AU607937B2 - 7-substituted cephalosporin compounds - Google Patents

Description

Australia Form PATENTS ACT 1952 COMPLETE SPECIFICATION

(ORIGINAL)

FOR OFFICE USE Short Title: Int. CI: 607937 Application Number: Lodged: Complete Specification-Lodged: Accepted: Lapsed: Published: Priority: This donument contains the amendnents made und'i Section 49 ard is correct for Spribl ing_. i Related Art: TO BE COMPLETED BY APPLICANT GLAXO GROUP LIMITED S-Name of Applicant: Address of Applicant: 0 Actual Inventor: Clarges House, 6-12 Clarges Street, London W1Y 8DH, United Kingdom.

CHRISTOPHER EARLE NEWALL and BRIAN EDGAR LOOKER.

CALLINAN AND ASSOCIATES, Patent Attorneys, of Address for Service: 48-50 Bridge Road, Richmond, State of Victoria, Australia.

Complete Specification for the invention entitled: f T'M "7 P A r r II S The'fo, opwing statement is a full description of this invention, including the best method of performing it known to V' Note he description is to be typed in double spacing, pica type face, in an area not exceeding 250 mm in depth and 160 mm in width, on tough white paper of guod quality and it is to be inserted inside this form.

Tsrl i L ~C j i -L L I RESEARCH LIMITED.

re Deltc. kr K44, rJmti@ 4. 'he basic applicationsreferred to in paragraph 2 is Dec ti was the first AppJication. applicationsmade in a Convention country in respe th inve o the subject of the application. I Inert plta an date of Declared at this da 19 SIGN Newsam l j la This invention relates to improvements in or relatinq to cephalosporins. More particularly it relates to new cephalosporin compounds and derivatives thereof having valuable antibiotic activity.

The cephalosporin compounds in this specification are named with reference to 'cepham' after J. Airier. Chem. Soc., 1962, 84, 3400, the term 'cephem' referring to the basic cepham structure with one double bond.

Cephalosporin antibiotics are widely used in the treatment of diseases caused by pathogenic bacteria in human beings and animals, .oOo and are especially useful in the treatment of diseases caused by bacteria which are resistant to other antibiotics such as penicillin 0 0 0 0 compounds, and in the treatment of oenicillin-sensitive patients. In 000o o 0° 0 many instances it is desirable to employ a cephalosporin antibiotic .o°o13 which exhibits activity against both Gram-positive and Gram-negative o o°n microorganisms, nd a significant amount of research has been directed 0 000 to the development of various types of broad spectrum cephalosporin antibiotics.

0oo Thus, for example, in our British Patent Specificationi No.

o02 0 1399086, we describe a novel class of cephalosporin antibiotics containing a 7p-(a-etherified oxyimino)acylamido side chain, the oxyimino group having the syn configuration. This class of antibiotic compounds is characterised by high antibacterial activity against a range of Gram-positive and Gram-negative organisms coupled with particularly high stability to P-lactamases produced by various S"Gram-negative organisms.

The discovery of this class of compounds has stimulated further research in the same area in attempts to find compounds which have improved properties, for example against particular classes of organisms, especially Gram-negative organisms. This interest is reflected in the very large numbers of patent applications which have been filed relating to cephalosporin antibiotics having particular substituents both on the 70-acylamido side chain and at the 3 -position of the cephalosporin nucleus.

ATT 0 F MCIT.I*

OWN

2 For example, British Patent Specification No. 1576625 contains a qeneric definition of cephalosporin antibiotics having a 7p-(aetherified oxyimino)acetarmico side chain wherein the etherifyinq qroup is an aliphatic hydrocarbon group which may have suitable substituents (including, amongst a large number of possibilities a carboxy or protected carboxy group and a phenyl group substituted by up to three hydroxy groups), which side chain is further a-substituted by a group which inter alia may be an aminothiazolyl group. However none of the compounds specifically exemplified contains a carboxyphenylalkyl etherifying group. The 3-position group may also be selected from a large number of alternatives but a hydrogen atom is not included among these.

0o00 In British Patent Specification No. 1604971 a wide variety of o000 cephalosporin antibiotics are generally disclosed in which the o 0 015 7p-position side chain may be selected from inter alia a 2-(2-amino- 0.

0 o 0 thiazol-4-yl)-2-(etherified oxyimino)acetamido group, in which the o oo etherifying group, amongst very many possible meanings, may be an S0o alkyl group methyl) substituted by both phenyl and carboxy, 0 000 although there is no specific exemplification of compounds having a 20 carboxyphenylalkyl group and the preferred etherifying group is stated 00 Sooo to be an unsubstituted methyl group. The 3-position group may also be o o0 selected from a large nurr.er of alternatives and possible 0 00 3-substituents within the generic definition include a hydrogen atom.

0000 "o In UK Patent Specification No. 2017702 the oxyimino etherifying group, according to the generic definition, may inter alia be an a-carboxyphenylmethyl radical. In the compounds specifically 0 0 exemplified, the carboxyphenylmethyl group is always combined with an o o acetoxymethyl group at the 3-position.

UK Patent Specification No. 2104888 generically discloses cephalosporin antibiotics in which the 7P-p-sition side chain is a aminothiazol-4-yl)-2-(etherified oxyimino)acetamido group. The oxyimino etherifying group may inter alia be a carboxyphenylmethyl group in which the phenyl group may be substituted by a hydroxy group.

The 3 -position group is an isothiazolylthiomethyl or iminoalkylidenedithiethane group.

-3- European Patent Specification No. 197409 generically discloses cephalosporin antibiotics in which the 7 -position side chain is a 2-(2-aminothiazol-4yl)-2-(etherified oxyimino) acetamido group. The oxyimino etherifying group may be inter alia a catechol carboxymethyloxyimino group. The 3-position substituent may be selected from a number of alternatives but these do not include a hydrogen atom.

