IE42705B1 - Substituted cephalosporin antibiotics - Google Patents

Abstract

A process is described for the preparation of novel cephalosporin compounds of the formula I, which are substituted by a ureido radical, by reaction of a compound of the formula II with a compound of the formula (III) R''-NH-CO-N(R')-CO-A. These can be converted into the esters of the formula IV which are likewise new. The substituents in the compounds of the formula I and IV have the meanings stated in Claim 1 and Claim 10 respectively. The compounds of the formula I and IV have a high activity and a broad spectrum of action as antibiotics and are particularly suitable for the treatment of infections caused by Gram-negative microorganisms.

Description

The present invention relates to ureido substituted cephalosporin compound which are highly active broad spectrum antibiotics especially useful in the treatment of infections attributable to the gram-negative micro5 organisms.

Cephalosporin compounds having a ureido or a substituted ureido substituent in the α-position of the 7acylamido side chain have been described. In O.S.

Patent 3,673,183 issued June 27, 1972, a-ureidocephalo10 sporanic acids are disclosed. Acyloxymethyl esters of aureidocyclohexadienylacetamido-cephalosporins are described in U.S. Patent 3,708,479 and of a-aminobenzylpenicillin in U.S. Patent 3,697,507. Penicillins and cephalosporins having an α-arylacetamido side chain are also described in U.S. Patents 3,634,405 and 3,646,024, respectively, α-3-Acylureidobenzylpenicillins also are known wherein a wide variety of acyl groups are attached to the terminal nitrogen Of the α-ureido group of the 6arylacetamido side chain.

The cephalosporin antibiotics described herein can be characterized as expanded spectrum cephalosporin antibiotics in that they not only possess the usual > - 3 43705 high level of activity against gram-positive microorganisms but they also possess a high level of activity against a broad spectrum of gram-negative microorganisms which the prior art compound did not possess.

This invention relates to new ureido substituted cephalosporin antibiotic compounds represented by the following general formula X wherein R is a ureido group represented by the formula Η O I » R—N—C—N— I R‘ wherein R is hydrogen, alkyl, allyl, propargyl, C,—C. cycloalkyl, phenyl, benzyl, or J o furfuryl,- R1 is hydrogen or methyl; is phenyl, hydroxyphenyl, dihydroxyphenyl, halophenyl, dihalophenyl, hydroxy substituted halo or di-halophenyl, thienyl or furyl; R^ is acetoxy.

-S.

N --— N S or z 48709 wherein 2 is C^-C^ alkyl; R3 is hydrogen, indanyl, phthalidyl, an acyloxymethyl group of the formula O g wherein Ϋ' is alkyl or phenyl, and when R^ is hydrogen, the pharmaceutically acceptable non-toxic salt thereof.

The term "halogen as used herein refers to fluoro, chloro, and bromo and preferably chloro.

Representative of the substituted phenyl groups, (R^) hydroxyphenyl, dihydroxyphenyl, halophenyl, dihalophenyl and hydroxy substituted halophenyl and 4-chlorophenyl, 4-bromophenyl, 3-fluorophenyl, 4-hydroxyphenyl, 3-hydroxyphenyl, 3-chloro-4-hydroxyphenyl, 2-chlorophenyl, 3,4-dichlorophenyl, 3,5-dichlorophenyl, 3,5-dihydroxyphenyl, 2-hydroxyphenyl, 4-hydroxy-3,5-dichlorophenyl, and 4-hydroxy-3,5-dibromophenyi.

The term C^—alkyl, represented by Z in the above formula, refers to methyl, ethyl n-propyl, iso20 propyl, n-butyl, sec-butyl, isobutyi and t-butyl.

The term C_—C, cycloalkyl refers to cyclopropyl, o o cyclobutyl, cyclopentyl and cyclohexyl.

The cephalosporin antibiotic compounds represented by the formula I, when R3 is hydrogen, can be prepared by acylating a 7-{D-a-amino-a-arylacetainido)-3-cephem-4carboxylic acid represented by the formula II, ΕΪΜΙΙΙ»·Ι· 42708 wherein and R^ have the previously defined meanings.

The compounds of formula X wherein R is the ureido group Η O I II R—N—C—»— (Formula I, R'=CH-) I CH3 can be prepared by acylating the cephalosporin compound II with the corresponding carbamoyl chloride, HO 0 I II II R—N—C—N—C—Cl I CH3 where R is as defined above.

Ihe compounds of the formula I wherein R is a ureido group H 0 I II R—N—C—N— (Formula I, R'=H) I H can be prepared by the acylation of II with a p-nitrophenyl carbamate 43705 The starting materials represented by the formula II wherein is a phenyl, substituted phenyl, or thienyl group and is a 1—alkyl-lH-tetrazol-55 yl group or a 5-C^_^ alkyl-1,3,4-thiadiazol-2-yl group are described by Ryan in U.S. Patent 3,641,021. Compounds of the formula II wherein R^ is a furyl group are prepared by the acylation of a 7-amino nucleus compound of the formula with the anhydride formed with a-(t-butoxycarbonyl)furanaoetic acid and isohutyl chloroformate. Following the acylation the t-butyloxycarbonyl protecting group is removed by known methods, for example, with trifluoro15 acetic acid in the cold, or alternatively with p-toluenesulfonic acid in acetonitrile as described by Chauvette in U.S. Patent 3,769,281.

The starting materials represented by formula II wherein is the acetoxy group are prepared by the acyla20 tion of 7-aminocephalosporanic acid with a phenyl, thienyl, or furyl glycine. The compound of the formula II wherein is phenyl and R2 is acetoxy is the well known antibiotic, cephaloglycin.

The acylation of a compound of the formula II with the ureidocarbamoyl chloride HO O I It II R N—C—N—C—Cl I CIi3 where R is as hereinbefore defined involves the acylation of the free a-amino group in tha 7-position side chain. The acylation is preferably carried out in an inert solvent in the presence of a hydrogen chloride acceptor at a temperature between -5°C. and 20°c. and more preferably at 0—5°C. Solvents such as acetonitrile, tetrahydrofuran, dimethylformamide and J,N-dimethylacetamide can be used in the acylation. A preferred solvent is acetonitrile. Should the starting material be insoluble or partly insoluble in the solvent, it can be solubilized by the addition of a silylating agent such as bis-(trimethylsilyl)acetamide (BSA) before the addition of the carbamoyl chloride.

Hydrogen chloride acceptors which can be used include the tertiary amines such as triethylamine and pyridine and the alkylene oxides such as propylene cxide or butylene oxide.

The carbamoyl chlorides HO 0 II R—N—C—N—C—Cl I CIi3 where R is as hereinbefore defined, are prepared b.z reacting the urea with phosgens in a dry, inert solvent such as dichloroethane, dichloromethane or tetrahydrofuran.

The reaction is preferably carried out in the cold at 43708 O—5°C.

The symmetrical 1,3-dimethylurea affords but one carbamoyl chloride, namely, N-methylcarbamoyl-N-methyl carbamoyl chloride HO 0 I II II CH—N—C—N—C—Cl (R=methyl) I CH3 since both nitrogen atoms of the urea are equivalent.

The unsymmetrical ureas, wherein R is a group other than methyl, can form two carbamoyl chlorides on reaction with phosgene, chloride The desired. N-methylcarbamoyl HO 0 I II II R-JJ—C-JJ-C-C1 CH_ is separated from the undesired isomeric chloride by fractional crystallization from mixtures of polar and non-polar organic solvents such as mixtures of diethyl ether and petroleum ether, and acetone or ethyl acetate with hexane or petroleum ether.

