US2802005A

US2802005A - S-eluorourace

Info

Publication number: US2802005A
Authority: US
Publication date: 1957-08-06
Anticipated expiration: 1974-08-06

Description

United States Patent ice Patented Aug. 6, 1957 S-FLUQRUURAQHL Charles Heidelherger, Madison, Wis, and Robert Duschinslry, Essex Fells, N. 3.,

No Drawing. Application September 26, 1956, Serial No. 612,032;

12 Claims. (Cl. 26li--260) RA represents a lower alkylating agent; preferably a lower alkyl ester of an inorganic mineral acid, such as diethyl sulfate, methyl bromide, ethyl iodide, and the like: in the preferred case, R represents a lower alkyl radical and A represents an anionic portion of said mineral acid.

M and M each represents an alkali metal, for example potassium or sodium.

R and R each represents a lower alkyl radical.

R represents a radical selected from the group consisting of lower alkyl and benzyl.

X represents a halo substituent selected from the group consisting of chloro and brorno.

It will be understood that none of the lower alkyl radicals represented by the symbols R R R and R need be identical; but any or all of such radicals can be identical alkyl radicals. Likewise, the alkali metals represented by the symbols M and M need not be identical; but such metals can be identical.

FLOW SHEET-PROCESS II FCHQOO OR" (21) R 0 0 0 0 0 0 R M(0r M-O-RD] i 0 R al FLOW SHEET-PROCESS III R-S-O G LE =l l H N I H FLOW SHEETPROCESS IV FLOW SHEET-PROCESS V PROCESS I As will be appreciated from the foregoing flow sheet for Process 1, a comprehensive embodiment of this aspect of the invention provides a process which comprises reacting alkali metal fluoroacetate (11) with lower alkylating agent (12), thereby producing lower alkyl fluoroacetate (13); subjecting the latter to Claisen condensation with lower alkyl formate (14) in the presence of alkali metal-containing condensing agent (15), thereby producing alkali metal enolate of lower alkyl fluoromalonaldehydate (16); condensing the latter under anhydrous conditions with a member (17) selected from the group consisting of S-(lower alkyl)-isothiourea and S-benzylisothiourea, thereby producing the corresponding member (18) of the group consisting of S-loWer alkyl ether of 2-thio-5-fiuorouracil and S-benzyl ether of 2- thio-S-fiuorouracil; and hydrolyzing said corresponding member, thereby producing S-fluorouracil (19).

The first stage of the comprehensive embodiment of Process I referred to above comprises reacting the alkali metal fluoroacetate (11) with a lower alkylating agent (12). Preferably the sodium or potassium salt of fluoroacetic acid is used as reactant (11); and preferably diethyl sulfate or dimethyl sulfate or methylbromide or ethyl chloride or the like is used as the lower alkylating agent (12). The reaction can be effected, for example, by heating the reactants together until completion of the reaction whereby the lower alkyl radical is exchanged for the alkali metal. An inert solvent can be employed, if desired, but its use is not required. The product can be purified, if desired, by conventional means, for example by distillation.

The second stage of said comprehensive embodiment comprises reacting the lower alkyl fluoracetate (13) in a Claisen condensation with lower alkyl formate (14), employing an alkali metal-containing condensing agent (15), such as an alkali metal itself, e. g. potassium or sodium, or an alkali metal lower alkoxide, e. g. potassium ethoxide or sodium methoxide. Preferably a potassium lower alkoxide, such as potassium ethoxide, is employed as condensing agent; the potassium enolate thereby produced as product can be easily isolated in well crystallized form. A suitable method of effecting this second stage of the overall process comprises heating the reactants (13), (14) and (15) together, in an anhydrous inert solvent, such as diethyl ether or toluene, until completion of the condensation. The product, represented by general formula (16) in the flow sheet, can be styled alkali metal enolate of lower alkyl fluoromalonaldehydate; alternative nomenclatures are, alkali metal salt of lower alkyl ester of fluoromalonaldehydic acid, or alkali metal salt of lower alkyl ester of forrnylfluoroacetic acid. Particularly when potassium lower alkoxides are used as condensing agents, the product 16) need not be purified by recrystallization for further use in the process.

The third stage of the comprehensive embodiment of Process I shown in the flow sheet comprises condensing the alkali metal enolate of lower alkyl fluorom'alonaldehydrate (16) with (17), i. e. S-(lower alkyl)isothiourea or S-benzylisothiourea, under anhydrous conditions. Preferably, the reaction is effected by heating the reactants together in an anhydrous inert solvent, such as methanol or ethanol, until the condensation reaction has been completed. It is recommended to use the alkali metal enolate reactant (16) as soon as possible after it has been prepared. It is preferred to use freshly prepared alkali metal enolate reactant (16), i. e. material which is not older than two (or at most three) days old; and preferably the alkali metal enolate should be usedas soon as it is prepared. The S-(lower alkyl)- isothiourea or S-benzylisothiourea is advantageously employed in the form of its acid addition salt with a mineral acid, e. g. S-methylisothiouronium sulfate or S- ethylisothiouroniurn bromide or S-benzylisothiouronium chloride, in the presence of at least the equivalent amount of alkali necessary to neutralize the mineral acid. The product, S-lower alkyl (or S-benzyl) ether of 2-thio-5- fluorouracil (18), can be purified byconventional means, e. g. by recrystallization from inert solvents.

The last stage of the comprehensive embodiment shown in the flow sheet for Process I comprises hydrolyzing the S-lower alkyl (or S-benzyl) ether of 2-thio-5-fiuorouracil (18) thereby producing S-fluorouracil (19). The hydrolysis can be conveniently eifected by conventional means, for example by heating the reactant (18) with an aqueous solution of a mineral acid, e. g. with concentrated aqueous hydrochloric acid. The S-fluorouracil obtained as product can be purified by conventional means, for example by recrystallization from inert solvents or by sublimation in vacuo.

