[1]United States Patent Office 3@7089480 3,708,480 3-CYANOMETHYL-DELTA-3-CEPHALOSPORIN ANTIBIOTICS AND INTERMEDIATES L Alan Webber and Earle M. Van Heyningen, Indianapolis, Ind., assignors to Eli Lilly and Company, Indianapolis, Ind. No Drawing. Continuation-in-part of applications Ser. No. 790,842, Jan. 13, 1969, and Ser. No. 802,643, Feb. 26, 1969, both being continuations-in-part of application Ser. No. 703,523, Feb. 7, 1968. This application Nov. 16, 1970, Ser. No. 90,085 Int. Cl. C07d 99124 U.S. Cl. 260-243 C 7 Claims ABSTRACT OF THE DISCLOSURE Certain 3-eyanomethyl, 3-oxymethyl, and 3-thiomethyl ether A3- cephem-4-carboxylic acids, zwitterions, salts, and esters, e.g., pmethoxybenzyl 7-amino-3-cyanomethyl-A3cephem-4- carboxylate, a nucleus ester, and 7-[D-a-aminociphenylacetamidol-3 - methoxymethyl-A3-cephem-4-carboxylic acid, and 7 - [D-m-amino-a-phenylacetamidol-3methyltbiomethylA3-cephem-4-carboxylic acid, new cephalosporin antibiotics. CROSS-REFERENCE This application is a continuation-in-part of our priorapplications (a) Ser. No. 790,842, ffled Jan. 13, 1969, and (b) Ser. No. 802,643, filed Feb. 26, 1969, each of which applications (a) and (b) being a continuation-in-part of our prior application Ser. No. 703,523, filed Feb. 7, 1968. INTRODUCTION T'his invention relates to A3-cephalosporin compounds, that is, to cephalosporin ester compour.-ds having a carbon to carbon double bond between the carbon atoms in the 3 and 4 positions of the dihydrothiazine ring moiety of the cephalosporin compounds. More particularly, this invention provides a group of new 3- cyanomethyl- and some 3oxymethyl and 3-thiomethyl ether-A3- cephem-4-carboxylic acids, zwitterions, salts and esters, which are useful generally as antibiotics or in the process route to antibiotically active A3-cephalosporanic acid derivatives. These compounds can now be readily prepared by the oxidation, reduction, cleavage, reacylation, and de-esterification of the A2- cephem products described in our prior application, Ser. No. 703,523, filed Feb. 7, 1968, and our application Ser. No. 790,886, filed January 13, 1970 both now abandoned. BACKGROUND OF THE INVENTION The semi-syntlietic production of 7- acylamidodesacetoxycephalosporin antibiotics from penicillin starting materials has become of importance recently, due to the process invention of Morin and Jackson (U.S. Pat. 3,275,626) who describe and claim a process for converting penicillin sulfoxide esters to desacetoxycephalosporanic acid esters and to the improvements on that Morin-Jackson process by Chauvette and FIynn (U.S. application Ser. No. 574,311, filed Aug. 23, 1966) who found that certain esters of the penicillin starting materials and of the resulting desacetoxycephalosporin ester products were more useful in the process in that they were more easily cleaved than those employed by Morin and Jackson. Further improvements 6f the Morin-Jackson process were claimed by Robin D. G. Cooper (U.S. applications, Ser. No. 636,629, Ser. No. 636,593, and Ser. No. 636,592, all filed May 8, 1967, and now replaced by Cooper application Ser. No. 838,697, filed July 2, 1969, now U.S. Pat. No. 3,647,787) who found that the use of certain solvents directed the Patented Jan. 2, 1973 2 heat rearrangement of the peniciIIin sulfoxide esters more specifically toward production of the corresponding desacetoxycephalosporin esters and permitted the use of lower temperatures. Some of the 7- acylamidodesacetoxycephalosporanic acid compounds have been found to have unique properties as antibiotics in their own right. For example, cephalexin, 7-[D-a-amino-phenylacetamidol-3-methylA3cephem-4-carboxylic acid zwitterion and pharmaceutically acceptable cationic and anionic salt forms thereof are use10 ful as oral antibiotics in combatting infections caused, e.g., by penicillin resistant strains of Staphylococcus aureus, and many other Gram-positive and Gram-negative microorganisms. One of the unique advantages of A3- desacetoxycephalo15 sporin compounds, that is, compounds of the general formula 0 s l@ 7 /1\ R-C-NH-CII-CH6 2CE12 20 @ O-NI 5 3@-CH3 8 4:@@' c @OORI (1) wherein R is the residue of the acylamido group in the 7- 25 position and R' is hydrogen, a salt forming cation, an ester grotip, or an anionic charge when the COO- forms a salt with a cation either within or outside of the molecule, is that such compounds can now be prepared from penicillin sulfoxide esters by the Morin-Jackson process, especially 30 as improved by the Chauvette-Flynn and R. D. G Cooper improvements. In efforts to improve and expand the properties and utilities of these penicillin derived semi-synthetic cephalosporin substances, efforts have been expended on changing 35 the 3-methyl group of the above Asdesacetoxycephalosporins to a group which gives the resulting cephalosporin compound enhanced antibiotic activity against one or more Gram-positive or Gram-negative microorganisms. However, to date at least, it has not been possible to convert 40 directly a A3-desacetoxycephalosporin, to a 3-methyl functionalized A3-cephalosporin in any significant yield. In our prior application, Ser. No. 703,523, filed Feb. 7, 1968, now abandoned, but replaced in part by application Ser. No. 790,886, Med Jan. 13, 1969, now U.S. Pat. No. 3,637,678, 45 we disclosed a method for converting A2-desacetoxycephal@Osporin ester compounds to 3-bromomethyl- and 3(functionalized methyl)-A2-cephalosporin esters and the Teason why it was necessary to do so in the preparation of some cephalosporins. It was there pointed out that 3- 50 cyanomethyl- and the 3-oxymethyl-A2-cephem-4-carboxylate products of the process of that invention were useful as intermediates in the preparation of new and known A3cephalosporin antibiotic compounds. Such A2- cephalosporin compounds can be converted to the corresponding 55 A3-compounds by the oxidation of the A2-CoMpound to the sulfoxide-A3-compound, and followed by reduction of the sulfoxide-A3-CoMpound to the A3-cephalosporin compound by procedures disclosed therein. These processes are also described in Cooper application Ser. No. 764,939, filed 60 Oct. 3, 1968, now U.S. Pat. No. 3,647,786, and in Murphy et al. application, Ser. No. 764,925, filed Oct. 3, 1968, now U.S. Patent No. 3,641,014. When the desired 3-cyanomethyl- or 3-oxymethylA3cephalosporin has been obtained as described above, any 65 acyl group in the 7-position may be removed by known methods such as described in Chauvette's application, Ser. No. 651,662, fiied July 7, 1967 now Pat. No. 3,549,628. It is intended that the 7-acyl group in some of the 3cyanomethyl- and 3-oxymethyl-ether A3-cephalosporin 70 products of this invention can be removed by methods such as are described in Chauvette U.S. Pat. No. 3,549,628, or by the method described in U.S. application, Ser. No,
