I i THE SYNTHESIS OF CERTAIN AMINOTETRAZOLE DEREVATWES AND A STUDY OF THEIR. 3ACTERIOCEDAL PROPERTIES Thesis for The Degree cf M. 5. MECHIGAN STATE COLLEGE Charles Francis Proberger WSO This is to certify that the thesis entitled THE SYNTHESIS OF CERTAIN AMINO TETRAZOLE DERIVATIVES AND A STUDY OF THEIR BACTERIOCIDAL PROPERTIES presented by Charles F. Froberger has been accepted towards fulfillment of the requirements for _;1L_5;__ degree in minim QM W. MW Major professor Date April 27, 1950 0-169 THE SYNTHESIS OF CERTAIN AMINOTETRAZOLE DERIVATIVES AKD A STUDY CF THEIR BACTERIOCIDAL PROPERTIES BY Charles Francis Froberger A THESIS Submitted to the School of Graduate Studies of Hichigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of TESTER OF SCIENCE Department of Chemistry 1950 WWI. T545 at; ' t l h- ’1 ACKI‘IOY‘.‘ EDGl'EI‘IT I wish to thank Doctor Robert M. Herbst for the assistance and encouragement he has given me during this work. *sxsesssss #******* **#*#* **** ** 4. conmrrg INTRODUCTION.............................................. HISTORICAL................................................ DISCUSSION................................................ EXPERIHENTAL.............................................. Preparation of l-n-Octyl-S-Aminotetrazole............ Preparation of l-Methyl-5-Ethylaminotetrazole........ Preparation of the Phenylthiourea Derivative of 1- Hethyl-S-Ethylaminotetrazole....................... Preparation of l-Nethyl-S-n-Octylaminotetrazole...... Preparation of the 3,5-Dinitrobenzamide Derivative of l-Wethyl-S-n—Octylaminotetrazole................... Preparation of l-n-Octyl-S-Methylaminotetrazole...... Preparation of the Phenylthiourea Derivative of l-n- Octyl-S-Nethylaminotetrazole....................... Preparation of l-EthylaS-n-Octylaminotetrazole....... Preparation of the 5,5-Dinitrobenzamide Derivative of l-Ethyl-5—n-Octylaminotetrazole.................... Preparation of l-n—Octy1-5-Ethylaminotetrazole....... Preparation of the p-Nitrobenzamide Derivative of 1- n-Octyl-S-Ethylaminotetrazole...................... Preparation of l-n-Ootyl-B-Benzylaminotetrazole...... Preparation of the 3,5-Dinitrobenzamide Derivative of l-n-Octyl—S—Benzylaminotetrazole................... Preparation of l-Benzy1-5-n-Octy1aminotetrazole...... Bacteriostatic Titers................................ Phenol Coefficients.................................. STun-TIY‘ARYOOOCOIOOIOOIOOOOOOOOOOOOOOOOOO0.0.000...00.0.0.0... BIBLIOGRApfflooooeoooooo0000000000.IOOOOOOOOOOOOOOOOOOOOOOO 16 16 17 18 19 19 20 21 21 22 23 24 24 25 25 26 30 33 34 TABLE I. II. III. IV. VI. VII. VIII. IX. XI. XII. XIII. XIV. XV. XVI. LIST OF TABLES PAGE l-Alkyl-5-AITIant8tI‘azoleso......................... 1-Alky1-5-Alkylamln0tetr320168.... 0.0. o 0000 0.000000 Derivatives of 1-Alkyl-5-Alkylaminotetrazoles...... Bacteriostatic Titer of 1-Alkyl-5-Alkylaminotetra- 2016 PydrOChlorideSCOO0.0.00.00.00.00..0.00.0.00... Phenol Coefficient of l-Alkyl-S-Alkylaminotetrazole HydrOChloridGSOOOOI...0.0.0.0000....COIOOOOOOOOOOOC l-Nethyl-S-Ethylaminotetrazole Hydrochloride....... 1-Nethyl-5-n-Octylaminotetrazole Hydrochloride..... l-n—Octyl-S-Methylaminotetrazole Hydrochloride..... l-Ethyl-S-n—Octylaminotetrazole Hydrochloride...... l-n-Octy1-5-Ethy1aminotetrazole Hydrochloride...... l-n-Octyl-S-Benzylaminotetrazole Hydrochloride..... Phenol Coefficient of l-Methyl-5-n-Octylaminotetra- zole mdrOChlorideOOOOOOOOO00....OOOOOOOOOOOOOOOOOQ Phenol Coefficient of l-n-Octyl-S-Methylamino Hydro- ChloridBOOOOOOOOOOOOOOOOICOOCOOOOOOOCCOOOOOOOOOI... Phenol Coefficient of l-Ethyl-S-n-Octylaminotetra- zole EfydrOChloridGOOOOIOOOOOOOOOOO0.000000000COOOOO Phenol Coefficient of l-n-Cctyl-S-Ethylaminotetra- ZOle HydI'OCthI'ide................................. Phenol Coefficient of l-n-Octyl-S—Benzylaminotetra- ZOIe I-IydrOChlori-dSCOOOOOOOOO000......0000000000000. 11 12 13 14 15 27 27 2B 28 29 29 31 32 52 INTRODUCTION The feasibility of preparing l-alkyl-5-alkylaminotetrazole deriva- tives by the alkylation of the corresponding l-alkyl-S-aminotetrazoles has recently been demonstrated (1). From an investigation of some of the pharnocological actions of an extensive series of these compounds, Gross and Featherstone (2) con- cluded that central nervous stimulatory action was common to many comp pounds of this group. Their observations indicated that maximum stimulatory action was exhibited by compounds in which the alkyl group in the l-position contained five or six carbon atoms, and that the activity decreased markedly when either larger or smaller alkyl groups were substituted in the same position. No other investigations of the biological effect of compounds of this type have been reported. Of particular interest was the observation that compounds in which a n-heptyl group was