One preferred embodiment of the overall Process I shown in the flow sheet comprises reacting sodium fluoroacetate with diethyl sulfate, thereby producing ethyl fluoroacetate; reacting the latter in a Claisen condensation with ethyl formate in thepresence of potassium ethoxide, thereby producing potassium enolate of ethyl fluoro- Inalonaldehydate; condensing freshly prepared potassium enolate of ethyl fluoromalonaldehydate under anhydrous conditions with S-methylisothiourea, thereby producing S-methyl ether of Z-thio-S-fiuorouracil; and hydrolyzing the latter, thereby producing -fluorouracil. Still another preferred embodiment of the overall Process I comprises the same operations in the same sequence; except that S-ethylisothiourea is employed in lieu of S- methylisothiourea in the penultimate step, S-ethyl ether of Z-thio-S-fluorouracil being thereby produced in lieu of S-methyl ether of 2-thio-5-fiuorouracil.

PROCESS II As will be appreciated from the foregoing flow sheet for Process II, a comprehensive embodiment of this second aspect of the invention provides a process which cornprises subjecting lower alkyl fluoroacetate (21) to Claisen condensation with di(lower alkyl) oxalate (22) in the presence of alkali metal-containing condensing agent (23), thereby producing alkali metal enolate of di(lower alkyl) fiuorooxalaeetate (24); condensing the latter with a member (25) selected from the group consisting of 8- (lower alkyl)-isothiourea and S-benzylisothiourea, thereby producing the corresponding member (26) selected from the group consisting of S-lower alkyl ether of 2-thio- S-fluoroorotic acid lower alkyl ester and S-benzyl ether of Z-thio-S-fiuoroorotic acid lower alkyl ester; hydrolyzing said corresponding member, thereby producing 5-fiuoroorotic acid (27); and decarboxylating the latter, thereby producing S-fluorouracil (28) The first stage of the comprehensive embodiment of Process II referred to above comprises reacting lower alkyl fluoroacetate in a Claisen condensation with di(lower alkyl) oxalate, in the presence of alkali metal-containing condensing agent. Preferably, ethyl fluoroacetate is employed with diethyl oxalate; or, alternatively, methyl fluoroacetate with dimethyl oxalate. As the alkali metalcontaining condensing agent, preferably potassium ethoxide is employed; but, alternatively, potassium methoxide or potassium can be employed. A suitable method of effecting this first stage comprises heating the reactants (21), (22) and (23) together in an anhydrous inert solvent such as ethanol, toluene or diethyl ether, until completion of the condensation. The product, represented by the general formula (24) in the flow sheet for Process II, can be styled alkali metal enolate of di(lower 6 alkyl) fluorooxalacetate; an alternative nomenclature is, alkali metal salt of lower alkyl ester of fluorooxalacetic acid. Particularly when ethyl fluoroacetate is condensed with diethyl oxalate in the presence of potassium ethoxide, the resulting product, diethyl potassio-fluorooxalacetate, need not be purified before further reaction in Process II.

I The second stage of the comprehensive embodiment of Process 11 referred to above comprises condensing alkali metal enolate of di(lower alkyl) fluorooxalacetate with S-(lower alkyl)isothiourea or S-benzylisothiourea. Advantageously, the S-(lower alkyl)isothiourea or S-benzylisothiourea is employed in the form of its acid addition salt with a mineral acid, e. g. S-methylisothiouronium sulfate or S-ethylisothiouronium bromide or S-benzylisothiouronium chloride, in the presence of at least the equivalent amount of alkali necesary to neutralize the mineral acid. The reaction is preferably eifected by heating the reactants together in an anhydrous inert solvent, such as methanol or ethanol, until the condensation reaction has been completed. It is recommended to use freshly pre pared alkali metal enolate reactant (24). The product (26), i. e. S-lower alkyl ether of 2-thio-5-fluoroorotic acid lower alkyl ester or S-benzyl ether of Z-thio-S-fluoroorotic acid lower alkyl ester, can be purified by conventional means, e. g. by recrystallization from inert solvents.

The third stage of the comprehensive embodiment of Process II comprises hydrolyzing the S-lower alkyl (or S- benzyl) ether of 2-thio-5-fluoroorotic acid lower alkyl ester. The hydrolysis can be conveniently effected by conventional means, for example by heating the reactant (26) with an aqueous solution of a mineral acid, e. g. with concentrated aqueous hydrochloric acid; alternatively, the reactant (26) can be saponified, e. g. by treating with an aqueous solution of a strong alkali such as aqueous KOH, followed by acid hydrolysis of the resulting solution of the alkali salt of S-lower alkyl (or S- benzyl) ether of 2-thio-5-fluoroorotic acid, e. g. by means of concentrated aqueous hydrochloric or hydrobrornic acid. The S-fluoroorotic acid (27) obtained as product can be purified by conventional means, for example by recrystallization from water or from an inert organic solvent.

The last stage of the comprehensive embodiment of Process II referred to above comprises decarboxylating 5- fluoroorotic acid (27) at its melting point, carbon dioxide being thereby liberated, and leaving a residue of 5-fluorouracil. For a larger scale preparation, it is preferable to heat the reactant (27), above its melting point, while dissolved in a high boiling inert solvent such as diphenyl, diphenyl oxide or mixtures thereof, until the evolution of carbon dioxide has ceased.

One preferred embodiment of the overall Process II shown in the flow sheet comprises condensing ethyl fluoroacetate with diethyl oxalate in the presence of potassium ethoxide, thereby producing potassium enolate of diethyl fluorooxalacetate; condensing the latter with S-ethylisothiourea, thereby producing S-ethyl ether of Z-thiO-S- fluoroorotic acid ethyl ester; refluxing the latter with con :centrated aqueous hydrochloric acid thereby producing S-fiuoroorotic acid; and heating the latter above its melting point, while dissolved in an inert solvent, thereby producing S-fluorouracil. In an alternative preferred embodiment, S-methylisothiourea is used in lieu of S-ethylisothiourea, S-rnethyl ether of Z-thio-S-luoroorotic acid ethyl ester being thereby produced in lieu of S-ethyl ether of 2--thio-5-fiuoroorotic acid ethyl ester.