The ureas are prepared by well known methods or are commercially available.

Illustrative N-substituted-carbamoyl-N-methyl20 carbamoyl chlorides are represented by the foregoing formula wherein R is ethyl, H^C^H—CHj—"(allyl), HC=C—CH2—(propargyl), phenyl, benzyl, 2-furfuryl, cyclopropyl and cyclohexyl.

The preparation of the compounds represented by the 25 formula I wherein R^ is hydrogen and R' is CH^ is illus4 37 0 5 trated by the following description of the preparation of 7 - [ct - (3 - methylcarbamoyl - 3 - methylureido)a - phenylacetamido] - 3 - [(1 - methyl - IH - tetrazol5 - yl)thiomethyl] - 3 - cephem - 4 - carboxylic acid.

- (D - a - Amino - a - phenylacetamido) - 3 - [(1methyl - 1H - tetrazol - 5 - yl)thiomethyl] - 3 - cephem4 - carboxylic acid is suspended in acetonitrile containing an excess of propylene oxide. A small excess of bis(trimethylsilyl) - acetamide is added to the suspension with stirring to effect solution. The solution is cooled to about 5°C. anda molar equivalent of N-methylaminocarbonyl - N - methylcarbamoyl chloride is added dropwise or portionwise with stirring. After stirring for 2—4 hours in the cold, the reaction mixture is allowed to warm to room temperature. The product is preferably isolated via extraction with an organic solvent such as ethyl acetate. The reaction mixture is poured into a mixture of ethyl acetate and water, the aqueous phase is separated, acidified, and the product extracted with ethyl acetate.

The compounds of the formula I wherein R' is hydrogen are prepared by acylating the a-aminocephalosporin starting material II with a p-nitrophenyl carbamate of the formula: I II I II NO. wherein R has the previously defined meanings. These esters are prepared by reacting urea or the mono-substituted urea, R—NH—C(0)—NH2 in an inert solvent such as THF with p-nitrophenyl chloroformate. For example, methylurea is reacted in dry THF at about O°C. - 10 48705 with p-nitrophenyl chloroformate to form p-nitrophenyl N-(methylcarbamoyl)carbamate.

As is the case in the preparation of the abovedescribed carbamoyl chlorides, the reaction of urea or' a mono-substituted urea with p-nitrophenyl chloroformate affords two isomeric p-nitrophenyl carbamates.

The desired carbamate is formed by the acylation of the (unsubstituted) urea nitrogen while the undesired carbamate ester is formed by the acylation of the N3 (substituted), or the H urea nitrogen. Usually the two products are formed in equal amounts.

The desired p-nitrophenyl N-(substituted carbamoyl)15 carbamates form isocyanates when treated with silylating agents such as bis-(trimethylsilyl)acetamide (BSA) or N(trimethylsilyl)acetamide (MSA). The reaction is illustrated by the scheme: The undesired p-nitrophenyl carbamate 43708 formed along with the above desired carbamate is incapable of forming an isocyanate with the silylating agent.

In the acylation of an a-aminoarylacetamido-cephalosporin, II, to form the cephalosporin I wherein R'=H., the mixture of both carbamates obtained as described above is conveniently used. Ihe acylation reaction is carried out in an inert dry solvent in the presence of an excess of a silylating agent such as BSA or MSA. The p-nitrophenyl N-(substituted carbamoyl)carbamate forms the isocyanate in situ which then reacts with the u-amino group of II to form the product.

Illustrative p - nitrophenyl carbamates useful in preparing the compounds of the formula I wherein R'=H are p - nitrophenyl N - (ethylcarbamoyl) - carbamate, pnitrophenyl N - (cyclopropylcarbamoyl)carbamate, pnitrophenyl N- (phenylcarbamoyl)carbamate, p- nitrophenyl N - (propargylcarbamoyl)carbamate, p - nitrophenyl N - (allylcarbamoyl)carbamate, p - nitrophen/1 N - (benzylcarbamoyl)carbamate and p nitrophenyl i(carbamoyl)carbamate (R=H).

The acylation of an α-aminoarylacetamidocepha Losporin, II, with the above p-nitrophenyl carbamates is conveniently carried out in dry acetonitrile at about room temperature (20—25°C.). In order to ensure a lhydrous conditions the acylation is preferably carried cut in an atmosphere of a dry inert gas such as nitrogen or arg48708 - 12 on. A silylating agent such as BSA or MSA is added in excess and serves two functions. It first serves to solubilize the ά-aminoarylacetamidocephalosporin II via formation of soluble silyl derivatives (for example a silyl ester of II) and secondly the excess reacts with, the p-nitrophenyl carbamate to generate, in situ, the isocyanate as described above.

The acylation reaction is preferably carried out as follows. A suspension of the a-aminoarylacetamido10 cephalosporin II, in dry acetonitrile is treated with excess BSA. After a homogeneous solution is obtained the mixture of p-nitrophenyl carbamates is added in an amount sufficient to provide at least one molar equivalent of the desired p-nitrophenyl carbamate isomer per compound II.

The reaction mixture is stirred at room temperature for between 1 and 3 hours after which the product is recovered.

The cephalosporin product (formula I, R'=H, R3=H) is conveniently recovered hy extracting the reaction mixture, after dilution with water, with a water-immiscible organic solvent such as ethyl acetate, amyl acetate or other suitable solvent. The extraction is carried out at acid pH and preferably at about pH 2.5. The ex25 tract is washed, dried and evaporated to yield the cephalosporin antibiotic of the invention.

In an exemplary preparation of the cephalosporihs of the formula I wherein R' is hydrogen, 7 - [a - aminoα - (2 - thienyl)acetamido] - 3 - [(5- methyl - 1,3,4— thiadiazol - 2 - yl)thiomethyl] - 3 - cephem - 4carboxylic acid is reacted in dry acetonitrile in the - 13 42705 presence of excess bis - (trimethylsilyl)acetamide with p_ — nitrobenzyl N - (methylcarbamoyl)carbamate to provide, after recovery, 7 - [a - (3 - methylcarbamoylureido) - « -(2- thienyl)acetamido! - 3 - [(5- methyl5 1,3,4 - thiadiazol - 2 - yl)thiomethyl] - 3 - cephem4 - carboxylic acid.

According to the above described preparative procedures the compounds of the formula I wherein R is a ureido group of the formula H 0 I il R—JJ—Q—N— (R'=CH ) I 3 ch3 wherein R is as hereinbefore defined are prepared with the above described carbamoyl chlorides. The compounds I, wherein R1 is hydrogen, are prepared as described above with a ^-nitrophenyl N-(substituted carbamoyl) carbamate, H 0 H o --ιι ι h 11 /Λ R -N-C-N-C-0—r _N02 Illustrative of the cephalosporin antibiotics of the formula I wherein R3 is hydrogen are the following.

H 0 ι « CH N—C—N— I "3 do do phenyl 4-hydroxyphenyl 4-chlorophenyl N --N do do 43705 Η CH3-N-C-N- 4-hydroxy- 3-chloro- phenyl _sjn I ch3 0h3 do 2-thienyl do do 2-furyl do do 3-thienyl do do phenyl do 4-hydroxy- phenyl do do 2-thienyl do do phenyl acetoxy do 4-hydroxy- phenyl do do 3- chloro- 4- hydroxyphenyl do do 2-thienyl do A* (R) (R‘) N_N phenyl H phenyl do ch3 do acetoxy 4370& (R") (R‘) i benzyl H phenyl f 1 —S—< N ch3 furfuryl H 4-hydroxyphenyl do allyl H phenyl do do ch3 do acetoxy propargyl II 3- chloro- 4- hydroxyphenyl do do H 2-thienyl do i r doCH3 do _J ί CH3 propargyl H phenyl — S— Il N \ / N cn3 cyclo- H do d·. propyl do H do acetoxy do ch3 do do R (R) cyclopentyl do do (R') phenyl 4-chlorophenyl 4-hydroxyphenyl acetoxy R.

