3,076,830 United States Patent Office Patented Feb. 5, 1963 2 3,076,830 and an organic acid residue derived from an amino cy WANADHUM COMEPOUNDS or 6-alkylthioacid. Representative of these acids is S John B. Conn, Westfield, N.J., assignor to Merck & Co., benzylcysteine, and representative formulae are Inc., Rahway, N.J., a corporation of New Jersey No Drawing. Original application Apr. 29, 1959, Ser. R. 2. Hi-R-C- O O-CO No. 809,59. Divided and this application Jan. 26, Y / 1960, Ser. No. 9,665 W 5 Claims. (C. 260-429) / N 4 This invention relates to inhibition of cholesterol syn O=C-O s--R-NH. thesis in mammals and provides a novel compound, O R2 (III) methods of preparation of compounds, novel composi and tions and methods for treatment of mammals to inhibit R2 R. O 24 cholesterol synthesis thereby. This application is a divi &– o O-6 sion of application S.N. 809,591 filed April 29, 1959. H. N. Cholesterol synthesis may occur in some mammals as 5 an incident of normal body functions and also occurs in HS-C { Dk Dic-sil, mammals as an incident of abnormal conditions such as g-o - the existence of tumors. The inhibition of the synthesis O R. R. (IV) is often desirable, particularly where it is occasioned by wherein R is hydrogen, alkyl or aryl and the R's can all abnormal condition, and for this purpose the use of 20 be alike or different. vanadium has been proposed. The introduction of vana Other materials among those referred to are zwitterionic dium in a convenient manner to provide it at the site of complexes of oxo-vanadium (IV) and an acid residue of the synthesis, without undesirable side effects, presents an amino a- or 3-hydroxyacid or thioacids. Representa a problem and it is a principal object of the invention tive acids are serine, cysteine, and aminosalicylic acid, to provide for the suitable introduction of the material. 25 and representative formulae are: It has been found that the synthesis can be inhibited by orally administering to a mammal capable of acting as a host for the synthesis a water soluble material including a complex of an organic acid residue and an oxo-vana -- dium group in which the valence of vanadium is 4 or 5. 30 C-O The vanadium is absorbed in the gut. Attempts to ob 0--R-NH, tain absorption in the gut from simple vanadium com (S) H. (W) pounds such as vanadyl sulfate and sodium metavanadate and were not successful. R3 (S) Materials included among those proposed for use ac 35 d-o O o cording to the invention are salts having as an anion a complex of an oxo-vanadium group in which the valence HŠ-off s ric-RH, of vanadium is 4 or 5 and an organic acid residue derived from an o- or 6-hydroxy acid or thioacid. Representa Yo-Yo-1s (S) Rs (VI) tive acids are glycollic, thioglycollic, lactic, hydraulic 40 wherein R3 is hydrogen, alkyl or aryl and the R's can malic, and thiomalic. Representative formulae for anionic be alike or different, and the notation (S) has the mean complexes formed from these acids are: ing assigned thereto hereinbefore. H (S) Other materials among those referred to are compounds R-(-0 O of the oxo-vanadium (V) group and an organic acid 45 residue derived from an alkylthioacid, for example S methylthioglycollic acid or S-methylthiosalicylic acid. A representative formula for these materials is (I) H i H 0. and 50 R-(- O O--R, R (S) Y/ (-0 O R 1. o=t-o? i-C=0 N R (VII) 55 wherein R is alkyl or aryl, and the R4's can be alike or s (II) different. where R1 is hydrogen, alkyl, aryl carboxy or carboxy Where the complex is the cation, the anion can be the substituted alkyl, and the R's can be alike or different, chloride or other physiologically innocuous anion, and and the notation (S) indicates that the adjacent oxygen 60 in the zwitterionic materials, other physiologically innocu atom (-O-) can be present in the complex as such or ous ions can replace the ammonia ion. can be replaced by divalent sulfur. Regarding organic acid residues present in ammonia Other cz- or B-hydroxy acids which are operable to complexes according to the invention, oxalic and malonic provide anionic complexes are oxalic, salicylic, thiosali acid which are representative of lower aliphatic decar cylic, malonic and citric. Complexes formed from these 65 boxylic acids operative according to the invention, are acids can have structural formulae as is indicated above herein considered to be a- and 3-hydroxy acids, respec by Formulae I and II. A preferred acid of the group tively, since the hydroxy group in the cy- or B-position ce- or B-hydroxy acids is tartaric. With tartaric acid, the is functionally the same as the hydroxy group in the cy mol ratio of the oxo-vanadium group and the tartaric or 3-position of acids such as lactic and hydracrylic acid. acid residue can be, and preferably is, unity. O A water soluble salt, for example the ammonium, so Other materials of the type referred to are Salts having dium or