[2]3)708;480 3 758,600, filed September 9, 1968, now U.S. Pat. No. 3,632,578, or other known methods, and then the resulting 7-amino-3-cyanomethyl- or 7-amino-3-oxymethyl ether-A3-cephem-4-carboxylate ester or the corresponding acid can be acylated with any desired acyl group. The ester 5 group can be removed by known acid hydrolysis or hydrogenation procedures to yield the 7-amino-3-cyanomethylor 3-oxymethyl ether-A3-cephem-4-carboxylic acid nucleus as such, or as the zwitterion, or as a cationic or anionic salt form thereof. Similar ester group removal can be accom- 10 plished on the 7-acylamido compounds. In U.S. Pats. 3,261,832 and 3,278,531 there are described a number of cephalosporin compounds substituted on the 3-methyl group with sulfur-containing nucleophiles. However, the compounds of 3,261,832 are said to have 15 no practical antibiotic utility. They are useful only as intermediates in the preparation of cephalosporin antibiotics substituted on the 3-methyl with a pyridinium group. The sulfur-containing substituents of the compounds of 4 notes hydrogen, or taken together with the carbons to which they are bonded, complete a benzo- ring, RIO-C-C-CH-C oil 11 @, N N 0 wherein each of R' and R2 iS hydrogen or methyl, I-sydnone-3-C, to C3-alkanoyl-, C, to Clo-alkanoyl-, C3 to CIO-alkenoyl, Cl tO C7-alkyl-X-C, to C3-alkanoyl where X is oxygen or sulfur, C2 tO C7-alkenyl-X-C, tO C3-alkanoyl where X is oxygen or sulfur, C2 to Clo-haloalkanoyl where the halogen is fluorine, chlorine, or bromine, and C2 tO CIO-cyanoalkanoyl; 3,278,531 are rather complex and not readily obtainable, 20 R' is eyano (-CN), --S-CH3, or -OY where Y is and many of the compounds may be administered only by C, to CIO-alkyl injection. In fact, it is stated that they are particularly valu- C3 to CIO-alkenyl able for the formulation of injectable preparations. C3 to Clo-alkynyl It is an object of this invention to provide a gro,up of C2 to CIOhaloalkyl, new cephalosporin antibiotic derivatives having improved 25 C3 to Clrhaloalkenyl, and stability. C3 to Clo-haloalkynyl where halogen in each of such It is a more specific object of this invention to provide haloalkyl, haloalkenyl, and haloalkynyl groups is certain new 3-eyanomethyl- and 3-oxymethyl ether A3- fluorine, chlorine, bromine, or iodine cephalosporin compounds. C5 to Cq-cycloalkyl, It is a further object of this invention to provide those 30 C5 to Cq-cycloalkyl-C, to C3-alkylin the cephalosporin antibiotic art with certain new 3- Cl tO C3-alkyl-X-C2 to C6-alkyl where X is oxygen cyanomethyl- and 3-oxymethyl ether A3- cephalosporin or sulfur, antibiotics, and intermediate compounds leading thereto C2 tO C4-alkanoyl-C, to C6-alkyl, which were not available by previously known methods. C2 tO C4-alkanoyloxy-C2 to C6-alkyl, Other objects, aspects, and advantages of this invention 35 3'-furyl will become apparent from the description and claims (2'-furyl)-C3-alkyl, which follow. tetrahydrofuryl-C, to C2-alkyl, (2'-thienyl)-C, to C3-alkyl DETAILED DESCRIPTION OF THE INVENTION phenyl, benzyl, and phenylethyl, and This invention provides compounds of the formula 40 phenyl, benzyl, and phenylethyl s substituted on ring carbon atoms thereof with C, to C3-alkyl, C, to C3-alkyloxy, Cl tO C3-alkyloxyR-NH-CH-CEI CH2 carbonyl, fluorine, chlorine, bromine, nitro, cyano, O=U-N @-CHz--R' or trifluoromethyl, or combinations thereof; and 45 R" is selected from the group consisting of @OOR" wherein R is selected from the group consisting of hydrogen 50 Q CH-cowhere X is amino, a protected amino, hydroxy, C, to C3alkyloxy, -COOH, or Cl tO C3- alkanoyloxy, 55 Q (CH2)y-X-(CH2).-COwherein X is oxygen or sulfur or a chemical bond, y is 60 an integer of from 0 to 2, and z is I to 3, Q <::3>-CH=CH-00- 65 where Q in each of the above phenyl rings is hydrogen, Cl tO C3-alkyl, Cl tO C3-alkyloxy, cyano, nitro, hydroxy, chloro, bromo, fluoro, trifluoromethyl, a-amino-C, tO C3alkyl, carboxymethyl (HOOC--CH2)-, or carboxamidomethyl (H2NC(O)CH2--), 70 Z-C-Cll 11 11- Z-C C CH2COx where X is oxygen or sulfur, and each Z separately de75 hydrogen, a zwitterionic charge, or a salt with a pharmaceuticoy acceptable cation or anion, and C4 tO C6-tert-alkyl, such as tert-butyl, tert-pentyl, and tert-hexyl, C5 tO C7-tert-alkenyl, such as tert-pentenyl, terthexenyl, and tert-heptenyl, C5 tO C7-tert-alkynyl, such as tert-pentynyl, terthexynyl, and tert-heptynyl, benzyl, methoxybenzyl, nitrobenzyl, benzhydryl, phthalimidomethyl, succinimidomethyl, trimethylsilyl, and phenacyl ester groups Certain of the above included 7-acylamido-3-eyanomethyl- and 3-oxymethyl ether-A3- cephem-4-carboxylate esters are prepared by oxidizing the corresponding A2compound to the A3-sulfoxide, and then reducing the A3-sulfoxide to the A3-cephalosporin ester compound. For example, p-methoxybenzyl 7-phenoxyacetamido-3- cyanomethyl-A3-cephem-4-carboxylate is prepared by oxidizing p-methoxybenzyl 7-phenoxyacetamido-3-cyanomethyl-A2cephem-4-carboxylate to the corresponding Ioxide (sulfoxide) e.g., with m-chloroperbeiizoic acid in an appropriate solvent, and reduciiig the sulfoxide with sodium dithionite or equivalent reducting a-ents in the presence of acetyl chloride.
[3]5 The 7-amino-3-cyanomethyl- and 3-oxymethyl etherA3- cephem-4-carboxylate esters, referred to as nuclei type compounds, are prepared by cleaving the 7-acyl group or other 7-amino-blocking group which was present during the above described oxidation and reduction steps. For example, p-nitrobenzyl 7- amino-3-ethoxymethyl-A3cephem-4- carboxylate is obtained by reacting p-nitrobenzyl 7 -phenoxyacetamido-3-ethoxymethylA3 - cephem4-carboxylate with PCI5 in the presence of a tertiary amine, followed by treatment with an alkanol such as methanol, and then with water to effect cleavage. The rernaining 7-acylamido-3-cyanomethyl- and 3-oxymethyl ether A3-cephem-4- carboxylate compounds are prepared by acylating the 7-amino-3-cyanomethyl or 3oxymethyl ether A3-compounds, either in the ester or acid form with the desired acyl group according to known cephalosporin acylation procedures. The 7-amino-3-cyanomethyl- and 3-oxymethyl ether A3 -cephem-4-carboxylic acids and the 7-acylamido-3cyanomethyl- and 3-oxymethyl ether -A3-cephem-4-carboxylic acids are prepared by de-esterifying the esters by known methods or by methods described in the above cited applications, either before or after the 7-amino group is re-acylated. our novel 3-thiomethyl ether compounds can be prepared by the acylation of 3- methylthiomethyl-7-aminoA3-cephen-4-carboxylic acid with phenylglycine, thienylglycine, or an appropriately substituted phenylglycine. T'hroughout this specification reference to phenylglycine is meant to include thienylglycine and such substituted phenylglycines. It is understood that the a-amino group Of phenylglycine is protected during the acylation step with any known protecting group commonly used in protecting amino groups in amino acid or peptide chemistry. A few examples of such protecting group@,, are t-butoxycarbonyl, benzyloxycarbonyl and 2,2,2- trichloroethoxycarbonyl. At the completion of the acylation step these groups are removable by treatment with an acid such as p-toluenesulfonic acid or by reductive cleavage. For the acylation the protected phenylglycine may be activated with a carbodiimide or used as the acid chloride or bromide, or a mixed anhydride, especially with a lower alkyl carbonic acid. Acylation procedures are well known to those skilled in the art. The 3 - methylthiomethyl-7-amino-A3-cephem-4-carbOxylic acid to be acylated may be prepared by any known method. For example, this acid can be prepared by treating 7- aminocephalosporanic acid with methyl mercaptan or treating 3-acetoxymethyl - 7 - phenyl-acetamido-A3-cephem-4-carboxylic acid or the corresponding 7- phenoxyacetamido acid with methyl mercaptan, protecting the carboxyl group by ester formation, and then cleaving the phenylacetyl or phenoxyacetyl group by reaction with phosphorous pentachloride in th.