PROCESS III 7 As will be appreciated from the foregoing flow sheet for Process III, a comprehensive embodiment of this 7 fluorouracil (32); and oxidizing the latter, thereby producing S-fluorouracil (33).

The first stage .of the comprehensive embodiment of Process III referred to above comprises splitting ofi the hydrocarbon radical attached to the mercapto sulfur atom of the compound (31). This scission can be effected by heating (31) with an anhydrous hydrogen halide. Preferably, anhydrous hydrogen iodide is employed. An alternative method comprises heating S-benzyl ether of 2- thio-S-fluorouracil with anhydrous aluminum bromide.

The second stage of the comprehensive embodiment of Process III referred to above comprises oxidizing the 2- thio-S-fluorouracil intermediate product (32). A suitable method of oxidation comprises heating the 2-thio-5-fluorouracil with hydrogen peroxide.

One preferred embodiment of the overall Process III shown in the flow sheet comprises heating S-lower alkyl ether of 2-thio-5-fluorouraci1 with anhydrous hydrogen iodide, thereby producing 2-thio-5-fluorouracil; and heating the latter with a concentrated aqueous solution of hydrogen peroxide, thereby producing S-fluorouracil.

PROCESS IV As will be appreciated from the foregoing flow sheet for Process IV, a comprehensive embodiment of this fourth aspect of the invention provides a process which comprises reacting a member (41) of the group consisting of S-lower alkyl ether of 2-thio-5-fiuorouracil and S-benzyl ether of Z-thio-S-fluorouracil with a phosphorus pentahalide selected from the group consisting of phosphorus pentachloride and phosphorus pentabromide, thereby producing the corresponding member (42) selected from the group consisting of 2-(lower alkyl)mercapto-4-chloro-5-fiuoropyrimidine, 2 (lower alkyl)mercapto 4 bromo-5fluoropyrimidine, 2-benzylmercapto-4- chloro-S-fiuoropyrimidine and 2-benzylmercapto-4-bromo- S-fluoropyrimidine; aminating said corresponding member, thereby producing the corresponding member (43) of the group consisting of 2-(lower alkyl)mercapto-4- amino-S-fluoropyrimidine and 2-benzylmercapto-4-amino- S-fiuoropyrimidine (42) obtained as the product of this stage can be purified by extracting with an inert solvent, such as diethyl ether.

The second stage of the comprehensive embodiment of Process IV referred to above comprises exchanging the 4-halo substituent in the intermediate product (42) for an amino group. This reaction can be effected conveniently by reacting the product (42) with liquid ammonia under heat and pressure. Alternatively, alcoholic ammonia can be employed. The resulting 2-(lower alkyl) mercapto (or 2-benzylmercapto-) 4-amino-5-fluoropyrimidine (43) can be purified by recrystallizaton from an inert solvent.

The third stage of the comprehensive embodiment of Process IV referred to above comprises hydrolyzing the intermediate product (43). veniently effected by conventional means, for example by heating the reactant (43) with an aqueous solution of a mineral acid, e. g. with concentrated aqueous hydrobromic acid. The resulting product, S-fiuorocytosine (44), can be purified, for example, by recrystallization from water.

The hydrolysis can be con- H The last stage of the comprehensive embodiment of and pressure, thereby producing S-ethylmercapto-4-amino S-fiuoropyrimidine; refluxing the latter with concentrated aqueous hydrobromic acid, thereby producing S-fluorocytosine; and reacting the latter with nitrous acid, thereby producing S-fluorouracil.

PROCESS V As will be appreciated from the foregoing flow sheet for Process V, a comprehensive embodiment of this aspect of the invention provides a process which comprises condensing alkali metal enolate of lower alkyl fluoromalonaldehydate (51) under anhydrous conditions with a member (52) selected from the group consisting of 2- lower alkyl-pseudourea and Z-benzyl-pseudourea, thereby producing the corresponding member (53) of the group consisting of 2-lower alkyl ether of S-fluorouracil and 2- benzyl ether of S-fluorouracil; and hydrolyzing said corresponding member (53), thereby producing 5-fluorouracil (54).

The first stage of the comprehensive embodiment of Process V referred to above comprises condensing the alkali metal enolate of lower alkyl fluoromalonaldehydrate (51) with (52), i. e. 2-lower alkyl-pseudourea or 2-benzyl-pseudourea, under anhydrous conditions. Preferably, the reaction is effected by heating the reactants together in an anhydrous inert solvent such as methanol or ethanol, until the condensation reaction has been completed. It is recommended to use the alkali metal enolate reactant (51) as soon as possible after it has been prepared. The 2-lower alkyl-pseudourea or 2-benzyl-pseudourea is advantageously employed in the form of its acid addition salt with a mineral acid, e. g. 2-methyl-pseudourea hydrochloride or 2-benzyl-pseudourea hydrochloride, in the presence of at least the equivalent amount of alkali necessary to neutralize the mineral acid. The product (53) of this stage, 2-lower alkyl (or 2-benzyl) ether of 5- fluorouracil, can be purified by conventional means, e. g. by recrystallization from inert solvents.

The second stage of the comprehensive embodiment of Process V shown in the flow sheet comprises hydrolyszing the ether (53), thereby producing S-fiuorouracil 4). conventional means, for example by heating the reactant (53) with an aqueous solution of a mineral acid, e. g. with concentrated aqueous hydrochloric acid. The 5- fluorouracil obtained as product can be purified by conventional means, for example by recrystallization from inert solvents or by sublimation in vacuo.