We have now discovered that by the selection of a (Z)-2-(2-aminothiazol- 4-yl)-2-(etherified oxyimino)acetamido group at the 7-position in combination with a hydrogen atom at the 3-position, and by the selection of a o-carboxy substituted 0 a 10 phenylmethoxyimino group as the etherified oxyimino grouping, cephalosporin o compounds having a particularly advantageous profile of activity (described in more o detail below) against a wide range of commonly encountered pathogenic organisms 00 00 0 and/or that are of use as intermediates in the preparation of other active 0 0 0 0 00 compounds, may be obtained.

00 YS 1 Accordingly, we provide cephalosporin compounds of formula (I) 0000 0 0 0000 °ooo 0 00 0 H I o H

H

0 00 0 11 0 0 S

I

00 00 atom or a carboxyl blocking group; represents a hydrogen atom or an amino protecting group; 'v R 3 represents a hydrogen atom or an amino protecting group; -3a

R

4 and R 5 each represents a hydroxy or a formyloxy group or a group of formula

-OCOR

6 (where R 6 is a C 1 8 alkyl group), a group -OCO 2

R

6 (where R6 is as defined above), a (C 1 4 alkyl) silyloxy group, or a group [-OB(OR 7 2 or

[-OP(O)(OR

7 2 (where R 7 represents C1- 4 alkyl) or R and R 5 together represent a C1- 20 alkylidenedioxy group optionally substituted by one or more phenyl,

C

1 4 alkoxy or oxo substituents, or R 4 and R 5 together represent

-OP(O)(OR

7 (where R 7 is as defined above) or a di(C 14 alkyl) silyldioxy group; %lt e& 00 00 ace0 000 0 0 Cco oo oo 0 000 0000 00000 JITc ti7'( -4- Z is >S or >S-0 or the dotted line bridging the and 4-positions indicates that the compound is a ceph-2-em or ceph-3-em compound; and non-toxic salts and solvates (especially hydrates) thereof.

In the compounds of formula where R and/or R 2 represent carboxyl blocking groups, the blocking group may be for example the residue of an esterforming aliphatic or araliphatic alcohol or of an ester-forming phenol, silanol or stannanol (the said alcohol, phenol, silanol or stannanol preferably containing from 1 to 20 carbon atoms) or a symmetrical or mixed anhydride blocking group derived from an appropriate acid. Particular examples of R or R include t-butyl, diphenylmethyl and p-nitrobenzyl.

•Where R is an amino protecting group, the protecting group may be S for example a C7- 20 aralkyl group (for example a triphenylmethyl or o S: 4-methoxybenzyl group), an acyl group, such as an optionally substituted C1g e o S alkanoyl group (for example a formyl or chloroacetyl group) or an optionally o oe substituted C1- 6 alkoxycarbonyl group (for example a t-butoxycarbonyl or 2,2,2,-trichloroethoxycarbonyl group), or a C7- 10 aralkyloxycarbonyl group (for So example a benzyloxycarbonyl group) or a silyl group (for example a tri-methylsilyl group).

0 In general, compounds of formula in which R is a hydrogen atom O a a a are preferred.

When R or R 5 represents a (Cl_ 4 -alkyl) silyloxy group, it may represent 00 00 0 a a group such as trimethylsilyloxy or t-butyldimethylsilyloxy.

Where R 4 and R together form an alkylidenedioxy group, having 1-20 carbon atoms, it may preferably be a methylenedioxy, ethylidenedioxy or isopropylidenedioxy group which may carry one or more substituents as hereinbefore defined, for example methoxymethylenedioxy, diphenylmethylenedioxy or carbonyldioxy groups. When R 4 and R 5 together form a di(C 1 4 alkyl)silyldioxy group, that is preferably a dimethylsilyldioxy group.

5 f==examp-iez2O:09 TQ or -OP(O)(OR 7 (where R 7 j s fe above) or a cyclic silyl ether q- ,-ep-.ga di(Cl4alkyl)silyldioxy group for In general, such silyloxy, borate or phosphate groups represent protected hydroxy groups which may be cleaved to provide a compound of formula having free hydroxyl groups.

In general, R 4 and R 5 is each preferably an acetoxy group or in particular a hydroxy group.

In the compounds of formula Z is preferably >S.

Ceph-3-em compounds of the invention are particularly preferred.

Where the compound is to be used in medicine any ester of the oooo oooo carboxyl groups in the molecule should be a non-toxic metabolically 0 labile ester function. Examples of non-toxic metabolically labile .oo ester derivatives include acyloxyalkyl esters, for example, lower alkanoyloxy-methyl or -ethyl esters such as acetoxy-methyl or -ethyl Sor pivaloyloxymethyl esters, and alkoxycarbonyloxyethyl esters, for

SO

o example, lower alkoxy- carbonyloxyethyl esters such as the ethoxy, bonyloxyethyl ester.

oo addition to the above ester derivatives, it will be 0 Q oooo understood that the present invention includes within its scope the 0 o. active compounds of the invention in the form of other physiologically ooo. acceptable equivalents, i.e. physiologically acceptable compounds o o 0 °25° which like the metabolically labile esters are converted in vivo into the parent antibiotic compounds of the invention.

o 00 Non-toxic salt derivatives which may be formed by reaction of the carboxyl group present in the compounds of formula include o° inorganic base salts such as alkali metal salts sodium and potassium salts) and alkaline earth metal salts calcium salts); amino acid salts lysine and arginine salts); organic base salts procaine, phenylethylbenzylamine, dibenzylethylenediamine, ethanolamine, diethanolamine and N-methylglucosamine salts). Other non-toxic salt derivatives include acid addition salts, e.g. formed with hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, formic and trifluoroacetic acids. The salts may also be in the form of i. i, i LI 6 resinates formed with, for example, a polystyrene resin or crosslinked polystyrene divinylbenzene copolymer resin containing amino or quaternary amino groups or sulphonic acid groups, or with a resin containing carboxyl groups, e.g. a polyacrylic acid resin. Soluble base salts alkali metal salts such as the sodium salt) of the compounds of formula may be readily used in therapeutic applications because of the rapid distribution of such salts in the body upon administration. Where, however, insoluble salts of compounds are desired in a particular application, e.g. for use in depot preparations, such salts may be formed in conventional manner, for example with appropriate organic amines.

