Technical field

The present invention relates to novel aminobenzoic acid N-glycoside derivatives of formula I

COOH

wherein R stands for 2-acetamido-2-deoxv-D- glucopyranosyl group, to the pharmaceutically acceptable esters and salts thereof, to pharmaceutical compositions comprising the same and a process for the preparation of the novel compounds. The novel compounds are suitable for the prevention or treatment of diseases caused by pathological free radical reactions.

Background art There are several amino benzoic acid derivatives known in the art which exhibit pharmaceutical activity. Belgian patent specification No. 898,359 describes such compounds of formula I wherein R represents a

SUBSTITUTE SHEET

residual group formed by the removal of -OH from the 1(alpha) or 1(beta) -position of arabinose, xylose, rhamnose, glucose, galactose, mannose or fructose and to the pharmaceutically active salts thereof. The compounds are suitable for the treatment of cerebral ischemia and inhibit thro bocite aggregation.

British published patent application No. 2,056,856 898,359 describes such compounds of formula I wherein R represents a residual group formed by the removal of -OH from the l(alpha) or 1(beta) -position of arabinose, xylose, rhamnose, glucose, galactose, mannose or fructose and to the C* ^* _| __4 alkyl esters and pharma¬ ceutically acceptable metal salts thereof. This derivative regulates prostaglandins or metabolites thereof.

When the mechanism of action of p-amino benzoic acid N-glycoside derivatives was examined, it has been stated that they modify prostaglandine level and metabolism and regulate the synthesis of cyclic nucleotides (12th International Congress of Chemotherapy, Abstracts 195, 19-24 July, 1981, Florence, Italy) .

The effect of so-called free radicals onto biological systems was thoroughly studied after the Hiroshima disaster. The experiments have given new results and a new explanation of several symptoms, pathological processes. It can be supposed that the free radicals play an important role in numerous illnesses, such as Parkinson's disease, thrombosis, athero¬ sclerosis, ischemia, essential hypertonia, diabetes mellitus, tumorous and inflammation illnesses (Tappel, A. L. : Lipid Peroxidation Damage to Cell Competents, Federation Proceedings, Vol. 32, No. 8, 1973; Har an, D.: The Free Radical Theory of Aging, E. Eds. Hayaishi and Mino Elsevier 385-393, 1987). The most accepted form of protection against

pathological free radical reactions is the preventive or curative administration of natural and synthetic anti¬ oxidants. These antioxidants may influence the different stages of lipidperoxidation, but one specific anti- oxidant may exhibit more activities. Such antioxidants include e.g. vitamin A, C and E, D-penicillamine, lipoic acid, lobenzarit disodium, 5,6-methylene-bis(2,2,3-tri- methyl)-1,2-dihydroquinoline.

Compound of formula I of the present invention has not been described yet and no reference has been made that similar compounds have antioxidant activity and may effect the pathological free radical reactions in living organisms.

Summary of the invention

The present invention is based on the recognition that compounds of formula I, their pharmaceutically acceptable esters and salts are suitable for the preven¬ tion or treatment of diseases caused by pathological free radical reactions.

The invention is based on the further recognition that the compounds of formula I can be prepared by reacting a ino benzoic acid with N-acetyl glucosamine. Hitherto the glycosilation of amino benzoic acids with N-acetyl glucosamine could have not been carried out with the known techniques.

Detailed description of the invention

The present invention relates to novel aminobenzoic acid N-glycoside derivatives of formula I

COOH

HEET

wherein R stands for 2-acetamido-2-deoxy-D-gluco- pyranosyl group, to the pharmaceutically acceptable esters and salts thereof.

The pharmaceutically acceptable esters include the Cι_ ₄ alkyl esters, i.e. methyl, ethyl, propyl, i-propyl, butyl, i-butyl, sec-butyl esters.

The pharmaceutically acceptable salts include the metal salts, preferably the alkaline metal salts, such as sodium or potassium salts. The present invention also covers the steric isomers of the compounds of formula I.

The present invention covers

4-(2-acetamido-2-deoxy-D-glucopyranosyl) benzoic acid and the pharmaceutically active esters and salts thereof,

3-(2-acetamido-2-deoxy-D-glucopyranosyl) benzoic acid and the pharmaceutically active esters and salts thereof,

2-(2-acetamido-2-deoxy-D-glucopyranosyl) benzoic acid and the pharmaceutically active esters and salts thereof, and the steric isomers thereof.

Preferred compounds are the 2-, 3- or 4-(2- acetamido-2-deoxy-D-glucopyranosyl) benzoic acid Cι__ ₄ alkyl esters, more preferred compounds are 2-, 3- or 4- -(2-acetamido-2-deoxy-D-glucopyranosyl) benzoic acid methyl ester and 2-, 3- or 4-(2-acetamido-2-deoxy-D- glucopyranosyl) benzoic acid ethyl ester.

