US2731494A

US2731494A - New and biologically active diphenylmethane derivatives and method of making the same

Info

Publication number: US2731494A
Application number: US308704A
Authority: US
Inventors: Hahn Ladislaus Arthur; Fekete Janos
Original assignee: Ferrosan AB
Current assignee: Pharmacia Animal Health Inc
Priority date: 1949-12-09
Filing date: 1952-09-09
Publication date: 1956-01-17
Anticipated expiration: 1973-01-17

Description

.been denominated mucooligosaccharase.

United States Ladislaus Arthur Hahn, Mairno, Sweden, and Janos Fekete, Lorena, Brazil, assignors to Aktiebolaget Ferrosan, Malmo, Sweden, a corporation of Sweden Application September 9, 1952, Serial No. 308,704

Claims priority, application SivedenDecer'nber 9, 1949 1 Claim. (Cl. 260-507) No Drawing.

This application is a continuation-impart of our copending application No. 199,564, filed December 6, 1950, now abandoned.

The present invention relatesto new biologically active diphenylmethane derivatives and to a method of makirig the same.

An enzyme which is capable of splitting mucopolysaccharides and amongst them particularly the hyaluronic acid has been discovered i. e. in the testicles and the sperm of mammals, furthermore in the leech, in serpents and insects poisons and in certain bacteria. The enzyme has been denominated mucinase but the name has later been changed to hyaluronidase, or mucopolysaccharase, respectively.

The substrate hyaluronic acid is rather common in the animal organism. Among the most important sources may be mentioned the interfibrillar substanceof mcsenchymal tissues, especially of the connective tissues, furthermore the vitreous body of the eye, the synovia and the gel that surrounds the ovum on its way through the oviduct. Another important substrate for the hy aluronidase, the condroitin sulfuric acid, is a substantial component of all sorts of cartilage, thus also of the cartilage of the joints.

The splitting effect of the hyaluronidase on the hyaluronic acid has been elucidated to a large extent: the highly polymeric acid is depolymerized and split into smaller units, the glucosidic bonds between the elements of the hyaluronic acid, i. e. the glucuronic acid and the N--acetyl glucos amine, being hydrolyzed. In the complete splitting up of the hyaluronic acid to monosaccharide units also other enzymes are active.

One of these enzymes has been isolated from bulls testicles and has The splitting of the hyaluronic acid and of other mucopolysaccharides, for instance the chondroitin sulfuric acid, through hyaluronidase may be experimentally shown and quantita tively determined for instance (a) by observing the decrease in viscosity of the substrate of (b) by determining the reducing groups which are liberated at the hydrolysis. There is also a biological method of determination which is based upon the influence of the hyaluronidase on the permeability of the skin, said influence being explained in the following.

The effect of the hyaluronidase in vivo corresponds to its efiect on mucopolysaccharides in vitro: for instance the hyaluronic acid of the skin is split by the action of hy- .aluronidase, which is brought into the tissue in one way mother, and looses its high viscosity. This causes the skin to become much more permeable. This increase of, the permeability is rather general and results therein, thatas well physiological as non-physiological substances,-

.such as water, salts, added chemical compounds of all .kinds, coloring matters, poisons, bacteria and virus will spread many times quicker in the tissue if hyaluronidase is present. This may easily be shown experimentally by intracutaneous injection of 0.2 millilitre of Indian ink 2,731,494 Patented Jan. 17, 1956 (I for instance on one side of the back of a rabbit, the same amount of ink to which there has been added some micrograms of hyaluronidase being injected on the other side. The ink to which no hyaluronidase has been added spreads only very slowly in the skin. The ink which has been mixed with the enzyme spreads in a few minutes over an area of 206 cm? or more. That which has bee said about the effect of the hyaluronidase upon the permeability of the skin holds true also in respect of other kinds of mesenchymal tissue.

The high viscosity of the syncvial fluid depends to a great extent on the hyaluronic acid and the same holds true i. e. of the vitreous body of the eye and of the gel that surrounds the ovum in the oviduct. The effect of the hyaluronidase upon such materials is to make them lose their viscosity so that they become fluent.

The hyaluronidase of certain bacteria is of great importance for various reasons. Some of the pathogenic bacteria containing hyaluronidase, such as streptococci, are connected with certain types of rheumatic diseases. Since the viscosity of the synovial fluid depends mainly on its content of hyaluronic acid and since the cartilage of the joints contains chondroitin sulfuric acid it was assumed that at least some forms of rheumatic and other joint diseases are caused directly or indirectly by hyaluronidase.

