US3905966A - Cephalosporin cleavage process - Google Patents

Abstract

A 7-(amic acid) cephalosporin is cleaved by conversion to the corresponding mixed anhydride and cleavage of the mixed anhydride to the corresponding 7-aminocephalosporin or to a corresponding 7-acylamidocephalosporin.

Description

United States Patent [1 1 Kukolja et al.

law" AVAILABLE eow [451 Sept. 16, 1975 CEPHALOSPORIN CLEAVAGE PROCESS Inventors: Stjepan P. Kukolja, Indianapolis; Steven R. Lammert, Greenwood, both of Ind.

Assignee: Eli Lilly and Company, Indianapolis,

Ind.

Filed: June 18, 1973 Appl. No.: 371,011

U.S. Cl. 260/243 C; 424/246 Int. Cl. C07D 501/04 Field of Search 260/243 C References Cited UNITED STATES PATENTS 3,487 U70 12/1969 Shcchan ..260/243C 3,487,074 l2/l969 Shcchan .i 26()/239.l 3 769,28l lO/l973 Chauvcttc 260/243 C Primary Examiner-Nicholas S. Rizzo Assistant Examiner.losc Tovar Attorney, Agent, or Firm-William C. Martens, Jr.; Everet F. Smith 5 7 ABSTRACT 12 Claims, No Drawings BACKGROUND OF THE INVENTION 7 This invention relates to a process for selectively cleaving an amic acid function from 11-7 5mm acid) cephalosporin. f i i It has been a customary practice for some time in the development of cephalosporin antibiotics to employ an imide substituent in the 7-position when that portion of the molecule was not the point of investigation. The presence of such a protective group, particularly the phthalimido group, tended to render the 7-position chemically quite inert and afforded the possibility to treat other portions of the molecule rather vigorously with the relative assurance that the 7-position would remain intact.

However, it has long been recognized that the presence of an imide function in the 7-position of a cephalosporin rendered the structure antibiotically only minimally active. Unfortunately, it has been impossible to successfully cleave a 7-imido group from a cephalosporin to liberate the protected amino group. Thus, the investigator was left with a stable substituent in the 7- position, and one which rendered a cephalosporin ex hibiting only minimal antibiotic activity. The use of such a substituent thus could be attractive commercially only if it could conveniently be removed at any desired point in a synthetic scheme. t

It is not intended by the above to say, in general, that it has been made impossible successfully to cleave an amide group. Several methods for'accomplishing this are recognized. The Japanese publication by Minoru Shindo, Cleavage Reactions of the Phthalimido Group, Yuki Gosei Ktlgaku Kyokai S/zi, 29 (5 1971') pp. 496-509, contains an extensive discussion of cleavage techniques. Any of these would be available in achieving cleavage of the imide function from a cephalospo'rin were this the only essential consideration. However, it is at least of equal importance to employ conditions which will accomplish cleavage without sacrificing the structural integrity of the cephalosporin molecule. To date, this has been impossible to achieve.

It has been possible to achieve a partial cleavage of the imide side chain of a cephalosporin structure to form the correspononding amic acid side chain (see, for example, Sheehan et. al., Journal of the American Chemical Society, 73, 1951 pp. 43674372; Sheehan et. al., Journal of the American Chemical Society, 78, (1956) pp. 368()3683; Perron ct al., Joarnul of ()rganic Chetnistt v, 7, (1964) pp. 483-487). The phthal imide function has been converted to the correspond ing phthalamic acid by alkaline hydrolysis such as is de scribed in the first Sheehan publication. However, as noted in the second Sheehan publication, all attempts to carry the cleavage beyond this pointhave met with failure, theB-lactam ring of the penicillin being preferentially opened with destruction of the penicillin.

Sheehan, US. Pat. No. 3,487,074, discloses the cleavage of 6-phthalimidor3-penamyl-carboxylic acid by treatment thereof with hydrazine hydrate in dioxane for 12 hours at room temperature. However, thismethod has been found to be unsuccessful when ap plied to penicillins and cephalosporins, although moderate success was experienced when this approach was appliedto a 7-phthalimido A -cephalosporin [see Spry,

D. 0., Joni-nu! 0f the American Chemical Society, 92,

Amethod has now been discovered by which an amic acid function of acep halosp-orin canbe cleaved with outtopening the ,B-lac tam ring. This invention comprises such a method. Normally, the amic acid function 5 will be obtained by partial cleavage of an imide function; however, this is by no means essential.

SUMMARY on THE INVENTION This invention relates to a. process for cleaving the amic acid function of a 7-(amic acid) cephalosporin having the formula 2O COOH in which R,, is an alkyl group having from 1 to 4 carbon atoms;

2. reacting the product mixture from the aforementioned chloroformate treatment with a hydrazine of i the formula j i R HNNHRi I in which R;, and R independently are hydrogen or methyl; and I i t i a. reacting the reaction mixture from the aforementioned hydrazine treatment with an acyl halide to produce the corresponding 7-acylarnido cephalosporin; or i b: when at least one of R and R is methyl, recovering the corresponding 7-amino cephalosporin from the reaction mix tureiof the aforementioned hydrazine treatment; or

c. when R;. and R are hydrogen. heating the reaction mixture from the aforementioned hydrazine treatment to a temperature of from about 50C. to about l()0C. to produce the corresponding 7-amino cephalosporin; or

(1. when R;, and R are hydrogen. reacting the reaction mixture from the aforementionedhydrazine treatment with acid to produce the corresponding 7-amino cephalosporin in the form of its acid addition salt.