One preferred embodiment of the overall Process V shown in the flow sheet comprises condensing freshly prepared potassium enolate of ethyl fluoromalonaldehydate under anhydrous conditions with 2-n1ethyl-pseudourea, thereby producing 2-methyl ether of S-fiuorouracil; and hydrolyzing the latter, thereby producing S-fiuorouracil.

It will be appreciated that the compounds represented in the foregoing flow sheets by the formulas (18), (19), (26), (27), (32), (44), and (53); and sometimes referred to in this specification, respectively, as S-lower alkyl ether of 2-thio-5-fluorouracil (or species thereof), andS-benzyl ether of 2-thio-5-fluorouracil, S-fluorouracil,

The hydrolysis can be conveniently etfected by can exist in tautomeric forms, resulting from the shifting of a proton between a nitrogen atom and an oxygen atom. The invention includes all of the tautomeric forms of said compounds.

The compounds sometimes referred to herein as S-lower alkyl ether of Z-thio-S-Iluorouracil (or species thereof), and S-benzyl ether of 2-thio-5fluorouracil,

S-fiuoroorotic acid,

2-thio-5-iuorouracil, and

2-lower alkyl ether of 5-fluorouracil (or and 2-benzyl ether of S-fluorouracil species thereof),

exhibit acidic properties, and form salts with bases. The invention includes salts obtained by reacting said compounds, respectively, with medicinally acceptable bases, e. g. alkali metal hydroxides, alkaline earth metal hydroxides, ammonia, non-toxic organic bases such as ethanolarnine, and the like.

The compounds sometimes referred to herein as 2-(lower alkyl)mercapto-4-amino-S-fiuoropyrimidine (or species thereof) and 2-benzyhnercapto-4-amino-S-fiuoropyrimidine, and

S-fiuorocytosine;

exhibit basic properties, and form acid addition salts. The invention includes acid addition salts formed by reacting said compounds with medicinally acceptable acids, e. g. mineral acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid; non-toxic organic acids, such as ethanesulfonic acid, toluenesulfonic acid, tartaric acid, citric acid; and the like.

The novel compound S-fluorouracil and its salts with medicinally acceptable bases are useful as germicidal agents, being active, for example, against gram-positive and gram-negative and other bacteria, such as Proteus vulgaris, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Bacillus megatherium, Sarcina lutea, Corynebacterium simplex and the like; against protozoa such as T etrahymena geleii and the like; against fungi such as Scopulariopsis brevicaulis and the like. They are also useful as antimetabolites, being active to inhibit the growth of Lactobacillus arabinosus, Lactobacillus leichmannii, Streptococcus faecalis, Saccharomyces carlsbergensis and the like, e. g. by interfering with the utilization of uracil, thymine, cytosine and thymidine by the microorganism. For example, S-fluorouracil can be applied locally, in solution in a physiologically inert liquid medium, e. g. saline solution, to lesions infected with Pseudomonas aeruginosa, as an antibacterial agent to terminate the development of the infection.

The invention is further disclosed in the following examples, which are illustrative but not limitative thereof. Temperatures are stated in degrees centigrade, corrected.

Example 1 A mixture of 200 g. (2 mols) of dry sodium fluoroaeetate and 442 g. (2.86 mols) of diethyl sulfate was refluxed for three and one-half hours in an oil bath. The reaction mixture was then distilled through a fractionating column, yielding 177.3 g. of crude ethyl fluoroacetate, having a boiling range of 116-l20. The material was redistilled through a fractionating column, yielding purified ethyl fluoroacetate boiling at 114118.

Example 2 i In a 2-liter, 3-neck, round bottom flask, provided with stirrer, dropping funnel and reflux condenser, was placed 880 ml. of absolute diethyl ether, and 47.6 g. (1.22 mols) of potassium, cut into 5 mm. pieces, was suspended therein. 220 ml. of absolute ethanol was added dropwise, while stirring, whereby the heat of reaction produced refluxing. In order to obtain complete dissolution of the potassium, the mixture was finally refluxed on a steam bath. The reaction mixture was then cooled in an ice bath, and a mixture of g. (1.22 mols) of ethyl fluoroacetate and 96.4 g. (1.3 mols) of freshly distilled ethyl formate was added dropwise, while stirring and cooling, over a period of two and one-half hours. Upon completion of the addition of the ethyl formate, the reaction mixture was stirred for an additional hour while cooling, and then was allowed to stand overnight at room temperature. At the end of this time the crystalline precipitate which had formed was filtered off with suction, washed with diethyl ether, and dried in a vacuum desiccator. The product comprised essentially the potassium enolate of ethyl fluoromalonaldehydate (alternative nomenclature, the potassium salt of fluoromalonaldehydic acid ethyl ester).

Example 3 A mixture of 103.6 g. (0.6 mol) of the freshly prepared potassium enolate of ethyl fluoromalonaldehydate, 83.4 g. (0.3 mol) of S-methylisothiouronium sulfate and 32.5 g. (0.6 mol) of sodium methoxide was refluxed with stirring in 1500 ml. of absolute methanol. At first the reactants dissolved to a great extent, but very shortly thereafter precipitation occurred. The reaction mixture was refluxed for two hours and at the end of this time was evaporated to dryness in vacuo. The residue was treated with 280 ml. of water; incomplete dissolution was observed. The mixture obtained was clarified by filtering it through charcoal. The filtrate was acidified (to a slight Congo acid reaction) by adding concentrated aqueous hydrochloric acid, containing 37% by weight HCl (48 ml. required). The material which crystallized from the acidified solution was filtered off, washed free of sulfates with water and dried at 100, yielding crude S-methyl ether of 2-thio-5fluorouracil, having a melting range from 202 to 221. The latter material was recrystallized by dissolving it in 2035 ml. of boiling ethyl acetate and cooling to minus 20, yielding S-methyl ether of 2-thio-5- fluorouracil, M. P. 230-237, in a sufficient state of purity that it could be used directly for the next step. A sample of the material was recrystallized from water (alternatively, from ethyl acetate) thereby raising the melting point to 24l243. For analysis the material was further purified by subliming it in vacuo at /0.1 mm.