The effect of the hyaluronidase upon connective tissues results therein that bacteria containing or producingthis enzyme will penetrate through the organism much easier than other bacteria so that the risk of infection is highly increased. The hyaluronidase-bearing bacteria need not necessarily be pathogenic themselves, or they may be pathogenic only to a small degree-nevertheless they may mean a great danger to the organism by facilitating the penetration of other non-physiological substances, such as other bacteria, virus etc., into tissues and organs. This is of particular importance since many highly pathogenic virus show a rather low penetration capability. If the organism is infected by hyaluronidase-producing bacteria infection with the said agents may result.

Since hyaluronic acid is present also in capillary walls the capillary permeability is influenced by hyaluronidase. The hyaluronidase may therefore promote or facilitate the penetration of inflective substances through the capillaries.

Certain substances inhibiting the effect of the hyaluronidase are known. As an example carbo y-pbenzoquinone, rutin, ascorbic acid, heparin, hexylresorcinol, certain sera fractions, and nitrates of .hyaluronic acid may be mentioned.

It has now been discovered that an inhibition of the action of hyaluronidase which is many times stronger than that of the above-mentioned known substances is developed by certain diphenylmethane derivatives. These derivatives are obtained by condensing with formaldehyde compounds selected from the group consisting of monosulfo and disulfo derivatives of dihydroxy benzoic acids and monosulfo derivatives of trihydroxy benzoic acids. Hyaluronidase inhibitory power of these compounds is more than 30 times higher than that of any inhibitory earlier described. This very high inhibitory activity on hyaluronidase makes it possible to use these compounds in the treatment of rheumatoid arthritis on one side and infectious diseases induced by hyaluronidase producing bacteria on the other side.

The inhibitors according to the invention may also be introduced-into the organism in a suitable manner, as'pcr nos, by superficial treatment or by. subcutaneous,-intra- 1 muscular or intraperitoneal injection, etc., in order to prevent the spreading-effect of the hyaluronidas'e in poisonings and infections. As mentioned above serpents and insects poisons and various pathogenic bacteria contain hyaluronidase and in part also other mucopolysaccharases. If these enzymes get into the organism such as by biting or infection they hydrolyse the hyaluronic acid of the skin and other tissues so that the poison or the bacteria are more easily infiltrated in the organism. Also infection with other infective substances which are free from hyaluronidase, such as bacteria and virus, spreads much quicker in the organism if the same is infected with hyaluronidase-bearing bacteria. The inhibitors according to this invention inhibit the action of the hyaluronidase of poisons and bacteria upon tissues and thus prevent or limit infection. The action ditfers principally from the treatment with anti-toxins or anti-sera (no specificity).

The compounds which are the matter of the present invention are prepared in the following manner:

The hydroxy-benzoic acid derivative is dissolved or suspended in a suitable solvent (for instance water, alcohol, dioxan, ether, acetic acid, hydrochloric acid, sulfuric acid). Then the formaldehyde solution is added either all at one time or successively in the course of the reaction. The latter mode of operation is used in certain cases in order to control the condensation reaction.

When using as starting material hydroxysulfo benzoic acids, which easily condense with formaldehyde the reaction will occur at room temperature and in absence of condensation agents. However, in most cases it is preferable, or necessary, that a condensation agent is present. The reaction then occurs either at room temperature, or at an elevated temperature, and the reaction period may vary from some minutes up to several hours depending upon the reactive power of the compound in question, the concentration of the condensing agent and other conditions. As condensation agents mineral acids (for instance hydrochloric acid, sulfuric acid) or alkalis (for instance alkali hydroxides) may be used.

If the starting material is sensitive to air oxygen the reaction should preferably be carried out in an inert atmosphere. Since the reaction often takes place in a heterogeneous phase the reaction mix should be vigorously stirred.

As generally the reaction product is considerably less soluble than the starting material, purification of the reaction product may often be carried out taking advantage of the said difference in solubility.

The production of certain of the contemplated substances may sometimes be carried out in such a manner that the carboxylic groups are introduced after the condensation has been effected.

The product of the condensation of dihydroxy-monosulfo-benzoic acid derivatives with formaldehyde under conditions here described was found to be a mixture of simple diphenylmethane derivatives and their polycondensation products. The presence of condensing agents, increasing concentration of mineral acids, increasing temperature and increasing time of reaction generally favour the production of polycondensed products. The separation of the simple diphenylmethane derivatives from the corresponding olycondensed products is rather difficult. Fractionationwith organic solvent, for example alcohol, gives in some cases good results.