substituted in the 1-position exhibited defi- nite soaplike characteristics, and appeared to be surface active sub- stances. Such characteristics suggested the possibility that compounds of this type might have interesting bacteriostatic and bacteriocidal properties. Although the bacteriological properties of quaternary ammonium compounds, in which one of the substituents on the nitrogen is usually a large alkyl group, have been extensively investigated (3,4,5), such action has not been associated with secondary amines or their salts. -1- The synthesis of a number of l-alkyl-S-alkylaminotetrazoles with large alkyl groups as substituents either in the l-position or on the amino group, and a preliminary study of their bacteriostatic and bac- teriocidal properties was therefore undertaken. The object of the synthetic portion of the investigation was to prepare a series of such tetrazole derivatives in which a large alkyl group was substituted in the 1-position while both large and small groups were introduced as substituents on the amino group. Conversely, a number of compounds with small alkyl groups in the l-position and large substituents on the amino group were prepared. The object was to learn which type of com, pound would exhibit the most favorable bacteriological properties. The bacteriostatic titer and the phenol coefficient were determined for each of the new compounds prepared during the course of the'work. HISTORICAL The preparation of 5-aminotetrazole was first reported by Thiele (6), who diazotized aminoguanidine. The resulting imide azide, guanyl azide, was heated with sodium carbonate or sodium.acetate, to bring about rearrangement to the 5-aminotetrazole. t-- -,.--T: I -'- -I H211 c m1 M2 H502 Has '(3 l5 Nazco3 I-tH 33H HN - C—NH 2 l I! N N s / N Cyanamide (7), or diqyandiamide (8) may also be treated with hydrazoic acid to form 5-aminotetrazole. The latter dissociates to cyanamide dur- ing the reaction. _ ' a - -m H? N CN f HHS .________9 EDI i 3H2 ‘Q / N Direct alkylation of S-aminotetrazole has resulted in the forma- tion of a complex mixture of alkylated products. Thiele and Ingle (9) have described a variety of products resulting from methylation, ethyla- tion, and benzylation of 5-aminotetrazole. After benzylation of 5-amino- tetrazole with benzyl chloride in.a1kaline medium, at least four products could be isolated. l—Vethyl-S-aminotetrazole was one of the alkylated products isolated after the methylation of 5-aminotetrazole with methyl iodide. -3- A more clear cut preparation of l-methyl-S-aminotetrazole, des- cribed by Stone/(10), was achieved by the interaction of methyl- thiourea and sodium azide in an atmosphere of carbon dioxide in the presence of lead carbonate. S H CH - N" - C - NH NaN CH - N - C -NH 3 n 2 3 i 3 , N 2 PbCO IE7 003 N N 2 a / N By application of this reaction to a variety of thiourea derivatives Stolle’(ll,12) prepared l-methyl-S-aminotetrazole and a number of l-aryl-5-aminotetrazoles. Stella/also correctly identified the come pound prepared by Oliveri-Handalh and Noto (13) by the interaction of hydrazoic acid and phenyl isothiocyanate in ethereal solution as l-phenyl 5-aminotetrazole. Oliveri-Mandala and Note had considered the compound as an addition product of hydrazoic acid and phenyl isothiocyanate but Stolle’showed that the sulfur had been eliminated during the reaction. The product was identical with the l-phenyl-5—aminotetrazole formed by interaction of phenylthiourea and sodium azide. A more attractive procedure that eliminates the use of lead car- bonate in the presence of sodium.azide or hydrazoic acid was developed by von Braun and Keller (14). The procedure involves the interaction of nitriles and hydrazoic acid in the presence of concentrated sulfuric acid. From the appropriate nitriles, l-phenyl, l-p-tolyl and l-benzyl- 5-aminotetrazole were prepared by these authors according to the follow- ing equation: -4- R-CN + 22m H so R-N - 0 -NH N e 2 4 5 , u 2 i 2 N N // \ N The mechanism postulated by von Braun and Keller (14) assumed the form- ation of a free imine radical (mm) by the evolution of nitrogen from hydrazoic acid upon contact with sulfuric acid, as postulated by Schmidt (15). The imine radical was assumed to add to the cyanide group, followed by rearrangement of the addition product to a structure analagous to the isocyanate grouping. A second molecule of hydrazoic acid then added to the rearrangement product, and'the resulting inter- mediate then underwent cyclization. HN H SO :5 2 4 HM * N2 --***—-9 _ ,yNH R-CN + )NH ___9 R-C . _..