The S-methyl ether of 2-thio-5-tluorouracil so obtained was an acidic material, which formed salts upon reaction with bases. For example, it dissolved, with salt formation, in dilute aqueous sodium hydroxide solution, in aqueous ammonia, and in an aqueous solution of ethauolamine.

Example 4 A solution of 10.0 g. of purified S-methyl ether of 2- thio-S-fluorouracil, M. P. 230237, in 150 ml. of concentrated aqueous hydrochloric acid (containing approximately 37% by weight HCl) was refluxed under nitrogen for four hours. The reaction mixture was then evaporated in vacuo. The crystalline brownish residue was recrystallized from water. The resulting recrystallized product was further purified by sublimation in vacuo at -200 (bath temperature)/O.1 mm. pressure. There was obtained 5-fiuorouracil, in the form of colorless or pinkishtan crystals, M. P. 282-283 (with decomposition).

The S-fluorouracil so obtained was an acidic material which formed salts upon neutralization with bases. It dissolved, with salt formation, in dilute aqueous potassium hydroxide solution, in aqueous ammonia and in aqueous ethanolamine.

Example A mixture of 17.3 g. (0.1 mol) of the freshly prepared potassium enolate of ethyl fluoromalonaldehydate, 18.5 g. (0.1 mol) of S-ethylisothiouronium bromide and a solution of 2.3 g. (0.1 mol) of sodium in 250 m1. of absolute ethanol was refluxed for two hours while stirring. The reaction mixture, consisting of a slightly yellowish crystalline material suspended in the liquid, was evaporated to dryness in vacuo. The residue was dissolved in ml. of warm water and the solution was clarified by filtering through charcoal. The filtrate was cooled in an ice bath and acidified (to a slight Congo acid reaction) by adding concentrated aqueous hydrochloric acid (5.8 ml. required). The material which crystallized from the acidified solution was filtered ofi, washed free of bromides with ice cold water and dried at yielding crude S-ethyl ether of 2-thio-5-fluorouracil, having a melting range from 164 to The latter material was suspended in a 1:4 (by volume) mixture of toluene and ligroin (boiling point 90120) and refluxed for a few minutes. Partial solution was obtained. The insoluble material was filtered from the boiling mixture and suspended in toluene. The suspension was brought to boiling and filtered hot. Upon cooling in an ice bath, the filtrate deposited crystals which were filtered off and dried at 100. There was thus obtained purified S-ethyl ether of 2-thio-5-fluorouracil melting from 181 to (alternative nomenclatures: 2-ethylmercapto-4-hydroxy-5-fluoropyrimidine, or Z-ethylmercapto-5-fluoro-4(3H)-pyrimidinone). The material was again recrystallized from ethyl acetate, yielding further purified compound melting at l91. This material was sufficiently pure to be used for the next step. For analysis a sample of 300 mg. was again recrystallized from 10 ml. of ethyl acetate to yield 210 mg. of pure compound melting at 192-l93. The S-ethyl ether of 2-thio-5-fluorouracil thus obtained was an acidic material, which formed salts upon reaction with bases. For example, it dissolved, with salt formation, in dilute aqueous sodium hydroxide solution and in aqueous ammonia.

Example 6 A solution of 0.52 g. of purified S-ethyl ether of 2-thio- S-fluorouracil, M. P. 190-191, in 10 ml. of concentrated aqueous hydrochloric acid (37% by weight HCl) was refluxed for four and one-half hours. The reaction mixture was then evaporated in vacuo. The crystalline tan residue was recrystallized from 4 ml. of water.. The crystals obtained were washed with cold water and then with methanol, yielding 5-fluorouracil, M. P. 281282 (with decomposition). Sublimation in vacuo (0.1 mm., 190 bath temperature) raised the melting point to 282- 283.

Example 7 A solution of 43 g. (0.25 mol) of freshly prepared ethyl potassio-fluoromalonaldehydate, 50.6 g. (0.25 mol) of S-benzylisothiouronium chloride and 13.5 g. (0.25 mol) of sodium methoxide in 640 ml. of methanol was refluxed for two hours while stirring. The reaction mixture was evaporated to dryness in vacuo, and the residue was taken up in 220 ml. of water. The resulting turbid solution was made alkaline to phenolphthalein by addition of 12 ml. of 2 N aqueous sodium hydroxide solution and was then extracted with three portions of diethyl ether, each portion consisting of 60 ml. The aqueous layer was then acidified with 16 ml. of concentrated aqueous hydrochloric acid and cooled with ice. The precipitated crystals were filtered and washed chlorine-free with water and then with diethyl ether. There was thus obtained crude S-benzyl ether of 2-thio-5-fluorouracil (alternative nomenclature: 2-benzylrnercapto-5-fluoro-4 3H) -pyrimidinone) having melting point 180-189. Upon recrystallization 12 from ethanol, the melting point was raised to 205-206.- A second recrystallization from ethanol raised the melting point to 216-218.

Example 8 A potassium ethoxide solution in toluene was prepared from 47.6 g. (1.214 mols) of potassium, 880 ml. of toluene and 190 ml. of ethanol, 300 ml. of toluene-ethanol mixture being distilled off after complete dissolution of the potassium. To the ice-cold solution, from which potassium ethoxide began to crystallize, there was added, under nitrogen, 355 g. (328 ml., 2.428 mols) of diethyl oxalate. A clear yellow solution resulted. While cooling and stirring, 135 g. (1.214 mols) of ethyl fluoroacetate was added dropwise during one and one-half hours. Stirring and cooling were continued for one hour, then the crystallizing mixture was allowed to stand at room temperature overnight. The readily settling colorless crystals were filtered, washed with diethyl ether and dried in vacuo, yielding potassium enolate of diethyl fluorooxalacetate (alternative nomenclature, diethyl potassio-fluorooaxalacetate).

