2,731,494 United States Patent Office Patented Jan. 17, 1956 2 for instance on one side of the back of a rabbit, the same 2,731,494 amount of ink to which there has been added some micro NEW AND BIOLOGICALLY ACTIVE DIPHENYL grains of hyaluronidase being injected on the other side. METHANE DERVATIVES AND METHOD OF The ink to which no hyaluronidase has been added MAKING THE SAME Spreads only very slowly in the skin. The ink which has been mixed with the enzyme spreads in a few minutes LadislausFekete, Lorena,Arthur Brazil,Hahn, assignorsMaimo, toSweden, Aktiebolaget and Jaros Fer over an area of 200 cm.2 or more. That which has bee rosan, Maimo, Sweden, a corporation of Swedeir Said about the effect of the hyaluronidase upon the per meability of the skin holds true also in respect of other No Drawing. Application September 9, 1952, 0. kinds of mesenchymal tissue. . . Serial No. 308,704 The high viscosity of the synovial fluid depends to a Claims priority, application Sweden December 9, 1949 great extent on the hyaluronic acid and the same holds trie i. e. of the vitreous body of the eye and of the gel 1 Claim. (CI. 260-507) that surrounds the ovum in the oviduct. The effect cf the 5 hyaluronidase upon such materials is to make them lose This application is a continuation-in-part of our co their viscosity so that they become fluent. pending application No. 199,564, filed December 6, 1950, The hyaluronidase of certain bacteria is of great in now abandoned. portance for various reasons. Some of the pathogenic The present invention relates, to new biologically ac bacteria containing hyaluronidase, such as streptococci, tive diphenylmethane derivatives and to a method of mak 20 are connected with certain types of rheumatic diseases. ing the same. Since the viscosity of the synovial fluid depends mainly An enzyme which is capable of splitting mucopoly on its content of hyaluronic acid and since the cartilage saccharides and amongst them particularly the hyaluronic of the joints contains chondroitin sulfuric acid it was acid has been discovered i.e. in the testicles and the assumed that at least some forms of rheumatic and other sperm of mammals, furthermore in the leech, in serpents' 25 joint diseases are caused directly or indirectly by hy and insects' poisons and in certain bacteria. The enzyme aluronidase. has been denominated mucinase but the name has later The effect of the hyaluronidase upon connective tissues been changed to hyaluronidase, or mucopolysaccharase, results therein that bacteria containing or producing this respectively...... enzyme will penetrate through the organisrin much easier The substrate hyaluronic acid is rather common in the 30 than other bacteria so that the risk of infection is highly animal organism. Among the most important sources increased. The hyaluronidase-bearing bacteria need not may be mentioned the interfibrillar substance of mesen necessarily be pathogenic themselves, or they finay be chymal tissues, especially of the connective tissues, fur pathogenic only to a small degree-nevertheless they may thermore the vitreous body of the eye, the synovia and mean a great danger to the organisin by facilitating the the gel that surrounds the ovum on its way through the 35 penetration of other non-physiological substances, such oviduct. Another important substrate for the hy as other bacteria, virus etc., into tissues and organs. This aluronidase, the condroitin sulfuric acid, is a substantial is of particular importance since many highly pathogenic component of all sorts of cartilage, thus also of the virus show a rather low penetration capability. If the cartilage of the joints. organism is infected by hyaluronidase-producing bacteria The splitting effect of the hyaluronidase on the hy 40 infection with the said agents may result. aluronic acid has been elucidated to a large extent: the Since hyaluronic acid is present also in capillary walls highly polymeric acid is depolymerized and split into the capillary permeability is influenced by hyaluronidase. smaller units, the glucosidic bonds between the elements The hyaluronidase may therefore promote or facilitate of the hyaluronic acid, i. e. the glucuronic acid and the the penetration of inflective substances through the N-acetyl glucos amine, being hydrolyzed. In the corn capillaries. plete splitting up of the hyaluronic acid to morosac Certain substances inhibiting the effect of the hy charide units also other enzymes are active. One of these auronidase are known. As an example carboxy-p- enzymes has been isolated from bulls testicles and has benzoquinone, rutin, ascorbic acid, heparin, hexylresor been denominated mucooligosaccharase. The splitting cinol, certain sera fractions, and nitrates of hyaluronic of the hyaluronic acid and of other mucopolysaccharides, 50 acid may be mentioned. for instance the chondroitin sulfuric acid, through hy It has now been discovered that an inhibition of the aluronidase may be experimentally shown and quantita