DETAILED DESCRIPTION OF THE INVENTION Unless otherwise specifically mentioned, as used herein, the terms cleavage". cleaving". and the like, are intended to refer to the removal of a substituent in the 7-position of a cephalosporin thereby to produce a free 7-amino cephalosporin. a free 7-amino cephalosporin in the form of its acid addition salt, or a 7-amino cephalosporin resulting from removal of the amic acid function followed by re-acylation to contain another acyl substituent in its 7-position.

ln accordance with one aspect of the process of this invention. a first step involves conversion of an amic acid function to produce a corresponding mixed anhydride. When. in the amic acid structure, R and R taken together with the carbon atoms to which they are at- The preparation of a compound having the above structure from the corresponding phthalimido compound is well-recognized in the art. and any of the known conditions can be employed. A typical method by which partial cleavage is effected involves an alkaline hydrolysis such as is described in Sheehan et al.. Journal of the Arm'rl'tun C/lt'lllifll/ Sucit'l). 73, 1951 pp. 43674372.

The conditions of alkaline hydrolysis which can be employed to accomplish partial cleavage to the amic acid include use of an alkali metal hydroxide or sulfide. such as sodium hydroxide, potassium hydroxide. lithium hydroxide. sodium sulfide. potassium sulfide, lithium sulfide. and the like. Generally. from about 1 to 2 equivalents of the alkali metal hydroxide or sulfide are employed, except in the instance in which the free acid of the cephalosporin is employed. in which case the free carboxyl itself will consume one equivalent of the alkaline reagent. and. therefore. an additional equivalent will be required.

ln general. the pH of the reaction medium ranges from about 9 to about 1 l. The hydrolysis is achieved generally by use of an aqueous medium containing an inert, water-miscible organic solvent. such as. for example. tetrahydrofuran. N.N-dimethylformamide. acetone. dimethylsulfoxidc, dioxanc. and the like.

The partial cleavage generally is quite rapid. typically being completed in from about 3 to about 30 minutes, and more typically in from about 5 to about 10 minutes. The temperature of reaction usually is from about l()C. to about room temperature, and preferably.

about 0C.

The amic acid can also have the formula COOR The above amic acid. as well as phthalamic acid de fined herein. are also available from sources other than their corresponding imide precursors. For example. the free amino compound can be reacted with the appropriate anhydride. for example. maleic anhydride. to produce the corresponding amic acid compound, specifically, in this case, the 3-carboxyacrylamido compound.

One step of the process of this invention comprises the conversion of the amic acid to a mixed anhydride. The mixed anhydride is not isolated in accordance with the process of this inventionphowever. it has the for mula R,, represents an alkyl group. preferably a lower alkyl group having from 1 to 4 carbon atoms.

The conversion of the amic acid to the mixed anhydride is accomplished by reacting the amic acid with an alkyl chloroformate. such as ethyl chloroformate. propyl chloroformate. t-butyl chloroformate, isobutyl chloroformate. and the like. Preferably. a lower alkyl chloroformatc is employed. typically one in which the alkyl group has from about l to about 4 carbon atoms. This reaction is carried out in the presence of a tertiary amine. such as pyridine. quinoline, triethylamine. N- methylmorpholine. N.N-dimethylaniline. and the like. Additionally. this reaction typically is carried out in the presence of an aprotic organic solvent. that is. one which does not offer or accept protons. A wide variety of such solvents are known to those skilled in the art and can be used in accordance with the process of this invention. Included as such solvents are N.N-dimethyl formamide, N,N-dimethylacetamide, tetrahydrofuran, dioxane, aliphatic nitriles, such as acetonitrile, propionitrile, and the like; aromatic hydrocarbons and halogenated derivatives, such as benzene, toluene, dichlorobenzene, and the like; and aliphatic halogenated hydrocarbons, such as methylene chloride, chloroform. bromoform, carbon tetrachloride, carbon tetrabromide', ethylene dichloride. ethylene dibromide, and the like. i

No more than one equivalent of the amine based upon the amic acid is employed, and it is highly preferred to employ a slightly deficient quantity of the amine. Any excess tertiary amine will tend to convert the amic acid to an imide, typically whatvmay have been the original starting material in the process of this invention. A slight excess of the ehloroformate can be employed; however, this is not preferred, since any excess will react with the hydrazine employed in the next step of the process of this invention;

The reaction is carried out for from about 5 to about 40 minutes, preferably from about 20 to about 30 minutes at a temperature of from about -20C. to about +5C., and preferably from about -20C. to about 5C. The mixed anhydride is maintained intact by retaining the reaction mixture at approximately the temperature at which the reaction was carried out.

The aforementioned mixed anhydride cephalosporin can then be selectively cleaved by a step-wise treatment thereof with a hydrazine, typically unsubstituted hydrazine, methyl hydrazine, or N,N'-dimethylhydrazine, followed by product recovery and/or further treatment depending upon the product which is desired and the particular hydrazine which is employed.

The hydrazine treatment involves the reaction of the mixed anhydride in an inert organic solvent such. as any of the aprotic solvents mentioned hereinabove with one equivalent of the hydrazine. Care must be taken 'to avoid the presence of any excess hydrazine. Therefore, in order to ensure the avoidance of such excess, up to one equivalent of the hydrazine per equivalent of the original amic acid is employed, and, typically, a slight deficiency of hydrazine is employed. The reaction'is carried out at relatively cold temperatures ranging from about -l0C. to about room temperature and preferably at about ice temperature (0C.). The hydrazine typically-is added to the mixed anhydride mixture while the mixture is at the relatively cold reaction temperature. Thus, the mixture of the mixed anhydride in the organic solvent is maintained at the temperature of reaction while the' hydrazine, previously cooled, is added. The reaction is rather rapid, generally being completed within from about 1 to about minutes, and the reaction generally is permitted to proceed for about an additional 5 minutes.

The particular treatment which the hydrazine reaction mixture then receives depends upon the structure of the hydrazine which is employed and the ultimate product which is desired.

The hydrazines which are used have the structure R HNNHR,

in which R;, and R independently are hydrogen or methyl. When either or both of R and R, are methyl, no further treatment is necessary since the free 7- amino cephalosporin is generated and can be isolated simply by applying techniques well recognized in the art.

When R5; and R in the hydrazine which is employed are both hydrogen, a complex of the free 7-amino cephalosporin and the by-product, diketophthalazine, forms, and this complex must be broken. This can be accomplished by heating the mixture or by treating the mixture with acid or, more'readily, by a combination of both heat and acid treatment.