The compounds according to the invention are syn isomers. The syn isomeric form is defined by the configuration of the 0000

-OCHCOOR

2 I oooo 00 00 oo 0 o o oo 0o R 4 ooo 0 000

R

oo group with respect to the carboxamido group. In this specification, G oo the syn configuration is denoted structurally as 0 .0 0 0 0 0 00

NHR

3 ooo o0 04 S N

ONI-

o tN OCHCOOR2 II I

SR

4 o a a S7 It will be understood that since the compounds according to the invention are geometric isomers, some admixture with the corresponding anti isomer may occur.

It will further be appreciated that in the oxime etherifying group, the carbon atom adjacent to the oxy group is chiral and may therefore exist in either the R or S configuration. The invention thus includes within its scope the individual R and S forms at this chiral carbon atom as well as mixtures (including diastereomeric mixtures) thereof. In general, compounds of formula in which this chiral carbon atom has the S-confiquration and R/S mixtures in which the S-isomer predominates are preferred.

The compounds according to the present invention may exist in tautomeric forms (for example in respect of the 2-aminothiazolyl .oo group) and it will be understood that such tautomeric forms, e.g. the o,°o 2-iminothiazolinyl form, are included within the scope of the oooo00 oo invention.

0 o 0 0o 0 As indicated previously, the compounds of the invention are o o active against a wide range of commonly encountered pathogenic o 0o organisms and/or are of use as intermediates for the preparation of other active compounds. In general, when the compounds of the o invention are to be used as intermediates the groups R 1 and R 2 will 0 000 often be carboxyl blocking groups; the group R 3 will often be an amino ooo° protecting group, and the groups R 4 and R 5 will often be protected oooo hydroxy groups such as silyloxy, borate phosphate groups, or together will be a cyclic protected diol group. Non-toxic derivatives wherein R 4 and/or R 5 represent acyloxy groups such as acetoxy groups o may serve as either intermediates or as active compounds.

00 In general, active compounds of the invention will be ceph-3-em compounds of formula in which R 1

R

2 and R 3 represent hydroqen atoms,Z represents >S and R 4 and Rb which may be the same or different represent hydroxy or CI-C 4 acyloxy groups e.g. acetoxy groups.

Important active compounds according to the invention have the formula (Ia) -~iil-i m rru--- 8 NH/ 2 S N H H S _C -CONH-- II I I I N N OCHCOOH 0 H 00H 7T i (Ia) (la)

R

a

R

wherein R4a is a hydroxy or acetoxy group; 00 R 5 a is a hydroxy or acetoxy group; o 015 and the non-toxic salts, solvates, hydrates and metabolically labile o esters thereof.

00o A particularly preferred compound of the invention is: .00 (6R,7R,2'Z,S)-7-[2-(2-aminothiazol-4-yl)-2-[(carboxy)(3,4-dihydroxy- 00 0 .oo phenyl)methoxyimino]acetamido]ceph-3-em-4-carboxylic acid; and the non-toxic salts, solvates, hydrates and metabolically labile esters oo thereof.

o t' o on° We have discovered that (6R,7R,2'Z,S)-7-[2-(2-aminothiazol- 0 00 0° o0 4-yl)-2-[(carboxy)(3,4-dihydroxyphenyl)methoxyimino]acetamido]ceph-3- 0.oo em-4-carboxylic acid can advantageously and consistently be prepared 0025 and isolated with a high degree of purity in crystalline form and this forms a further aspect of the invention. In particular we have o. prepared and isolated crystalline hydrated "0 aminothiazol-4-yl)-2-[(carboxy)(3,4-dihydroxyphenyl)methoxyimino]o acetamido]ceph-3-em-4-carboxylic acid. This new material is superior not only in its crystallinity and increased purity but is also obtained in high yields and has increased stability on storage even at a high temperature over extended periods. These properties of the crystalline material render it of particular value in pharmaceutical use.

9 The above hydrated crystalline material may be characterised by the infra-red spectrum and/or by its x-ray powder diffraction pattern.

IR Spectrum (Nujol) vmax 3700-2100 (broad), 1760, 1720, 1660, 1555, 1520, 1350, 1300, max 1290, 1240, 1215, 1170, 1155, 1120, 1030, 1000, 920, 860 and 755cm- 1 X-ray Diffraction Pattern (given as d spacings in Angstrom units and percentage intensities I).

12.5 9.97 9.51 6.79 6.33 5.29 4.76 4.63 (100); 4.39 4.32 4.18 4.09 3.91 3.78 3.69 3.61 3.52 3.48 3.42 oo 3.33 3.15 3.07 3.02 2.96 2.85 2.78 S.00 2.75 2.65 2.59 2.50 2.44 2.38 15 2.31 2.27 2.20 2.16 2.10 2.05 2.01 0000 O 0 0 0 00 0 °o Compounds according to the invention exhibit broad spectrum 0° antibiotic activity against both Gram-positive and Gram-negative organisms, including many p-lactamase producinq strains. The 20 compounds also possess high stability to P-lactamases produced by a 00 0 ooo range of Gram- negative and Gram-positive organisms.

0o 00 Compounds acccording to the invention have been found to 0 00 exhibit high activity against strains (including penicillinase- 0000 o producing strains) of Gram-positive bacteria such as Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus species. This is coupled with excellent activity against Pseudomonas species, and also with high activity against various members of the Enterobacteriaceae strains of Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Serratia marcescens, Proteus mirabilis and indole-positive Proteus organisms such as Proteus vulgaris, Proteus morganii and Providence species), and strains of Haemophilus influenzae and Acinetobacter calcoaceticus. This combination of high activity against Gram-positive organisms with high activity against Gram-negative organisms, more particularly against Pseudomonas, that is possessed by the compounds of the invention is unusual and particularly advantageous.

Cephalosporin derivatives described herein have been found to possess a desirable long serum elimination half life in vivo.

Compounds of the invention may be used for treating a variety of diseases caused Dy pathogenic bacteria in human beings and animals, such as respiratory tract infections and urinary tract infections.