Example 9 Under nitrogen, to a solution of 6.8 g. (0.295-mol) of sodium in 750 ml. of ethanol there was added 72.5 g. (0.295 mol) of the potassium enolate of diethyl fiuorooxalacetate and 54.6 g. (0.295 mol) of S-ethylisothiouronium bromide. Almost complete dissolution was observed, followed by crystallization. T he mixture was refluxed under nitrogen while stirring for two hours, and then was evaporated to dryness in vacuo. The residue was dissolved in 200 ml. of ice-cold water, the solution obtained was clarified by filtration through charcoal and extraction of some oil by two portions of diethyl ether, each containing 50 ml. The aqueous layer was cooled in ice and acidified to pH 2 by addition of 50 ml. of dilute aqueous hydrochloric acid (19% by weight HCl). The crystallized material was filtered, washed chlorine-free with water and dried at 100, yielding Sethyl ether of 2-thio-5-fluoroorotic acid ethyl ester (alternative nomenclature: Z-ethylmercapto-4-hydroxy-S-fluoro--pyrimidinecarboxylic acid ethyl ester) M. P. 166167. Recrystallization of the product from toluene while cooling to -20 yielded purer material having M. P. l67168. For analysis, 0.74 g. of the latter material was again recrystallized from 10 ml. of toluene to yield 0.7 g., M. P. 168l69.

Example 10 Under nitrogen, 4 g. (0.0162 mol) of S-ethyl ether of 2-thio-5-fluoroorotic acid ethyl ester, M. P. 166-167, was refluxed in 70 ml. of concentrated aqueous hydrochloric acid (containing 37% by weight HCl) for four hours. The mixture was cooled in ice and the crystallized acid was filtered, washed chlorine-free with water and dried at 100. There was thus obtained S-fluoroorotic acid monohydrate. The compound retained its water of crystallization very tenaciously. The acid melted with decarboxylation at 255; the residue, which was 5- fluorouracil, solidified and then melted at 278279. For analysis, a sample of S-fluoroorotic acid monohydrate was recrystallized from ca. 35 volumes of water.

A samle of 5-fluoroorotic acid monohydrate was sublimed in vacuo at 260270 (bath temperature) and 0.1

d mm., yielding the anhydrous acid, which melted with decarboxylation and resolidification like the monohydrate.

The S-fluoroorotic acid (in both the anhydrous and the monohydrate forms) thus obtained was an acidic mate rial. Upon reaction with bases, it formed salts. Thus, it dissolved in aqueous sodium hydroxide and potassium hydroxide solutions, forming sodium and potassium salts, respectively.

S-fluoroorotic acid is useful as an antimetabolite, being active to inhibit the growth of L. leichmannii, L. Cassi and S. faecalis, for example. It is also active as a germicidal agent, against Pseazlomonas aeruginosa and similar gram-negative organisms.

Example 11 Example 12 A mixture of 100 g. (0.41 mol) of diethyl potassiofiuorooxalacetate, 57.2 g. (0.21 mol) of S-methylisothiouronium sulfate and a solution of 44.3 g. (0.82 mol) of sodium methoxide in 1100 ml. of methanol was refluxed under nitrogen and worked up as described in Example 9 above for the ethylmercapto ester. In this case, transesterification took place and S-methyl ether of 2-thio-5- fiuoroorotic acid methyl ester was obtained; M. P. 195 197. For analysis, a sample was recrystallized from 60 volumes of toluene: M. P. 199 201 Example 13 A solution of 1.84 g. (0.08 mol) of sodium in 150 ml. of ethanol, 8.25 g. (0.04 mol) of diethyl potassio-fluorooxalacetate and 5.57 g. (0.02 mol) of S-methylisothiouronium sulfate was refluxed for two hours and worked up as described in Example 9 above, yielding S-methyl ether of Z-thio-S-fiuoroorotic acid ethyl ester, M. P. 177 178. For analysis, the compound was recrystallized from 25 volumes of toluene: M. P. 183l84.

Example 14 T o a solution of g. (0.031 mol) of S-methyl ether of 2- thio-S-fiuorouracil in 128 ml. of acetic acid and 32 ml. of acetic anhydride, which was stirred and refluxed, was added dropwise a mixture of 8 ml. (0.106 mol) of 55% hydriodic acid (d=1.7), 120 ml. of acetic acid and 40 ml. of acetic anhydride. Refluxing was continued for an hour after all the hydriodic acid had been added. The reaction mixture was then evaporated in vacuo to a volume of ml., and the concentrate was cooled in ice. The resulting crystalline precipitate was filtered off, washed with a mixture of acetic acid and petroleum ether and finally with petroleum ether. There was thus obtained 2-thio-5-fiuorouracil, M. P. 219-221. Recrystallization from water raised the melting point to 222224, and a second recrystallization from water raised the melting point to 225-226.

9.5 g. of S-ethyl ether of 2-thio-5-fiuorouracil was refluxed with hydriodic acid, in the same manner indicated above except that the refluxing time was extended to two hours. There was thus obtained 2-thio-5-fiuorouracil.

Example 15 To a solution of 0.614 g. (0.0042 mol) of 2-thio-5- fluorouracil in 19 ml. of water was added 3 ml. of hydrogen peroxide. The mixture was cooled in ice, and 3 ml. of 5 N sodium hydroxide was added slowly. The reaction mixture was allowed to warm up to room temperature slowly and then was refluxed for one and onehalf hours. It was then concentrated in vacuo to a volume of 4 ml, cooled, and acidified with 0.5 ml. of concentrated hydrochloric acid. The acidified reaction mixture was evaporated to dryness and the crystalline residue was extracted with four portions of hot methanol, each portion consisting of 5 ml. The combined methanolic extracts were evaporated and the residue was dissolved in 0.5 ml. of hot water. Upon cooling, crude S-fluorouracil (M. P. 258) crystallized. Recrystallization raised the melting point to 274. Admixture of the recrystallized i the mixture for 30 minutes, the oil became crystalline.