When heat is employed, the complex typically can be broken by'subjecting the reaction mixture to a temperature of from about 50C. to about C. for from about 5 to about 20 minutes, and the free amino compound recovered by recognized techniques.

The diketophthalazine complex can also be broken by treating th e reaction mixture with an acid. Virtually any acid, organic or inorganic, can be used. Typical such acids include, for example, hydrochloric acid, hy-. drobromic acid, phosphoric acid, p-toluenesulfonic acid, sulfuric acid, methanesulfonic acid, and the like.

An equivalent or a moderate excess of the acid, typically up to about two equivalents of the acid, based upon the amic acid, is employed. Preferably, acid is employed in conjunction with heat. and, therefore, the resulting reaction mixture is heated to a temperature of from about 50C. to about l()0C., and the decomposition of the complex is permitted or proceed. Depending upon the relative temperature which is employed, the reaction typically will be completed within from about 5 to about l0 minutes. When an acid is employed. the free 7-amino cephalosporin in the form of its acid addition salt is thereby produced and is recovered in accordance with known techniques.

It is also possible to form a 7-acylamido cephalosporin "by subjecting the hydrazine reaction mixture to treatment with an acyl halide which contains an acyl function which, in combination with the 7-amino cephalosporin, will form the desired acylamido function. The use of an acyl halide obviates any necessity for heat or acid treatment to decompose the diketophthalazine complex, should such have formed, since the acyl halide itself is sufficiently acidic to accomplish the necessary decomposition. Any of the typical acyl fune' tions can be thereby introduced into the 7-position of the cephalosporin molecule simply by selection of the appropriate acyl halide, preferably the corresponding acyl chloride. The resulting 7-acylamido cephalosporin can be readily recovered by techniques well recognized in the art.

Typical acyl halides, each of which can be employed to produce the ultimate 7-acylamido product, are those of the formula in which Y is a halogen, such as chlorine, bromine, or iodine, and R, is 7 C to C -alkanoyl;

C to C -ehloroor bromoalkanoyl;

azidoacetyl;

cyanoacetyl;

2-syndrone-3-C to C -alkanoyl;

in which m is zero, 1, or 2;

i in which each Q is hydrogen or methyl. and Ar is 2- thienyl. "3-thienyl. 2-furyl. 3-furyl; 2-pyrrolyl, 3-.

pyrrolyl. phenyl. or phenyl substituted with chlorine.

bromine. iodine. fluorine. trifluoromethyl. hydroxy. C;

to C -alkyl. C, to C -alkyloxy, eyano. or nitro'. g

Ar-XCH C(O in which X is oxygen or sulfur. and Ar is as defined above; or Ar is 4-pyridyl and X is sulfur; or

or the enamine from methyl acetoacetate or acetylacetone; OH; or OH protected by esterification'with a C, to C,;-alkanoic acid: C OOH. or CQOH protected by esterification with a C, to C.;-alkanol; -N;,: CN. or C(O)NH:.

The cephalosporin used as starting material in the process-of this invention has the following fomiula:

R, in the above formula as well as in the various products of the process of this invention denotes a carboxy protecting groupl'lhe nature of the carbosy protecting group is not important. and any of those recognized in the art can he used. Preferably. however. this group is the residue of an ester function which is removable by acid treatment or by hydrogenation. Preferred carbosy pivaloyloxymethyl, 'propionoxymethyl, phenacyl, pchlorophenacyl. p-bromop'henacyl, and the like.

Highly preferred ester residues are t-butyl. benzyl, p-nitrobenzyl. I p-methoxybenzyl, benzhydryl. and 2.2.2-trichloroethyl; most preferably. the ester residue is' p -nitrobenzyl.

In the above formula. the 7-position of the cephalosp'orin contains'a 2-carboxybenzamido group (typically derived from a phthalimido'group) or a maleamido P- i I Thus. the process of this invention proceeds stepwise from a '7-( 2 carboxybenzamido)- to an unisolated mixed anhydride to a 7-aminoor 7-acylamidocephalosporin. I

It is also possible to begin the process of this invention with maleamido cephalosporin. This step-wise sequence includes conversion of 7-maleamidoto an unisolated' mixed anhydride to a 7-aminoor 7- position will contain one of the following: methyl. ace- The following are representative of the product conversions which are available in accordance with the process ofv this invention. it will be understood. however. that the ratio of products may vary depending upon the particular reactants which are employed. the relative quantities of reactants. and the conditions of reaction.

Methyl 7-( 2-carboxybenzamido )-3-methyl-3- cephem-4-carboxylate to methyl 7-amino-3-methyl-3- ccphem-4-carboxylate.

2.2.2'-'l'richloroethyl' acetosymethyl-3-cephem-4-carboxylate to trichloroethyl 7-amino-3-acetoxymethyl-3-cephem-4- carboxylate.

p-Nitrobehiyl 7-( Z-carboxybenzamido )-3- methoxymethyl-3-cepl'iem-4-carboxylate to pnitrobenzyl 7-amino-3 methoxymethyl-3-cephem 4- car'boxylate.

Benzy'l 7-(2-carboxybenzamido)-3- methylthiomethyl-3cephem-4-carboxylate to benzyl 7-amino-3-methylthiomcthyl-3-ccphem-4-carbosylate.

Benzhydryl 7-( l-ca'rboxybenzamido )-3-( S-methyll .3.4-tl'-iadiazol-2-yl )thiom'ethyl- 3cephem-4- carboxylate to ben'zhydryl 7-amino-3-( 5-methyl-l.3.4- thiadiazol-l-yl )thiomethyl-3-cephem4-carbosylate.

t Butyl 7-( Z-carboXybenZ'amido )-3-( I methyl-l H- tetmzol-S-yl )thiomethyl-3-cephem-4-carboxylate to t-hut'yl 7-amino-3-( l-methyl-l H-tetrazol-S- yl )thiomethyl3-cephem-4-carboxylate.

p-Nitrobenayl V 7-( Z-caibosybenzamido )-3- acetoxymethyl-3-cephem-4 carboxylate to pnitrobenzyl 7amino-3-acetoxymethyl-3-cephem-4- carboxylatc. V V

psNit'robenzyl 7 Z-czirboxybenzamido )-3-methyl-3- cephcm-4-carboxylate I to p-nitrob'enzyl 7-amino-3- methyl-3cephem-4-carhoxylate.