action of hyaluronidase which is many times stronger tively determined for instance (a) by observing the de than that of the above-mentioned known substances is crease in viscosity of the substrate of (b) by determining developed by certain diphenylmethane derivatives. These the reducing groups which are liberated at the hydrolysis. derivatives are obtained by condensing with formaldehyde There is also a biological method of determination which compounds selected from the group consisting of mono is based upon the influence of the hyaluronidase on the sulfo and disulfo derivatives of dihydroxy benzoic acids permeability of the skin, said influence being explained and monosulfo derivatives of trihydroxy benzoic acids. in the following. Hyaluronidase inhibitory power of these compounds is The effect of the hyaluronidase in vivo corresponds to 60 more than 30 times higher than that of any inhibitory its effect on mucopolysaccharides in vitro: for instance earlier described. This very high inhibitory activity on the hyaluronic acid of the skin is split by the action of hy hyaluronidase makes it possible to use these compounds aluronidase, which is brought into the tissue in one way in the treatment of rheumatoid arthritis on one side and or other, and looses its high viscosity. This causes the infectious diseases induced by hyaluronidase producing skin to become much more permeable. This increase 65 bacteria on the other side. of the permeability is rather general and results therein, The inhibitors according to the invention may also be that as well physiological as non-physiological substances, introduced into the organism in a suitable mainner, as per ... such as water, salts, added chemical compounds of all os, by superficial treatment or by, subcutaneous, intra kinds, coloring matters, poisons, bacteria and virus will muscular or intraperitoneal injection, etc., in order to spread many times quicker in the tissue if hyaluronidase 70 prevent the spreading effect of the hyaluronidase in is present. This may easily be shown experimentally by poisonings and infections. As mentioned above serpents' intracutaneous injection of 0.2 millilitre of Indian ink and insects' poisons and various pathogenic bacteria con 2,781,494 3 4. tain hyaluronidase and in part also other mucopolysac droxy-4-sulfo-benzoic acid). Relative hyaluronidase in charases. If these enzymes get into the organism such hibitory power: 4000. (Salicylic acid=1.) as by biting or infection they hydrolyse the hyaluronic Example 2 acid of the skin and other tissues so that the poison or the bacteria are more easily infiltrated in the organism. 5 in 90 grams of cold 50% sulfuric acid 20 grams of Also infection with other infective substances which are gentisic acid are suspended, whereupon 4 grams of a 40% free from hyaluronidase, such as bacteria and virus, formaldehyde solution are added. The reaction mix is spreads much quicker in the organism if the same is in boiled during 5 hours with vigorous agitation. The warm fected with hyaluronidase-bearing bacteria. The inhibi product is filtered through a glass filter and the precipi tors according to this invention inhibit the action of the O tate is pulverized and boiled with water, whereupon the hyaluronidase of poisons and bacteria upon tissues and same is washed several times with hot water. The sub thus prevent or limit infection. The action differs prin stance is easily soluble in alcohol, acetone and alkalis, cipally from the treatment with anti-toxins or anti-sera but very little soluble in water. Its decomposition point (no specificity). is above 260° C. 11 grams of the condensation product The compounds which are the matter of the present and 26 grams of Na2SO2:7H2O are dissolved in 150 ml. invention are prepared in the following manner: of water and boiled during 2 hours. The solution is fil The hydroxy-benzoic acid derivative is dissolved or Sus tered and to the filtrate 13.0 grams of oxalic acid are pended in a suitable solvent (for instance water, alcohol, added. The Solution is boiled to drive off SO2. The solu dioxan, ether, acetic acid, hydrochloric acid, sulfuric tion is then evaporated successively when sodium oxalate acid). Then the formaldehyde solution is added either 20 crystallizes. This is removed, whereupon the solution is all at one time or successively in the course of the re evaporated to dryness. The residue is treated twice with action. The latter mode of operation is used in certain 50 ml. of alcohol to remove free oxalic acid. The residue cases in order to control the condensation reaction. not soluble in alcohol is dried at 80° C. Yield: 72% of When using as starting material hydroxysulfo benzoic the theory. The product obtained is methylenedi-(2.5- acids, which easily condense with formaldehyde the re dihydroxy-4-sulfo-benzoic acid) and contains some poly action will occur at room temperature and in absence of condensed products. Relative inhibitory activity: 3800. condensation