___.9 R-N'-'-C-"-NH ENS \N< _________9 R-N : C - NH2 _______9 R - N - 3 - NH2 N3 N N Q- / :r' N The existence of a radical of the imine type seems rather doubtful, and a more logical explanation is the mechanism.suggested by Herbst, Roberts, and Harvill (1). This involves the addition of hydrazoic acid directly to the cyanide linkage, followed by an acid catalyzed rearrange- ment, similar to the Curtius rearrangement of acid azides, accompanied by the elimination of nitrogen. The same rearrangement product, postu- lated by von Braun, is assumed to result and, after addition of another -5- mole of hydrazoic acid, cyclizes to the l-alkyl-S-aminotetrazole. 4.NH . "' I l- 1",T=: R CN.+H13.____9 ac H2304 RNCNH+NZ —————-> ~ N3 R-N:C:NH 4. ENS—.9 R-N:c':-NH2__>R-N-c-NHZ I ll N3 N N w / N By this reaction, Herbst, Roberts, and Harvill have prepared an exten- sive series of l-alkyl-S-aminotetrazoles (l). -6- DISCUSSION In this work, a number of l-alkyl-S-aminotetrazoles were prepared by the method of von Braun and Keller (14). These l-alkyl-S-amino- tetrazoles were methylated, ethylated, and benzylated to give the cor- responding l-alkyl—5-alky1aminotetrazoles, which, for convenience, were separated as the hydrochlorides. H 30 R - CN 7‘ ZHN3 ——2—-——-—l-}-—9 N2 1- R - N - C -NH2 Alkylation7‘ o u N N 0 / N R - N - C - NH R' 1 l N N s / All of the l-alkyl-S-alkylaminotetrazoles were characterized as phenyl- thiourea, p-nitrobenzamide, or 3, 5-dinitrobenzamide derivatives. Of the l-alkyl-S-aminotetrazoles prepared, l-n-octyl-S-aminotetra- zole was the only one which had not been previously reported. It was synthesized by the action of hydrazoic acid on nonanenitrile, accord- ing to the method of von Braun and Keller (14). Although the l-alkyl- 5-aminotetrazoles are represented as primary amines, the basic proper- ties of the amino group are masked, possibly due to the character of the heterocyclic ring (1). They are relatively high melting solids, moderately soluble in hot water. Their aqueous solutions are usually neutral or slightly acid. They can be acylated and alkylated, but only under considerably more drastic conditions than are usually required -7- for primary amines. For example, the alkylation of l-n-octyl-S-amino- itetrazole with dimethylsulfate is accomplished only after heating to about 100°C, when an exothermic reaction is initiated and the tempera- ture continues to rise to 160°C. On the other hand, the alkylation of n—octylamine occurs almost immediately upon the addition of dimethyl- sulfate to the amine at room temperature. The method of synthesis of l-n-octyl-S-aminotetrazole, which was completely analogous to that used for the preparation of l-n—heptyl, l-n-nonyl and l-n-undecyl-5-aminotetrazole (1), makes it reasonable to assume that the structure may be written as follows: This assumption is further supported by the formation of secondary amines upon alkylation and the formation of typical derivatives of the secondary amines, all of which.gave results in conformity with the calculated values upon quantitative determination of the nitrogen con- tent. Alkylation of the l-alkyl-S-aminotetrazole may conceivably follow two distinct courses. In addition to the formation of secondary amines, the possibility of interaction of the alkylating agent with the ring nitrogen to form a quaternary salt must be considered. The formation of quaternary compounds has been observed with several pentamethylene tetrazole derivatives (16). If a quaternary salt had been formed by alkylation of the 5-aminotetrazole derivative, it would be anticipated -8- that the quaternary base liberated, upon the addition of alkali, would be extremely soluble in water and difficult to extract with the common immiscible organic solvents. Furthermore, distillation of quaternary bases usually is accompanied by extensive decomposition. However, if alkylation occurred on the amino group of the l-alkyl-S-aminotetrazole, the base liberated, upon addition of alkali would be relatively insolu- ble in water, capable of being extracted with ether or benzene, and distillable without decomposition. The alkylated product should also form derivatives characteristic of secondary amines, such as substituted thioureas and amides. Since the bases liberated on treatment of the alkylated products with alkali were insoluble in.water, soluble in ether or benzene, and distillable under reduced pressure without decom- position, it is reasonable to assume the products are the 5-alkylamino derivatives. This assumption is further substantiated by the formation of a phenylthiourea or nitrobenzamide derivative in each instance. Nitrogen determinations upon all products and derivatives were in con- formity with these conclusions. In alkylating the various l-alkyl-S-aminotetrazoles, several pro- cedures were employed. The methylation, ethylation, and benzylation 'were accomplished by heating;a mixture of equimolecular quantities of the amine and dimethylsulfate, diethylsulfate, or benzyl chloride, to a sufficiently high