The antibiotic compounds of the invention may be formulated for administration in any convenient way, by analogy with other antibiotics, and the invention therefore includes within its scope pharmaceutical compositions comprising an antibiotic compound in accordance with the invention adapted for use in human or veterinary medicine. Such compositions may be presented for use in conventional manner with the aid of any necessary pharmaceutical carriers or excipients.

.000 o^e The antibiotic compounds according to the invention may be 0 0 0 formulated for injection and may be presented in unit dose form, in o o Sampoules, or in multi-dose containers, if necessary with an added O 0oo preservative. The compositions may also take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising and/or Soo dispersing agents. Alternatively the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use.

25 If desired, such powder formulations may contain an appropriate non-toxic base in order to improve the water-solubility of the active ingredient and/or to ensure that when the powder is reconstituted with water, the pH of the resulting aqueous formulation is physiologically acceptable. Alternatively the base may be present in the water with which the powder is reconstituted. The base may be, for example, an inorganic base such as sodium carbonate, sodium bicarbonate or sodium acetate, or an organic base such as lysine or lysine acetate.

The composition may also be presented in a form suitable for absorption by the gastro-intestinal tract, for example, tablets, capsules, syrups or suspensions for oral adminstration, and suppositories.

1 11 Compositions for veterinary medicine may, for example, be formulated as intramammary preparations in either lonq acting or quick-release bases.

The compositions may contain from 0.1% upwards, e.g. 0.1-99% of the active material, depending on the method of administration. When the compositions comprise dosage units, each unit will preferably contain 100-3000 mg of the active ingredient e.g. 200-2000 mg. The daily dosage for adult human treatment will preferably range from 200 to 12000 mg e.g. 1000-9000 mg per day, depending inter alia on the nature of the infection and the route and frequency of administration.

In general, intravenous or intramuscular administration will be employed, for example using 400 to 4000 mg per day of the active ingredient in adult human treatment. It will be appreciated that in some circumstances, for example, in the treatment of neonates, smaller 16 dosage units and daily dosages may be desirable.

oThe antibiotic compounds according to the invention may be

F

o administered in combination with other therapeutic agents such as antibiotics, for example penicillins, cephalosporins or other p-lactam Scompounds.

The compounds of the invention may be prepared by a number of processes, discussed below.

Thus, according to another embodiment of the invention we j provide for the preparation of an antibiotic compound of general S1 formula as hereinbefore defined or a non-toxic salt, solvate or hydrate thereof by acylating a compound of the formula (II) H H Z HN- (II) 0 H I H

COOR

1 (wherein R 1 Z and the dotted line are as defined for general formula or a salt, e.g. an acid addition salt (formed with, for example, a mineral acid such as hydrochloric, hydrobromic, sulphuric, nitric or phosphoric acid or an organic acid such as methanesulphonic or i i i: ,~rll 12 toluene-p-sulphonic acid) or a 7-N-silyl derivative thereof, with an acid of formula (III)

NHR

3

(III)

S N

COOH

II

N

OHCOOR

2

R*

(wherein R 2

R

3 R and R 5 are as defined above) or a salt thereof, or o with an acylating agent corresponding thereto; ii) reduction of a compound wherein Z is to orm ompound 0wherein Z is >S, oo 20 ii) reduction of a compound wherein Z is >S0 to form a compound wih wherein Z is >S, a 0 0 iii) conversion of a carboxyl group into a non-toxic metabolically labile ester function, .0 iv) formation of a non-toxic salt or solvate, v) removal or any carboxyl blocking and/or N-protecting groups, and vi) removal of any hydroxy r'locking groups.

The above reactions i) to vi) may be carried out in conventional manner.

In the acylation process, the starting material of formula (II) is preferably a compound wherein Z is >S and the dotted line represents a ceph-3-em compound.

Acylating agents which may be employed in the preparation of compounds of formula include acid halides, particularly acid chlorides or bromides. Such acylatinq agents may be prepared by Complete Specification for the invention entitled: "CHEiiCAL COIMPUDS" The owing statement is a full description of this invention, including the best method of performing it known to m:i^ o JTT 1 Note lhe description is to be typed in double spacing, pica type face, in an area not exceeding 250 mm in depth and 160 mm in width, on tough white paper of good quality and it is to be inserted inside this form, 13 reacting an acid (III) or a salt thereof with a halogenating agent e.g. phosphorus oxychloride, thionyl chloridd or oxalyl chloride.

Acylations employing acid halides may be effected in aqueous and non-aqueous reaction media, conveniently at temperatures of from -50 to +50 0 C, preferably -40 to +30 0 C, if desired in the presence of an acid binding agent. Suitable reaction media include aqueous ketones such as aqueous acetone, aqueous alcohols such as aqueous ethanol, esters such as ethyl acetate, halogenated hydrocarbons such as methylene chloride, amides such as dimethylacetamide, nitriles such as acetonitrile, or mixtures of two or more such solvents. Suitable acid binding agents include tertiary amines triethylamine or dimethylaniline), inorganic bases calcium carbonate or sodium bicarbonate), and oxiranes such as lower 1,2-alkylene oxides (e.g.

ethylene oxide or propylene oxide) which bind hydrogen halide o liberated in the acylation reaction.

Bo oAcids of formula (III) may themselves be used as acylating O 0 agents in the preparation of compounds of formula Acylations employing acids (III) are desirably conducted in the presence of a condensing agent, for example a carbodiimide such as N,N'-dicyclohexylcarbodiimide or N-ethyl-N'-y-dimethylaminopropylcarbodiimide; a oo carbonyl compound such as carbonyldiimidazole; or an isoxazolium salt such as N-ethyl-5-phenylisoxazolium perchlorate; or N-ethoxycarbonylo 2-ethoxy-1,2-dihydroquinoline.