agents. However, in most cases it is pref Example 3 erable, or necessary, that a condensation agent is present. The reaction then occurs either at room temperature, or 0.2 mols of 3.4.5-trihydroxy-2-sulfo-benzoic acid are at an elevated temperature, and the reaction period may 30 dissolved in 150 ml. of water. After addition of 0.1 mol vary from some minutes up to several hours depending of 40% formaldehyde solution the mixture is boiled dur upon the reactive power of the compound in question, ing 2 hours. The solution is then neutralized with ammo the concentration of the condensing agent and other con nium carbonate and evaporated to dryness. Yield: 82% ditions. As condensation agents mineral acids (for in of the theoretical value. The product is 2.2.3.3.4.4'- stance hydrochloric acid, sulfuric acid) or alkalis (for hexahydroxy-6,6'-dicarboxy-5.5'-disulfo-diphenylmethane. instance alkali hydroxides) may be used. Relative inhibitory activity: 3000. If the starting material is sensitive to air oxygen the Example 4 reaction should preferably be carried out in an inert at mosphere. Since the reaction often takes place in a In 180 grams of cold 50% sulfuric acid 0.2 mol of heterogeneous phase the reaction mix should be vigor 2.4-dihydroxy-sulfo-benzoic acid is dissolved. 0.1 mol of ously stirred. a 40% formaldehyde solution is then added. The mix As generally the reaction product is considerably less ture is boiled during 5 hours with vigorous agitation. soluble than the starting material, purification of the re After cooling the reaction mixture is neturalized with action product may often be carried out taking advantage ammonium carbonate and then evaporated to dryness. of the said difference in solubility. Yield: 77% of the thoretical value. The product ob The production of certain of the contemplated sub tained is polycondensed methylenedi-(2,4-dihydroxy-sulfo stances may sometimes be carried out in Such a manner benzoic acid). Relative inhibitory power: 3000. that the carboxylic groups are introduced after the con Example 5 densation has been effected. Polycondensed methylenedi (3.4-dihydroxy-5 -sulfo The product of the condensation of dihydroxy-mono 50 benzoic acid) is obtained by treating 3.4-dihydroxy-5- sulfo-benzoic acid derivatives with formaldehyde under sulfo-benzoic acid in the similar manner as described for conditions here described was found to be a mixture of simple diphenylmethane derivatives and their polycon 2.4-dihydroxy-sulfo-benzoic acid in Example 4. Yield: densation products. The presence of condensing agents, 81% of the theoretical value. Relative inhibitory active increasing concentration of mineral acids, increasing tem ity: 3500. perature and increasing time of reaction generally favour Example 6 the production of polycondensed products. The separa In a mixture of 80 grams of 60% sulfuric acid and tion of the simple diphenylmethane derivatives from the 40 grams of acetic acid 15.4 grams of 2.3-dihydroxy corresponding polycondensed products is rather difficult. benzoic acid are dissolved. The solution is warmed Fractionation with organic solvent, for example alcohol, 60 on a boiling water bath and 4 grams of a 40% formal gives in some cases good results. dehyde Solution are added in the course of half an hour. We found that also the polycondensed products men Heating is continued during 8 hours. The warm prod tioned above show very pronounced inhibitory action on uct is filtered through a glass filter and the precipita hyaluronidase. In most cases the inhibitory power of tion is pulverized and washed several times with hot the polycondensed product is somewhat higher than the water. Yield: 11.9 grams. 11 grams of the condensa inhibitory power of the corresponding diphenylmethane tion product and 26 grams of Na2SO 7H2O are dis derivatives. solved in 150 ml. of water and boiled during 2 hours. Example 1 The solution is filtered and to the filtrate 13.0 grams of oxalic acid are added. The solution is boiled to drive 0.2 mols of 2.5-dihydroxy-4-sulfo-benzoic acid are dis of SO2. The solution is then evaporated successively solved in 150 ml. of water, whereupon 0.1 mol of a 40% when sodium oxalate crystallizes. This is removed, formaldehyde solution is added and the mixture boiled whereupon the solution is evaporated to dryness. The during two hours. The solution is neutralized with am residue is treated twice with 50 ml. of alcohol to re monium carbonate and then evaporated to dryness. move free oxalic acid. The residue not soluble in alco Yield: 88% of the theoretical value. The product ob hol is dried at 80° C. Yield: 72% of the theory. The tained is the ammonium salt of methylenedi-(2,5-dihy 75 product obtained is 3.3.4.4'-tetrahydroxy-5.5'-dicarboxy 2,781,494 5 6 disulfo-diphenylmethane being a mixture of the simple di After addition of 4 grams of 40% formaldehyde solu phenylmethane derivative and of polycondensed products tion the mixture is boiled during 5 minutes. The iso of varying molecular size. Relative inhibitory activity: lation of the reaction product is carried out according 3300. to Example 3. Yield: 14.0 grams of 2.2.5.5'.6.6'- Example 7 hexahydroxy-3.3'-dicarboxy diphenylmethane. 