potassium salt, having as its anion-oxo-tartrato as a cation a complex of an oxo-vanadium (IV) group vanadate (IV), is suitable for cholesterol synthesis inhi 3,076,880 3 4. bition according to the invention. Thus, the following grams per unit. The water-soluble medicinal materials compound is suitable: can be incorporated in animal feeds for veterinary use. Suitable solid carriers are sugar, talc, alginic acid and Diammonium oxy-tartratovanadate (IV) of formula: starch. Lubricants, binders, and coating agents com monly used in the art can be employed in formation of tablets. A suitable combination of inerts for tableting. Examples of other compounds within the scope of the is lactose, talc, and magnesium stearate. Gelatin cap above description are: sules, powder mixtures, or liquid unit dosages can also Triammonium dioxy - bis - oxalatovanadate (V) of be used. A suitable liquid unit is a water solution of the formula: 10 active ingredient. Techniques well known in the art can (NH4)3VO2(CO4)2 (1) be employed for the formulation of unit dosages. Diammonium dioxy-tris-oxalatodivanadate (IV) of for The invention provides a novel and improved method mula: for the production of compound (5), i.e. diammonium (NH4)2(VO2(CO4)3] (2) oxo-tartratovanadate (IV), and this method can be em 5 ployed for the production of related compounds. Thus, Diammonium oxy-bis-oralatovanadate (IV) of formula: according to the invention, there can be produced a water (NH4)3VO (CO4)2) (3) soluble vanadium (V) salt having as its anion a com Diammonium oxy-bis-malonatovanadate (IV) of for plex of an oxy-vanadium group in which the vanadium mula: is vanadium (V), such as is indicated by structural (NH4)2CVO (CH2O4)2 (4) 20 Formulae I and II above, and an organic acid residue of the organic acids a- and 6-hydroxy acids and thioacids Diammonium oxy-bis-salicylatovanadate (IV) of for e.g. organic acid residues such as are indicated by struc mula: tural Formulae I and II above. These acids contain at (6) least two functional groups which are bonded to different (NH); (VO(CHOCO.), 25 carbon atoms and include a carboxyl group and a second Oxo-bis-(p-aminosalicylato) vanadium (V) (8) carboxyl group or a hydroxyl group. Suitable acids are The structural formula for compound (8) is set forth those mentioned above as being contained in anionic com hereinafter. plexes of the invention. The vanadium (IV) salts can be In complexes according to the invention, variation in the made by a two step process. In the first step hydrazine, mol ration of the oxo-vanadium group and the organic acid 30 hydroxylamine, or a hydrazine derivative containing an residue is possible. This will be apparent when the For amino group, an organic acid, and a metavanadate salt mulae 1, 2 and 3, above are considered. are admixed and the vanadyl salt of the organic acid is The invention provides as a novel compound, oxo-bis formed. In the second step this salt is admixed with a (p-aminosalicylato) vanadium (IV) which has the follow base of the vanadium (IV) salt to be produced and an ing structural formula: 35 ce- or (3-hydroxy acid, and the desired vanadium (IV) salt is formed. NH The first step of the method is preferably carried out in an aqueous medium and at a slightly elevated tem O perature, e.g. 60-70° C. While solevent mediums other C-O O 40 than water can be employed, as a practical matter water is preferred. Other suitable mediums are aqueous o/No-COYC methanol, ethanol or glycols. Desirably, to assure com pletion of reaction, about twice the theoretical amount of the acid and hydrazine or other amine compound is em NH ployed. During the reaction, nitrogen is evolved and ter This novel compound is zwitterionic and offers inter mination of evolution of nitrogen can be conveniently esting possibilities for use in inhibiting cholesterol Syn used to signal the end of the reaction. The metavanadate thesis according to the invention. Salt can be added gradually to the reaction medium con The novel zwitterionic compound of the above struc taining the amine compound and organic acid so that turial formula can be made by reacting p-amino-salicylic 50 evolution of nitrogen is gradual. acid with a vanadyl salt of an organic acid. An aqueous The vanady compounds produced by the first step are medium is preferred for the reaction. Other liquid me a convenient source for other vanadyl compounds so that diums in which the reactants are suitably soluble can be the first step of the method has usefulness beyond com employed. The acid from which the vanadyl salt is de bination thereof with the second step of the method. rived can be any organic acid providing the salt as solu 55 The second step can be carried out by adding to the ble in the reaction medium. The salt can be derived reaction product of the first step the base and acid em from an aliphatic acid, for example a lower aliphatic ployed as reactants in the second