temperature to initiate an exothermic reaction. From the resulting homogeneous melt, the l-alkyl-S-alkylaminotetrazoles 'were isolated. Alkylation with n-octyl chloride was brought about by reaction in a sealed tube. -9- Phenol coefficients and bacteriostatic titers were determined for each of the secondary amines by the usual standard procedures, us- ing 24-hour cultures of Staphylococcus aureus and Eberthella typhosa, which had been transferred for three consecutive days prior to use. A blank, using distilled water, was run along with each series of tests. Organic matter interfered with the bacteriocidal action of the compounds. From the data recorded in Table IV, it will be noted that the l-alkyl-5-alkylaminotetrazoles are more effective against Gram negative organisms than.against Gram positive organisms. It is noteworthy that this effect is the reverse of that usually exhibited by the quaternary salts. Although the data which has been assembled are not sufficient to determine the optimum.structure for maximum bacteriological activity, it does appear to be essential that one of the alkyl groups should be rather large. Furthermore, whether the large alkyl group is present as a substituent in the l-position or on the amino group seems to be immaterial. Since the introduction of small alkyl groups as substituents on the amino group during the alkylation process could be accomplished more easily and with better yields than the introduction of large alkyl groups, it would be more profitable in any extension of this investigation, to direct efforts toward the preparation of l-alkyl- 5-a1kylaminotetrazoles with the large group in the l-position. -10- .osewcnoop messanoaowe an eoewsnopoe comOApmz ** .oopoenuoo one mpcwom wprHoE Haw * eH tn- nu- new .eoe.ewfl-m.emfi sesame amoemeo Hanson e.me m.mm was .eom.eeflteefi mamfimmo‘ anemone Hapooue H tn- -n- was .ooeeasm.eefl cabana memo Hague oH.o -u- -u- ae.ee .ooemmte.amm sesame one Hanson .eom oceoa .ofloo once» .uo onescoa m oHoeeceoeoeaaoum ..z a eeeaaeea ..c«: -Haaaenfl z ' \ ¢ z a N z ~ le o I Z I m noHoweapopcn«EHqu HHH mqmda coo.ONIH ooo.omuH oeHeoHaococuenHaeoouenH ooo.OH-H 4| ooomnH ocHeoHaceoumuHApooucuH 11. ooo.oH-H oooHuH noeHaoHapoouesmuHaeponH ooo.oH-H ooomuH 1, -oeHEoHaEeostmnHapootcuH ooomnH oooHuH -oeHseHauooncumuHaeeoshH 11‘ once econ IoaHEeHzrpotthhnpoETH smonmmu eflflmmwwepm unease mSoQOQOstmmflm nopHp OHpspmoHnopoem ooHcoHeooeoae oHoNeepoeoeHeoHanHoumuHanHoaH z \ z = Hum..mm23 o I 0. z i z I m eoeHcoHeoocoam oHoceceopocHeoHaaHetmnHHaHeuH mo copHH oHseeooHcoeoem >H mAm £9; EXPE RI Iii-EN TAL All of the l-alkyl-S-aminotetrazoles were prepared following the procedure of von Braun and Keller (14). The l-metnyl, l-ethyl, and l- benzyl-S-aminotetrazoles have been previously described (1,9,10,14). The melting points of these compounds compared favorably with those re- corded in the literature. Since l-n-octyl-S-aminotetrazole has not been previously described, the procedure employed for its preparation will be given in detail. The other l-alkyl-S-aminotetrazoles were pre- pared in a completely comparable manner. Preparation of l-n-Octyl-Squinotetrazole N N \\ / N Concentrated sulfuric acid (300 ml.) was added dropwise to a me- chanically stirred solution of 69.6 g. (0.5 mole) of nonanenitrile and 55 g. (1.25 mole) of hydrazoic acid in about 500 ml. of benzene. After addition of about 75 ml. of sulfuric acid, the solution turned chalky white, and the temperature rose to 40° C. The solution was cooled and kept between the temperature range of 30-550 C. Addition of the sulfuric acid was accompanied by vigorous evolution of nitrogen. After about two hours, the addition of the sulfuric acid was completed, and the result- ing solution was cautiously warmed to 40° C. The stirring was continued for 6-7 hours after complete addition of the sulfuric acid. After separation from benzene, the acid layer was poured onto about 300 g. of ice and neutralized with about 600 g. of sodium carbonate. The resulting -16.. solution was evaporated to dryness on a steam.bath, with the aid of a stream.of air blowing across the top of the beaker. The solid left, after evaporation of the water, was extracted re- peatedly with 1000 ml. of boiling isopropyl alcohol, using the mother liquor for successive extractions. The resulting crude product was recrystallized from aqueous isopropyl alcohol. Yield 66 g.-67§. M. p. 164-164.5° c. (cor.). Preparation of l-Nethyl-S-Ethylaminotetrazole CH3 - N - C - NH-Cdz-CH3 l N N \\ / A mixture of 34 g. (0.34 mole) of l-methyl-S-aminotetrazole and 56.4 g. (0.34 mole) of diethylsulfate was heated on a steam bath with occasional stirring. 