7-( 2-carboxybenzamido )-3- tion of the 7-amino function. cleavage of the ester function by known techniques. Deesterification can be achieved by treatment of the ester with an acid such as trifluoroacetic acid. hydrochloric acid. and the like. or with zincv and acid. such as formic acid. acetic acid. or hydrochloric acid. It can likewise be accomplished by hydrogenating the ester in the presence of palladium. rhodium. or a compound thereof. in suspension or on a carrier such as barium sulfate. carbon. alumina. or the like.

The following examples are provided to further illustrate this invention. It is not intended that this invention be limited. in scope by reason of any of these examples.

tone and 20 ml. of water. 5 g. (0.05 mol.) of KHCO were slowly added. The resulting solution was evaporated to dryness. and 38 ml. of DMF. and 5 ml. of

methyl iodide were added to the residue. The mixturewas stirred for 3 hrs. at room temperature. To this mixture were then added I g. of ice. and the resulting solid product was filtered. The product was crystallized from a mixture of I00 ml. of 2-propanol and I00 ml. of acetone. Yield: 7.9I of crystals. m.p. l8.7-I88; ir (CHCl I790 and l735 cm". nmr (CD0 8 2.31 (s.

3. CH3). 3.0 and 3.75 (ABq. 2. .I=l5 Hz). 3.85 (s. 3.

CH 515 (d. l. J=-4.4 Hz). 5.74 (d. I. .I=4.4 Hz) and 7.73 (m. 4. ArH).

Anal. calcd. for C H N O S: C. 56.98: H. 3.94; N. 7.82: S. 8.95. Found: C. 56.75; H. 3.66; N. 7.53; S. 8.8966.

PREPARATION B.

t-Butyl 7-phthalimido-3-methyl-3-cephcm-4carhoxylate.

A mixture of I3.76 g. mmol.) of 7-phthalimido- 3-mcthyI-3-cephem-4-carhoxylic acid. l0 ml. of cone. HQ SO I00 ml. of dry dioxane and 50 ml. of liquid isobutylene was stirred at room temperature in a sealed pressure bottle and then poured into an excess of ice phthalic anhydride and 25 ml. of benzene was refluxed for 2 hrs. using a Dean-Stark water collector. The solution was cooled, washed with NaHCO 1.68 g. in 20 ml. of H O).'water. and brine. and then dried. The solvent was evaporated to give 1.22 g.- of a neutral product. The product was chromatographed over silica gel using a gradient mixture of benzene and ethyl acetate. Fraction 54-87 gave 330 mg. of the phthalimido compound which was recrystallized from dichloromethane/ether; prisms. m.p. l76l78; [aj 43.4 (MeCN); ir (CHCI I800. 1785 and I735 cm;

EtOH 260 my. (t=|0,000 nmr.(CDCl,,) a 1.55 (s, 9. t-Bu); 2.l (s, 3. CH 3.5 (s. 2. CH 4.9 and 5.3 (ABq. 2. .l =l4 Hz); 5.1 (d. l, .l Hz); 5.82 (d. I.

4.5 Hz). and 7.82 (m, 4. ArH).

Anal. calcd. for: C H N O S: C, 57.63; H. 4.84; N, 6.1 I; O. 24.43; S. 6.99. Found: C. 57.56; H. 4.60; N. 6.3 I; O. 24.60; S. 6.90%.

After removal of the neutral product the aqueous portion was acidified to pH 3.6 and the acid mixture was extracted with ethyl acetate. Evaporation of the ethyl acetate gave 2.9 g. of t-butyl 7-(2- carboxybenzamido) cephalosporanate. This material was dissolved in ml. of benzene. l5 mg. of imidazole was added. and the mixture was refluxed for 30 min.. 7 using a Dean-Stark water collector. After work up prowere heated forv only 15 min.. about I mg of the phthalimido compound and about 4.34 g. of the phthacold aqueous NaHCO (44 g. Extraction ofthe resulting mixture with ethyl acetate and evaporation of the solvent gave a crude ester which was crystallized from C HC I The first crop gave 3.34. g. of crystals. m.p. I89-I9'I. and the second crop 'I.'72 g.. m.p. l8l-"I83: (ab. 77.7" (MeCN); ir (CHCM I800. I785. and I735 cm: nmr (CDCl 8 1.55 (.v. 9. t-Bu); 2.23 (s. 3. CH;.). 3.05 and 3.6 (ABq. 2. J l6 Hz).'5. l. (d. l.'.] 4.5 Hz). 5.72 (d. I. J 4.5 Hz) and 7.8 (m. 4. ArH

Anal. calcd. for: C ..,H. ..,N- -O -,S: C. 59.99.; H. 5.03; N.

Found: C. 60.27: H. 4.9lz" N.

7.00;:0. I998: 5. 8.0L 7.04: O. 20.06: S, 7.74%.

Iamic acid compoundwould have been obtained.

PREPARATION o.

p-Methoxyh enzyl 7-phthalimido-3-methyI-3-cephern-4-carboxylate.