R. '2 cyclopentyl CII^ phenyl 4370S -sCH.

The cephalosporin antibiotics of the formula I wherein R^ is hydrogen are converted to the acyloxy5 methyl esters, wherein R^ is represented by the group 0 —CII, wherein Y is as hereinbefore defined by reacting an alkali metal salt of the cephalsporin carboxylic acid, for example, the lithium, sodium, or potassium salt, with an acyloxymethyl halide of the formula 0 x-ch2· wherein X is chlorine or bromine and Y' has the same meanings as previously defined. Acyloxymethyl halides which can be employed include chloromethyl acetate, bromomethyl acetate, bromomethyl propionate, chloromethyl pivaloate, and chloromethyl benzoate.

The preparation of the acyloxymethyl esters of the formula I is carried out by reacting the alkali metal salt of the parent acid in an inert solvent with a slight molar excess of the bromo or chloromethyl ester, e.g., bromomethyl acetate at room temperature or at slightly elevated temperatures up to 40—45°C. Solvents such as acetone, tetrahydrofuran, dioxane, dimethylformamide, and methylene chloride can be used.

The indanyl esters of the formula I are prepared by reacting the appropriate indanol, for instance 5indanol, in an inert solvent such as dioxane or tetrahydrofuran with the free acid form of a compound of the formula I wherein is hydrogen, in the presence of a condensing agent such as a diimide, for example, dicyclo hexylidiimide. The reaction is preferably carried out with stirring at 20—35°C. for 6 to 8 hours. The indanyl ester is isolated by first diluting the reaction mixture with water and filtering the reaction mixture to remove the insoluble dicyclohexylurea. The ester is then extrac ted from the filtrate.

Alternatively, the indanyl esters can be prepared by reacting a mixed acid anhydride formed with a cephalo15 sporin acid of the formula I and acetic acid with 5indanol.

The phthalidyl esters of the formula I wherein R^ is the phthalidyl group are obtained by reacting bromophthalide of the formula with a salt of a cephalosporin acid of the formula I.

The esterification can be carried out in dimethylformamide, N,N-dimethylacetamide, dioxane, tetrahydrofuran or mixtures thereof by slowly warming a mixture of equimolar amounts of the cephalosporin acid salt, for example, the sodium or potassium salt and bromophthalide.

Illustrative esters of the formula I are: acetoxymethyl 7 - [a - (3 - methylcarbamoyl - 3methyl - ureido) - a - phenylacetamido] - 3 - [(1methyl - lH - tetrazol - 5 - yl) thioraethyl] - 3 - cephem4 - carboxylate; pivaloyloxymethyl 7 - [a - (3 - methylcarbamoyl3 - methylureido] - a -(4- hydroxyphenyl)acetamido]3 - [(5- methyl - 1,3,4 - thiadiazol - 2 - yl) thiomethylj - 3 - cephem - 4 - carboxylate; phthalidyl 7 - [a - (3 - methylcarbamoyl - 3methylureido) -a - phenylacetamido] - 3 - [(1 - methyl1H - tetrazol - 5 - yl)thiomethyl] - 3 - cephem - 4carboxylate; acetoxymethyl 7 - [a - (3 - methylcarbamoyl - 3methylureido) - a - (3 - chloro - 4 - hydroxyphenyl)acetamido] - 3 - [(1- methyl -IH - tetrazol - 5 - yl)thiomethyl| - 3 - cephem - 4 - carboxylate; acetoxymethyl 7 [ rr. — (3 - methylcarbamoylureido) - 20 42708 α - (2 - thienyl)acetamido] - 3 - [(1- methyl - 1Htetrazol - 5 - yl)thiomethyl] - 3 - cephem - 4 - carboxylate; and ' pivaloyloxymethyl 7 - [a - (3 - methylcarbamoyl5 ureido) - a - phenylacetamido] - 3 - [(1 - methyl - 1Htetrazol - 5 - yl)thiomethyl] - 3 - cephem - 4 - carboxy late.

The cephalosporin compounds of this invention, in the free acid form (formula I, = Η), form pharmaceuti10 cally acceptable salts with inorganic bases such as the alkali metal carbonates and bicarbonates. For example, the sodium and potassium salts can be formed with sodium and potassium carbonate by following conventional procel dures.

Salts can also be prepared with basic organic amines, such as methylamine, diethylamine, cyclohexylamihe, dicyclohexylamine, ethanolamine, diethanolamine, and tris-(hydroxymethyl)aminomethane. Such salts can be used to formulate the antibiotics into suitable pharmaceu tical forms for parenteral administration.

The cephalosporin antibiotics of this invention are highly effective in inhibiting the growth of a wide spectrum of pathogenic microorganisms of both the gram-positive and gram-negative type.

A number of cephalosporin antibiotics are known which are effective against gram-positive microorganisms but are limited in the spectrum.of activity against the gram-negative microorganisms. Other cephalosporin antibiotics which have been synthesized have'demonstrated enhanced gram-negative activity; however, they· possess either a lower spectrum of activity against the gram42705 - 21 positive organisms or a decreased level of activity against these organisms. The antibiotics described herein have enhanced gram-negative acticity both with respect to spectrum and level of activity as well as activity against the gram-positive microorganisms. Thus, the antibiotics described herein can be characterized as cephalosporins having an expanded spectrum of activity.

These cephalosporin antibiotics exhibit high levels of activity against the Pseudomonas, Enterobacter sp., indole (+) and (-), Proteus sp., Aerobacter, Serratla. and Klebsiella gram-negative microorganisms. They also are effective in controlling the growth of penicillin resistant Staphylococcus as well as the Streptococcus D group, e.g,, S. faecalis.

