those with the amino group in the ortho position. In particular, even though sodium p-aminobenzoate-N-L-rhamnoside is administered orally, 500T! The fact that 19/K9 administration has a high proliferation inhibition rate of 72.5% is an astonishing fact, considering that no side effects are observed. In addition, p-aminobenzoic acid mono-N-D-xyloside, p-aminobenzoic acid-
For ND~glucoside, p-aminobenzoic acid-NL-rhamnoside, the growth inhibition rate (1.R.%) was determined in the same manner by oral administration of 1000 Immediately/K9, and 42.1, 40, 3,
I got 65.2.

Next, we will discuss the formulation of this substance.

When used as an antitumor agent, the substance can be used in any convenient form to obtain its medicinal effect 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.

Thus, forms for oral or parenteral administration are optionally provided. 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, ambles, 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 wt. %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, and intravenous injections;
Including intravenous fluids, etc. The dosage of the medicine of the present invention depends on whether it is an animal or a human, and is influenced by age, individual differences, medical conditions, etc., so in some cases, doses outside the ranges listed below may be administered, but generally when the drug is administered to humans. , the oral dosage of this substance is 0.1 to 500 η per kg body weight, preferably 1 to 500 η per day.
250η, the parenteral dosage is also 0.01-200
That is, preferably 0.1 to 100η is administered in 1 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 mm.

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 mm, 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-N-L-arabinoside mono-Na salt P-aminobenzoic acid 4.67, L-arabinose 57, ammonium chloride 0.5f7 407TL194
% ethyl alcohol under reflux.

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-like 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 monosodium salt Anthranilic acid 2.3t. 30m2 of L-arabinose 2.57, ammonium chloride 0.27
Heat condensation in 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-NL-arabinoside was gradually dissolved in a 1% aqueous solution containing a calculated amount of NaOH, the insoluble material was filtered off, the oral liquid was concentrated under reduced pressure, and a large excess of acetone was added. After dehydration and drying, colorless crystals were obtained.

The yield was 100%, and the total yield was 61.3%.

Production Example 3Production of P-aminobenzoic acid-ND-xyloside monosodium salt P-aminobenzoic acid 2.37, D-xylose 2.5f1 ammonium chloride 0.057 to 251
IL1 is heated and condensed in 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%. Obtained in this way,
P-aminobenzoic acid-N-D-xyloside in the calculated amount of N
It was gradually dissolved in a 1% aqueous solution containing aOH, the aqueous solution 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%, and the TOtal yield was 73.7%.

Production example 40-Aminobenzoic acid-N-D-xyloside monosodium salt production method Ansura [2.3y. 2.57 g of D-xylose and 0.27 g of ammonium chloride are heated and condensed in 35 ml of ethyl alcohol under reflux.

After the reaction, the mixture was concentrated to about 1/2 under reduced pressure, and when left at room temperature, crystals were observed 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-ND-xyloside was gradually dissolved in a 1% aqueous solution containing a calculated amount of NaOH, the insoluble material was filtered off, the oral liquid was concentrated under reduced pressure, and a large excess of acetone was added. After dehydration and drying, colorless crystals were obtained.

The yield was 100%, and the TOtal yield was 76.4%.

Production example 5p-aminobenzoic acid-N-D-glucoside N
Production method of a salt p-aminobenzoic acid 5f7, D-glucose 6,47,
0.5y of ammonium chloride is heated and condensed in 50ml of 94% ethyl alcohol under reflux.

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 gelatinous. 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%.

The thus obtained P-aminobenzoic acid mono-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 TOtal yield was 33.7%.

Production example 60-aminobenzoic acid-N-D-glucoside-
Production method of Na salt Anthranilic acid 4,6y. D-glucose 6.0y, ammonium chloride 0.51 40ml119
Heat condensation in 5% ethyl alcohol under reflux.

After the reaction, the mixture was concentrated to about 1/2 under reduced pressure and left in the refrigerator overnight to observe the precipitation of crystals.

The reaction solution was poured into the mouth, and 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 anthranilic acid-N-D-glucoside thus obtained 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 acetone was removed. In addition, the mixture was dehydrated and dried to obtain colorless crystals.

The yield was 100%, and the TOtal yield was 4.6%.

Production Example 7 Production method of p-aminobenzoic acid-N-D-galactoside-Na salt P-aminobenzoic acid 1.57, D-galactose 2y1 ammonium chloride 0.17 in 30ml
The mixture is heated and condensed in 94% 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.

The yield was 18.1%. Obtained in this way, P
-Aminobenzoic acid-N-D-galactoside in the calculated amount of N
The mixture was gradually dissolved in a 1% aqueous solution containing aOH, filtered to remove insoluble matter, and the oral liquid was concentrated under reduced pressure. A large excess of acetone was added, dehydrated, and dried to obtain colorless crystals.

The yield was 100%, and the TOtal yield was 18.1%.

Production example 80-Aminobenzoic acid-N-D-galactoside-Na salt production method Anthranilic acid 2.4f7, D-galactose 3.07, ammonium chloride 0.2f 30m
Heat condensation in 195% ethyl alcohol under reflux.

After the reaction, the mixture was concentrated to about 1/2 volume under reduced pressure, and when it was left at room temperature, crystals were observed 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 a 1% aqueous solution containing a calculated amount of NaOH, the cotton cloth was passed through the mouth, the oral fluid was concentrated under reduced pressure, and a large excess of acetone was removed. In addition, the mixture was dehydrated and dried to obtain colorless crystals.

The yield was 100%, and the TOtal yield was 16.4%.

Production example 9P-aminobenzoic acid-N-L-rhamnoside N
Production method of a-salt 37 of P-aminobenzoic acid, 47 of L-rhamnose, and 0.1y 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 was passed through the mouth, the crystals were 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 thus obtained P-aminobenzoic acid N-L-
Rhamnoside 0.27 was gradually dissolved in a 1% aqueous solution containing the calculated amount of NaOH, the insoluble matter was filtrated, the oral fluid was concentrated under reduced pressure, a large excess of acetone was added, and after dehydration, it was dried to form colorless crystals. I got it.

The yield was 100%, and the TOtal yield was 30.9%.

Production Example 100-Aminobenzoic acid-N-L-rhamnoside monosodium salt production method Anthranilic acid 2.3y. ．． 25ml of L-rhamnose 2.87, ammonium chloride 0.2V
Heat condensation in methyl alcohol under reflux.

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%.

The thus obtained anthranilic acid-NL-rhamnoside 0.27 was gradually dissolved in a 1% aqueous solution containing a calculated amount of NaOH, the insoluble material was sifted, 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 drawings]

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

Claims (1)

[Claims] 1 At least one aminobenzoic acid derivative 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) Antitumor agent containing seeds. 2 General formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (However, R is arabinose, xylose, glucose,
The antitumor agent according to claim 1, which is a galactose or rhamnose residue. 3. The antitumor agent according to claim 1 or 2, which is in an oral administration form.