step. The amount of acid having 2 to 4 carbon atoms. The acetate salt is pre acid reactant and base can be the theoretical amount, ferred. Reaction time, temperature and pressure are not i.e. the stoichiometric equivalent of the vanadyl salt pro critical. The proportion of reactants is not critical and 60 duced in the first step and of the metavanadate salt used can be stoichiometric, i.e. two moles per mol of salt. in the first step. The temperature for the reaction is not The novel compositions of the invention comprise the critical and can be room temperature to about 60° C. water soluble materials described above in combination The steps of the method each involve rapid reactions with a physiologically acceptable carrier such as those and a distinctive feature of the method of the invention commonly used in pharmacy. The resulting compositions 65 is the speed of the reactions involved, particularly the can be compounded into unit-dosage forms suitable for speed of the second step. The reactions go to completion. use in the manner of the invention. For example, tablets, Preferably, the reactions of the first and second step are capsules, pills, suspensions and powders, containing any carried out separately by permitting the reaction of the suitable concentration of the active ingredient can be em first step to go to completion before commencing the ployed. Such compositions can contain a mere trace of 70 second step. the active ingredient but for practical treatment is is de The vanadium (IV) salts can be recovered from the sirable to use compositions containing at least 0.05% by reaction product of the second step by evaporation where weight and up to 100% by weight of the active ingredient. upon crystals of the product are formed. Desirably, if Desirably, unit-dosage forms contain from about 10 to 50 evolution of acid is detected, by for example, odor, addi milligrams, and preferably from about 20 to 30 milli 75 tional base of the vanadium (IV) salt to be produced 8,076,830 5 6 should be added to suppress the evolution. The crystals solution was transferred to an evaporating dish and con can be washed with methanol to remove ammonium centrated on the steam cone; purple crystals soon began acetate, and then they can be dried. to separate. Whenever, during the evaporation, any odor The crystals can be hydrates containing varying of acetic acid was noted, more ammonia, was added. amounts of water depending on the particular compound First crop 193 g; second crop 37 g. (total 230 g.-86%. produced and the extent of the drying. aS In a preferred embodiment of the invention, a lower aliphatic alcohol such as methanol, ethanol, or isopro (NH4)2CVO (CHOs)).H2O) panol, is added to the second step reaction product and Analysis for CHNOV.H.O., F.W. 267.11.-Calcd.: functions as a precipitant causing crystallization of the O C, 17.96; H, 4.53; N, 10.49. Found: C, 18.24; H, 4.50; vanadium complex. By working up the product in this N, 10.73. - manner, fine crystals are obtained and these are suitable, Diammonium oxo-tartratovanadate (IV) has been as such, for tableting, e.g. combination with carriers and found to be effective to inhibit cholesterol synthesis, by formation into tablets. When an alcohol is not em brain tumors in man. The drug has been administered ployed, the crystals, obtained are large and size reduction 5 orally in doses of 50 to 75 mg. daily. Urinary levels of by, for example grinding, is required, in order to adapt 200 meg./24 hours are obtained after a few days and them for use in making tablets. this suggests the drug is cumulative and that maintenance Concerning materials suitable for use in the two-step dosage for prolonged administration may be lower. Such method, in the first step, any organic acid can be em treatment when extended over a period of 2-8 weeks, ployed. The organic acid should be soluble in the re 20 results in significant lowering of cholesterol synthesis by action medium and when water is employed, the acid is brain tumors as evidenced by blood analysis. No toxicity preferably a weak, lower aliphatic, water soluble acid was observed. such as a carboxylic aliphatic acid containing from about The compounds (1) to (4) and (6) above, can be 2 to 4 carbon atoms. Acetic acid and propionic acid are produced by known methods. suitable. Acetic acid is preferred. The metavandate salt is preferably one whose base is EXAMPLE II the same as the base of the vanadium (IV) salt produced Preparation of Oxo-Bis-(p-Aminosalicylato) in the second step. Thus, where diammonium oxo-tartra Vanadium (IV) tovanadate (IV) is produced, ammonium metavanadate To a solution of 180 ml. glacial acetic acid and 10 ml. is preferably employed. The salt can be organic or in 30 85% hydrazine hydrate in 1 1. distilled water was added organic and should be soluble in the reaction medium. 