'When the temperature of the mixture approached 1000 C., a rapid exothermic reaction took place, causing the tempera- ture to rise to 170° C. After the reaction mixture had been heated on the steam.bath for an additional 30 minutes, it was allowed to cool to room temperature. The base was isolated by adding a warm solution of the crude ethosulfate in 100 ml. of absolute ethanol to a warm solution of 10 g. of sodium in 100 ml. of absolute ethanol. After refluxing the resulting mixture for ten minutes,'the ethanol was distilled off under reduced pressure. The residue was suspended in 100 ml. of water, and after addition of about 200 g. of anhydrous potassium carbonate, the base was extracted from the sludge with six 200 ml. portions of benzene. The benzene extracts were combined and the solvent removed by distilla- tion. The residue remaining after removal of the solvent, was distilled under reduced pressure. The yield of product distilling at 115-1190 at 25 mm was 24 g. or 53%.~ The hydrochloride was prepared by dissolv- ing the entire quantity of base in 75 ml. of absolute isopropyl alcohol, and adding dry hydrogen chloride in excess. After recrystallization from absolute isopropyl alcohol, 25.2 g. (45%) of pure hydrochloride was obtained. M. p. 203-2040 C. (cor.). Preparation of the Phenylthiourea Derivative of l-Nethyl-S-Ethylamino- tetrazole __._.__.__._._ CH2 - CH / CH3 - N - c - N 3 l H ‘c - NHCGHS N N u s / S N About 1 g. of the l-methy1-5-ethylaminotetrazole hydrochloride was dissolved in a minimum.amount of water. The water solution.was saturated with potassium.carbonate and extracted with ether. After evaporation of the ether, one gram of phenyl isothiocyanate was added to the residue and the solution warmed on a steam bath. After cooling, the partially solid mixture was filtered. To the filtrate, a solution of equal volumes of ethanol and ligroin was added, and the resulting precipitate was filtered off by suction. The combined precipitates were recrystallized twice from 95% ethanol. M. p. l49-l49.5o C. (cor.). A mixed melting point of the phenylthiourea derivative with an authentic preparation (1) showed no depression of the melting point. -18- Preparation of l-Methyl-S-n-Octylaminotetrazole ..I.. .. .- CH3 a % NH C8 H17 N N Iii / A mixture of 10 g. (0.1 mole) of 1-methyl-5-aminotetrazole, 15 g. (0.1 mole) of n-octyl chloride and 25 ml. of absolute ethanol was sealed into a long combustion tube. The sealed tube was placed in an oven for 48 hours at a temperature of 180° C. At the end of this time, the tube was opened and the contents evaporated to dryness on.a steam bath. The residue was taken up in a minimum amount of water, a few drops of concentrated hydrochloric acid added, and the solution ex- tracted with ether. The water solution was saturated with potassium carbonate, and the free base extracted with ether. Excess dry hydrogen chloride was bubbled into the ethereal solution. The precipitate of hydrochloride was filtered off, and recrystallized twice from.absolute isopropyl alcohol. The yield of the hydrochloride was 5.5 g. (22%). N. p. 197-197.50 c. (cor.). Preparation.of the 3,5-Dinitrobenzamide Derivative of l-Nethyl-S-n- Octylaminotetrazole C H CH - N - c - N ’ 8 17 n ‘c-/ No2 N N u e / O N \ About 1 g. of l-methyl-5-n-octylaminotetrazole hydrochloride was 102 dissolved in a minimum.ammunt of water. The water solution was saturated with potassium carbonate and extracted with ether. After evaporation of the ether, the free amine was dissolved in 10 ml. of dry benzene and -19- added to a solution of l g. of 3,5-dinitrobenzoyl chloride in 5 ml. of benzene. The resulting solution was refluxed for 15 minutes, and then allowed to cool. The solution was filtered, and the precipitate washed with 10 m1. of warm benzene, the washings being added to the original filtrate. The benzene solution was then washed with 10 ml. of 2% sodium carbonate, followed by 10 ml. of 2% hydrochloric acid, and final- ly 10 ml. of distilled water. The benzene was evaporated and the resi- due recrystallized four times from.di1ute ethanol. N. p. 70.5-71o C. (cor.). Preparation of l-n-Octyl-S-Nethylaminotetrazole C8 H17 - N - fi - NHCH3 N N s.‘ f N A mixture of 19.7 g. (0.1 mole) of 1-n-octyl-5-aminotetrazole and 12.6 g. (0.1 mole) of dimethylsulfate was heated in an oil bath to a temperature of 1700 C. Heating was continued for one hour, after which the reaction mixture was allowed to cool to room.temperature. The base was isolated by adding a warm solution of the crude methosulfate in 175 ml. of absolute ethanol to a warm solution of 2.5 g. of sodium.in 100 ml. of absolute ethanol. After refluxing the resulting mixture for one hour, the ethanol was distilled off under reduced pressure. The residue was suspended in 150 ml. of water and after addition of about 300 g. of anhydrous potassium.carbonate, the base was extracted from the sludge with six 200 ml. portions of benzene. The