12 grams 0.2 mol of 2.6-dihydroxy-3.5-disulfo-benzoic acid is of the condensation product and 26 grams of 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 are dissolved in 150 ml. of water and boiled during 2 ammonium carbonate and then evaporated to dryness. O hours. The solution is filtered and to the filtrate 13.0 Yield: 85% of the theoretical value. The product ob grams of oxalic acid are added. The solution is boiled tained is an ammonium salt of 3.3'.5.5'-tetrahydroxy to drive off SO2. The solution is then evaporated suc 4.4'-dicarboxy - 2.2'.6.6’ - tetrasulfo - diphenylmethane. cessively, when sodium oxalate crystallizes. This is re Relative inhibitory power: 4100. moved, whereupon the solution is evaporated to dry 5 ness. The residue is treated twice with 50 ml. of alcohol Example 8 to remove free oxalic acid. The residue not soluble in In 80 grams of diluted hydrochloric acid (1:1) 18.1 alcohol is dried at 80° C. Yield: 72% of the theory. grams of 3.5-dihydroxy-benzoic acid are suspended. The product obtained is methylenedi-(2.4.5-trihydroxy After the addition of 4 grams of 40% formaldehyde sulfo-benzoic acid). Relative inhibitory power: 3100. solution the mixture is boiled during 12 hours. The 20 Example II reaction product is isolated in the same manner as in Example 1. Yield: 12.6 grams. 11 grams of the con In 100 grams of cold 50% sulfuric acid 17.0 grams densation product and 26 grams of Na2SO27H2O are of 2.4.6-trihydroxy benzoic acid are suspended, where dissolved in 150 ml. of water and boiled during 2 hours. upon 4 grams of a 40% formaldehyde solution are added. The solution is filtered and to the filtrate 13.0 grams 25 The reaction mixture is boiled during 5 minutes with of oxalic acid are added. The solution is boiled to vigorous agitation. The warm product is filtered through drive off SO2. The solution is then evaporated succes a glass filter and the precipitate is pulverized and washed sively when sodium oxalate crystallizes. This is re with hot water until free from sulfuric acid. The prod moved, whereupon the solution is evaporated to dry uct obtained is 2.2.4.4.6.6'-hexahydroxy-3.3'-dicarboxy ness. The residue is treated twice with 50 ml. of alco 30 diphenylmethane. 12 grams of the condensation product hol to remove free from oxalic acid. The residue not and 26 grams of Na2SO2:7H2O are dissolved in 150 soluble in alcohol is dried at 80° C. Yield: 72% of ml. of water and boiled during 2 hours. The solution is filtered and to the filtrate 13.0 grams of oxalic acid the theory. The product obtained is methylenedi-(3.5- are added. The solution is boiled to drive off SO2. dihydroxy-sulfo-benzoic acid) and its polycondensed The solution is then evaporated successively when so products. Relative inhibitory activity: 3000. 35 dium oxalate crystallizes. This is removed, whereupon Example 9 the solution is evaporated to dryness. The residue is 2 mols of 2.3.4-trihydroxybenzoic acid are mixed treated twice with 50 ml. of alcohol to remove free with water, and on a boiling water bath there is added oxalic acid. The residue not soluble in alcohol is dried at 80° C. Yield: 74% of the theory. The product ob with agitation and in a nitrogen atmosphere a molar tained is 2.2.4.4.6.6'-hexahydroxy-5.5'-dicarboxy-3.3'- weight of formaldehyde. In absence of a condensation disulfo-diphenylmethane. Relative inhibitory power: agent the reaction takes place when heating during 3 4200. hours. The mixture is then cooled and the precipitate What we claim is: - is filtered off and washed with cold water. 12 grams A compound selected from the group consisting of of the condensation product and 26 grams of 45 methylene-di-(2.5-dihydroxy-mono-sulfo - benzoic acid) and its polycondensation products all of which com are dissolved in 150 ml. of water and boiled during pounds are condensation products of monosulfo gentisic 2 hours. The solution is filtered and to the filtrate acid and formaldehyde. 13.0 grams of oxalic acid are added. The solution is boiled to drive off SO2. The solution is then evaporated 50 References Cited in the file of this patent successively when sodium oxalate crystallizes. This is UNITED STATES PATENTS removed, whereupon the solution is evaporated to dry ness. The residue is treated twice with 50 ml. of alcohol 706,354 Summers ------Aug. 5, 1902 to remove free oxalic acid. The residue not soluble in al 2,335,136 Thuau ------Nov. 23, 1943 cohol is dried at 80° C. Yield: 81% of the theory. The 55 FOREIGN PATENTS product obtained is methylenedi-(2.3.4-dihydroxy-sulfo 584,196 Great Britain ------Jan. 9, 1947 benzoic acid). Relative inhibitory power: 3300. OTHER REFERENCES Example 10 Beilstein, vol. 10, page 594 (1927). In 100 grams of diluted hydrochloric acid (1:1) 17.0 60 grams of 2.4.5-trihydroxybenzoic acid are suspended.