- presence of ,i tertiary amine followed by treatment with a lower alkano'l, then with water or aqueous acid to form the 3-Tnethylthiomethyl-7- amino-A3-cephem-4-carboxylic acid ester. After acylation with the phenylglycine the ester group may be removed to regenerate the free carboxylic acid. Our novel compounds can also be prepared from a peniciIIin starting material such as penicillin V by a multistep process starting with the process described in Morin and Jackson U.S. Pat. 3,275,626 for the conversion of a penicillin sulfoxide ester to a desacetoxycephalosporanic acid ester. This @A3-des acetoxycephatoporin ester is then isomerized to a A2-cephalosporin which can be converted to the corresponding 3- bromomethyl derivative by treatment with N-bromosuceinimide. Treatment of this 3- bromomethyl compound with methyl mercaptan results in the formation of the corresponding 3-methylthiomethYl compound. The double bond in the 2-position can then be isomerized to the 3-position by base isomerization. The acyl group in the 7- position can be removed as described above to yield the3-methylthiomethyl-7-aminoA3-cephem-4-carboxylic acid or ester. Arylation of this 7-amino 3)7081480 6 compound with phenylglycine yields the desired acid or ester. The amino protecting or blocking group represented by R in the above formula may be, e.g., triphenylmethyl (trityl), trimethylsilyl, or any other amino protecting group such as tert-butoxycarbonyl or benzyloxycarbonyl, but most practically, and preferably, it is an acyl group. Many acyl groups suitable as intermediates for the purpose of preparing compounds of this invention are already known 10 in the penicillin and cephalosporin art. In the first steps of preparing compounds of this invention, R is preferably a phenylacetyl, phenoxyacetyl, phenylmercaptoacetyl, or other acyl group that occurs in penicillins available by biosynthesis as described in the applications cited 15 above. This acyl group is thus one that is economical, stable to the various chemical reactions to which the molecule is subjected in the intermediate steps, but which is readily cleaved by the procedures defined above at the desired stage in the preparation of the cephalosporin anti20 biotics of this invention. Preferably, it is derived from the penicillin that was used several steps prior for conversion to a cephalosporin material. To place the preparation of the new compounds of this invention in perspective relative to earlier discoveries in 25 the conversion of penicillins to cephalosporin compounds a summary of steps is given, using phenoxymethyl penicillin (-.penicillin V) as an example. (1) Phenoxymethylpenicillin is converted to the sulfoxide and esterified with an ester group which can be 3o easily removed by acid hydrolysis methods or hydrogenation methods later in the process. Alternatively, the penicillin can be esterified first and converted to the pencillin sulfoxide thereafter; (2) The phenoxymethylpenicillin sulfoxide ester is 35 heat treated according to the Morin-Jackson process referred to above as improved by the Cooper improvements to form the corresponding A3-cephalosporin ester, 7- phenoxyacetamido-3-desacetoxy-A3-cephem-4-carboxylate; the Alcephalosporin ester is hydrolyzed to the correspond40 ing acid by basic hydrolysis to the A2-acid which issubsequently converted into the acid chloride and A2-cephalosporin ester, by the Murphy method described in U.S. application Ser. No. 759,490, filed Sept. 12, 1968, now abandoned, but replaced with application Ser. No. 151,- 45 555, filed June 9, 1971. (3) The A3-cephalosporin ester is treated according to our described procedure referred to above to form the corresponding 7-phenoxyacetamido - 3 - bromomethyl-A2cephem-4-carboxylate ester, and then with the appropri50 ate nucleophilic reagent, e.g., methanol to form, e.g., pmethoxybenzyl 7- phenoxyacetamido-3-methoxymethyl-A2cephem-4-carboxylate as product. (4) The product of step (3) is then oxidized in a suitable solvent with mchloroperbenzoic acid, or other ox55 idizing agent, to form the p-methoxybenzyl 7-phe noxyacetamido-3-methoxymethyl - A3 - cephem-4-carboxylateI-oxide (the sulfoxide); (5) The sulfoxide is thereafter treated with an approptiate reducing agent such as sodium dithonite in the 60 presence of aii acyl halide such as acetyl chloride to form thefirst product of this invention, p-methoxybenzyl 7-phenoxyacetamido-3-methoxymethyl - A3 - cephem-4-carboxylate. This new product is useful as anintermediate as described in the steps which follow for the preparation 65 of a valuable new cephalosporin antibiotic. (6) The product from step (5) can be de-esterified by known methods to produce 7-phenoxyacetamido-3-methoxymethyl-A3-ce phem-4-carboxylic acid, an antibiotic. We prefer, however, to cleave the 7-acyl group from the 70 ester by treating the ester first with PCI5 in the presence of pyridine, then with methanol, and then with water to form the p-methoxybenzyl 7-amino-3- methoxymethyl-A3cephem-4-carboxylate ester nucleus, as such, or preferably as the salt of a C6 tO C12 aromatic hydrocarbon sul75 fonic acid, such as the p-toluenesulfonate (tosylate) salt,
[4]7 (7) The new ester nucleus or its tosylate salt from step (.6) can then be de-esterified to obtain the nucleus acid zwitterion, but the nucleus ester is preferably re-acylated with any of a variety of desired acylating agents, containing one of the acyl groups set forth above, e.g., with an isobutyl or sec-butyl mixed anhydride form of D-a,-(blocked amino)-a-phenylacetic acid to form the pmethoxybenzyl 7-[D-o:-(blocked amino) - a - phenylacetamido]-3-me thoxymethyl-A3-cephem-4- carboxylate ester. A suitable blocking group is tert-butoxycarbonyl, benzyloxycarbonyl, or the enamine formed with methyl acetoacetate. (8) The ester from step (7) is treated by known methods to remove the amino blocking and p-methoxybenzyl ester groups to otbain 7-(D-cc-amino-,%- phenylacetamido)3-methoxymethyl-A3-cephem-4-carboxylic acid, as such, or in the zwitterionic or pharmaceutically acccptable salt form such as the sodium, potassium, cationic salts, or as anionic salts with suitable strong acids having a pKa of less than 4 such as hydrochloric, hydrobromic, sulfuric or like acids. Examples of nucleus-type compounds of this invention which can be made by the general procedure described above include: 7-amino-3-cyanomethyl-A3-cephem-4-carboxylic acid, 7-amino-3-n-hexyloxymethyl-A3-cephem-4-carboxylic acid, 7-amino-3-allyloxymethyl-A3-cephem-4-carboxylic acid, 7-amino-3-(I-methyl-3-butynyloxymethyl)-A3-cephem4-carboxylic acid, 7-aniino-3-(cyclohexyloxymethyl)-A3-cephem-4-carboxylic acid, 7-amino-3-(cyclopentylmethyloxymethyl)-A3-cephem-4- carboxylic acid, 7-amino-3-(3-bromopropyloxymethyl)-A3-cephem-4- carboxylic acid, 7-amino-3-(2-phenylethoxymethyl)-A3-cephem-4- carboxylic acid, 7-amino-3-phenoxymethyl-A3-cephem-4-carboxylic acid, 7-amino-3-methylthiomethyl-A3-cephem-4-carboxylic acid, 7-amino-3-(4'-nitrophenoxymethyl)-A3-cephem-4- ca,rboxylic acid, 7-amino-3- (ethylthioethoxymethyl) -As-cephem-4- carboxylic acid, 7-amino-3-(propionyloxymethoxymethyl)-A3-cephem-4- carboxylic acid, 7-amino-3-(butanoylpropoxymethyl)-A3-cephem-4- r-arboxylic acid, 7-amino-3-(pentyloxycarbonylethoxymethyl)-A3-cephem4-carboxylic acid, 7-amino - 3 - (2'-thienylethoxymethyl)-A3 -cephem-4-carboxylic acid, as zwitterions or in the ester forms with an easily removable ester group. Examples of 7-acylamido-3-cyanomethyl- and 3-oxymethyl ether A3-cephem-4- carboxylic acid compounds of this invention,'which can