We found that also the olycondensed products mentioned above show very pronounced inhibitory action on hyaluronidase. In most cases the inhibitory power of the polycondensed product is somewhat higher than the inhibitory power of the corresponding diphenylmethane derivatives.

' Example 1 0.2 mols of 2.5-dihydroxy'4-sulfo-benzoic acid are dissolved in 150 ml. of water, whereupon 0.1 mol of a 40% formaldehyde solution is added and the mixture boiled Off S02- droxy-4-sulfo-benzoic acid). Relative hyaluronidase inhibitory power: 4000. (Salicylic acid=1.)

Example 2 V In grams of cold 50% sulfuric acid 20 grams of gentisic acid are suspended, whereupon 4 grams of a 40% formaldehyde solution are added. The reaction mix is boiled during 5 hours with vigorous agitation. The warm product is filtered through a glass filter and the precipitate is pulverized and boiled with Water, whereupon the same is washed several times with hot water. The substance is easily soluble in alcohol, acetone and alkalis, but very little soluble in water. Its decomposition point is above 260 C. 11 grams of the condensation product and 26 grams of NazSO2-7H2O are dissolved in ml. of water and boiled during 2 hours. The solution is filtered and to the filtrate 13.0 grams of oxalic acid are added. The solution is boiled to drive off $02. The solution is then evaporated successively when sodium oxalate crystallizes. This is removed, whereupon the solution is evaporated to dryness. The residue is treated twice with 50 ml. of alcohol to remove free oxalic acid. The residue not soluble in alcohol is dried at 80 C. Yield: 72% of the theory. The product obtained is methylenedi-(2.5- dihydroxy-4-sulfo-benzoic acid) and contains some polycondensed products. Relative inhibitory activity: 3800.

Example 3 0.2 mols of 3.4.5-trihydroxy-2-sulfo-benzoic acid are dissolved in 150 ml. of water. After addition of 0.1 mol of 40% formaldehyde solution the mixture is boiled during 2 hours. The solution is then neutralized with ammonium carbonate and evaporated to dryness. Yield: 82% of the theoretical value. The product is 2.2'.3.3.4.4'- hexahydroxy-6.6-dicarboxy-5 .5 -disulfo-diphenylmethane. Relative inhibitory activity: 3000.

Example 4 In grams of cold 50% sulfuric acid 0.2 mol of 2.4-dihydroxy-sulfo-benzoic acid is dissolved. 0.1 mol of a 40% formaldehyde solution is then added. The mixture is boiled during 5 hours with vigorous agitation. After cooling the reaction mixture is neturalized with ammonium carbonate and then evaporated to dryness. Yield: 77% of the thoretical value. The product obtained is Polycondensed methylenedi- (2.4-dihydroxy-sulfobenzoic acid). Relative inhibitory power: 3000.

Example 5 Polycondensed methylenedi -(3.4 dihydroxy -5 -sulfobenzoic acid) is obtained by treating 3.4-dihydroxy-5- sulfo-benzoic acid in the similar manner as described for 2.4'dihydroxy-sulfo-benzoic acid in Example 4. Yield: 81% of the theoretical value. Relative inhibitory activity: 3500.

Example 6 In a mixture of 80 grams of 60% sulfuric acid and 40 grams of acetic acid 15.4 grams of 2.3-dihydroxybenzoic acid are dissolved. The solution is warmed on a boiling water bath and 4 grams of a 40% formaldehyde solution are added in the course of half an hour. Heating is continued during 8 hours. The warm product is filtered through a glass filter and the precipitation is pulverized and washed several times with hot water. Yield: 11.9 grams. 11 grams of the condensation product and 26 grams of NazSO-7Hz0 are dissolved in 150 ml. of water and boiled during 2 hours. The solution is filtered and to the filtrate 13.0 grams of oxalic acid are added. The solution is boiled to drive The solution is then evaporated successively when sodium oxalate crystallizes. This is removed, whereupon the solution is evaporated to dryness. The residue is treated twice with 50 ml. of alcohol to remove free oxalic acid. The residue not soluble in alcohol is dried at 80 C. Yield: 72% of the theory. The product obtained is 3.3.4.4'-tetrahydroxy-5.5'-dicarboxydisulfo-diphenylmethane being a mixture of the simple diphenylmethane derivative and of polycondensed products of varying molecular size. Relative inhibitory activity: 3300.