To a suspension of I3.4 g. (38 mmol.) of 7- phthalimido-3-methyl-3-cephem-4-carboxylie acid in 20 ml. of dioxane and 10 ml. of water were slowly added 3.8 g. of KHCO;.. The solution was evaporated to dryness. and' I00 ml. of DMF and 8.8 g. of pmethoxybenzyl bromide were addedto the potassium salt residue. The mixture was stirred for 2 hrs. and then poured onto 200 g. of ice. The resulting mixture was extracted twice with ethyl acetate. The extract was washed with water and brine. dried. and the solvent was then evaporated. The residue was recrystallized from ethyl acetate. Yield: 4.I g. of large crystals. m.p. ll8-I2I: second crop I.8 g.; lu],,'+ 41.2" (MeCN). ir ((HCLn I800. I785. I745 and I735 cm". nmr (CDCl 6 2.l5 (x. 3. CH 3.0 and'3.7 (AM. 2.! 151-11.). 3.8 .t. 3. CH3). 5.! I' 1. I. .l 4.5 Hz). 5.28

' 2. CH 5.75 (d. l. .l 4.5 Hz). 6.8-7.8 (m. 8).

Anal. calcd. for: C H b1 0 8: C. 62.06; H. 4.34; N. 6.03: O. 20.67: S 6.90. Found: C. 6215; H. 4.3l; N. (1.32; 0. 20.88; s. 6.82%.

mmol.) of Na S.9H O and 32 ml. of ice water. After 7 minutes at C., ml. of 1N HCl were added to the mixture. The volume was reduced in vacuo to about 40 ml.. and the resulting aqueoussolution was washed with ethyl acetate. The pH of the aqueous layer was adjusted to 4.5 with 1N HCl and then was extracted with ethyl acetate (40 ml.). The ethyl acetate layer was washed with brine (30 ml.), dried over MgSO and evaporated to give 1.8- g. of methyl 7-(2- carboxybenzamido)-3-methyl-3-cephem-4-carboxylate as an amorphous colorless solid. Recrystallization from acetone gave an analytical sample; m.p. l82184.5 (dec.); ir (KBr) 1768., 1630, 1610 (shoulder) and 1665 cm; nmr (CDCl DMSO 2.08 (s. 3. CH 3.12 and 3.52 (ABq, 2. .1 HZ). 3.8 (s, 3. CH ester). 5.06 (d,

l, J 4.5 Hz), 5.86 (dd, 1, J 4.5 and .I 8.0 Hz) and 7.4-8.08 (m, 4. ArH).

Anal. calcd. for C H N O S: C, 54.25; H. 4.28; N. 7.44; S, 8.52. Found: C. 53.98; H, 4.18; N. 7.73; S.

The pH of the aqueous layer from above was lowered to pH 2.5 with 1N HCl and then was extracted with ethyl acetate (2 X 30 ml.). The ethyl acetate extracts were combined. washed with brine (30 ml.) and dried over MgSO The ethyl acetate was evaporated in vacuo during which time a colorless crystalline product crystallized. when the volume had been reduced to ca. 10 mL. the solution was filtered giving 190 mg. (6.571 of 7( 2-ca rboxybenzamido )-3-methyl-2-cephem-4- carboxylic acid; m.p. 196-198 (dec.); ir (KBr) 1773. 1700. and 1658 cm; nmr (DMSO 1.88 (s. 3. CH 4.64 (s. l. C -H). 5.15 (d. l. .I'=4.0 Hz). 5.5 (dd. 1. J 4.0 and 8.0 Hz).'6.l5 (s, l. C. ,H) and 7.4- 8.06 (m. 4. ArH).

Anal. calcd. for C H N O S: C. 53.03; H. 3.89; N. 7.73; S. 8.85. Found: C. 52.76; H. 3.85; N. 7.68; S. 8.77.

Evaporation of the filtrate from above gave an additional 600 mg. of methyl 7-(2-carboxybenzamido)-3- methyl-3-cephem-4-carboxylate; total yield-8092.

EXAMPLE 2 Methyl 7-amino-3-methyl-3-cephem-4-carboxylate hydrochloride To a suspension of methyl 7( 2-carboxybenzamido)- 3-methyl-3-cephem-4-carboxylate (752 mg.. 2 mmol.) in 35 ml. of tetrahydrofuran at 0C. was added triethylamine (0.28 ml., 2 mmol.). After minutes. ethyl chloroformate (0.2 ml.. 2 mmol.) was added. and then after one-half hour at 0C.. anhydrous hydrazine (0.07 ml. 2.2 mmol.) was added to the reaction mixture. After 15 minutes, the mixture was filtered, and the filtrate was evaporated in vacuo to dryness. The crude product was taken up in ml. of chloroform. reluxed for 90 minutes, and then allowed to stir at about C. overnight. Filtration gave 180 mg. of diketophthalazine (m.p. 340343). The filtrate was evaporated to dryness in vacuo. The resulting crude product was taken up in 3 ml. of 1N HCl plus 2 ml. water and washed with ethyl acetate (2X7 ml.). Evaporation of the aqueous layer in vacuo gave a yellow amorphous solid which was recrystallized from ethanol/diethyl ether to give 115 mg. of methyl 7-amino-3-methyl-3-cephem-4- carboxylate hydrochloride.

EXAMPLE 3 t-Butyl 7( 2-carboxybenzamido )-3 -acetoxymethyl-3 -cephem- 4-carboxylate A solution of 458 mg. l mmol.) of t-butyl 7- phthalimido-3-acetoxymethyl-3-cephem-4-carboxylate in 10 ml. of THF was cooled in an ice water bath, and 1.1 ml. of 1N NaOH were then added. After stirring for 5 min.. 10 ml. of water and 30 ml. of ethyl acetate were added. The ethyl acetate layer was separated. and mg. of starting material were recovered therefrom. The aqueous layer was acidified to pH 4.0, and the acidified layer was extracted with ethyl acetate. After workup, 330 mg. (83% of the desired phthalamic acid were obtained [at 1 26.37 (MeCN); EtOH 260 my. (a 8800)"; ir (CHCl 1785, 1730 and 1685 cm; nmr (CDCLQ 8 1.55 (s. 9. t-Bu). 2.05 (S. 3. Ac). 3.3 and 3.6 (ABq, 2. .1 =17 Hz). 4.72 and 5.2 (ABq, 2. .1 =14 Hz). 4.98 (d. l. .1 =4.5 Hz). 5.9 (dd. 1. .1 4.5 and 9 Hz). and 7.5-8 (m. 4. ArH).