The antibiotic activity of the cephalosporin compounds of the formula X is illustrated by the data presented in Table I for representative compounds. The values in the table are the minimum inhibitory concentrations (MIC) for the test compounds against the indicated micro20 organisms. The MIC values were obtained in the Gradient Plate in vitro method for determining antibiotic activity. 487 OS co O' H ro n CN CN cn in m CN TABLE I ANTIBIOTIC ACTIVITY OF UREIDO SUBSTITUTED CEPHALOSPORINS Minimum Inhibitory Concentration (meg./ml.) ϋ O o u 4J (0 0) H <3· i—1 ι—ί f—1 ro t—1 o co Ο CQ ro O' ’«Ι· Ό* CN 00 CN cn co O r-I rd o o m o o o 00 f-d rd ro I—J rd 00 co O' ι—1 Φ rt •d 0) Φ tn d Φ rt 01 0 fi 0] es fi XI •d fi o Φ rd tn Φ •H tn Φ fi 0 H P 0 Ό d 01 0 a) d •d Φ Φ O e Φ Φ rt 0 • ft rt x; d fil rt 0) rt in •d id k rt rt g XJ td Φ rd fi rt □ rd 4J rd o rt «d •d Φ 0 Φ s •d rd d •d rt fi 0 •P Φ 01 Λ g T3 rt tn XS XI 0 § fi d •d 0 0) d λ Φ d XJ in fd Φ rt 01 Φ ω w w < ω CM W X4OO >20 >20 >20 >20 - 23 Ο Ο to ιη Ο Ο ιη rd Γ-1 m Ch cd CM tn Ο m o uo CM CO & KΓσ α 3 • 0 O rd ft ni ε ε to ο O • υ i-l 0' υ Ρ X Γ; 10 ni — φ N O n β Ο •rd Γ-ι '0 M Ρ Φ Eh rti 3 O Ρ c Ed Ρ -d M β P H Φ β CO □ 0 CQ β u β> Ο co υ H o >1 ω Q Id Ρ 0 -I W Ρ 03 ni •Η o Λ Pd o tn l·* Id > H H υ u Id A O H cq M H Z •Η χ: η Η 1 •d β •Η in m in ΓΟ m d ΓΊ Μ* Γσι in tn tn ιο lO to «—J id σι O O rd in tn vp σι rd O LH rd rd CM m m rn in in cm m rd fd CM (M O to rO cm in O (3h m rd Ο N m rd O CM Λ m o cm /' O rn O O mom CM A to rin ο O m to o O O ci d1 to O cm A O rd CM A (0 -d td β Cn P O P CO Φ td iae to Φ cn Ρ φ φ σι 0 β to β β λ •d β ο Φ rd Cn φ •d cn Φ β ϋ rd 0 Ό Ρ ω 0 φ Ρ •Η Φ Φ ϋ α Φ Φ «3 ϋ ft Λ Χί ρ ά ιϋ (0 >ϋ ω •rl nJ Ρ nJ nj ε PC rd Φ rd β ιϋ ϋ rd Ρ rd 0 φ rd Ή Φ ϋ Φ ε Η rd Ρ d Φ β 0 Ρ Φ φ σι Λ 0 Ό β tn χί Χί 0 ε β Ρ •d ϋ φ Ρ d φ Ρ Χί to Ή φ ίϋ to φ cn ω <5 cn Cd cn CM O m xp co > X > to a ϋ ϋ Ο ο rd r*: .il P nl P cn -P β ίϋ P ω •d ω 0) P β ϋ •d β Φ ft 0) P ra d* > ΰ ftf CM m :-400 is a methicillin resistant Staphylococcus d1 > ω ε ω •d c nJ Cn P 0 P co Φ &d X Ed 4370» - 24 A. 7 - [α τ (3 - methylcarbamoyl - 3 - methylureido) - α - (4 - hydroxyphenyl)acetamido] - 3 - [(1methyl - lH - tetrazol- 5 - yl)thiomethyl] - 3 cephem - 4 - carboxylic acid.

B. 7 - [a - (3 - methylcarbamoyl - 3 - methylureido) - a - (4 - hydroxyphenyl)acetamido] - 3 - [(5methyl - 1,3,4 - thiadiazol - 2 - yl)thiomethyl] - 3cephem - 4 - carboxylic acid.

C. 7 - [ct - (3 - methylcarbamoyl - 3 - methyl10 ureido) - a - phenylacetamido] - 3 - acetoxymethyl 3cephem - 4 - carboxylic acid.

D. 7 - [a - (3 - methylcarbamoyl - 3 - methylureido) - a - phenylacetamido] - 3 - [(1 - methyl - IHtetrazol - 5 - yl)thiomethyl] - 3 - cephem - 4 - carboxy lie acid.

E. 7 - [a - (3 - carbamoylureido) - a - phenyl acetamido] - 3 - [(1 - methyl - lH - tetrazol - 5yl)thiomethyl] - 3 - cephem - 4 - catboxylic acid.

F. 7 - [a - (3 - methylcarbamoylureido) - ct20 (2 - thienyl) - acetamido] - 3 - [(1 - methyl - 1Htetrazol - 5 - yl)thiomethyl] - 3 - cephem - 4 - carboxy lie acid.

G. 7 - [ct - (3 - methylcarbamoylureido) - aphenylacetamido) - 3 - [(1 - methyl - lH - tetrazol25 5 - yl)thiomethyl] - 3 - cephem - 4 - carboxylic acid.

H. 7 - [a - (3 - methylcarbamoyl - 3 - methylureido) - a - (2 - thienyl) - acetamido] - 3 - [(1methyl - IH - tetrazol - 5 - yl)thiomethyl] - 3cephem - 4 - carboxylic acid.

I· Sodium 7 - [a - (3 - methylcarbamoylureido)42705 - 25 -(4 - hydroxyphenyl) - acetamido] - 3 - [(5methyl - 1,3,4 - thiadiazol - 2 - yl)thiomethyl] - 3cephem - 4 - carboxylate.

J. 7 - [ a - (3 - phenylcarbamoylureido) - aphenylacetamido]' - 3 - [ (1 - methyl - IH - tetrazol5 - yl)thiomethyl] - 3 - cephem - 4 - carboxylic acid.

In the standard agar dilution method, the MIC values for the above test compounds A through E against Staphylococcus aureus were as follows: A, 2 B, 1? C, 0.5; meg./ml. 02;E,0.S MCG/ML The cephalosporin antibiotics of the formula I, wherein is hydrogen, and the pharmaceutically acceptable salts thereof are useful in combating infections in warm blooded mammals when administered parenterally in non-toxic doses between 10 and 500 mg./kg. The indanyl, phthalidyl and acyloxymethyl esters of the formula I are useful antibiotics when administered orally in non-toxic doses of between 50 and 750 mg./kg. of body weight.

According to one aspect of the invention then· is provided a pharmaceutics I formulation comprising a compound of formula I, or a pharma ceutically-acceptable salt thereof, associated with a pharmaceutically acceptable carrier therefor. The nature of suitable carriers will be well-known to those skilled in the art.

A preferred group of cephalosporin antibiotics of this invention is represented by the formula I wherein R is 48705 - 26 0 I CH -N-C-N31 11 II R R' is H or methyl; R^ is phenyl, hydroxyphenyl, hydroxy-substituted halophenyl, or 2-thienyl; R2 is N_Ν N-N R^ is hydrogen and the pharmaceutically acceptable salts thereof.

A further preferred group of antibiotics is re10 presented by the formula I when R is I CH,—S—C—»— II H R’ R' is hydrogen or methyl; R^ is phenyl, hydroxyphenyl, or hydroxy substituted halophenyl or 2-thienyl; R2 is 4370S i - 27 Ν--------Ν Ν-Ν R3 is hydrogen and the pharmaceutically acceptable non-toxic salts thereof.

Exemplary of the preferred antibiotics are 7 - f u(3 - methylcarbamoyl - 3 - methylureido) - n - phenylacetamido] - 3 - [(1- methyl - IH - tetrazol - 5 - yl)thiomethyll - 3 - cephem - 4 - carboxylic acid, - [o.- (3 - methylcarbamoyl - 3 - methylureido) - [e - (3 - methylcarbamoyl - 3 - methylureido)a - (4 - hydroxyphenyl) - acetamido] - 3 - [(1 - methyl1H - tetrazol - 2 - yl)thiomethyl] - 3 - cephem - 4carboxylic acid. - [ u - (3 niethylcarbiimoy I ureido) - a - pheny l.H'l'l.iiiii ι|ιι| ! - | (I nielliyl III Icli.iznl ’> yl) l Ιι i omi' I hy I j i - cephem Ί - riir boxy I i e acid, - | ii - (3 - methylcarbamoyl - 3 - methylureido)a -(2- thienyl) - acetamido] - 3 - [(1- methyl - 1Htetrazol - 5 - yl)thiomethyl] - 3 - cephem - 4 - carboxylic acid, - [n - (3 - methylcarbamoylureido) - a - (2thienyl) - acetamido] - 3 - [(1 - methyl - lH - tetrazol 1- 5 - yl) thiomethyl] --3- cephem - 4 - carboxylic acid. 43705 - 28 7 - [ιι.- (3 - methylcarbamoyl - 3 methylureido)(i - (3 - chloro - 4 - hydroxyphenyl)acetamido -3[(1- methyl - IH - tetrazol - 5 - yl)thiomethyl] - 3cephem - 4 - carboxylic acid, 7 - [a - (3 - methylcarbamoyl - 3 - methylureido)a - (3,5 - dichloro - 4 - hydroxyphenyl)acetamido] - 3[(1- methyl - 1H - tetrazol - 5 - yl) thioiriethyl] - 3cephem - 4 - carboxylic acid, and the pharmaceutically acceptable, non-toxic salts thereof.