be prepared by acylating nuclei of the above types by known methods include: 7-(D-a-amino-a,-phenylacetamido)-3-cyanomethyl-A3- cephem-4-carboxylic acid, 7-(D-a-amino-o:-(2'-thienyl)acetamido)-3-methoxymethyl-A3-cephem-4-carboxylic acid, 7-(2'-sydnone-3'-acetamido)-3-tert-bittoxymethyl-A3- cephem-4-carboxylic acid, 7- (a-carboxy-a-phenylacetamido) -3-methoxymethylA3cephem-4-carboxylic acid, 7-octanoylamido-3-allyloxymethyl-A3-cephem-4-carboxylic acid, 7-(2'-heptenoylamido)-3-propynyloxymethyl-A3-cephem4-carboxylic acid, 7-ce-amino-a-thienylacetamido)-3-methylthiomethyl-A3- cephem-4-carboxylic acid, 3,708,480 8 7-hexylthioacetamido-3-cyclopentyloxymethyl-A3-cephem4-carboxylic acid, 7-(2-butenyloxyacetamido)-3-isopropoxymethyl-A3- cepheni-4-carboxylic acid, 7- (3'-hydroxyphenylacetamido) -3-ethoxymethyl-A3- cepheni-4-carboxylic acid, 7-(4'-bromophenoxyacetamido)-3-(2'-phenylethoxymethyl)-A3-cephem-4-carboxylic acid, 7 - [p-(3'-chlorophenyl)propenoylamido]-3-cyanometh10 yl-A3-cephem-4-carboxylic acid, and esters of such compou[ids with ester groups removable with trifluoroacetic acid, acetic or formic acids, by dilute hydrochloric or sulfuric acid, or by hydrogenation in the presence of a palladium on carbon, barium sulfate, or alumina carrier with 15 or without acid. The preferred ester groups for this purpose are those listed above. Although the new compounds of this invention can be generally classed as A3- cephalosporin compounds, specific starting materials, intermediates, and products are often 20 named herein, for convenience, by use of the "cephem" nomenclature system. "Penam" nomenclature for the penicillins is described by Sheehan, Henery-Logan, and Johnson in the Journal of the American Chemical Society (JACS), 75, 3292, footnote 2 (1953), and has been 25 adapted to the cephalosporins by Morin, Jackson, Flynn, and Roesko [JACS, 84 3400 (1962)]. In accordance with these systems of nomenclature "penam" and "cepham" refer, respectively, to the following saturated ring 30 systems: 6 7 s CHa-CH5 2CH2 CH@C@6 \2CH2 35 0=@-k4-3@H@ 0=@-N5 3@H, 7 8 \4/ CH2 Penam Cepham 40 "Cephem" refers to the cepham ring structure containing a double bond, the position of which is indicated by a prefixed "A" with asuperscript integer denoting the lowest numbered atom to which the double bond is connected, or by the word "delta" with the same number 45 relationship. Sometimes, the position of the double bond in the "cephem" is indicated by the number alone without using the "A" or "delta" terms. Thus, for example, penicillin V, 6-phenoxymethylpenicillin can be named 6- phenoxyacetamido-2,2-dimethyl-penam - 3 - carboxylic 50 acid, and a suitable cephalosporin starting material for preparing compounds of this invention can be named 4-nitrobenzyl 7 -phenoxyacetamido-3- methoxymethyl-A2cephem-4-carboxylate. Similarly, a cephalosporin antibiotic of this invention can be named sodium 7-(2'-thien55 ylacetamido)-3-cyanomethyl-A3-cephem - 4 - carboxylate salt. An oxidation procedure for converting 3-nucleophile methyl-A2-cephem products to sulfoxides is disclosed in United States patent application, Serial No. 764,939, filed 60 Optober 3, 1968. A procedure for reducing cephalosporin sulfoxides is described in United States Patent No. 3,641,- 014. The invention is further illustrated by the following detailed examples which are intended to be illustrative 65 only, and not as limiting the scope of new compounds of this invention. EXAMPLE 1 t-Butyl 7-amino-3-cyanomethyl-A3-cephem-4- 70 carboxylate p-toluenesulfonate To a solution of 2.15 gm. (5 mM) t-butyl 7-phenoxya cetamido-3-cyanomethyl - A3 - cephem-4-carboxylate in 10@O cc. dry benzene was added (590 m.@. 1.5 eq.) dry pyridine and (1.56 gm., 1.5 eq.) phosphorus pentachlo75 ride. This mixture was heated under N2 for two hours
[5]3)708,480 9 at 56' C. The benzene was removed and 100 cc. of icecold methanol was added. After standing at room temperature overnight, the methanol was removed under reduced pressure, 75 cc. T'HF added, and, after cooling, 75 cc. cold pH 4.5 buffer solution. After standing 20 minutes, the THF was removedunder reduced pressure and ethyl acetate and some water was added. The pH of this mixture was adjusted to 6.5 with -sodium bicarbonate. The organic layer was separated, washed twice and N-aCl solution, dried over MgSO4, filtered and evaporated to a small volume. To a solution of this tert.-butyl 7@amino-3eyanomethyl-A3-cephem-4-carboxylate in 50 cc. ethyl acetate was added a solution of 951 mg. of p-toluenesulfonic acid in 50 cc. ethyl acetate. The ptoluenesixlfonate salt was allowed to crystallize overnight and then collected by suction filtration, 856 mg., M.P. 177' C. (d). The structure was cotlflrmed by I.R., U.V., N.M.R., and elemental analysis. EXAMPLE 2 t-Butyl 7-amino-3-methoxymethyl-A3-cephem-4carboxylate p-toluenesulfonate To a solution of 364 mg. (0.84 mM) of oily tert-butyl 7 - phenoxyacetamido-3- methoxymethyl-A3-cephem-4-carboxylate dissolved in 30 cc. dry benzene was added 83 mg. dry pyridine and 218 mg. phosphorus pentachloride (1.25 eq.). The mixture was heated for 2 hours under M2 at 56-58' C. The benzene was removed and 35 cc. of icecold methanol added. This solution was allowed to stand for 24 hours at room temperature. The methanol was removed under reduced pressure and 20 cc. THF added. After cooling, 20 cc. pH 4.5 buffer was added, and the mixture allowed to -stand 20 minutes at room temperature. After removing the THF under reduced pressure, dilute NaCl solution and ethyl acetate were added, and the pH adjusted to 6.5 with solid NaHCO3. The organic layer was removed, washed twice with saturated NaC] solution, dried over MgSO4, filtered, and evaporated to a small volume. To a solution of this tert-butyl 7- amino-3methoxymethyl-A3-cephem-4-carboxylate in 20 cc. ethyl acetate was added a solution of 160, mg. p-toluenesulfonic acid monohydrate. The p-toluenesulfonate salt of tertbutyl 7-amino-3-methoxymethyl - A3 - cephem - 4 - carboxylate crystallized spontaneously giving 177 mg. (45 percent yield). This solid salt was recrystallized from isopropyl alcohol to give a more pure sample, M.P. 183187' C. whose structure was confirmed by I.R., U.V., N.M.R., and elemental analysis. EXAMPLE 3 t-Butyl 7-(21-thienylacetamide)-3-cyanomethyl-A3- cephem-4-carboxylate To a cooled mixture of 467.5 mg. (1 mM) of t-butyl 7amino-3-cyanomethyl - A3 - cephem - 4 - carboxylate ptoluenesulfonate, (420; mg., 5 mM) solid NaHCO3, and (30 cc.) dry acetone was added 48 mg. (3 mM) of distilled thiophene-2-acetyl chloride in 10 cc. dry acetone. The reaction mixture was stirred one hoar in the cold (0'-5' C.) and 3 hours at room temperature. The acetone was removed under reduced pressure and benzene added. This mixture was washed successively with sodium bicarbonate solution, cold 5 percent HCI solution, five times with NaHCI3 SOlUtiOn, then a NaCl solution, dried over MgSO4, filtered and evaporated to give 481 mg. of product. This product was crystallized from carbon tetrachloride to give 271 mg. (66 percent), of tert-butyl 7- (2'thienyl)acetamido - 3 - cyanomethyl - A3 - cephe m-4-carboxylate, M.P. 164-166' C. The structure was confirmed by I.R., U.V., N.M.R. and elemental analysis. EXAMPLE 4 7-(2'-Thienyl)acetamido-3-eyanomethyl-A3- cephem-4-carboxylic acid - 10 cyanomethyl-A3-cephem-4-carboxylate in 40 cc. of 98-100 percent formic acid was stirred under N2 at room temperature for ZI/2 hours. The formic acid was