EXAMPLE 4 t-Butyl 7-amino-3-acetoxymethy1-3-cephem-4-carhoxylate In accordance with the procedure of Example 2. tbutyl 7( 2-carhoxybenzamido )-3-acetoxymethyl-3- cephem-4-carboxylate is converted to t-hutyl 7-amino- 3-acetoxymethyl-3-cephem-4-carhoxylate hydrochloride. which is converted to the free amine by treatment with NaHCO and extraction with chloroform. ir and nmr spectra are in agreement with an authentic sample prepared according to the method of R. .l Stedman. J. Med. ('Iwm.. 1966) p. 444.

EXAMPLE 5 t-Butyl 7( 2-carhoxybenzamido )-3-methyl-3-cephem-4- carboxylate A solution of 800 mg. (2 mmol.) of t-hutyl 7- phthalimido-3-methyl-3-cephem-4-carboxylate in 25 ml. of tetrahydrofuran and 8 ml. of water was cooled in an ice bath. To the solution 660 mg. of Na S.9H. ,O'

were added. and the mixture was-stirred and cooled for 10 min. At the end of this period 10 ml.,of water were added. and the mixture was extracted with 40 ml. of ethyl acetate. The extract was discarded. The aqueous portion was acidified to pH 4.3 with 1N H 50 and then extracted with ethyl acetate. The ethyl acetate extract was dried and evaporated. giving 700 mg. of the title compound. The product was recrystallized from chloroform/cyclohexane; m.p. l78'l79; ir (nujol) 1770, 1735. and 1680 cm: nmr (CDCl DMSO,,.;)

8 1.5 (s. 9. t-Bu). 2.1 (s. 3. CH 3.2 and 3.5 (ABq.' 2. .1 =18 HZ). 5.02 (d. 1. .1 =45 HZ). 5.82 (dd. 1. .I

4.5 9H2). and 7.4-8 (m. H. ArH).

Anal. calcd. for C H N O sz C. 57.40; H. 5.30; N. 6.69; O. 22.94 and S. 7.66. Found: C. 57.70: H. 5.20; N. 6.52; O. 22.72 and S. 7.53%.

The identical (nmr. ir. m.p.) substance also can be obtained in 94% yield from phthalic anhydride and the t-butyl ester of 7-ADCA.

EXAMPLE 6 t-Butyl 7 amino-3-methyl-3-cephem-4-carboxylate In accordance with the procedure of Example 2. t

' methoxybenzyl EXAMPLE 7 p-Me'thoxybe'nzyl V '7-( 2-carboxyben7amido )-3 methyl-3-'cep hem-4- v A r carboxylate v A solution of 930 mg. (2 mmol.) of p-methoxybenzyl 7-phthalimido-3-methyl-3 cephem-4-carboxylate in 25 ml. of THF-and 8 ml. of water was cooled in an ice water bath. and 660 mg. of"Na- ,S.9H. .O were then added. The mixture was stirred for IS min.. and ml.

of water and 40 ml. of ethyl acetate were added. The layers were separated. and I40 mg. of a neutral material' wcre obtained from the ehtyl acetate layer. The

aqueous layer was acidified to pH 4.3 with lN H 50 and extracted twice with ethyl acetate. The ethyl acctate extract was washed. dried. and evaporated to give 660 mg. 68% of the phthalamic acid as an amorphous solidsldln 85.6'(MeCN); ir (CHCl l78l. 1740 and 1710 cm"; nmr (CDCl;,) 8 2.08 (x. 3. CH 3.] and 3.43 (ABq. 2. .l 17 Hz). 3.79 (s. 3. CH 5.0 (d.

1. .I 4.5 Hz). 5.1 2, CH2). 5.x (dd. 1; J 4.5 Hz);

-Ana l. calcd. for C H. ,N- ,O;S; C. 59.74; H. 4.60:, N. 5.8l; O. 23.21: S. 6.65. Found: C. 59.8l; H. 4.32; N.

EXAMPLE 8 p-Methoxybenzyl 7-amino-3-methyl-3-cephem-4-carboxylate. p-toluene sulfonic acid salt ln accordance with the procedure of Example 2. pmethosyhenzyl 7-( 2-carboxybenzmido )-3-methyl-3- cephcm-4-carboxylate' is converted to p- 7-amino-3-methyl-3-cephem-4- carboxylatc, p-toluenesulfonic acid salt.- using p-toluenc'sullonic acid monohydrate. instead of hydrochloric acid as in Example 2. This material is identical with the salt described by C hauvette et. al.. J. Org. (71mm. 36. 1265 I97] EXAMPLE 9 4 p-Nitrobenzyl 7-( Z-carboxybenzamido )-3-methyl3-cephem-4- carboxylate Toan ice cooled solution ol'480 mg. 1 mmol.) of pnitrobenzyl I 7-phthalimido-3-methyl-3-cephem-4- carboxylate in 25 ml. of tctrahydrofuran and ml. of

waterwere added 340 mg. of sodium sulfide (N11 59- H o The mixture was stirred at pH I I5 for 7.min.. arid 40 ml. of ethyl acetate and I0 ml. of water were added. The layers were separated. and the. organic layer was washed with 5 ml. of water and 5 ml. of brine to give l50 mg. of a neutral material. The aqueous layer was acidified to pH 4.5 with lN sulfuric-acid. and the resulting emulsion was. extracted with ml. of ethyl acetate. The extract was washed with brine. dried and evaporated to dryness to give 290 mg. of the crude product. from which I50 mg. (309'?) of pure product was obtained. 7

carboxylate.

' The same compound also can be prepared by the a]- temate procedure of refluxing phthalic anhydride and the p-nitrobenzyl ester of 7-ADCA in acetontrile for min.

A sample was recrystallized from dioxane/water. and

colorless crystals were obtained melting at l92l93;

nmr (DMSO-d..) 8 2.04 (s. 3QCH3). 3.35 and 3.68 (ABq,2. .l 18 Hz). 5.2 (d, l, .l 4 Hz. .H6). 5.4 (.s. 2. CH2). 518((1, d. l. J =4.5 and 9 Hz), 8 (m, 8 Ari-I).