Other preferred group of compounds is the 2-, 3- or 4-(2-acetamido-2-deoxy-D-glucopyranosyl) benzoic acid alkaline metal salts from which 2-, 3- or 4-(2- acetamido-2-deoxy-D-glucopyranosyl) benzoic acid sodium salt is the most preferred.

The compounds of formula I are prepared by reacting a compound of formula II

COOR ¹

I

wherein R is the same as defined hereinabove, R ¹ is hydrogen or alkyl group, with N-acetyl glucosamine and if desired, transforming the compound thus obtained into pharmaceutically active ester or salt in a manner known per se.

The compounds of formula I can be prepared by glycosilating the corresponding amino benzoic acid derivative with N-acetyl glucosamine in water. The amino benzoic acid N-glycoside derivatives thus prepared can be recovered in the form of pure anomers with good yield.

According to a preferred embodiment of the invention o-, m- or p-amino-benzoic acid and N-acetyl glucosamine are suspended in water,' the reaction mixture is heated to a temperature of 60 to 95 °C, kept at the same temperature for some minutes, then cooled to room temperature and the free acid is recovered. The metal salt of the carbonic acid can be prepared in a manner known per se.

If the ester derivative of o-, m- or p-amino benzoic acid is used as starting material, the reaction mixture is slightly acidified by the addition of a mineral acid, preferably hydrochloric acid and then heated. The target compound crystallizes from the mixture after cooling and if desired it can be recrystallized from ethanol.

Acute toxicity

The acute toxicity of compounds according to Example 1 to 4 was measured by using the conventional test method on CFLP mice by oral administration. The LD5 ₀ value of all of the compounds was higher than 10 g/kg,

SUBSTITUTE SHE

it means that the compounds of the invention are not toxic.

Antioxidant and free radical scavenger activity The antioxidant and free radical scavenger activity of the compounds of the invention was examined on the basis of the in vitro test worked out by Stocks at al. (Stocks, I. et_al, Clin. Sci. Mol. Med. 215-22, 223- -33, 1974) . The following compounds were used in the test:

A) Compounds of the invention

1) 4-(2-acetamido-2-deoxy-D-glucopyranosyl)-benzoic acid methyl ester

B) Comparative compounds 1) p-amino benzoic acid

2) p-amino benzoic acid methyl ester

3) N-acetyl glucosamine

4) p-amino benzoic acid N-xyloside sodium salt.

In the above test the C50 value (that concentration which is necessary to reduce the initial autooxidation with 50 %) of the compounds was measured. The C50 value is characteristic for the antioxidant properties of the compound; the less is C50 the better radical scavenger the compound is. According to the test 4-(2-acetamido-2-deoxy-D- glucopyranosyl) benzoic acid methyl ester exhibits surprisingly high antioxidant activity and inhibits lipid peroxidation depending on the dose. Its C50 value is 0.66 mM. According to the test results the comparative compounds did not influence the lipidperoxidation. At a concentration of 0.66 mM they reduced the initial auto¬ oxidation in an extent of less than 25 %.

The C50 value of vitamine E, which is generally used in therapy against pathological free radical reactions,

is 0 . 45 mM.

The advantage of the compounds of the invention over vitamin E is that they are water and lipid soluble, while vitamin E is only lipid soluble. Dosage forms suitable for internal administration contain from about 1 milligram to about 500 milligrams of active ingredient per unit. The dosage administered vary depending upon known factors such as the mode and route of administration, age, health and weight of the recipient; nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the effect desired. In the pharmaceutical compositions the active ingredient will ordinarily be present in an amount of about 0.5 to 95 % by weight based on the total weight of the compositon.

The pharmaceutical compositions comprising the compounds of the invention as active ingredient can be administered via any of the accepted modes of administration for therapeutic agents. These methods include oral, parenteral, transdermal, rectal, subcutaneous and other systemic modes. When the intended route of administration is parenteral, the pharma¬ ceutical composition should, of course, be in a sterile form. Thus the compositions may be in the form of solid dosage forms such as capsules, tablets, coated tablets, powders, suppositories or liquid dosage forms such as syrups, emulsions, injections, elixirs, suspensions, emulsions, etc. For solid compositions, conventional non-toxic solids include, for example, pharmaceutical grades of manitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like may be used. The active compound may be formulated as suppositories

using, for example, polyalkylene glycols, such as propylene glycol, as carrier.

Liquid dosage forms can, for example, be prepared by dissolving, dispersing, suspending, emulsifying, etc. an active compound and optional pharmaceutical adjuvants in an excipient, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol and the like.

If desired, the pharmaceutical composition may also contain minor amounts of nontoxic auxiliary substances such as wetting agents, pH buffering agents, preservatives, flavouring agents, etc., for example, sodium acetate, sodium lauryl sulphate, sorbitan mono- laurate, triethanolamine sodium acetate, triethanolamine oleate, etc. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art; see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975.