. The in vivo effective dose (Εϋ^θ) in mg./kg. for representative antibiotics of the formula I has been determined in experimental infections in mice. Table XI lists the Εϋ^θ dose vs Streptococcus pyogenes and Escherichia coli infections. In the table, the anti15 biotics are identified with the R, R', and R^—Rg terms of Formula I.

TABLE II.

Test CompoundR3 ED5Q(mg/kg S.pyogenes x 2)i.p. E.coli R R‘R1R2 ch3 H phenyl tet1 H 0.7 72 "3CH3 4-hydroxy phenyl tet H 0.7 72 H H phenyl 4-hydroxy tet H 0.7 <72 ch3 H phenyl thiad H 0.7 <72CS3 H 2-thienyl tet H <7.2 <72 ch3 ch3: phenyl acetoxy H <7.2 <72 •tet = (l-methyl-lH-tetrazol-5-yl)thio-’thiad = (5-methyl-l,3,4-thiadiazol-2-yl)thio-, The following non-limiting Examples 3 to 7, 9 to 11, 13 to 15, and 17 are provided for the purpose of specifically describing methods which may be utilised to prepare compounds of value in accordance with the invention. 43705 - 29 Examples 1, 8, 12. and I describe the preparation of starting materials.

In the Examples the following abbreviations have the meaning indicated below.

BSA — bis - (trimethylsilyl)acetamide TUP — tetrahydrofuran DMF —~ dimethylformamide NMR — nuclear magnetic resonance spectrum IR — infrared absorption spectrum UV — ultraviolet absorption spectrum,λ max being in nm Example 1 N-Methylcafbamoyl-N-methylcarbamoyl chloride.

To a cold suspension of 22 g. (0.25 m.) of symdimethylurea in diehloroethane was added dropwise with stirring a cold solution of 30 g. (0.3 m.) of phosgene in 90 ml. of diehloroethane. After the addition of the phosgene solution was complete, the reaction mixture was allowed to stir at room temperature for one hour and was then heated to 80°C. and purged with nitrogen for one hour. The reaction mixture was evaporated under reduced pressure and the residual gum was extracted twice with 350 ml. portions of ether. The extracts were combined and evaporated to provide 25 g. of the carbamoyl chloride.

Example 2 Preparation of 7 - [a - (3 - methylcarbamoyl - 3 methylureido) a - (4 - hydroxyphenyl)acetamido] 3 - [(1- methyl - IH - tetrazol - 5 - yl)thiomethyl] - 3 - cephem - 4 - carboxylic acid To a suspension of 483 mg. (1 mmole) of 7-[D-(x-amino-«(4-hydroxyphenyl)acetamido] -3-[(l-methyl-lH-tetrazol-542705 -30yl)thiomethyl]-3-cephem-4-carboxylic acid in 8 ml of dry acetonitrile containing 2 ml. of propylene oxide maintained under an atmosphere of dry argon was added 1 ml. of bis-(trimethylsilyl)acetamide. When solution was obtained, the reaction vessel and solution were cooled to O—5°C. A solution of 150 mg. (1 mmole) of N-methylcarbamoyl-N-methylearbamoyl chloride in 2 ml. of dry acetonitrile was added dropwise to the cold solution with stirring. The reaction mixture was stirred for 2 hours and was allowed to warm to room temperature. The reaction mixture was poured into a mixture of water and ethyl acetate and the pH of the mixture was adjusted to pH 9. The aqueous layer was separated and relayered with fresh ethyl acetate. The pH of the aqueous layer was adjusted to pH 2.5 and the ethyl acetate layer was separated, was washed with brine and dried over sodium sulfate. The dried ethyl acetate solution contain ing the reaction product was evaporated to dryness in vacuo to obtain the product as a faintly yellow powder.

The product was dissolved in ethyl acetate and in part precipitated by adding petroleum ether to the solution. The precipitated product was filtered and dried to ! yield 138 mg. Additional product was recovered by evaporation of the filtrate.

NMR (60 MHz., DMSO άθ): 9.8 (d, J=7, IH), 9.3 (d, J=8, IH), 7.4-6.5 (m, 5H), 5.85—5.50 (g, IH), 5.50— 5.30 (d, J=7, IH), 4.9 (d, J=5, IH), 4.3 (broad, 2H), 3.5 (ε, 3H),. 3.6 (broad 2H), 3.1 (s, 3H) and 2.65 (d, J=3, 3H) delta.

Elemental analysis (percent) for ^2^25^9^7821 Theory: C, 44.66; H, 4.26; N, 21.31 Pound: C, 44.32; H, 4.34; N, 19.32. - 31 .Example 3 Preparation of 7 - |o - (3 - methylcarbamoyl -3methylureido) - a -(4- hydroxyphenyl)acetamido] 3 - [(5 - methyl - 1,3,4 - thiadiazol - 2 - yl)thiomethyl] - 3 -,cephem - 4 - carboxylic acid.

To a suspension of 2.12 g. (4 mmole) of 7 - [Οη - amino - a - (4 - hydroxyphenyl) - acetamido] - 3[(5- methyl - 1,3,4 - thiadiazol - 2 yl)thiomethyl]3 - cephem - 4 - carboxylic acid in 32 ml. of dry aceto. I nitrile containing 8 ml. of propylene oxide maintained under an atmosphere’of dry argon was added 4 ml. of bis(trimethylsilyl)acetamide with stirring. When solution had occurred, the solution was cooled to 0°C. and 600 mg. (4 mmole) of N-methylcarbamoyl-N~methylcarbamoyl chloride in 8 ml. of dry acetonitrile were added dropwise with stirring. The reaction mixture was htirred for 2 hours during which time the temperature of the mixture was allowed to rise to room temperature. The reaction mixture was poured into a water-ethyl acetate mixture and the pH of the agueous layer was adjusted to pH 8.5. The aqueous layer was separated and layered with fresh ethyl acetate The pH of the aqueous layer was then adjusted to about pH 2.5 and the ethyl acetate layer was separated, washed with brine and dried over sodium sulfate. The dried solution was evaporated to dryness under reduced pressure to yield about 1.6 g. of the product as a light yellow solid. The product was triturated with ethyl acetate and the insoluble product (645 mg.) was filtered. The filtrate was concentrated by evaporation to yield 423 mg. of precipitated product. The latter filtrate was evaporated to dryness to yield 560 mg.

The above three product fractions were shown to be the same when chromatographed on silica gel thin layer chromatograms using chloroform:methanol (7:3, v:v) for - 32 development and.either iodine vapors or ultraviolet light for visualization of the developed plates.

Elemental analysis (percent) for C„_H„c.N 0 S_: 773 Theory: C, 45.46; H, 4.15; N, 16.13 Found: C, 45.53; H, 4.47; N, 14.85.