Anal. calcd. for C H N O S: C, 55.53; H. 3.85; N, 8.45; 0.25.73; S. 6.45. Found: C. 55.67; H. 3.94; N, 8.49; O. 25.89; S. 6.47%.

The subsequent acidification of the aqueous solution to pH 2.0 followed by ethyl acetate extraction produced 120 'mg. of p-nitrobenzyl 7-(2- carboxybenzamido )-3-methyl-3-cephem-4 EXAMPLE l0 p-Nitrobenzyl 7-arnino-3-methyl-3-cephem-4-carb xylate,p-toluene sulfonate salt A solution of 1.0 g. (2 mmol.) of p-nitrobenzyl 7-( 2- carboxybenzamido )-3-methyl-3-cephem-4-carboxylate in 30 ml. of dry tetrahydrofuran was cooled in an icesalt bath. and-0.28 ml. (2mmol.) of triethylamine and 0.20 ml. (2 mmol.) of ethyl'ehloroformate were added. After'cooling' and stirring for 20 min.. 0.15 ml. of 85.71

hydrazine hydrate was added. and stirring was continv ued for 10 min. The Et;;N'.HCl salt was filtered. and the filtrate was evaporated todryriess. The residue was dissolved in a mixture of 25 ml. of ethyl acetate and 10 ml; of water. The organic extract was separated from the aqueous and washed with NaHCO solution. water and brine. After drying. the solvent was evaporated. The residue was dissolved in l0 ml. of acetonitrile. and the solution was refluxed for 50 min. and then cooled. To. this solution 380 mg. of p-toluenesulfonic acid hydrate and 2.5 ml. of water were added. the precipitate (160 mg.) was filtered. and most of the acetonitrile was evaporated from the filtrate. Upon cooling and scratching. crystallization began. Two hours later 680 mg. (63% of the title compound were collected. The purity of product was tested by thin-layer chromatography (tlc) using silica plate and MeOHtBenzene (1:3) system. A sample was recrystallized from methanol-ether. m.p. l-l 74 decfl. nmr (DMSO 8 2.20 S.3.CH., 2.30 (s.3.CH;,). 3.6 '.\'.2.SCH 5.22 (s.2.CHl ester). 5.4 (.sylazetidinone 'Hs). and 7.1-8.2. ("1.8 ArHs); ir (KBr) l780 ('azetidinone CO) and. l730 cmester Anal. calcd for: C H ,N;,O,.S. (50.66: H. 4.45:, N; 8.06; S. l2.30. Found: C. 5l.()3: H. 4.27; N. 819: S.

l L) l7r.

EXAMPLE l l p- Nitrobenzyl 7phenylacetamido-3 methyl-3-cephem-4-carboxylate To a suspension of p-nitrobenzyl 7-( 2- carboxyben'lamido )-3-methyl-3-cephem-4-carboxylate (-162 mg... l mmol.) in tetrahydrofuran at 0C. is added triethylamine (0.14 ml.. 1 mmol.). After 15 minutes.

ethyl chloroforrnate (0.l ml.. 2 mmol.) is added. and then after 0.5 hourat 0C.. anhydrous hydrazine (0.03 ml.. l mmol.) is added to the reaction mixture. After 15 minutes. the mixture is filtered. and the filtrate is evaporated to dryness in vacuo. The residue is taken up in acetone ml.) and tetrahydrofuran (15 ml.), and phenylacetyl chloride (0.l3 ml., 1 mmol.) is added.

After refluxing for 30 min., the mixture is cooled and i evaporated in vacuo to dryness. The product is taken EXAMPLE l2 7 t-Butyl 7-( 2 '-carboxy )acrylamido-3-acetoxymethyl-3-cephem- 4-carboxylate A solution of 656 mg. (2 mmol. of t-butyl 7-amino- 3-acetoxymethyl-3-cephem-4-carboxylate and 196 mg. (2 mmol.) of maleic anhydride in ml. benzene was refluxed for one-half hour, cooled, and evaporated in vacuo to dryness. Tlc indicated no starting material and one slow moving product: nmr (CDCl 94 (s, 9, t-Bu), 127 (s, 3, OAc), 207 and 216 (ABq, 2, J=20 Hz), 293 and 307 (ABq, J=l4.0, CH OAc), 305 (l, d, J=4.5, azetidinone H), 350 (l, q, Jflj and 8.0 Hz, azetidinone H), 390 (2H, q, J=l2 and 2.0 Hz), 534 (1, d, J=8.0, NH), and 8061-12 (1, broad s, COOH).

EXAMPLE l3 t-Butyl 7-amino-3-aeetoxymethyi-3-cephem-4-carboxylate EXAMPLE l4 Benzyhdryl 7-( 2-carboxybenzamido)3-( S-methyll ,3,4-thiadiazol- Z-yl )thiomethyl-3-cephem-4-carboxylate A solution of benzhydryl 7-phthalimido-3-(S-methyl- 1,3 ,4-thiadiazol-2-yl )thiomethyl3-cephem-4- carboxylate (2 mmol.) in ml. of THF and 8 ml. of water is cooled in an ice-water bath, and 660 mg. of Na S.9H O are then added. The mixture is stirred for 15 minutes, and 10 ml. of water and 40 ml. of ethyl acetate are added. The layers are separated, and a neutral material is obtained from the ethyl acetate layer. The aqueous layer is acidified to pH 4.3 with 1N sulfuric acid and is extracted twice with ethyl acetate. The ethyl acetate extract is washed, dried, and evaporated to give the title compound.