58.5 g. (0.5 mole) ammonium metavanadate as in pre The preferred amino compound is hydrazine. Other vious example. To the hot solution of vanadyl acetate suitable compounds are hydroxylamine and hydrazine thus obtained, there was next added 153 g. (1 mole) p derivatives containing an amino group, for example alkyl aminosalicylic acid all at once, with rapid stirring. The hydrazines in which one of the hydrazine nitrogens is 35 p-aminosalicylic acid dissolved at first, but in a few sec unsubstituted. A lower alkyl hydrazine such as methyl onds the solution became dark-colored, and a purple, hydrazine can be used. crystalline, insoluble solid rapidly separated. The sus As indicated above, suitable cz- and 3-hydroxy acids pension was cooled, filtered, and the solid was thoroughly are the following: oxalic, malonic, tartaric, salicylic, lac washed with distilled water, after which it was dried in tic, citric, glycollic, thioglycollic, thiosalicylic, hydracrylic, 40 a desiccator. The purple solid underwent efflorescence malic and thiomalic. during drying, reverting to a blue-gray powder; it is ap The base employed in the second step corresponds with parently an unstable hydrate. Yield, 177 g. analysis the base of the vanadium (IV) salt to be produced. The refers to the anhydrous compound, obtained by drying in latter compound, when employed for inhibition of choles WaCO. terol synthesis according to the invention, is water sol 45 For C4H1NOV, F.W. 371.20: uble and can include as its base ammonia, sodium, or potassium or other base suitable to provide the water soluble vanadium (IV) salts and which is physiologically Calc, Found innocuous. Thus, the base employed as a reactant in the - 45.28 44. 70 second step can be the hydroxides or carbonates of am 50 H--- 3.26 3.80 N-- 7.55 7.58 monia, sodium or potassium. Drying Loss, percent. 13.8 in a preferred embodiment of the invention, the two step method is employed for the production of diam monium oxo-tartratovanadate (IV). Ammonium meta The compound is insoluble in water, but soluble in vanadate and acetic acid are employed in the first step 55 either dilute acid or base. and tartaric acid is employed in the second step. The What is claimed is: reactions are indicated by the following equations: 1. The method of producing a water-soluble vanadium (IV) salt having as an anion a complex of an oxo-vana 4NHVO--12HOAc--NH-> dium group having the vanadium in the tetravalent state 60 with an organic acid residue derived from an organic complexing acid selected from the group consisting of (NH4) 2 VO (C4H2Os) -- 2NHOAc glycollic, thioglycollic, lactic, hydracrylic, malic, thio EXAMPLE I malic, oxalic, salicylic, thiosalicylic, p-amino-salicylic, malonic, citric and tartaric acids which comprises heating A mixture of 360 ml. glacial acetic acid, 640 ml. water, 65 to 60-70° C. an aqueous medium containing a lower and 20 ml. commercial 85% hydrazine hydrate was aliphatic carboxylic acid of 2 to 4 carbons, a reducing placed in a 2 liter beaker which was mounted on the compound selected from the group consisting of hydroxyl steam cone. The mixture was heated to 65° C. under amine, hydrazine, and lower alkyl hydrazines in which stirring, and 117 g. (1 mole) ammonium metavanadate one of the hydrazine nitrogens is unsubstituted, and am was gradually added. The solid dissolved to a deep blue 70 monium metavanadate, the said organic acid and said solution of oxo-vanadium (IV) acetate, with evolution reducing compound being present in stoichiometric ex of nitrogen and heat. When all solid was in solution cess, until evolution of nitrogen has substantially ceased and evolution of nitrogen had ceased, 150 g. (1 mole) and adding to said reaction mixture, while holding said tartaric acid was introduced, followed by 450 ml. 20% reaction mixture at a temperature from ambient to 60 ammonia water. The solution now became purple. The 75 C., at least stoichiometric amounts of a base selected from 3,076,830 7 8: - - the group consisting of hydroxides and carbonates of complexing acid is tartaric acid and the said lower ali alkali metal and NH4+ cations, which cations become phatic alcohol is methanol. the cations of the salt produced, and an organic complex- 5. The method of claim 1 in which the said organic ing acid of the above defined group," and isolating the complexing acid is p-aminosalicylic acid. said water-soluble vanadium (IV) salt. 5 F w . . 2. The method of claim 1 in which said isolation of References Cited in the file of this patent the said vanadium (IV) salt is carried out by the addition UNITED STATES PATENTS of a lower aliphatic alcohol to precipitate the said salt 2,795,549. Abbott et al.------June 11, 1957 as a fine powder. 3. The method of claim 1 in which the said organic O OTHER REFERENCES complexing acid is tartaric acid. Sidgwick: "Chemical Elements and Their Compounds,' 4. The method of claim. 2 in which the said organic vol. I, Clarendon Press, 1950, pp.-822-823.