removed under reduced pressure. Ethyl acetate and water were added and the pH was adjusted to about 7.5 with sodium bicarbonate. To the aqueous layer was added ethyl acetate, the mixture cooled, and acidified to pH 2.8 with 20 percent HCI. The organic layer was removed, washed twice with saturated N-aCl solution, dried over MgSO4, filtered, and evaporated 10 to give 234 mg. of a golden foam. This foam material was taken up in boiling ethyl ether (10 mg. insoluble). After evaporation to a small volume, the desired acid 7-( 2'-thienyl)acetamido-3- cyanomethylA3-cephem-4-carboxylic acid, crystallized, 97 mg., M.,P. 15 114-117' C. The structure was confirmed by I.R., U.V., and N.M.R. It gave an active antibiotic zone on a bioautograph of a paper chromatogram which was slightly faster moving than sodium cephalothin. EXAMPLE 5 20 p-Methoxybenzyl 7-amino-3-methoxymethyl-A3- cephem-4-carboxylate p-toluenesulfonate salt To a solution of 996 ma (2 mM) of p-methoxybenzyl 7 - phenoxyacetamido-3- m@e'thoxymethyl - A3 - cephem-4- 25 carboxylate and 221.5 mg. dry pyridine (1.4 eq.) in 75 cc. dry benzene was added 601 mg. (40 percent excess) of phosphorus 13entachloride. The mixture was heated under N2 for two hours at 50' C. The benzene was removed by evaporation under reduced pressure and 100 cc. of 30 ice-cold methanol was added. After standing at roomtemperature overnight the methanol was removed by evaporation under reduced pressure. To the residue was added 40 cc. THF, and, after cooling, 40 cc. water was added. This homogeneous mixture was allowed to stand 35 25 minutes; THF was removed under reduced pressure, ethyl acetate and some water added, and the pH adjusted to 6.5 with solid NaHCO3. The organic layer was removed, washed twice with saturated NaCl solution, dried with MgSO4, filtered, and evaporated to a small volume. 40 To a solution of the crude p-methoxybenzyl 7-ami no-3methoxymethyl-A3-cephem4-carboxylate obtained therefrom in a total of 80 cc. of ethyl acetate was added a solution of 380 mg. (2 mM) p-toluenesulfonic acid monohydrate in 10 cc. ethyl acetate. The p-toluenesulfonate 45 salt began to crystallize within a few minutes. After 3 hours, the crystals were collected by suction filtration: 781 mg. (75 percent), M.P. 160-164' C., structure confirmed by I.R., U.V., N.M.R., and elemental analysis. 50 EXAMPLE 6 tert-Butyl 7-(2'-sydnone-3-acetamido)-3-methoxymeth yl-A3-cephem-4- carboxylate O-CH-CH 55 N-CHaCO-NH-CH-CH CH2 I 0=@- I O-N N C 60 @00-t-butyl The tosylite salt of tert-butyl 7-amino-3-methoxyme thYI-A3-cephen-4- carboxylate (236 mg., 0.5 millimole) was stirred in a water/ethyl acetate mixture. The pH was 65 adjusted to 7 with Na2HP03. The ethyl acetate layer was shaken with saturated sodium chloride solution, dried with magnesium sulfate, and then evaporated. The residue was dissolved in 10 ml. of tetrahydrofuran (THF) and added to the tert-butyl mixed anhydride of 2-sydnone-3-acetic 70 acid prepared at -5' C. by adding 50, mg. (0.07 ml.) of triethylamine and 0.06 ml. of pivaloyl chloride to 80 mg. of 2-sydnone-3-acetic acid in 10 ml. of dry tetrahydrofuran. After stirring in an ice bath for one hour and for two hours at room temperature, the reaction mixture A solution of 455 mg. t-butyl 7-thiopheneacetamido-3- 75 was diluted with 100 ml. of water and extracted with ethyl
[6]317081480 acetate. The ethyl acetate solution was shaken with pH 8 phospbate buffer, dried with magnesium sulfate, and concentrated. ne residue was slurried with ethyl ether, collected, washed with ether and vacuum dried, yielding 95 mg. of the above named ester. A nuclear magnetic 5 resonance (NMR) analysis confirmed the structure. EXAMPLE 7 7-(2'-sydnone-3'-acetamido)-3-methoxymethyl-A3- cephem-4-carboxylic acid 10 An 86 mg. portion of the tert-butyl 7-(2'-sydnone-3- acetamido)-3-methoxymethyl-A3-cephem - 4 - carboxylate was dissolved in 2 ml. of 98 percent formic acid and stirred at room temperature for two hours. The solution was evaporated to &yness. The semi-solid residue which 15 resulted was triturated with ethyl ether and the solid was then collected and vacuum dried, yielding 52 mg. of 7- (2'-sydnone - 3' - acetamido) - 3 - methoxymethyl - A 3- cephem-4-carboxylic acid. Elemental microanalysis and ultraviolet (U.V.) analysis data were consistent with t s 20 structure. EXAMPLE 8 tert-Butyl 7-(2'-carboxy-2-phenylacetamido)-3methox ymethyl-A3-cephem-4- carboxylate 25 A solution of 328 mg. (1.4 mmoles) of the mono-tertbutyl ester of phenylmalonic acid and 0.21 ml. (156 m@., 1.54 nimoles) of triethylamine in 30 ml. of THF was cooled to -5' C. and 168 mg. (0.168 ml., 14 mmoles) of pivaloyl chloride was added to form the tertbutyl 30 12 ample 8 was stirred in one ml. of cold trifluoroacetic acid. After 15 minutes the solution was evaporated. The residue was stirred in 5 ml. of a 1: 1 v. /v. mixture of petroleum hydrocarbon (B.P. 60-68' C.): ethyl ether for 1 hour, and then collected and vacuum dried, yielding 56.1 mg. of 7-(2'-carboxy - Z' - ph enylacetamido)-3-methoxymethyl-A3- cepbem-4-carboxylic acid. The elemental analyses and UV spectrum were consistent with this structure. EXAMPLES 10 TO 16 A mixture of 268 mg. (0.5 mmole) of the tosylate salt of p-methoxybenzyl 7-amino-3-methoxymethyl-3-cephem4-carboxylate and 336 mg. (2.0 mmoles) of sodium bicarbonat, was stirred in 25 ml. of acetone while cooling the reaction mixture in an ice-bath. After adding 130 mg. (0.62 mmole) of 2-benzothienylacetyl chloride, the reaction mixture was stirred for one hour in the cold, followed by a further two hours at room temperature. The reaction solution was then diluted with 100 ml. of H20- The precipitated solid was collected, washed with water and vacuum dried at 45', yielding 250.0 mg. (93 percent), M.P. 182-5' C. The NMR spectrum and microanalysis confirmed the structure of this compound. It was shown by thin layer chromatography (TLC) to contain only one product, p-methoxybenzyl 7-(2'- benzothienylacetamide)3-methoxymethyl-A3-cephem-4-carboxylate. A summary of analytical data from this and other new compounds prepared as described above is given below: 0 11 s RLC)N 0 CH2OR2 I'@@O CH3 Caled. Found Example Ri R2 C. C H N 0 H N 10 --------- 2- benzothienylmethyl ----------- CH3 182- 5 60.22 4.87 5.20 60.02 4.97 5.19 11 --------- 4-nitrobenzyl -------------------- CH3 201-5 56.92 4.78 7.97 56.92 4.83 8.05 12 --------- 3-chlorobenzyl ------------------- CH3 171- 4 58.07 4.87 5.42 57.82 4.91 5.36 13 --------- 3- chlorophenylthioinethyl ------- CH3 138- 143 54.68 4.59 5.10 64.84 4.81 5.32 14 --------- (4@inetboxy1,2,5-oxadiazolyl-3) CH3 158- 165 52.38 4.80 11.11 50.35 5.04 10.00 methyl. 15 --------- a-Formyloxy-aphenylmethyl --- CH3 149- 150 59.77 4.24 5.36 59.70 4.06 5.19 16 --------- 2-thienylmethyl ----------------- -C2H5 159- 162 57.37 5.22 5.58 57.64 5.33 5.35 mixed anhydride. After 20 minutes, a solution of 342 mg. (1.4 mmoles) of 7-amino3-methoxymethyl-A3-cephem-4- 55 carboxylic acid (7-AMCA) prepared by hydrolysing the tert-butyl 7-amino-3-methoxymethyl-A3-cephem - 4 - carboxylate ester in formic acid, in a mixture of 10 ml. of water-10 ml. of THF, containing an equivalent amount of triethylamine was added to the mixed anhydride solu- 60 tion. After stirring the mixture for 1 hour in the cold (0'-5' C.) and 2 hours at room temperature, the reaction solution was cooled, overlayered with ethyl acetate, and the pH was adjusted to 2.0 with 6 N HCI. The etbyl acetate solution was dried and concentrated. The residue 65 was taken up in ethyl ether, then re-precipitated with a commercially available petroleum hy&ocarbon fraction, mostly hexane (B.P. 60-68' C.), yielding 88.0, mg. of the tert-butyl 7-(2'-carbo xy-2'-phenylacetamido)-3-methoxymethyl-A3-cephem-4-carboxylate. 