Acylation may also be effected with other amide-forming derivatives of acids of formula (III) such as, for example, an activated ester, a symmetrical anhydride or a mixed anhydride (e.o formed with pivalic acid or with a haloformate, such as a lower alkylhaloformate). Mixed anhydrides may also be formed with phosphorus acids (for example phosphoric or phosphorous acids), sulphuric acid or aliphatic or aromatic sulphonic acids (for example toluene-p-sulphonic acid). An activated ester may conveniently be formed in situ using, for example, 1-hydroxybenzotriazole in the presence of a condensing agent as set out above. Alternatively, the activated ester may be preformed.

Acylation reactions involving the free acids or their abovementioned amide-forming derivatives are desirably effected in an W-ii1 1 ii i i- H Y 0ai1ii uu OXU"' UIC.L 11 allu a I IC Cl l -1 '-YJ UO.LL..LUII of the cephalosporin nucleus.

i- -14anhydrous reaction medium, e.g. methylene chloride, tetrahydrofuran, dimethylformamide, acetonitrile, dimethylacetamide or dimethyl sulphoxide.

An alternative method of activation is, for example, by reacting an acid of formula (III) with a solution or suspension preformed by adding a carbonyl halide, in particular oxalyl chloride or phosgene, or a phosphoryl halide such as phosphorus oxychloride to a solvent such as a halogenated hydrocarbon, for example methylene chloride, containing a lower acyl tertiary amide such as N,N-dimethylformamide. The activated form of the acid of formula (III) may then be reacted with a 7-amino compound of formula (II) in a suitable solvent or mixture of solvents for example halogenated hydrocarbons e.g. oichloromethane; alcohols such as an alkanol, e.g. ethanol or industrial methylated spirits; esters, e.g. ethyl acetate; ethers, oo7 5 e.g. tetrahydrofuran or dioxan; ketones, e.g. acetone; amides, e.g.

000 dimethylacetamide; acetonitrile; water ar.1 mixtures thereof.

00 The acylation reaction may conveniently be effected at 'oso temperatures of from -50 0 to 50 0 C, preferably -40 0 to +30 0 C, if 00 cI°o desired in the presence of an acid binding agent, for examnle as described above dimethylaniline, triethylamine or sodium Sbicarbonate).

0 If desired, the above acylation reactions may be carried out in o0 0" the presence of a catalyst such as 4-dimethylaminopyridine.

The acids of formula (III) and acylating agents corresponding 0" 25 thereto may, if desired and where appropriate, be prepared and employed in the form of their acid addition salts. Thus, for example, acid chlorides may conveniently be employed as their hydrochloride salts, and acid bromides as their hydrooromide salts.

For use as starting materials for the preparation of compounds of general formula according to the invention, compounds of general formula (III) and the amide forming derivatives thereof, such as acid halides and anhydrides corresponding thereto, are preferably used in their syn isomeric form or in the form of mixtures of the syn isomers and the corresponding anti isomers containing at least 90% of the syn isomer.

oLinietnane group.

Acids of formula (III) and their derivatives may be prepared by etherification of a compound of formula (IV)

NHR

3

I

8

(IV)

N

OH

(wherein R 3 is as hereinbefore defined and R 8 represents a hydrogen atom or a carboxyl blocking group) or a salt thereof, by selective reaction with a compound of general formula (V) 41* T.CHCOOR 2 o o (wherein T is a chloro, bromo or iodo atom; a sulphate roup; or a .sulphonate group, such as tosylate), followed by removal of any carboxyl blocking group R 8 The etherification reaction is conveniently carried out in the Spresence of a base, e.g. potassium carbonate or sodium hydride, and is preferably conducted in an organic solvent, for example dimethylsulphoxide, a cyclic ether such as tetrahydrofuran or dioxan, oor an N,N-disubstituted amide such as dimethylformamide.

Separation of isomers may be effected either before or after such etherification. Under the conditions described above the configuration of the oxyimino group is substantially unchanged by the etherification reaction.

When the compound of formula (IV) is employed in the form of a free acid or a salt with a base, the etherification reaction is generally carried out in the presence of a strong base e.g. potassium t-butoxide, sufficient base being added to form a dianion.

Furthermore, the reaction should be effected in the presence of a base Furthermore, the reaction should be effected in the presence of a base wherein R 1 and R 2 which may be the same or different, each represents a hydrogen atom or a carboxyl blocking group; S, R 3 represents a hydrogen atom or an amino protecting group; lr 1- Lli I i. 1 I Fl I 16 if an acid addition salt of a compound of formula (IV) is used, the amount of base being sufficient to neutralise rapidly the acid in question.

Acids of formula (III) may also be prepared by reaction of a compound of formula (VI)

NHR

3 00 .0.COOR 8

(VI)

(wherein R 3 and R 8 are as hereinbefore defined) with a compound of formula (VII)

H

2 N.O.HCOOR2 o 00 0 0 o 0 00 o0 0 o 0 25 4 t I t T

R

(VII)

(wherein R 2

R

4 and R 5 are as hereinbefore defined) followed Dy removal of any carboxyl blocking group R 8 and where necessary the separation of syn and anti isomers.

The reaction is conveniently carried out in a solvent such as dimethylformamide, dimethylacetamide, dimethyl sulphoxide, tetrahydrofuran or methanol, all optionally in the presence of water, at a temperature of -200 to +50 0 C, preferably 00 to 30 0

C.

The acids of formula (III) may be converted into the corresponding acid halides and anhydrides and acid addition salts by conventional methods, for example as described hereinabove.

Intermediates of formula (VII) may be prepared by treating compounds of formula (VIII) i, I -4

(VIII)

R (wherein Y is an imido group, for example a phthalimido, succinimido or maleimido group) with a hydrazine reagent such as hydrazine hydrate or an alkyl hydrazine such as methyl hydrazine. The reaction will generally be performed in an inert solvent, for example a haloqenated hydrocarbon such as methylene chloride at a low temperature, for 00 Intermediates of formula (VIII) may be prepared by alkylation 0000 with compounds of formula (IX) -017- Y.O.CHCOOR2

I(IX)

4

(VIII)

R

I (wherein Hal is a n i mido group, for exampl a phthaine imido, succinimido o r maleimido group) with a hydrazine reagent such as hydrazine hydrate Sor an alkyhydroxysuccinimide such as methyl hydrazine. The resence of a basewill generally be performed in an inert solvent, for example a halogenated hydrocarbon such as methylene in a solvent such a acetlow temperrile ature, for example example -70 0 to Intermediates of formula (VIII) a re either known compounds or may be prepared using methods analogous to those used for the preparation of the known compounds.