Electrometric titration in 66 percent DMF Showed the presence of two titratable groups: pK 4.8 and 12.2 r a and an apparent molecular weight as calculated from the 10 titration data of 587 (calculated MW=6O7). The infrared absorption spectrum (mineral oil mull) showing the characteristic β-lactam carbonyl absorption at about 2920 wave numbers and the NMR spectrum were in agreement with the expected product. ov absorption spectrum (methanol) max 303 ζ = 9.246 max 275 i- ξ = 9,273 max 229 t ξ =16,254 Example 4 Preparation of 7 - [a - (3 - methylcarbamoyl - 3methylureido). - a - phenylacetamido] - 3 - acetoxymethyl - 3 - cephem - 4 - carboxylic acid.

The above-named compound was prepared by the reaction of 7-(D-a-amino-a-phenylacetamido)-3-acetoxy25 methyl-3-cephem-4-carboxylic acid (cephaloglycine) with N-methylcarbamoyllN-methylcarbamoyl chloride by following the reaction conditions employed in Example 3. The product (158 mg.) was obtained as a white crystalline solid. 4370S - 33 Nuclear magnetic resonance spectrum (60 MHz., DMSO dr): 7.45 (s, 5H), 5.9—5.4 (m, 2H) , 5.2—4.4 (m, b 3H), 3.5 (broad', 2H), 3.15 (s, 3H), (s, 3H) and 2.05 (s, 311) delta. < Example 5 Preparation of Sodium 7 - [a - (3 - methylcarbamoylureido) - a - (4 - hydroxyphenyl)acetamido] - 3f(5 - methyl - 1,3,4 - thiadiazol - 3 - yl]thiomethyl] - 3 - cephem - 4 - carboxylate.

To a suspension of 2.88 g. (6 mmole) of sodium - [D - a - amino - ct - (4 - hydroxyphenyl)acetamido]3 - [ (5 - methyl - 1,3,4 - thiadiazol - 2 - vl)thiomethyl] - 3 - cephem - 4 - carboxylate in 48 ml. of dry acetonitrile at room temperature were added 6 ml. of BSA. When an homogeneous solution was obtained 5.2 g. of p-nitrophenyl N-(methylcarbamoyl)carbamate were added. The reaction mixture of water and ethyl acetate. The pH was adjusted to 6 and the ethyl acetate phase was separated and replaced with fresh ethyl acetate. The pH of the aqueous phase was adjusted to pH 2.5. The aqueous layer was separated and discarded.

The ethyl acetate phase was washed with diluted hydrochloric acid (pH 2.0) and fresh water was added. The pH was finally adjusted to 5.5 and the aqueous phase lyophilized to yield 1.3 g. of the product as a light yellow powder.

NMR (DMSO—d6): 2.7 (s, 6H, NHCH^ & thiadiazole CH3), 3.5 (broad, 2H, c2—H2), 5.0 (d, J=5, IH, C6—H), 5.4— —9 (m, 2H, C7—H & side chain CH), 6.9 (d, J=9, 2H, aromatic) and 7.4 (d, J=9, 2H, aromatic) delta. - 34 Example 6 Preparation of Sodium 7 - [a - (3 - methylcarbamoyl3 - methylureido) - a - phenylacetamido] - 3[(5 - methyl - 1,3,4 - thiadiazol - 2 - yljthio5 methyl] - 3 - cephem - 4 - carboxylate.

To a suspension of 2.78 g. of sodium 7 - (D - examine - a - phenylacetamido) - 3 - [(5 - methyl1, 3,4 — thiadiazol - 2 - yl)thiomethyl] - 3 - cephem4 - carboxylate in 48 ml. of dry acetonitrile contain10 ing 12 ml. of propylene oxide were added 6 ml. of bis(trimethylsilyl)acetamide. When a homogeneous solution was obtained the solution was cooled to about O°C. and a solution of 6 mmoles of N - methylcarbamoyl - Nmethylcarbamoyl chloride in 12 ml. of dry acetonitrile was added. The reaction mixture was stirred for 2 hours.

The product was recovered by following the workup procedures described in Example 5 to yield 1.1 g. of the sodium salt.

IR (mull): β-lactam carbonyl absorption at about 2920 cm NMR (DMSO d6) : 2.7 (Broad, 6H, NHCH3 & Thiadiazole CH ), 3.1 (s, 3H, N—CH3), 4.4 (Broad, 2H, 0(3')H2), 4.9 (d, J=4.5, IH, C(6)H), 5.4-5.9 (m, 2H, C(7)H Side Chain CH), 9.4 (d, J=9, IH, NH) and 10.0 (d, J=7, IH, NH) delta.

Example 7 Preparation of 7 - [a- (3 - methylcarbamoyl - 3methylureido) - a - (2 - thienyl) -acetamido - 330 [(1- methyl - IH - tetrazol - 5 - yl)thiomethyl]3 - cephem - 4 — carboxylic acid.

To a suspension of 234 mg. of 7 - [a - amino43705 - 35 ι/ -(2- thienyl)acetamido| - 3 - [(1 - methyl - 1Htetrazol - 5 - yl) thiomethyl] - 3 - cephem -- 4 - carboxylic acid in 4 ml. of dry acetonitrile containing 1 ml. of propylene oxide was added under argon 0.5 ml. of BSA to form a homogeneous solution. The solution was cooled to 0°C. and a solution of 75 mg. of Nmethylcarbamoyl-N-methylcarbamoyl-chloride in 1 ml. of acetonitrile. The reaction mixture was stirred for 2 hours and was then poured into a mixture of water ethyl acetate. The pH of the aqueous phase was adjusted to pH 6 and the organic phase separated. Fresh ethyl acetate was added to the aqueous phase and the pH adjusted to 2.5 with dilute hydrochloric acid. The organic phase was separated, dried and evaporated to dryness to yield the product.

Elemental analysis for C Η N 0 S : Au A3 963 Theory : C, 41.30; H, 3.99; N, 21.68 Found : C, 41.78; H, 4.14; N, 21.73.

UV (methanol): λ max 238 ζ 17,475 λ max 270 ζ 9,000 Example 8 Preparation of p-nitrophenyl N-(methylcarbamoyl)carbamate.

Methylurea (3.7 g., 50 mmole) and ^-nitrophenyl chloroformate (50 mmole) were allowed to react under nitrogen in 10 ml. of dry THF. The reaction mixture became clear initially and the product began to precipitate from the clear solution. The mixture was stirred for about 18 hours and the precipitated product was filtered. The product was washed with water and diethyl ether to yield 5.9 g. of approximately a 50:50 - 36 mixture of p-nitrophenyl N-(methylcarbamoyl)carbamate an.d p-nitrophenyl carbamoyl-N-methylcarbamate as shown by NMR.

Example 9 Preparation of 7 - [a - (3 - methylcarbamoylureido)a - phenylacetamido] - 3 - [(1 - methyl - 1Htetrazol - 5 - yl) thiomethyl] - 3 - cephem - 4carboxylie acid.

A suspension under argon of 2 mmoles of 7 - (aamino - a - phenylacetamido] - 3 - [(1- methyl - 1Htetrazol - 5 - yl)thiomethyl] - 3 - cephem - 4 - carboxylic acid in 16 ml. of dry acetonitrile was solubilized by adding 2 ml. of ESA. With stirring, 4 mmoles of the product mixture obtained as described by Example 12 (mixture of p-nitrophenyl N-(methylcarbamoyl)carbamate and p-nitrophenyl N-carbamoyl-N-methylcarbamate) were added. The reaction mixture became clear in a few minutes and was stirred for about 2 hours.