EXAMPLE l5 Benzhydryl 7-amino-3-( S-methyll ,3,4-thiadiazol2-yl thiomethyl-3-cephem-4-carboxylate A solution of benzhydryl 7-(2-carboxybenzamido)3- (5-methyl-l ,3 ,4-thiadiazol2=yl )thiomethyl-3-cephem- 4-carboxylate (2 mmol.) in 30 ml. of dry tetrahydrofuran is cooled in an ice-salt bath, and 0.28 ml. (2 mmol.) of triethylamine and 0.20 ml. (2. mmol.) of ethyl chloroformate are added. After cooling and stirring for 20 minutes, 0.106 ml. (2 mmol.) of N- methylhydrazine in 5 ml. of tetrahydrofuran is added. Stirring is continued for about 10 minutes. The Et N.HCl salt is filtered, and the filtrate is evaporated to dryness. The residue is taken up in about 15 m1. of chloroform, and the mixture is allowed to stand at room temperature for about one hour during which time methylphthalhydrazide (m.p. 243-245C.) precipitates. Filtration and evaporation in vacuo of the fil trate gives crude benzhydryl 7-amino-3-(5-methyll ,3,4-thiadiazol-2-yl )thiomethyl-3-cephem4- carboxylate.

EXAMPLE l6 2,2,2-Trichloroethyl 7-( Z-carboxybenzamido )-3-( l-methyll tetraxol-5- yl )thiomethyl-3-cephem-4-carboxylate,

In accordance with the procedure of Example 14. 2,2,2-trichloroethyl 7 -phthalimido,3-.( 1 -methyl- 1 H- tetraZol-S-yl)thiomethyl-3-cephem4rcarboxylate is converted to 2,2,2-trichloroethyl I carboxybenzamidoJ-B -(l-methyl l H-tetrazol-S- yl )thiomethyl-3-cephem-4-carboxylate.

' EXAMPLE 17 that in which R and R are hydrogen, or R and R taken together with the carbon atoms to which they are attached represent an orthophenylene ring; R is a carboxy protecting group; and R is hydrogen, acetoxy, methoxy, methylthio, (5- methyl-l,3,4-thiadiazol-2-yl)thio, or l-methyllH-tetrazol-S-yUthio; which comprises the steps of l. reacting said 7-(amic acid) cephalosporin with an alkyl chloroformate in the presence of a tertiary amine to form the corresponding mixed anhydride having the formula in which R,, is an alkyl group having from 1 to 4 carbon atoms;

2. reacting the product mixture from the aforementioned chloroformate treatment with a hydrazine of the formula about 20C. to about +5C.

3. Process of claim 2, in which the alkyl chloroformate is ethyl chloroformate.

4. Process of claim 1, in which the product mixture from the chloroformate treatment is first reacted with up to I equivalent of the hydrazine per equivalent of the amic acid at a temperature of from about -l0C. to about room temperature for from about 1 to about 10 minutes to produce the corresponding 7-amino cephalosporin.

5. Process of claim 1, in which R is C,-C alkyl, 2,2.2-trichloroethyl, benzyl, p-nitrobenzyl, pmethoxybenzyl, benzhydryl, C C alkanoyloxymethyl. phenacyl, or p-halophenacyl.

6. Process of claim I, in which R and R,, are hydrogen.

7. Process of claim I, in which R and R taken together with the carbon atoms to which they are at tachcd represent an ortho-phenylene ring.

8. Process of claim 1, in which R: is p-nitrobenzyl.

9. Process of claim 1, in which R is hydrogen.

10. Process of claim 1, in which R; is acetoxy.

11. Process of claim 1, in which R is (5methyl- 1,3,4-thiadiazo-2-yl )thio.

12. Process of claim 1, in which R is (l-methyl-l H- tetrazol-5-yl)thio.

Claims (17)

1. A PROCESS FOR CLEAVING THE AMIC ACID FUNCTION OF A 7(AMIC ACID) CEPALOSPHORN HAVING THE FORMULA

1. REACTING SAID 7-(AMIC ACID) CEPHALOSPORIN WITH AN ALKYL CHLOROFORMATE IN THE PRESENCE OF A TERIARY AMINE TO FORM THE CORRESPONDING MIXED ANHYDRIDE HAVING THE FORMULA

2. REACTING THE PRODUCT MIXTURE FROM THE AFOREMENTIONED CHLOROFOMATE TREATMENT WITH A HYDRAZINE OF THE FORMULA

2. reacting the product mixture from the aforementioned chloroformate treatment with a hydrazine of the formula R3HNNHCH3 in which R3 is hydrogen or methyl; and

2. Process of claim 1, in which the amic acid cephalosporin is reacted with an alkyl chloroformate in the presence of a tertiary amine at a temperature of from about -20*C. to about +5*C.

3. Process of claim 2, in which the alkyl chloroformate is ethyl chloroformate.

3. recovering the corresponding 7-amino cephalosporin from the reaction mixture of the aforementioned hydrazine treatment.

3. RECOVERING THE CORRESPONDING 7-AMINO CEPHALOSPORIN FROM THE REACTION MIXTURE OF THE AFOREMENTIONED BYDRAZINE TREATMENT.

4. Process of claim 1, in which the product mixture from the chloroformate treatment is first reacted with up to 1 equivalent of the hydrazine per equivalent of the amic acid at a temperature of from about -10*C. to about room temperature for from about 1 to about 10 minutes to produce the corresponding 7-amino cephalosporin.

5. Process of claim 1, in which R1 is C1-C4 alkyl, 2,2,2-trichloroethyl, benzyl, p-nitrobenzyl, p-methoxybenzyl, benzhydryl, C2-C6 alkanoyloxymethyl, phenacyl, or p-halophenacyl.

6. Process of claim 1, in which R and Ra are hydrogen.

7. Process of claim 1, in which R and Ra taken together with the carbon atoms to which they are attached represent an ortho-phenylene ring.

8. Process of claim 1, in which R1 is p-nitrobenzyl.

9. Process of claim 1, in which R2 is hydrogen.

10. Process of claim 1, in which R2 is acetoxy.

11. Process of claim 1, in which R2 is (5-methyl-1,3,4-thiadiazo-2-yl)thio.

12. Process of claim 1, in which R2 is (1-methyl-1H-tetrazol-5-yl)thio.