Example 7 0.2 mol of 2.6-dihydroxy-3.S-disulfo-benzoic acid is dissolved in 130 ml. of water. 0.1 mol of a 40% formaldehyde solution is then added and the mixture boiled during 2 hours. The solution is neutralized with ammonium carbonate and then evaporated to dryness. Yield: 85% of the theoretical value. The product obtained is an ammonium salt of 3.3'.5.5-tetrahydroxy- 4.4 dicarboxy 2.2.6.6' tetrasulfo diphenylmethane. Relative inhibitory power: 4100.

Example 8 In 80 grams of diluted hydrochloric acid (1:1) 18.1 grams of 3.5-dihydroxy-benzoic acid are suspended. After the addition of 4 grams of 40% formaldehyde solution the mixture is boiled during 12 hours. The reaction product is isolated in the same manner as in Example 1. Yield: 12.6 grams. 11 grams of the condensation product and 26 grams of NazSOz-7Hz0 are dissolved in 150 ml. of water and boiled during 2 hours. The solution is filtered and to the filtrate 13.0 grams of oxalic acid are added. The solution is boiled to drive off S02. The solution is then evaporated successively when sodium oxalate crystallizes. This is removed, whereupon the solution is evaporated to dryness. The residue is treated twice with 50 ml. of alcohol to remove free from oxalic acid. The residue not soluble in alcohol is dried at 80 C. Yield: 72% of the theory. The product obtained is methylenedi-(3.5- dihydroxy-sulfo-benzoic acid) and its polycondensed products. Relative inhibitory activity: 3000.

Example 9 2 mols of 2.3.4-trihydroxy benzoic acid are mixed with water, and on a boiling water bath there is added with agitation and in a nitrogen atmosphere a molar weight of formaldehyde. In absence of a condensation agent the reaction takes place when heating during 3 hours. The mixture is then cooled and the precipitate is filtered off and washed with cold water. 12 grams of the condensation product and 26 grams of NazSOa 7H2O Example In 100 grams of diluted hydrochloric acid 1:1) 17.0 grams of 2.4.5-trihydroxy benzoic acid are suspended.

6 After addition of 4 grams of 40% formaldehyde solution the mixture is boiled during 5 minutes. The isolation of the reaction product is carried out according to Example 3. Yield: 14.0 grams of 2.2.5.5'.6.6- hexahydroxy-3.3'-dicarboxy diphenylmethane. 12 grams of the condensation product and 26 grams of NazSOz 7H2O are dissolved in 150 ml. of water and boiled during 2 hours. The solution is filtered and to the filtrate 13.0 grams of oxalic acid are added. The solution is boiled to drive ofi S02. The solution is then evaporated successively when sodium oxalate crystallizes. This is removed, whereupon the solution is evaporated to dryness. The residue is treated twice with 50 ml. of alcohol to remove free oxalic acid. The residue not soluble in alcohol is dried at C. Yield: 72% of the theory. 1

The product obtained is methylenedi-(2.4.5-trihydroxysulfo-benzoic acid). Relative inhibitory power: 3100.

Example 11 In grams of cold 50% sulfuric acid 17.0 grams of 2.4.6-trihydroxy benzoic acid are suspended, whereupon 4 grams of a 40% formaldehyde solution are added. The reaction mixture is boiled during 5 minutes with vigorous agitation. The warm product is filtered through a glass filter and the precipitate is pulverized and washed with hot water until free from sulfuric acid. The product obtained is 2.2'.4.4'.6.6-hexa hydroxy-3.3-dicarb0xy diphenylmethane. 12 grams of the condensation product and 26 grams of Na2SO2-7H2O are dissolved in ml. of water and boiled during 2 hours. The solution is filtered and to the filtrate 13.0 grams of oxalic acid are added. The solution is boiled to drive otl' S02. The solution is then evaporated successively when sodium oxalate crystallizes. This is removed, whereupon the solution is evaporated to dryness. The residue is treated twice with 50 ml. of alcohol to remove free oxalic acid. The residue not soluble in alcohol is dried at 80 C. Yield: 74% of the theory. The product obtained is 2.2.4.4.6.6-hexahydroxy-5.5-dicarboxy-3.3'- disulfo-diphenylmethane. Relative inhibitory power: 4200.

What we claim is:

A compound selected from the group consisting of methylene-di-(2.5-dihydroxy-mono-sulfo benzoic acid) and its polycondensation products all of which compounds are condensation products of monosulfo gentisic acid and formaldehyde.

References Cited in the file of this patent UNITED STATES PATENTS 706,354 Summers Aug. 5, 1902 2,335,136 Thuau Nov. 23, 1943 FOREIGN PATENTS 584,196 Great Britain Ian. 9, 1947 OTHER REFERENCES; Beilstein, vol. 10, page 594 (1927).