benzene ex- tracts were combined and the solvent removed by distillation. The resi- due was distilled under reduced pressure and the fraction coming over -20- at 147-1510 at 4 mm. was collected. The yield was 9.5 g. (45%). The hydrochloride was prepared by dissolving the entire quantity of base in 25 ml. of absolute isopropyl alcohol, and adding dry hydrogen chloride in excess. After recrystallization from absolute isopropyl alcohol, 8.8 gm. (38%) of pure hydrochloride was obtained. N. p. 200.5-201° c. (cor.). Preparation of the Phenylthiourea Derivative of l-n-Octyl-5-Nethyl- aminotetrazole ’,CH3 08 H - N - c - N 17 c " \ cs - NHC6H5 N N 0 / N About 0.5 g. of the l-n-octyl-S-methylaminotetrazole hydrochloride was dissolved in a minimum amount of water. The water solution was saturated with potassium carbonate and extracted wdth ether. After evaporation of the other, 0.5 g. phenyl isothiocyanate was added to the residue. When the reaction was complete, the resulting solution was cooled in an ice-salt bath and ligroin added. The solution was fil- tered and the precipitate was crushed and washed with ligroin. The precipitate was recrystallized twice from 95% ethanol. N. p. 83.5-840 C. (cor.). Preparation of l-Ethyl 5-n-0ctylaminotetrazole C H - N - C - NH-C H 2 5 ‘ u 8 17 t l A mixture of 5.6 g. (0.05 mole) of l-ethyl-S-aminotetrazole and 7.4 g. (0.05 mole) of octyl chloride was heated together on an oil bath -2 1- to a temperature of 180° C. Heating was continued for one hour, after which the reaction mixture was allowed to cool to room temperature. The solid mass was recrystallized four times from.hot ethyl acetate. The weight of pure hydrochloride recovered was 5.6 g. (29%). M. p. 162.5-163.5° c. (cor.). Preparation of the 5,5-Dinitrobenzamide Derivative of 1-Ethyl-5—n- Cctylaminotetrazole C H c H - N - c - N 8 17 2 5 i I \ c - -No2 N N u c / 0 N N0 2 About 1 g. of l-ethyl-S-n-octylaminotetrazole hydrochloride was dissolved in a minimum amount of water. The water solution was satu- rated with potassium carbonate and extracted with ether. After evapora- tion of the ether, the free amine was dissolved in 10 ml. of dry benzene and added to a solution of l g. of 3,5-dinitrobenzoyl chloride in 5 ml. of benzene. The resulting solution was refluxed for 15 minutes, and then allowed to cool. The solution was filtered, and the precipitate washed with 10 ml. of warm benzene, the washings being added to the original filtrate. The benzene solution was then washed with 10 ml. of 2% sodium carbonate, followed by 10 ml. of 2% hydrochloric acid, and finally 10 ml. of distilled water. The benzene was evaporated and the residue recrystallized four times from dilute ethanol. M. p. 57-57.5o C. (cor.). -22- Preparation of l-n-Octyl-S-Ethylaminotetrazole - - - N} _ C8 H17 N C 11 CZ H5 I u N N t / N A mixture of 19.7 g. (0.1 mole) of l-n-octyl-S-aminotetrazole and 15.4 g. (0.1 mole) of diethylsulfate was heated on a steam.bath with occasional stirring. ‘When the temperature of the mixture approached 950 C., an exothermic reaction took place, causing the temperature to rise to 120° 0. Heating was continued for thirty minutes on the steam bath, after which the reaction mixture was allowed to cool to room tem- perature. The base was isolated by adding a warm solution of the crude ethosulfate in 100 ml. of absolute ethanol to a warm solution of 2.5 g. of sodium in 75 ml. of absolute ethanol. After refluxing the resulting mixture for one hour, the ethanol was distilled off under reduced pressure. The residue was suspended in 150 ml. of water, and after addition of about 300 g. of anhydrous potassium carbonate, the base was extracted from the sludge with six 200 ml. portions of ben- zene. The benzene extracts were combined and the solvent removed by distillation. .The residue remaining after removal of the solvent, was distilled under reduced pressure. The yield of product distilling at 160-1640 at 8 mm. was 18 g. or 69%. The hydrochloride was prepared by dissolving the entire quantity of base in 50 m1. of absolute isopropyl alcohol, and adding dry hydrogen chloride in excess. After recrystal- lization from.absolute isopropyl alcohol, 12 g. (4Q% of pure hydro- chloride was obtained. N. p. 165.5-166° C. (cor.). -23- Preparation of the p-Nitrobenzamide Derivative of l-n-Octyl-S-Ethyl- aminotetrazole c H /2 5 c a -N - c - N 8 17 ‘ H \c-/ N N n \\ / 0 130 N \ 2 About 0.6 g. l-n-octyl-S-ethylaminotetrazole hydrochloride was dissolved in a minimum amount of water. The water solution was satu- rated with potassium.carbonate and extracted wdth ether. After evap- oration of the ether, the free amine was dissolved in 5 ml. of dry ben- zene and added to a solution of 0.6 g. of p-nitrobenzoyl chloride in 10 ml. of benzene. The resulting solution was refluxed for 15 minutes, and then allowed to cool. The solution was filtered, and the precipi- tate recrystallized three times from dilute ethanol. M. . 