14 product with an authentic sample of S-fluorouraoil resulted in no depression of the melting point.

Example 1 6 To a solution of 4.3 g. (0.016 mol) of anhydrous alu minum bromide in 28 ml. of dry toluene was added, While stirring, 3.65 g. (0.0154 mol) of S-benzyl ether of 2-thio- 5-fiuorouracil, M. P. 2'l6-218. The mixture was heated to 60 for six hours while stirring continuously, then was cooled to 25. 5 ml. of water was added, resulting in the formation of an oily layer; but upon stirring The crystals were filtered off and washed with cold water, then dissolved in 30 ml. of hot N hydrochlori acid. The solution was cooled to room temperature, and was extracted with three portions of butanol, each consisting of 16 ml. The combined butanol extracts were evaporated, leaving a residue of. Z-thio-S-fluorouracil, M. P. 209- 211. TWO recrystallizations from water raised the melting point to 221223.

Example 17 A mixture of 10 g. of S-ethyl ether of 2-thio-5-fiuorouracil and 12 g. of phosphorus pentachloride was heated on a steam bath until the mixture was liquefied to a clear solution. The phosphorus oxychloride formed Was removed by heating in vacuo on a steam bath. To the oily residue was added crushed ice, whereupon crystallization was observed. The reaction mixture was extracted with three portions of diethyl ether, each consisting of 25 ml.; and the ethereal solution was dried over sodium sulfate. The dried solution upon evaporation yielded an oil comprising essentially Z-ethylmercapto 4 chloro 5 fluoropyrimidine, which crystallized only below room temperature.

Example 18 The oil obtained in Example 17 above was autoclaved for 12 hours with 120 ml. of liquid ammonia, in a boiling water bath. The ammonia was then evaporated, and the semisolid reaction product was taken up with 100 ml. of water and 10 ml. of ethanol. The crystals which separated were filtered and washed chlorine-free with water, leaving a residue of 2-ethylmercapto-4-amino-5- flnoropyrimidine, M. P. 94-95. A sample was recrystallized from 30 volumes of ligroin (B. P. 90 -120) the recrystallized product melted at 94 95.

The 2-ethylmercapto-4-amino-5-fluoropyrimidine, obtained as described above, was a basic material, which formed acid addition salts upon reaction with acids. Thus, it dissolved in dilute aqueous hydrochloric acid, thereby forming the hydrochloride salt.

Example 19 8.53 g. of crude 2-ethyhnercapto-4-amino-5-fiuoropyrimidine, M. P. 9091, was refluxed for four hours with ml. of concentrated aqueous hydrobromic acid (containing 48% by weight HBr) in a nitrogen atmosphere. The solution was evaporated in vacuo, and the residue was twice taken up with water and re-evaporated. The final residue was dissolved in 25 m1. of hot water and the solution was clarified by filtration through charcoal. Upon addition of 11 ml. of concentrated ammonia, the base precipitated. After cooling in ice, it was filtered and washed with cold water and ethanol, yielding S-fluorocytosine (alternative nomenclature: 2-hydroxy-4-arnino S-fiuoropyrimidine). The product melted with decomposition at approximately 297.

S-fluorocytosine is a basic material, and forms acid addition salts upon reaction with acids. Thus, it dissolved in dilute aqueous hydrochloric acid and dilute aqueous hydrobromic acid, with formation of S-fiuorocytosine hydrochloride and S-fiuorocytosine hydrobromide, respectively.

S-fiuorocytosine and its salts are useful as antimeta- 15 bolites,- being active, for example, to inhibit the growth of L. leichmannii, L casei, S. faecalz's, and the like.

Example 20 To a solution of 0.26 g. (0.002 mol) of S-fluorocytosine in ml. of water and 5 ml. of acetic acid there was added a solution of 0.69 g. (0.01 mol) of sodium nitrite in ml. of water in three portions over a period of one hour. The mixture was allowed to stand overnight, and then was evaporated on a steam bath. The semi-solid residue obtained was dissolved in 10 ml. of Water, the solution was rendered alkaline by addition of 3 ml. of 3 N sodium hydroxide and then was passed through a 1.8 cm. x 20 cm. column of Dowex 1X4 (Dow Chemical Co., Midland, Michigan: an anion exchange resin consisting of a crosslinked copolymer of styrene with divinyl benzene [4% of the latter], containing quaternary ammonium groups as the functional groups), 100-200 mesh size, previously saturated with formate ion by washing with 0.1 N aqueous formic acid. After washing the column with 250 ml. of water, elution was performed with 0.1 N aqueous formic acid, 50 ml. fractions being taken and examined for ultraviolet absorption at 265 III/1.. Only fraction No. 5 contained substantial amounts of absorbing material. This fraction upon evaporation to dryness yielded S-fluorouracil, which was identified by mixed melting point and comparison of the ultraviolet spectrum at pH=1 and 7.2 with an authentic specimen.

Example 21 A solution of 11 g. (0.0638 mol) of potassium enolate of ethyl fiuoromalonaldehydrate, 7.05 g. (0.0638 mol) of Z-methylpseudourea hydrochloride and 3.78 g. (0.07 mol) of sodium methoxide in 200 ml. of methanol was refluxed with stirring for two hours. The resulting cloudy liquid was evaporated to dryness in vacuo on the Water bath, the residue obtained was taken up with 18 ml. of water, the solution was filtered from undissolved material, cooled in ice and acidified to congo paper with 2.2 ml. of concentrated hydrochloric acid. The crystalline precipitate was filtered and washed with water and then with diethyl ether. There was thus obtained the 2- methyl ether of S-fluorouracil (alternative momenclature, 2 methoxy 5 fluoro 4(3H) pyrimidinone); M. P. 190-l92. The crude material was recrystallized from water: M. P. 205 206. A second recrystallization raised the melting point to 206-207. The substance was sublimed at 0.1 mm. and 155 l60 bath temperature, but there was no change in the melting point.