70 EXAMPLE 9 The tert-butyl 7-(2'-carbox y-2'-phenylacetamido)-3- methoxymethyl-A3-cephem-4-carboxylate ester from Ex- 75 EXAMPLES 17 to 23 A solution of 120 mg. (0.23 millimole) of p-methoxybenzyl 7-(4'- nitrophenylacetamido)-3-methoxymethyl-A3cephem-4-carboxylate, 0.4 ml. of trifluoroacetic acid and -20 drops of anisole in 10 ml. of benzene was allowed to stand at room temperature for 1.5 hours. The reaction solution was then added to 100 ml. of pH 7 buffer. After separation of layers, the aqueous layer was overlayered with ethyl acetate and the pH was adjusted to 1.5 with 6 N HCI. The ethyl acetate layer was washed with water and with saturated HaCl solution, dried (HgSO4) and concentrated. The residue was dissolved in 15 ml. of ethanol and 0.4 ml. of a I N solution of sodium acetate in methanol was added. After two hours at O', the solid was collected, waghed with ethanol and dried in vacuum at 45', yielding 76 mg. (85 percent) of crystalline product 7 - (4'- nitrophenylacetamid o)-3-methoxy-methyl-Aecephem-4- carboxylic acid, sodium salt. The NMR and UV spectra confirmed the structure of this compound. The microanalysis was also in agreement withcalculated values. A summary of the physical chemical data ob-
[7]3,708,480 13 14 tained from this and other salts of the formula below is acetate, cooled and the pH adjusttd to 2.0 with 6 N HCI. given: The ethyl acetate layer was dried with MgSO4 and evap. 0 Ri8N :L--.f s CH2OR2 02Na Caled. Found TTV Example RI Rs c H N c H N X 217 (10,800) 17 --------------- 4-nitrobenzyl -------------------------- CH3 47.54 3. 75 9. 78 47.70 4.15 9. 51 211 (11,,Ill) 227 (17,000) 18 --------------- 2-benzothienylmetbyl ----------------- CH3 51.81 3.89 6.36 51. 91 3.73 6.30 258 (9,650) 297 (2,000) 19 --------------- 3-chlorobenzyl --- ---------------------- CH3 49.08 4.76 6. 02 48.91 4.69 5. 73 258 (7,000) 20 --------------- 3-chlorophonylmercaptomethyl -------- CH3 45.28 3.57 6. 21 44.99 3.78 5. 92 252 (12,300) 21 --------------- (4-methoxy-1,2,5-oxadiazolyl-3) methylOHS 41.37 3.72 13 .79 41.40 4.01 13 .50 ---------------- 22 --------------- (D@-hydroxyl-a-phenyl)methyl ------ CHa 50.99 4.28 7.00 45.56 4.76 6.37 258 (6,800) 23 --------------- 2-thienylmethyl - ---------------------- -C2H5 57.37 5.22 5.58 57.64 5.33 5.35 ---------------- -----do --------------------------------- CH,- 48.89 4.37 7.60 48.60 4.50 7.63 - --------------- - The last compound in this table was analyzed as the free acid. The above new cephalosporins gave characteristic Rf values in paper chromatography and were tested in in vitro microbiological assays wherein all - compounds demonstrated excellent activity against several strains of 30 staphylococci. The derivatives after oral administration effectively protected mice from death by streptococcal infections. EXAMPLE 24 35 7-Amino-3-methoxymethyl-A3-cephem-4-carboxylic acid To a 268 mg. (1 millimole) portion of the p-toluenesulfonate salt of p-methoxybenzyl 7-am ino-3-methoxymethyl-A3-cephem-4-carboxylate, stirred in water, there 40 was added disodium orthophosphate to pH 7. while overlaying the solution with ethyl acetate. The ethyl acetate solution was dried over magnesium sulfate and evaporated to dryness. The oily residue was dissolved in 25 ml. of benzene containing 40 drops of anisole and 0.8 ml. of trifluoroacetic acid. After stirring the mixture for 2 hours 45 at room temperature, the benzene was evaporated and the residue was taken up in 10 ml. of water overlayered with 10 ml. of ethyl acetate. The pH was adjusted to 3.6 by the addition of triethylamine. After reftigerating the mixture overnight, the solid was collected, washed with 50 water and ethyl acetate and vacuum dried at 45' C. yielding 75 mg. of 7-amino-3-methoxymethy l-A3-cephem-4- carboxylic acid. The structure was confirmed by infrared (IR), ultraviolet (UV), and NMR spectra. A simiiar procedure was used to prepare 7-amino-3- 55 ethoxymethyl-A3-cephem-4-carboxylic acid from its pmethoxybenzyl ester. EXAMPLE 25 60 7-(D-2-Amino-2-phenylacetamido)-3-methoxymethyl-3-cephem-4-carboxylic acid A solution of i85 mg. (0.74mmole) of D-2-t-butoxy c arboxamido-2-phenylacetic acid in 15 ml. of tetrahydro6 5 fu ran (THF) was stirred and cooled to -5'. After adding 6 2 mg. (0.81 mmole) of triethylamine and 1,10 mg. (0 .81 mmole) of isobutyl chloroformate, the reaction mixtu re was stirred for 20 minutes. To this resulting mixed a nhydride solution was added a solution of 180 mg. 7 0 (0 .74 mmole) of 7-amino-3-methoxymethyl-3-cephem-4- c arboxylic acid (7-AMCA), in 5 ml. of water, 5 ml. of T HF and 0.103 ml. of triethylamine. After stirring the rea ction mixture for one hour in an ice-bath and for two h ours at room temperature, it was overlayered with ethyl 7 5 orated to dryness. The residue was taken up in ethyl ether. After crystallization began, the flask was refrigerated for several days. The solid was then collected and vacuum dried, yielding 95.5 mg. of 7-(D-2-t-butoxycarboxamido2 - phenylacetamide) - 3 - methoxymethyl - 3-cephem-4carboxylic acid (T-BOC compound). A second crop was obtained by dilution of the filtrate above with a petroleum fraction (mostly hexane), B.P. 60-68' C. A cold (0') solution of 150 mg. of the T-BOC compound in 2 ml. of trifluoroacetic acid was stirred for 5 minutes, then evaporated to dryness. The residue was taken up in 2 ml. of -water. To this solution was added 25 percent "Amberlite LA-1" liquid anion exchange resin (acetate form) in methyl isobutyl ketone (MIRK) to pH 4.5. The aqueous layer was given a second treatment with the liquid anion exchange resin, then washed with MIBX. The aqueous layer was then evaporated to about 0.5 ml. @Lnd diluted with acetonitrile. The gel which precipitated from solution slowly crystallized. The solid was collected, washed with acetonitrile and vacuum dried, yielding 42.0 mg, of 7 - (D - 2'-amino-2'-phenylacetamido)-3- methoxymethyl-A3-cephem-4-carboxylic acid. The structure was confirmed byUV and NMR spectra and by microanalysis. EXAMPLES 26 to 29 7-[D-2'-amino-Z'-(3"-hydroxyphenyl)acetamidol3-meth oxymethyl-A3-cephem-4- carboxylic acid The acylation of 7-AMCA with D-2-t-butoxycarboxamido - 2 - (3-hydroxyphenyl)acetic acid was performed using the same mixed anhydride procedure as the preceding example. The removal of the T-BOC group and isolation of the 7-ED-Z'-amino-2'(3"-hydroxyphenyl) acetamido] - 3 - methoxymethyl-A3-cephem-4-carboxylic acid as the zwitterion product was completed as follows: A solution of 513 mg. of the T-BOC protected compound was prepared in 6, ml. of cold (0') trifluoroacetic acid. After 5 minutes, the excess trifluoroacetic acid was evaporated. The residue was dissolved in 2 ml. of water overlayered with 10 ml. of MIBK. The pH was adjusted to 5 by the addition of tributylamine. The aqueous layer was separated, washed with MIBK and evaporated to about 0.5 ml. The solution was diluted with 5 ml. of acetonitrile and refrigerated. After several hours, the solid was collected, washed with acetonitrile and vacuum dried at 50', yielding 249.1 mg. of product. In the following table are listed phenylglycine derivatives prepared in the same manner as Example 26.