Swith compounds of formula (II) used as starting materials in the acylation process are known from the literature e.g. British Patent Application No. 2159817 or ca be prepared according to information-CHCOOR 2 from the literature.

II I (IX) o (wherein Hal is a halogen atom such as a chlorine or bromine atom) of an appropriate N-hydroxyimde, N-nydroxyphthalimide, Nhydroxysuccinimide or N-hydroxymaleimide) in the presence of a base 1 such as triethylamine in a solvent such as acetonitrile at for example -10° to Intermediates of formula (IX) are either known compounds or may be prepared using methods analogous to those used for the preparation i! of the known compounds. ,1 Compounds of formula (II) used as starting materials in the acylation process are known from the literature e.g. British Patent Application No. 2159817 or can be prepared according to information ij from the literature, l "z I 18 A A 2 -cephalosporin ester derivative obtained in accordance with the above process of the invention may be converted into the corresponding desired A 3 -derivative by, for example, treatment of the

A

2 -ester with a base, such as pyridine or triethylamine.

A ceph-2-em reaction product may also be oxidised to yield the corresponding ceph-3-em 1-oxide, for example by reaction with a peracid, e.g. peracetic or m-chloroperbenzoic acid; the resulting sulphoxide may subsequently be reduced as described hereinafter to yield the corresponding desired ceph-3-em sulphide.

Where a compound is obtained in which Z is >S-O this may be converted into the corresponding sulphide by, for example, reduction of the corresponding acyloxysulphonium or alkoxysulphonium salt prepared in situ by reaction with e.q. acetyl chloride in the case of Oooo 0000o an acetoxysulphonium salt, reduction being effected by, for example, S135° sodium dithionite or by iodide ion as in a solution of potassium 0000 iodide in a solvent e.g. acetic acid, acetone, tetrahydrofuran, 0 0 0 O dioxan, dimethylformamide or dimethylacetamide. The reaction may be oo effected at a temperature of from -200 to +50 0

C.

oo 0 o S0oo In the oxidation and reduction processes described above, the groups R 4 and R 5 in the starting materials are desirably other than :o hydroxyl groups.

"o 0° Metabolically labile ester derivatives of the carboxyl groups o o" in the compounds of formula may be prepared by reacting a compound 0 a4of formula or a salt or protected derivative thereof with the appropriate esterifying agent such as an acyloxyalkyl halide or alkoxycarbonyloxyalkyl halide iodide) conveniently in an inert o organic solvent such as dimethylformamide or"acetone, followed, where ooo necessary, by removal of any protecting groups.

oo Base salts of the compounds of formula may be formed by reacting an acid of formula with an appropriate base. Thus, for example, sodium or potassium salts may be prepared using the respective acetate, 2-ethylhexanoate or hydrogen carbonate salt. Acid addition salts may be prepared by reacting a compound of formula (I) or a metabolically labile ester derivative thereof with the appropriate acid.

19 Where a compound of formula is obtained as a mixture of isomers, the syn isomer may be obtained by, for example, conventional methods such as crystallisation or chromatography.

It should be appreciated that in some of the above transformations it may be necessary to protect any sensitive groups in the molecule of the compound in question to avoid undesirable side reactions. Examples of suitable protecting groups are given in "Protective Groups in Organic Synthesis" by Theodora W. Greene (John Wiley and Sons, 1981).

For example, during any of the reaction sequences referred to above it may be necessary to protect the NH 2 group of the aminothiazolyl moiety, for example by tritylation, acylation (e.g.

chloroacetylation or formylation), protonation or other conventional oo method. The protecting group may thereafter be removed in any o0ec convenient way which does not cause breakdown of the desired compound, o 0o e.g. in the case of a trityl group by using an optionally halogenated o carboxylic acid, e.g. acetic acid, formic acid, chloroacetic acid or S0 otrifluoroacetic acid or using a mineral acid, e.g. hydrochloric acid or mixtures of such acids, preferably in the presence of a protic 20 solvent such as water, or, in the case of a chloroacetyl group, by aa tcreatment with thiourea.

Similarly, the hydroxy groups of the catechol moiety may need to be protected during any of the above reaction sequences. Hydroxy protecting groups which may be removed under mild conditions will generally be suitable, for example acetyl or silyl groups. Such groups Smay be introduced in conventional manner and, when desired, removed such that breakdown of the product does not occur. For example in the case of an acetyl group the group may be removed by solvolysis with an aqueous solvent such as aqueous methanol or aqueous ethanol in the presence of a base for example sodium bicarbonate, ammonium hydroxide, ammonium carbonate or ammonium carbamate. A trimethyl silyl group may be cleaved, for example by treatment with a dilute aqueous acid.

Carboxyl blocking groups used in the preparation of compounds of formula or in the preparation of necessary starting materials are desirably groups which may readily be split off at a suitable stage in the reaction sequence, conveniently at the last stage. It _r ii 20 may, however, be convenient in some instances to employ non-toxic metabolically labile carboxyl blocking groups such as acyloxy-methyl or -ethyl groups acetoxy-methyl or -ethyl or pivaloyloxymethyl) and retain these in the final product to give an appropriate ester derivative of a compound of formula Suitable carboxyl blocking groups are well known in the art, a list of representative blocked carboxyl groups being included in British Patent No. 1399086. Preferred blocked carboxyl groups include aryl lower alkoxycarbonyl groups such as p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl and diphenylmethoxycarbonyl; lower alkoxycarbonyl groups such as t-butoxycarbonyl; and lower haloalkoxycarbonyl groups such as 2,2,2-trichloroethoxycarbonyl. The carboxyl blocking group may subsequently be removed by any of the appropriate methods disclosed in the literature; thus, for example, acid catalysed hydrolysis or reduction is applicable in many cases, as is .oa enzymically-catalysed hydrolysis.