Example 22 Fifty mg. of 2-methoxy-5-fluoro-4(3H)-pyrimidinone, M. P. 206-207, was refluxed for four hours with 5 ml. of concentrated aqueous hydrochloric acid (37% by weight HQ) in a nitrogen atmosphere. The solution was evaporated to dryness, taken up with water and reevaporated, yielding 5-fiuorouracil, M. P. 282283. The compound was further identified by mixed melting point with an authentic specimen.

Example 23 In a 3 liter flask provided with stirrer, reflux condenser, dropping funnel, thermometer and gas inlet tube by which a continuous stream of nitrogen was provided during all operations, was placed 880 ml. of toluene and then 47.6 g. (1.214 mol) of potassium. The flask was heated in an oil bath to ca. 80 until the potassium melted. The metal was finely divided by stirring. Then 190 ml. of ethanol was dropped in at such a rate (ca. 30 minutes required) that slight refluxing resulted. Heating was continued until all potassium was dissolved (additional 45 minutes required). Then ca. 250 ml. of tolueneethanol mixture was distilled oil. Upon cooling in an ice bath. some crystallization of potassium ethoxide was observed. 146 g. (2.428 mol, 149 ml.) of dry methyl formate (distilled over CaCO3) was added, whereupon a clear colorless solution resulted. With continuous cooling in an ice bath and stirring, g. (1.214 mol) of ethyl fluoroacetate was added dropwise within two hours. After one hour of further stirring, the ice bath was removed and the crystallizing mixture was allowed to stand at room temperature overnight. The almost colorless crystals were filtered, Washed with ether and dried in vacuo at room temperature, yielding potassium enolate of ethyl fiuoromalonaldehydate (alternative nomenclature, potassio ethyl formylfluoroacetate).

Example 24 To a solution of 6.7 g. (0.29 mol) of sodium in 720 ml. of absolute ethanol, prepared in a 2-liter, 3-neck flask provided with stirrer and reflux condenser, was added 50 g. (0.29 mol) of the above potassium enolate (from Example 23) and 53.6 g. (0.29 mol) of ethyl isothiouronium bromide. An almost complete solution resulted which soon became cloudy by crystallization. The mixture was refluxed with continuous stirring on a water bath for two hours. The alcohol was distilled off in vacuo; the residue obtained was dissolved in ml. of water, the solution was clarified by filtering through charcoal, cooled in an ice bath and acidified (to Congo paper) by addition of 22 ml. of concentrated hydrochloric acid. The crystalline precipitate was filtered, washed chlorine free with water and dried at 100, yielding 2-ethylmercapto-5-fluoro-4(3H)-pyrimidinone, M. P. 179. The crude material was dissolved in 710 ml. of boiling ethyl acetate, the hot solution was filtered and cooled to l0. The crystals were filtered and washed with cold ethyl acetate. The melting point was thus raised to 189l90.

We claim:

. 1. A compound selected from the group consisting of 5-fluorouracil and salts thereof.

3. A process of making S-fluorouracil which comprises hydrolyzing a member of the group consisting of S-lower alkyl ether of 2-thio-5-fluorouracil and S-benzyl ether of 2-thio-5-fiuorouracil.

4. A process of making S-lower alkyl ether of 2-thio- 5-fluorouracil which comprises condensing freshly prepared alkali metal enolate of lower alkyl fluoromalonaldehydrate under anhydrous conditions with S-(lower alkyl) isothiourea.

5. A process of making S-benzyl ether of 2-thio-5 fluorouracil which comprises condensing freshly preparen alkali metal enolate of lower alkyl fluoromalonaldehydate under anhydrous conditions with S-benzylisothio'urea.

6. A process of making S-lower alkyl ether of 2-thio S-fluorouracil which comprises condensing freshly prepared potassium enolate of lower alkyl fluoromalonalde hydate under anhydrous condition with S-(lower alkyl) isothiourea.

7. A process according to claim 6 wherein freshly prepared potassium enolate of ethyl fluoromalonaldehydate is employed.

8. A process of making S-fluorouracil which comprises decarboxylating S-fluoroorotic acid.

9. A process which comprises splitting oi the S-hy drocarbon substituent from a member selected from the group consisting of S-lower alkyl ether of 2thio-5- fluorouracil and S-benzyl ether of 2-thio-5-fiuorouracil, thereby producing 2-thio-5-fluorouracil; and oxidizing the latter, thereby producing 5-luorouracil.

10. A process of making S-iluorouracil which comprises oxidizing 2-thio-5-fluorouracil.

11. A process of making 5-fluorouraci1 which comprises reacting 5fluorocytosine with nitrous acid.

12. A process of making 5-luorouracil which comprises hydrolyzing a member of the group consisting of 7 8 2-l0wer alkyl ether of S-fluorouracil and 2-benzyl ether 2,666,764 Lanzillotti et al. Jan. 19, 1954 of S-fluorouracil. 2,753,347 Miller July 3, 1956 2,753,348 Miller July 3, 1956 References Cited in the file of this patent r OTHER REFERENCES UNITED STATES PATENTS a Johnson: Amer. Chem. Journal, vol. 40, pp. l9-36 2,138,756 Boese Nov. 29, 1938 (1908). 2,405,820 Faith Aug. 13, 1946 Johnson: Jour. Amer. Chem. Soc., 65 1218-20 (1943). 2,585,615 Barrett Feb. 12, 1952 Hilbert et al.: Jour. Amer. Chem. Soc., 56 1349 2,609,372 Ziegler Sept. 2, 1952 10 (1934).

Claims

1. A COMPOUND SELECTED FROM THE GROUP CONSISTING OF 5-FLUOROURACIL AND SALTS THEREOF.