[8]357082480 16 SUMMARY OF PHENYLGLYCINE DERIVATIVES NH2 @H <;D>60NII s RI "I --@ I 0= -N \,/,-CI12OR2 02H Microanalyses Caled. Found Example RI R2 c H N c H N x 26 ----------- OH ClI3 49.62 5.15 10.21 49.76 5.27 10.21 263 (7400) 27 ----------- H CH3 54.11 5.08 11.14 54.28 5.20 11.27 258 (7:700) 28 ----------- Cl CH3 49.67 4.40 10.20 49.36 4.63 10.10 260 (7,400) 29 ----------- H - C@@115 52.79 5.66 10.26 52.61 5.49 10.09 269 (7,500) EXAMPLE 30 20 A series of acylations were run as described in the following example:, A solution of 11 mg. of 7-AMOA and II mg. of NaHC03 was prepared in 5.5 ml. of water. Aliquots (0.75 ml.) of this solution were stirred in an 25 ice-bath and aliquots of acid chloride solutions of the indicated R group (containing 1.5 mg. of acid chlorride) were added. After stirring for 30 minutes, the acetone was evaporated and each solution rediluted with H 20 tO 1.5 ml. These solutions were then tested against a broad 30 spectrum of micro-organisms. It was also found that direct testing of the reaction solution without removal of acetone was possible. The compounds were also submitted for paper chromatography and bioautographs thereof. 35 The table below sunimarizes the antibiotic data obtained on the solutions, side chains used, and Rf values obtained from the bioautographs. An agar disc zone size of greater than 8 mm. is an indication of antibiotic act vity of the test compoun aga st the test organism in this qualitative test. EXAMPLE 49 A suspension of 0.353 g. of 3-methyl-7-phenoxyacetamido-A3-cephem4-carboxylic acid (prepared as described in U.S. Pat. No. 3,275,636) in 40 ml. of benzene is cooled in an ice bath and stirred while 0.256 g. of oxalyl chloride and I drop of dimethylformamide are added. The reaction mixture is stirred at about 5' C. for 45 minutes and the solvent removed under reduced pressure to yield the desired acid chloride of the starting A3-cephem acid. EXAMPLE 50 A solution of 1.75 g. of the acid chloride of Example 49 in 70 ml. of methylene chloride is added dropwise to a stirred solution of 0.72 g. of triethylamine and 4.0 g. of 3- ANTIBIOTIC ACTIVITY OF COMPOUNDS OBTAINED IN ACYLATIONS 0 R8N s 20CIT3 A g ar di sc te st (z o ne si ze , m m. ) R RE* G+SA G+Xl2 G+XI86 Tb X85 G-X45 G-XI42 G-XI61 G-X48 Example Number: 1.1 32 20 30 ------------ 11 --------------------- --------- 30 --------------- C112(CH2)s 31 ------------ CH3(CH2)4--CH=CH1.5 27 22 32 ------ ------ 12 ---------------------------------- 32 --------------- ci 0.8 24 26 31 ------------ 14 ------------------ ---------------- CICH2u.Li33 --------------- ci 0.9 29 28 35 ------------ 17 ------------ Tr - ---------- CHsCH2CH 34 --------------- Br 2. 2 30 29 45 ------------ 13 ----- ---------------------------- CHs(UJ-i2)7-C-Li35 --------------- CH3(CH2)3SCH2-- 1.0 30 33 40 ---- -------- 14 ----- ------ 16 ---------- 36 --------------- (CI:13)2CH(CH2)2SCH2-- 1.4 35 34 39 ------------ 16 ------------ 16 --- ----- 37 --------------- CH=-CHCH2SCH2- 0. 9 25 21 29 ------------ 17 Tr - ---------------------- 38 --------------- p-CH30-C3H4-CH4- 0.8 29 34 40 -- ---------- 16 ------------ 15 ---------- 39 --------------- P-NO2-C3H@-CH2CH2-- 1.0 32 20 37 -- ---------- 12 ------------ 11 ---------- 40 --------------- m-Cl-C3H4-OCHa1.5 36 34 42 -- ---------- 15 ------------ 13 --- ------ 41 --------------- P-Cl-C3H@OCHz--- 1.1 40 35 37 -- ---------- 13 ------------ Tr. 42 --------------- M-HO2 --- C3H@OCH2--- 1.0 35 35 40 -- ---------- 12 ------------ 12 ------ 43 --------------- M-CH3-C3H4-OCH2- 1.8 34 34 40 -- ---------- 12 ------------ Tr - ---------- 44 --------------- CoH@CH20CH2-- 1.0 27 25 32 ---- -------- 14 ---------------------------------- 45 --------------- p-F-C2H@CH2SCHr1.3 35 32 45 Tr. 15 ------------ 14 ---------- 46 --------------- C2H5-CH2CH2SCHa1.4 29 34 40 ------ ------ 14 ------------ 16 ---------- 47 --------------- CoH5(CH2)2SCHr1.7 31 34 37 ------ ------ 10 ------------ 13 ---------- 48 --------------- CfiHs --- CH=CH1.0 25 17 20 --------- ------------------------------------------- 'R. is defmed as the ratio between the distance of the respective spot (bioautograph) from the origin and the distance between the spot made by the standard antibiotic, sodium cephalothin, and the origin. The RK ratios were determined from bioautograms of paper chrornatographs of the test copi) alosporin compound relative to the standard compound, wbich was Keflin (sodium cephalothin, Lilly), designated by "K" sab3cript, a comniercially available cephalosporin antibiotic. NCTE.-SA=Staphylococcu8 aureus: X12=B. subtilis; X186=Sare@,'na sp.; X85=Sacharomyce3 pastorianu7n,, X45=Proteu8 vulagris; X142=Sal7nonella galli2iarum; X161=Lscherichia coli; X48=Pseudo7noizas aeruginosa.