Where a particular enantiomer of a compound of formula is o°o required starting materials having the desired stereochemical o. configuration should be used in the above processes. Such intermediates may be obtained using conventional resolution processes.

Thus for example enantiomeric intermediates of formula (VIII) wherein .o R 2 is a hydrogen atom in which the chiral carbon atom is in the or 0 o configuration may be obtained by reaction of a mixture of .Ot enantiomers with a resolving agent such as a chiral organic base [e.g.

R-(+)-a-methylbenzylamine] in a solvent such as acetone or acetonitrile to form the corresponding diastereoneric salts. The salts may then be separated by known methods and the desired chiral intermediate of formula (VIII) regenerated by treatment with an S aqueous acid, e.g. aqueous hydrochloric acid, at for example room temperature.

The crystalline (6R,7R,2'Z,S)-7-[2-(2-aminothiazol- 4-yl)-2-[(carboxy)(3,4-dihydroxyphenyl)methoxyimino]acetamido]ceph- 3-em-4-carboxylic acid as its hydrate can conveniently be prepared from an aqueous solution of a salt of the above cephalosporin, or a solvate thereof.

L. i I, i i i; I r 1 21 Thus, according to a further embodiment of the invention we provide a process for preparing crystalline hydrated (6R,7R,2'Z,S)-7-[2-(2-aminothiazol-4-yl)-2-[(carboxy)(3,4dihydroxyphenyl)methoxyimino]acetamido]ceph-3-em-4-carboxylic acid by crystallising said acid from aqueous solution, preferably an aqueous solution of an acid addition or base salt thereof or a solvate of said salts, to form said crystalline hydrated product. Crystallisation will normally be followed by isolating and drying the product.

Suitable base salts include, for example, inorganic base salts, such as alkali metal salts and alkaline earth metal salts, and organic base salts, as described above. A particularly suitable Dase salt is the sodium salt.

Starting acid addition salts may be formed with an organic or inorganic acid. Organic acids which may be used include carboxylic and sulphonic acids, for example, formic, trifluoroacetic and methanesulphonic acids. Inorganic acids which may be used include e mineral acids, for example hydrochloric, hydrobromic or sulphuric acids.

According to a further aspect of the invention the desired material may be crystallised from a solution of an acid or base salt of (6R,7R,2'Z,S)-7-[2-(2-aminothiazol-4-yl)-2-[(carboxy)(3,4dihydroxyphenyl)methoxyimino]acetamido]ceph-3-em-4-carboxylic acid, or a solvate thereof, in an aqueous medium by adjusting the pH to 1.0 to For example, a hydrate may be crystallised from an aqueous solution of a base salt on addition of an organic or inorganic acid to a pH in the above ranges. Suitable acids which may be used in the crystallisation include, for example, hydrochloric acid and sulphuric acid. Alternatively, the desired hydrate may be crystallised from an aqueous solution of an acid addition salt by adding an organic or inorganic base to an appropriate pH in the above ranges. Bases which may be used include, for example, alkali or alkaline earth metal hydroxides, carbonates, bicarbonates or acetate, for example sodium hydroxide, sodium carbonate, sodium bicarbonate or sodium acetate.

22 The aqueous medium may contain a water-miscible organic solvent such as an alcohol, for example ethanol or isopropanol, an ether, for example tetrahydrofuran or dioxan, an amide, for example N,N-dimethylformamide or N,N-dimethylacetamide, a ketone, for example acetone, or a nitrile, for example acetonitrile.

The crystallisation is desirably effected at ambient temperature, for example from 0 0 to 30 0 C, preferably from 15-25 0

C.

The crystalline product may be recovered by filtration and washed and dried in conventional manner, for example by air drying or by careful drying under reduced pressure.

The following Examples illustrate the invention. All temperatures are in OC. Nujol and Sorbsil are trade marks. Sorbsil is silica gel manufactured oy Joseph Crosfield and Son of '0 Warrington, Cheshire, England. Unless otherwise stated NMR spectra were recorded in DMSO-d 6 X-ray diffraction measurements were made on Sa Siemens D-500 diffractometer, using CuK- radiation. X-ray intensities were measured at 0.02 0 increments of 20 for 5 second 'o 0 intervals, using a scintillation counter, between values of 20 and o 900. Results were stored on a computer for processing.

Intermediate 1 2-(3,4-Dioxycarbonylphenyl)-2-(phthalimidooxy)acetic acid A solution of N-hydroxyphthalimide (14.6g) and triethylamine in acetonitrile (50ml) was added to a stirred suspension of 2-(3,4-dioxycarbonylphenyl)-2-chloroacetic acid (20.5g) in acetonitrile at -20 over 20 minutes. After a further hour at about 00, a solution of concentrated hydrochloric acid (7.5ml) in water (100ml) was added rapidly. Water (100ml) was added slowly and the initial oil liberated crystallised, aided by seeding. The mixture was filtered and the filter-cake was washed with water, and ethyl acetate:petroleum ether (bp 40-60 0 and dried to give the title compound (30g), m.p. 183 to 1850; 6 5.84 OCH), 7.53 catechol 5-H and 7.69 catechol and 7.85 phthalimido protons).

_i 1 i. i 23 Intermediate 2 (R)-(+)-a-Methylbenzylamine salt of (S)-2-(3,4-dioxycarbonylphenyl)-2-(phthalimidooxy)acetic acid A solution of R-(+)-a-methylbenzylamine (16.3ml) in acetone (100ml) was added rapidly to a magnetically stirred solution of Intermediate 1 (45g) in acetone (1.25 litres) at 210 under nitrogen.

After 30 minutes the mixture was filtered and the precipitate was washed thoroughly with acetone to yield the title compound (16.57q); [a]21 2420 (c 1.07, EtOH); 6 1.46 3 7Hz, CH 3 CH), 5.48 OCH), 7.3 to 7.6 aromatic protons) and 7.80 phthalimido protons).