The reaction mixture was poured into a mixture of water-ethyl acetate and the pH adjusted to 6, The ethyl acetate layer was separated and discarded. The aqueous phase was relayered with fresh ethyl acetate and the pH of the aqueous phase was adjusted to pH 2.5. The organic layer was separated, washed with water, dried and evaporated in vacuo. The product residue was triturated with diethyl ether to yield 600 mg. of prodi ct as a faintly yellow powder.

Elemental analysis for C2XH23Ng°6S2! Pound : C, 44.91; H, 4.13; 22.45; s, 11.42 : C, 44.66; H, 4.34; 22.29; s, 11.28. - 37 UV (methanol): /, max 272 r 11,091 NMR (DMSO d6) : 2.6 (d, J5H2, 3H, NHCII ), 3.9 (S, 311), —CH3 on tetrazole), 3.55 (Broad, 211, CH2), 5.0 (d, J=5, IH, C(6)H), 5.4—5.9 (m, 2H, C(7)H Side Chain CH), 7.3 (S, 5H, Q), 8.4 (d, J=7, IH, NH), 8.8 (S, III, NH), and 9.4 (d, J=9, IH, NH) delta.

Example 10 Preparation of 7 - [nr - (3- methylcarbamoylureido)a - (4 - hydroxyphenyl) - acetamido] - 3 - [(5methyl - 1,3,4 - thiadiazol - 2 -yl) thiomethyl] 3 - cephem - 4 - carboxylic acid.

To a suspension of 988 mg. (2 mmole) of 7 - [uamino - rt - (4 - hydroxyphenyl)acetamido] - 3 - [(5methyl - 1,3,4 - thiadiazol - 2 - yl)thiomethyl] - 3cephem - 4 carboxylic acid in 16 ml. of dry acetonitrile were added 2 ml. of BSA under argon. An homogeneous solution was obtained and 1.43 g. (6 mmole) of the product mixture-containing 50% of p-nitrophenyl N(methyIcarbamoyl) - carbamate (Example 8) were added. After stirring for one hour the product was recovered from the reaction mixture by following the work-up procedures described in Example 9. The product, 524 mg., was obtained as a light yellow powder.

UV (methanol) : λ max 232 ζ 16,072 λ max 275 ξ 14,309 IR (mull) : β-lactam carbonyl absorption at about 2900 cm 1 Example 11 Preparation of 7 - [« - (3 - methylcarbamoylureido)ί. -(2- thienyl)acetamido] - 3 - | (1 - methyl - 1Htotrazol - 5 - yl) thiomethyl| - 3 - cephem - 4carboxylic acid. - 38 To a suspension of 435 mg. (0.93 mmole) of 7[D - ra - amino - ra - (2 - thienyl)acetamido] - 3[(1- methyl - IH - tetrazol - 5 - yl)thiomethyl] - 3cephem - 4 carboxylic acid in 8 ml. of dry aceto5 nitrile was added 1 ml. of bis - (trimethylsilyl)acetamide. After an homogeneous solution was obtained 1.4 g. of £ - nitrophenyl N - (methylcarbamoyl)carbamate were added.

The reaction mixture was stirred at room tempera10 ture for 90 minutes. The product was recovered from the reaction mixture by following the work-up procedures described in Example 7. The product was obtained as a white powder weighing 341 mg.

Elemental analysis for C29H21N9°6S3: Theory : C, 40.20; H, 3.73; N, 22.21; S, 16.95 Pound ; C, 39.86; H, 4.02; N, 22.88; S, 14.66.

IR (mull) β-lactam carbonyl absorption at about 2920 cm-1.

UV (methanol): λ max 235 ξ 15,459 λ max 272 ξ 9,360 MIR (DMSO d6): 2.6 (d, J=4.5, 3H, NHCHj) , 3.6 (Broad, 2H, C(2)B2), 3.9 (5, 3H, tetrazole CH3), 4.25 (Broad, 2H, C(3')H2), 5.1 (d, J=5, IH, C(6)H), 5.6—5.9 (m, 2H, C(7)H & Side Chain CH), 6.9—7.6 (m, 4H, Thiophene & 1NH), 8.4 (d, J=8, IH, NH), 8.9 (S, IH, NH) and 9.5 (d, J=8.5, IH, NH) delta. - 39 Example 12 Preparation of p-nitrophenyl N-(phenvlcarbamoyl)-carbamate To a stirred solution of 6.8 g. of phenylurea in 50 ml. of dry THF maintained at 0°C. under nitrogen were added 5.05 g. of p-nitrophenyl chloroformate. The reaction mixture was allowed to warm to room temperature and was stirred for about 18 hours. The mixture was evaporated to dryness and the residue dissolved in ethyl acetate. The solution was washed twice with water, twice with brine, and was filtered through sodium sulfate. The filtrate was evaporated to dryness to yield 4.9 g. of product as a white product.

Example 13 Preparation of 7 - [a - (3 - phenylcarbamoylureido) - aphenylacetamido] - 3 - [(1 - methyl - IH - tetrazol 5 - yl)thiomethyl] 3 - cephem - 4 - carboxylic acid.

A suspension of 950 mg. (2 mmole) of 7 - (D - aamino - ct - phenylacetamido) - 3 - [(1- methyl - 1Htetrazol - 5 - yl)thiomethyl] - 3 - cephem - 4 - carboxylic acid in 16 ml. of dry acetonitrile at 0°C. was solubilized under nitrogen with 2 ml. of BSA. With stirring 1.2 g. of p-nitrophenyl N-(phenylcarbamoyl)carbamate, prepared as described in Example 12., were added to tin· sol id: imi. Tin· ro.ietioii mixture w-ι.·: οΐίπ··<1 Im mu· hour and Ihe product recovered by Iollowing the work up procedures described in Example 7. The product was obtained as a light yellow powder weighing 839 mg.

NMR (DMSO d6): 3.6 (Broad, 2HC(2)—H), 3.95 (S, 3H, Tetrazole-CHg), 4.3 (Broad, 2H, C(3')—H ), 5.0 (d, J=5, IH, C(6)—Η) , 5.5—5.9 (m, 2H, C(7)—II & Side Chain CH), 7.0—7.6 (Broad, 10H, Aromatic), 8.4 (d, J=7.5, IH, NH), 9 1 (s, IH, NH) , 9.6 (d, J=9, IH, NH), 9.8 (S, IH, NH). 43705 - 40 Example 14 Preparation of 7 - [a - (3 - benzyIcarbamoylureido)a - phenylacetamido] - 3 - [(1- methyl - 1Htetrazol - 5 - yl)thiomethyl] - 3 - cephem - 4carboxylic acid. · By following the procedure described in Example 13, 2 mmoles of 7 - (a - amino - a - phenylacetamido)3 - [(1 - methyl - IH - tetrazol - 5 - yl)thiomethyl] 3 - cephem - 4 - carboxylic acid were dissolved in acetonitrile with BSA and reacted with 1.26 g. of £10 nitrophenyl N-(benzylcarbamoyl)carbamate to yield the title compound.

NMR (DMSO d6)i 3.6 . (Broad, 2H, C(3)H2), 3.9 (S, 3H, Tetrazole CH^), 4.3 (Broad, 4H, C(3‘)—H2 and benzyl CH2), 5.0 (d, J=5, lH, C(6)H, 5.4—5.9 (m, 2Η» C(7)H Side Chain CH), 7.2—7.6 (m, 10H, Aromatic), 7.8 (t, J=5.5, IH), 8.4 (d, J=7, lH, NH), 8.95 (S, IH, NH), 9.5 (d, J=8, IH, NH).