[9]8,708,480 17 methyl-l-buten-3-ol in 50 ml. of methylene chloride maintained at ice temperature. Upon completion of the addition (about 90 minutes), the organic solution is washed successively with water and 3 percent hydrochloric acid and evaporated to dryness. The residue is dissolved in 5 ethyl acetate, washed with 5 percent sodium bicarbonate solution, and treated with activated charcoal. The mixture is filtered and the solvent is removed from the filtrate under reduced pressure to give 0.51 g. of 2-(2-methyl-3- butenyl) 3-methyl - 7 - phenoxyacetamid o-A2-cephem-4- 10 carboxylate. The structure is confirmed by the nuclear magneiic resonance spectrum. The neutral , and basic washes yield 0.82 g. of a mixture of A2 and A3 ac@ids. EXAMPLE 51 15 A solution of 4.16 g. of the ester from Example 50 in one liter of carbon tetrachloride is stirred and purged with nitrogen for 20 minutes. To the solution are then added 2.67 g. of N-bromosuccinimide and 50 mg. of azobis-isobutyronitrile. The solution is stirred and heated 20 under reflux until a negative starch-iodide paper test is obtained (approximately 41/2 hours). The solution is cooled overnight in a refrigerator, the excess N-bromosuccinimide is removed by filtration through a-cotton plug, and the carbon tetrachloride is evaporated to- yield 2-(2- 25 methyl-3-butenyl) 3 - bromomethyl-7-phenox acetamido'Y A2-cephem-4-carboxylate which is used in subsequent reactions. EXAMPLE 52 A solution of about 4 miliimoles of the 3- bromeimethyl 30 product from Example 51 in 25 g. of methyl mercaptan is stirred in a pressure bottle at 25' C. for on6 hour. The excess methyl mercaptan is allowed to evaporate and the residue is dissolved in methylene chloride. The - methylene chioride is washed with 5 percent sodiiim bicai-bonate 35 solution and water, &ied over sodium sulfate, and evaporated to dryness to give 1.4 g. of crude product. This crude product is chromatographed over 100 g. of silica gel containing 15 percent water using 6-8 percent ethyl 40 acetate in benzene as solvent. @In this manner there is obtained 100 mg. of pure 2-(2-methyl-3-butenyl) - 3-methylthiomethyl - 7 - phenoxyacetamido-A2-cepheni - 4 - carboxylate. The structure is confirmed by the nuclear magnetic resonance spectrum. 45 EXAMPLE 53 The A2 product from Example 52 is dissolved in 8 ml. of isopropanol, 2 ml. of triethylamine is added, and the mixture is stirred at 25' C. for one hour. The solvents are removed under reduced pressure. A nuclear magnetic 50 resonance spectrum confirms that isomerization to the A3 compound has occurred. EXAM-PLE 54 A mixture of 750 mg. of 2-(2-methyl-3-butenyl) 3- 55 methylthiomethyl - 7 - phenoxyacetamido - A3 - - cephem-4- carboxylate from Example 53, 580 mg. of phosphorous pentachloride, and 221 mg. of pyridine in 75 ml. of dry benzene is stirred at 55' C. After three hours the reaction mixture is evaporated, the residue is dissolved in 100 ml. 60 of methanol, and the solution is refrigerated overnight. T'he methanol is evaporated and the residue is - dissolved in a chilled mixture of 50 ml. of tetrahydrofuran and 50 ml. of a pH 4.5 citrate buffer. After 35 minutes the tetrahydrofuran is evaporated. The aqueous solution is over- 65 layered with ethyl acetate and the pH is adjusted to 7.0 by the addition of a solution of dibasic sodium orthophosphate. The ethyl acetate solution is washed with - saturated salt solution, dried over magnesium sulfate, and the ethyl acetate evaporated. The crude 2-(2-methyl-3-butenyl) 3- 70 methylthiomethyl-7-amino-A3-cephem - 4 - carboxylate is redissolved in 20 ml. of ethyl acetate and a solution of ptoluenesulfonic acid in ethyl acetate is added until the resulting solution is shown to be acidic on pH paper. After cooling for 30 minutes in an ice bath, the crystalline pre- 75 18 cipitate is collected, washed with ethyl acetate, and dried in a vacuum oven at room temperature yielding 370 mg. of 2-(2-methyl-3-butenyl) 3 - methylthiomethyl-7- aminoA3-cephem - 4 - carboxylate as the p-toluenesulfonic acid salt. The structure is confirmed by the nuclear magnetic resonance spectrum and elemental analysis. EXAMPLE 55 A solution of 412.7 mg. of the product from Example 54 in 5 ml. of formic acid is allowed to stand at room temperature for two hours. The reaction mixture is evaporated to &yness and the oily residue is taken up in a mixture of 10 ml. of ethyl acetate and 6 ml. of water. While cooling, the pH of this mixture is adjusted to 8.5 with dilute ammonia solution. The aqueous layer is separated and the pH is adjusted to 3..8 with,6 N hydrochloric acid. After cooling for two hours, the white crystalline precipitate that has sep@rated is collected and dried in a vacuum oven at 35' C., yielding 166.3 mg. of 3- methylthiomethyl-7-amino-A3-cephem - 4 - carboxylic acid. The structure is confirmed by the nuclear magnetic resonance spectrum and elemental analysis. Analysis.-Calculated for C9Hl2N203S2 (percent): C, 41,54; H, 4.65; N, 10.77. Found (percent): C, 41.70; H, 4.90; N, 10.52. EXAMPLE 56 A solution of 160 mg. of N-(t-butoxycarbonyl)phenyl@lycine in 25 ml. of tetrahydrofuran is cooled to -5' C. and 65 mg. of triethylamine and 87 mg. of isobutyl chloroformate are added. To this cold solution is added a solution of 150 mg. of the product from Example 7 and 0.08 ml. of triethylamine in 5 ml. of water and 2 ml. of tetrahydrofuran. This mixture is stirred for three hours while being allowed to warm to room temperature. The tetrahydrofuran is then removed on a rotary evaporator. The remaining mixture is dissolved in water, overlayered with ethyl acetate, cooled in an ice bath, and the pH adjusted to 2.0 by the addition of 6 Nhydrochloric acid. The ethyl acetate layer is separated, washed with saturated sodium chloride solution, dried over magnesium sulfate, and evaporated to leave a guminy residue. This residue is dissolved in diethyl ether and @t petroleum fraction boiling at 60' to 68' C. is added to precipitate 167..3 mg. of 3-methylthiomethyl - 7 - (a-t-butoxyc arboxamido-a-phenyl)acetamido-A3- cephem-4-caiboxylic acid. This N-blocked material is dissolved in, 3 ml. of cold trifluoroacetic acid. After stirring for fiv6 minutes, the solution is evaporated and the residue is taken up in 5 ml. of water and 5 ml. of methyl isobutyl keton - e. After adjusting the pH to 4.5 with tributylamine, the water layer is separated and concentrated to about 0.5 ml. This solution is diluted with about 15 ml. of acetonitrile and refrigerated overnight. The solid is collected, washed with acetonitriie, and dried in vacuo yielding 64.6 mg. of 3-methylthiomethyl-7-(aa mino-oc-phenyl)acet.amido-A3- cephem-4-carboxylic acid. Following the procedure of Example 56 using thienylglycine or the appro@riately substituted phenylglycine the following compounds are prepared: 3-methylthiomethyl-7-[a-amino-a-(m-chlorophenyl) acetamido]-A3-cephem-4-ca.-boxylic acid; 3-methylthiomethyl-7-[a-amino-,%-(p-methoxyphenyl) acetamido]--A3-cephem-4-carboxylic acid; 3-methylthiomethyl-7-[,x-amino-a-(o-butylpbenyl) acetamido]-A3-cephem-4-carboxylic acid; 3-methylthiomethyl-7-[a-amino-a-(p-nitrophenyl) acetamido]-A3-cephem-4-carboxylic acid; 3-methylthiomethyl-7-[a-amino-a-(p-cyanophenyl) acetamido]-A3-cephem-4-carboxylic acid; and 3-methylthiomethyl-7- (a-amino-a-thienyl) acetamidoA3-cephem-4-carboxylic acid.