The invention is further illustrated by the following, non-limiting examples.

Example 1

4- (2-acetamido-2-deoxy-β-D-glucopyranosyl) benzoic acid methyl ester 1.51 g (10 millimoles) of p-amino benzoic acid methyl ester and 4,42 g (20 millimoles) of N-acetyl-D- -glucoseamine are suspended in 8 ml of water. Then 0.2 ml of 2 N aqueous hydrochloric acid are added and the reaction mixture is kept at a temperature of 90 °C for 3 minutes. After cooling the precipitated crystals are filtered off and dried on air. The crude product _' thus obtained is recrystallized from 40 ml of ethanol. Yield: 1.521 g (42.9 %) .

[ ^a ] _n ° = -74.9° (c = 0.79, dimethyl formamide) Elementar analysis for formula Ci ₆ ^H ₂₂ ^N 2° ₇ (354.36):

Calculated: C: 54.23 % H: 6.26 % N: 7.91 o I Found: C: 54.32 % H: 6.29 % N: 7.83 C-NMR (DMSO-d6) : Enclosure 1

Example 2

4-(2-acetamido-2-deoxγ-β-D-glucopyranosyl) benzoic acid sodium salt

1.74 g (10 millimoles) of 4-amino benzoic acid and 3.3 g (15 millimoles) of N-acetyl-D-glucoseamine are suspended in 8 ml of water. The reaction mixture is kept at a temperature of 80 °C for 5 minutes. After cooling the precipitated material is filtered off. The filtrate is slightly alkalified by the addition of 20 % aqueous sodium hydroxide solution, then 40 ml of ethanol are added. The oily residue is dissolved in 10 ml of water and the product is precipitated by the addition of 50 ml of ethanol. Yield: 1.42 g (39.2 %) . [ ]2 ⁰ = +6.8° - +6.4° (c = 0.7, water) Elementar analysis for formula C ₁ 5H1 ₉ N2 ₀ O ₇ Na (362.32): Calculated: C: 49.73 % H: 5.29 % N: 7.73 %, Found: C: 49.64 % H: 5.22 % N: 7.80 %. C-NMR (DMS0-d6) : Enclosure 2

Example 3 4-(2-acetamido-2-deoxy-β-D-glucopyranosyl) benzoic acid ethyl ester

16.52 g (0.1 mole) of p-amino benzoic acid ethyl ester and 33.18 g (0.15 moles) of N-acetyl-D-glucose¬ amine are suspended in 80 ml of water. Then 1 ml of 2 N aqueous hydrochloric acid are added and the reaction mixture is thoroughly mixed at a temperature of 80 °C for 60 minutes then at a temperature of 95 °C for 5 minutes. The homogenous solution is left to cool. After 2 hour cooling the precipitated crystals are filtered off and dried on air. The crude product thus obtained

SUBSTITUTE SHEET

(30.1 g; 81.7 %) is recrystallized from 400 ml of ethanol comprising 1 ml of concentrated ammonia solu¬ tion. Yield: 20.2 g (54.8 %) . Melting point: 220-221 °C. [ ]2 ⁰ = -103.6° (c = 0.93, dimethyl formamide)

Elementar analysis for formula Ci ₇ H ₂₄ N ₂ o° ₇ (368.39): Calculated: C: 55.43 % H: 6.57 % N: 7.6 %, Found: C: 55.51 % H: 6.59 % N: 7.55 %. C-NMR (DMSO-d6) : Enclosure 3

Example 4

2-(2-acetamido-2-deoxy-beta-D-glucopyranosyl) benzoic acid methyl ester

1.51 g (10 millimoles) of 2-amino benzoic acid methyl ester, 4,42 g (20 millimoles) of N-acetyl-D- glucoseamine, 8 ml of water and 0.3 ml of 2 N aqueous hydrochloric acid are heated to at a temperature of 90 °C for 3 minutes. After cooling the crystallization starts upon mechanical intervenion. After 1 hour standing the precipitated crystals are filtered off and dried on air (1.39 g; 39.3 %). After one night standing further 0.44 g (12.5 %) is precipitated from the mother liquor. Total yield: 51.8 %. The crystalline crude product is recrystallized from 40 ml of ethanol contain- ing 1 drop of ammonia solution. Thus 1.21 g (34.2 %) of product are obtained. Melting point: 228-229 °C. [ ] ²⁰ = -74.2° (c = 0.30, dimethyl formamide).

Example 5

Tablet

The following tablets were prepared: compound of Example 1 microcrystalline cellulose corn starch talcum

500 g

Example 6

Capsule

The following mixture was prepared by homogenizing the ingredients then the mixture was filled into hard gelatine capsules.

Compound of Example 2 200 mg lactose 100 mg corn starch 50 mg talcum 15 mg magnesium stearate 0.5 mg

380 mg

SUB3TETUTΞ SHEET