56.5-57O C. (cor.). Preparation of l-n-Cctyl-5-Benzylaminotetrazole C H - N - C - NEG- H e 17 H206 5 A mixture of 19.7 g. (0.1 mole) of l-n-octyl 5-aminotetrazole and 12.6 g. (0.1 mole) of benzyl chloride was heated in an oil bath to a temperature of 1700 C. Heating was continued for two hours, after which the reaction mixture was allowed to cool to room temperature. The solid mass was recrystallized twice from absolute isopropyl alcohol. The melting point of the solid was 119-1200 C. The solid was recrystal- lized four times from boiling water. The weight of the pure hydro- chloride recovered was 8 g. (25%). M. p. 167-167.5o C. (cor.). -24- Preparation of the 3,5-Dinitrobenzamide Derivative of l-n-Octyl-S- Benzylaminotetrazole “:T . c H - N - c - N "C“2 C5 H5 8 17 u \ c _ -ro N N u 2 \\ / O 1* N0 About 1 g. of 1-n-octyl-5-benzylaminotetrazole hydrochloride was suspended in about 10 ml. of 50% potassium hydroxide solution. The aqueous layer was saturated with potassium.carbonate and extracted with ether. After evaporation of the ether, the free amine was dissolved in 10 ml. of dry benzene and added to a solution of 1 g. of 3,5-dinitro- benzoyl chloride in 5 ml. of benzene. The resulting solution was re- fluxed for 15 minutes, and then allowed to cool. The solution was filtered, and the precipitate washed with 10 m1. of warm.benzene, the washings being added to the original filtrate. The benzene solution was then washed with 10 ml. of 2% sodium carbonate followed by 10 ml. of 2% hydrochloric acid, and finally 10 m1. of distilled water. The benzene was evaporated and the residue recrystallized four times from dilute ethanol. x. p. 58-590 c. (cor.). Preparation of l-Benzyl-5-n-0otylaminotetrazole C6 H5 CH2 - N - f - hnCB d17 N N \‘ / N A mixture of 4.4 g. (0.025 mole) of l-benzyl-5-aminotetrazole and 4.8 g. (0.025 mole) of octyl bromide and 15 ml. of mesitylene was heated in an oil bath until the two layers disappeared. After cooling, -25- the mass was recrystallized repeatedly from water, isopropyl alcohol, and ethyl acetate. Weight of hydrobromide recovered was one gram. M. p. 148-1510 C. The hydrobromide was dissolved in a minimum amount of water. The water solution was saturated with potassium carbonate and extracted with ether. To the ethereal solution, dry hydrogen chloride was added and a white precipitate was formed. The hydrochlor- ide was recrystallized four times from hot water. The weight of hydro- chloride recovered was about 0.2 g. H. P’ 154-1570 C. (cor.). Deter- mination of nitrogen showed that the hydrochloride was still impure. Alkylation by a sealed tube reaction was also unsuccessful. Baoteriostatic Titers Baoteriostatic titers were determined for each of the secondary amines in the following manner: Dilutions of the secondary amines were made up with distilled water. To each dilution, 0.5 ml. of a 24-hour culture of §, aureus or E. typhosa which had been transferred for three consecutive days prior to use, was added. The dilutions remained at room temperature for 3%-4 hours. After this interval, one loopful of each dilution was transferred to 10 ml. of FDA broth. The broth cultures were incubated at 37° C., and readings were taken at 24, 48, and 72 hours. The highest dilution showing no growth after 72 hours incubation was the bacteriostatic titer. Tables VI-XI show the bacteriostatic titers for the compounds tested. -25- TABLE VI l-Uethyl-S-Ethylaminotetrazole Hydrochloride S. aureus E. typhosa 24 hours 48 hours 72 hours 24 hours 48 hours 72 Heurs 1-100 + + + + + + + + -L + + +- 1-200 + r t + l- + j + + + + + 1-500 + +~+ 11’ + + + + + j + 14000 + + f + + + L + '+ 1- L+ 'i'ater J + t + + + + + + + + 1- TABLE VII l-Vethyl-5-n-0ctylaminotetrazole Hydrochloride S. aureus E. typhosa 24 hours 48 hours 72 hours 24 hours 48 hours 72 hours 1-1000 - —— -— A —- _. _. 1-2000 f + f + -— .. .— 1-5000 - + + + - + + + 1-10,000 + + 4 + + + - + + + l-20,000 + + + + + + + + + + + + water + + + + + t + + + 4 + + -27- TABLE VIII l-n—Octyl-S-Vethylaminotetrazole Hydrochloride ‘ S. aureus E. typhosa 24 hours 48"hours 72fihours 24 Hours 48 hours I72 hours 1-10,000 faint + + + ~ —— .. .. l-20,000 + + i f + + + + + + +Mt water + f + + + f f + + + {#1 TABLE IX 1-Ethyl-5-n-Octy1aminotetrazole Hydrochloride S. aureus E. typhosa 24 hours 48 hours 72’hours 24Ihours 48 hours 72*hours 1-1000 -- -- .— .. .— 1-2000 -— faint + —- —. _. 1-5000 + + + + + f —— —- _— 140,000 + 1' + + + 1- -- - - 1-2o,ooo + + 4 + + + + + + 1» ++ Water + + + 4 + 4 + 4- + + + 4 -23- TABLE X l-n-O ctyl-S-Ethylami not etrazo 1e Hydrochlo ride S. aureus E. typhosa 24 hours 48 hours 72 hours 24 hours 48 hours 72 hours 1-5000 —— — .- -... _- .