Example 15 Preparation of 7 - [a - (3 - furfutylcarbamoylureido)20 a - phenylacetamido] - 3 - [(1- methyl - 1Htetrazol - 5 - yl) thiomethyl] - 3 - cephem - 4carboxylic acid.

By following the procedure described in Example 13 2 mmoles of 7 - (a - amino - a - phenylacetamido) - 3[(1- methyl - IH - tetrazol - 5 - yl)thiomethyl] - 325 cephem - 4 - carboxylic acid were reacted at 0°C. in dry acetonitrile with BSA and 1.22 g. of £-nitrophenyl N(furfurylcarbamoyl)carbamate to obtain the title compound.

NMR (DMSO d6): 3.55 (Broad, 2H, C(2)—H2), 3.9 (S, 3H, Tetrazole CH^), 4.3 (Broad, 4H, C(3')H2 & Thiophene CH2), 5.0 (d, J=5, IH, C(6)H), 5.4-5.9 (m, 2H, C(7) — - 41 Η & Side Chain CH), 6.3 (m, 2H, thiophene aromatic), 7.2—7.9 (m, 7H, aromatic NH), 9.4 (d, J=7, IH, NH), 8.9 (s, IH, NH), 9.4 (d, J=8, IH, NH) .

Example 16 Preparation of p-nitrophenyl N-(carbamoyl)carbamate To a mixture of 1.20 g. (20 mmole) of urea and 2.02 g. (10 mmole) of p-nitrophenyl chloroformate in a dry flask under nitrogen were added 10 ml. of dry acetonitrile. The reaction mixture momentarily became clear and then a precipitate formed. The mixture was stirred for 18 hours at room temperature and the precipitate was filtered, washed with water and dried under vacuum. The dried product was triturated with ether and redried to yield 1.0 g. of crystalline product.

Example 17 Preparation of 7 - [« - (3 - carbamoylureido) - θ'pheny lacetamido] - 3 - [ (1 - methyl - IH - tetrazol5 - yl)thiomethyl] - 3 - cephem - 4 - carboxylic acid.

To a suspension of 2 mmole of 7 - (a - amino - aphenylacetamido) - 3 - [(1- methyl - IH - tetrazol - 5yl)thiomethylj - 3 - cephem - 4 - carboxylic acid in 20 ml. of dry acetonitrile under argon were added 3/4 ml. of BSA. When an homogeneous solution was obtained it was evaporated to dryness. The residual dry off-white powder was dissolved in DMF and the solution added under argon with stirring to a solution of 20 mmole of pnitrophenyl N-(carbamoyl)carbamate (Example 16}in 10 ml. of DMF containing 20 mg. of 1-hydroxybenzotriazole monohydrate. The reaction mixture was stirred for 72 hours at room temperature and then was diluted with water and the product extracted with ethyl acetate at a pH of about 2.5. The extract was washed, dried and evaporated to yield the product as an amorphous powder.

Elemental analysis for C_H.NO.-S.

Theory Found '20 21 9 6 2' C, 43.87; H, 3.87; N, 23.02; S, 11.71 C, 43.77; H, 4.00; N, 22.86 S, 11.41.

UV (methanol) ί λ max 250 ξ 8,957 NMR (DMSO d6) j 3.55 (Broad, 2H, 0(2)¾) , 3.95 (S, 3H, tetrazole—CHg), 4.45 (Broad 2h, 0(31)¾), 5.0 (d, J=5, IH, C(6)H), 5.4—5.9 (m, 2H, C(7)H & Side Chain CH), 6.75 (Broad S, 2H, —¢1¾), 7.4 (S, 5H, phenyl), 8.51 (d, J=7, IH, NH), 8.8 (S, IH, NH) and 9.5 (d, J= 10, IH, NH) delta. - 43 43705 Following the preparative methods described in the above examples, 7 - [a - (3 - methylcarbamoyl - 3 methylureido) -it - (4 - hydroxyphenyl)acetamido]- 3 [(1 - methyl - 1,2,3 - triazol - 5 - yl)thiomethyl] - 3 5 cephem - 4 - carboxylic acid is prepared.

The per se claims 1 to 50 of Patent Specification No. 39294 in the name of the Beecham Group Limited, published after our priority date, are directed to certain cephalosporins of formula (I) or a pharmaceutically acceptable salt or readily hydrolysable ester thereof:- iooil wherein Y is oxygen or sulphur, R1 is an organic radical 2 containing up to 20 carbon atoms; R is alkyl having from 1 2 to 3 carbon atoms, or benzyl; or R and R together with the carbon and nitrogen atoms to which they are attached form a 5,6, or 7 membered ring; R is cyclohexadienyl, phenyl, phenyl substituted by one or more functional groups selected from hydroxy, halogen, nitro, alkoxy containing from 1 to 3 carbon atoms, and amino groups, 2- or 3- thienyl, cycloalkyl having from 3 to 7 carbon atoms or alkyl having from 1 to 4 carbon atoms; R^ is acetoxy, benzoxazole-2-thio, sulpho, or a group of formula, Claim 51 of that Patent is directed to processes for preparing those cephalosporins. - 45 The compounds disclosed and claimed in the present Specification, although they fall within the scope, of the claims of the Beecham Patent have not been specifically disclosed therein:-

Claims (9)

1. CLAIMS:I. A compound of the formula I OH OH III M R-C-N-CH-C-N CH-C-W -j-> ! N^^ CH 2 R 2 COOR, wherein ii is a ureido group represented by the formula H 0 I II R' ' ——N-C-NI R' wherein R' 1 is hydrogen, C^-C-j alkyl, allyl, propargyl, Cg-Cg eycloalkyl, phenyl, benzyl, or furfuryl; and R* is hydrogen or methyl; is phenyi, hydroxyphenyl, dihydroxyphenyl, halophenyl, dihalophenyl, hydroxy substituted halophenyl, thienyl or furyl; R 2 is acetoxy, -O r wherein Z is C^-C^ alkyl; 4270S - 47 Rg is hydrogen, indanyl, phthalidyl, or an acyloxymethyl group of the formula wherein Y' is c f~ c ^ alkyl or phenyl; or when Rg is hydrogen, a pharmaceutically acceptable non-toxic salt thereof.

2. A compound of formula I as claimed in claim 1, wherein R' and R' 1 are methyl, or Y is hydrogen and R is other than Z or when R^ is hydrogen, a pharmaceutically acceptable nontoxic salt thereof.

3. The compound of Claim 1 wherein R is H (J I II R< -.JJ-C -JI R' 15 wherein R' and R' 1 are as defined in claim 1, and Rg is hydrogen.

4. The compound of claim 1 or 3 wherein R'' and R' are both methyl.

5. The compound of claim 1 or 3 wherein R 1 ' is 20 methyl and R 1 is hydrogen. - 48

6. A process for preparing a ureido substituted cephalosporin compound of formula I, which comprises reacting a compound of formula II COOH wherein R^ and R 2 are as defined in claim 1, with a compound of the formula wherein R'' is as defined in Claim 1; and if desired esterifying the acid s,o obtained wherein is hydrogen LO to give the corresponding ester wherein R 3 is other than hydrogen.

7. A process for preparing a ureido substituted cephalosporin of formula I, as claimed ih claim 1, substantially as hereinbefore described. i LS

8. A compound as claimed in claim 1 whenever prepared by a process according to claim 6 or 7. - 49

9. A pharmaceutical formulation comprising a compound of formula I, as claimed in any one of claims 1 to 5 or 8 or a pharmaceutically acceptable salt thereof, associated with a pharmaceutically acceptable carrier therefor.