— _ l-l0,000 -- + + + ~— -— .- ’1-20,000 + + + + +- + + + + + +1 Water 1- i + + + + + + + + + 1: TABLE XI l-n-Octyl-5-Benzylaminotetrazole Hydrochloride S. aureus E. typhosa 24 hours 48 hours 72 hours 24 hours 48 hours 72 hours 1-10,000 - u— -— —— ._ .- 1-20,000 -— —— -— —— -— .— 1-5o,ooo -- + + + + + + + 4 + 1-100,ooo + + + + + + + + + 4- at Water + ++ +++ ? ++ +++ -29- I: 'lt‘ul 4n fl it'll] Phenol Coefficients Phenol coefficients were determined for each of the secondary amines by the method recommended by the United States Food and Drug Administration (17). This method was applied using g, aureus, strain 209. The following data was obtained. TABLE XII Phenol Coefficient of l-Metmyl-S-n-Octylaminotetrazole Hydrochloride S. aureus E. typhosa Phenol 5 min. 10 min. 15 min. Phenol 5 min. 10 min. 15 min. 1-70 - - - 1-90 - - ‘- 1-80 + - -— 1-100 + —— .. 1-90 j- + - 1-110 + + t Tetrazole Tetrazole 1-600 -— - -— 1-1200 -— -— - 1-800 + —- - 1-1500 —— -— -— 1-1000 1- -— -- 1-1700 4 -- -- 1-1200 f + f 1-1800 + + f -30- TAB LE XI II Phenol Coefficient of l-n—Octyl-S-Methylamino Hydrochloride w S. aureus E. typhosa Phenol 5 min. 10 min. 15 min. Phenol' 5 min. 10 min. 15 min. 1-70 -— -— - 1-90 -— -— .— 1-80 f .. -___ 1-100 + -— - 1-90 + +' + l-llO + + + Tetrazole Tetrazole 1-600 -- -- -- ‘ l-lOOO -- -- -- 1-800 + " -— - 1-1200 + -— -— 1-1000 + + + 1-1500 + + + TABLE XIV Phenol Coefficient of l-Ethyl-S-n-Octylaminotetrazole Hydrochloride S. aureus E. typhosa PhenoI’ 5 min. 10 min. 15 min. PhenoI’ 5 min. 10 min. 15 min. 1-70 a, — — 1—90 + — .. 1-80 + —- - 1-100 + .. .— 1-90 + + + 1-1 10 1. + f- _Tetrazole Tetrazole 1-1000 + -- -— __1_-1500 —- —- -- 1-1200 + -— -— 1-1700 f - -— 1-1500 + + + ' 1-1800 + + + . - - a _ l u ‘ t I v , -_ - __ _. I TABLE XV Phenol Coefficient of l-n-Octyl-5-Ethylaminotetrazole Hydrochloride S. aureus E. typhosa PhenoI 5 min. 10 min. 15 min. Phenol .5 min. 10 min. 15 min. 1-70 f - -— 1-90 f .. ._ 1-80 + - -— 1-100 tgr -— __ 1-90 + + + l-110 + t_ J: Tetrazole Tetrazole 1-1000 + -— -— 1-1500 - - - 1-1200 4- — - 1-1700 r j + 1-1500 + + _+ 1-1800 J- ’r '- TABLE XVI Phenol Coefficient of l-n-Octyl-5-Benzylaminotetrazole Hydrochloride S. aureus Phenol 5 min. 10 min. 15 min. 1-70 + -— —- 1-80 t, __ .. 1-90 1‘ j- r Tetrazole 1-10,000 + + -f 1-15,000 + + } 1-20,000 + + t E. typhosa _;Phenol 5 min. 10 min. 15 min. 1-90 -— . -— .- 1-100 + — .— 1-110 + + _. Tetrazole 1-10,000 -+ 4- + 1-15,000 + + L 1-20,000 + 1.. + i ST? '7 1491' 1) The following 1-alkyl-5-aminotetrazoles were prepared: a) l-methyl-5-aminotetrazole b) l-ethyl-S-aminotetrazole c) l-n-octyl-S-aminotetrazole d) 1-benzyl-S-aminotetrazole Of these, l-n—octyl-S-aminotetrazole is a new compound. 2) The following l-alky1-5-alkylaminotetrazoles were prepared and iso- 03 v n5 \J lated as hydrochlorides: a) l-methyl-S-ethylaminotetrazole b) l-methyl-S-n-octylaminotetrazole c) 1-n-octy1-5-methylaminotetrazole d) l-ethyl-5-n-octylaminotetrazole e) l-n-ootyl-S-ethylaminotetrazole f) l-n-octyl-S-benzylaminotetrazole Of these, only the l-methyl-5-ethylaminotetrazole has been previous- ly described. Derivatives of all the l-alkyl-S-alkylaminotetrazoles were prepared. Phenol coefficients and bacteriostatic titers were determined for all of the secondary amines. Pronounced bacteriostatic and bacteriocidal action was observed in all of the secondary amines which carried a large alkyl group as a substituent either in the ring or on the amino group. The l-alky1-5-alkylaminotetrazoles were more effective against Gram negative organisms than against Gram positive organisms. -33- 1. 2. 3. 4. 5. 6. B. 9. 10. ll. 12. 13. 14. 15. 16. 17. BIBLIOGRAPHY Herbst, Harvill, and Roberts, J. Org. Chem., in press Gross and Featherstone, J. Pharmacol. Exptl. Therap., 88, 353 (1946) Shelton et al., J. Am. Chem. Soc., 68, 753-5 (1948) Shelton et al., J. Am. Chem. 800., 68, 755-7 (1948) Shelton et al., J. Am. Chem. $00., 38, 757-9 (1948) Thiele, Ann., 319, 54 (1892) Hantzash and Vogt, Ann., 314, 339-369 (1901) Stolle: Ben, 52, 1118-26 (1929) Thiele and Ingle, Ann., 287, 232-265 (1895) Stclle’, J. prakt. Chem., 134, 282-309 (1932) Stone: J. prakt. Chem., 3.2.4., 261-300 (1900) Stolle,’ 8er., 251: 1287-97 (1922) 01iveri-iranda13 and Note, Gazz. chem. ital., 43 I, 304 (1913) von Braun and Keller, Ber., 65, 1677 (1932) Schmidt, Ber., g, 704 (1924) Harvill, Roberts, and Herbst, J. Org. Chem., 15, 58-67 (1950) Ruehle and Brewer, U. 8. Dept. Agr. Dept. Circ., 198 (1931) -34- OCT 3 0 '50 “MU-.11 - ”'7: fl; 0 S} ; T545.9 234500 ' F922 Froberger T545.9 F922 Froberger ' The synthesis of certain aminotetrazole derivatives and a study of their bact- eriocidal properties. 254500 1 f g #2:»: f;._,(:,.(‘/. ' 08f 8 0 ’50 i( k 1 )0 . v e- g; lllllllllllill‘lliHHHIIHH||1|lHI!Hlllllllllltlltlllllllll 31293 02446 7304 .