EtI S^ Diphenylmethyl (S)-2-(3,4-dioxycerbonylphenyl)-2-(phthalimidooxy) acetate 2M Hyorochloric acid (10ml) was added to a stirred suspension o 0o o°o° of Intermediate 2(a) (5.0g) in water (30ml) under nitrogen at 210 00 o°o After 2 minutes, a solution of diphenyldiazomethane containing one equivalent in methylene chloride (11ml) was added. After stirring vigorously for 35 minutes, the organic layer was separated and added S2o dropwise to stirred ethanol (150ml). The mixture was stirred at 210 0 oo for 10 minutes and then stored at 40 for 1 hour. The crystals were o collected by filtration, washed with ethanol and dried to give the title compound (3.59g), m.p. 135 to 135.50; [a]21 112.60 (c 1.18, ethyl acetate).

o Intermediate 3 0 'I 4 -Dihydroxyphenyl)(diphenylmethoxycarbonyl)methoxyamine Intermediate 2(b) (5.71g) was stirred in methanol (390ml) with M-hydrochloric acid (5.5ml) at ca 400 for 4.5 hours. The solution was concentrated and mixed with methylene chloride. After washing twice with water, the solution was dried over magnesium sulphate and evaporated to a foam. A solution of this foam in methylene chloride (176ml) was cooled to -50 0 with stirring under nitrogen and hydrazine hydrate (1.5ml) added. The mixture was allowed to warm slowly to 210 and stirred. After 5.75 hours the mixture was filtered ano the filter-cake was leached with methylene chloride. The combined i j L-Jf .L -II .Lu ,IJ U suppositories.

-24 filtrates were diluted with ethyl acetate and washed with citric acid solution and brine. After drying over magnesium sulphate, evaporation gave the title compound (4.35g) as a foam; [a] 21 17.8 0 (c 1.03, methanol); 6 5.02 OCHCO), 6.68 and 6.81 thiazole H, OCH and catechol protons) 7.1 to 7.4 aromatic protons) 9.04 OH).

Intermediate 4 (Z (2-Aminothiazol-4-yl)-2-[(3,4-dihydroxyphenyl)(diphenylmethoxycarbonyl)methoxyimino]acetic acid 2-(2-Aminothiazol-4-yl)qlyoxylic acid (1.91q) was added over 3 S'minutes to a solution of Intermediate 3 (4.20g) in N,N-dimethyl- !i formamide (22ml) at 30 with stirring. After a further 30 minutes with i ,,ice-water cooling, the solution was allowed to warm to 210 over i 1"i5 hours and then added dropwise to an ice-water mixture (110g) with oi °stirring for 20 minutes. The precipitate was collected by filtration, S °o4o washed with water and dried. It was resuspended in methylene chloride and stirred for 15 minutes before filtration. The filter-cake was washed with methylene chloride 7nd dried to give the title compound (3.76g); S[a] 2 1 25.40 (c 1.02, methanol);

D

8oi 6 5.59 OCHCO), 6.6 to 6.9 thiazole H, OCH, and catechol S' protons), 7.0 to 7.5 NH 2 and phenyl protons) and 9.06 and 9.13 (2s, OH).

Intermediate (Z,S)-2-(2-Formamidothiazol-4-yl)-2-[(3,4-dihydroxyphenyl)(diphenylmethoxycarbonyl)methoxyimino]acetic acid 2-(2-Formamidothiazol-4-yl)glyoxylic acid (6.96q) was added to a stirred solution of [(3,4-dihydroxyphenyl)(diphenylmethoxycarbonyl)]methoxyamine (12.73g) in N,N-dimethylformamide (100ml) at ambient temperature. After 1.5 hours more N,N-dimethylformamide was added, then at 2.5 hours the resulting solution was poured into water (700ml). The products were extracted into ethyl acetate (3 x 250ml). The combined extracts were washed with water (2 x 250mi), dried over magnesium sulphate, then evaporated to a solid. This was i..

25 treated with ethyl acetate (20ml) then with dichloromethane (200ml), giving a suspension. After refrigeration, the solid was collected by filtration, washed with dichloromethane, and dried to give the title compound (11.38g)m.p. 125-1270; [a]D+22.2 0 (c 0.99, methanol).

Example 1 Diphenylmethyl (6R,7R,2'Z,S)-7-[2-(2-aminothiazol-4-yl)-2-[(3,4dihydroxyphenyl)(diphenylmethoxycarbonyl)methoxyimino]acetamidojceph- 3-em-4-carboxylate The tosylate salt of diphenylmethyl (6R,7R)-7-aminoceph-3-em- 4-carboxylate (1.24g) was converted into the free amine by partitioning between methylene chloride and aqueous sodium bicarbonate 5 solution. The organic layer was washed with water and dried with "oO magnesium sulphate. Evaporation gave a foam which was mixed with °o Intermediate 4 (1.0g) in THF (25ml) with stirring and N,N'-dicyclohexylcarbodiimide (830mg) and nydroxybenzotriazole hydrate (290mg) were added. After stirring at 210 for 16 hours, the mixture 00 0 o20 was filtered and the filter-cake was leached with ethyl acetate. The o a combined organic solutions were evaporated to a foam which was redissolved in ethyl acetate and filtered through a column of Sorbsil 0a0 (100g) in ethyl acetate. Evaporation of the appropriate eluate gave the crude product which was redissolved in chloroform and loaded onto a column of Sorbsil U30 (100g) set up in chloroform. The column was eluted with chloroform and then 2% and 3% of methanol in chloroform. Evaporation of the appropriate eluate gave the title compound (800mg) as a foam; 21 +36.250 (c 1.17 DMSO); D (E 1 0t11) 1( Xinfl (EtOH) 230.8 (E 1 m303), 290 (E 1117) and 300.4nm (E '84).

1cm cm 1cm .i uiuuiu.ues or oromides. Such acylatinq agents may be prepared by 26 Example 2 (6R,7R,2'Z,S)-7-[2-(2-Aminothiazol-4-yl)-2-[(carboxy)(3,4-dihydroxyphenyl)methoxyimino]acetamido]ceph-3-em-4-carboxylic acid, trifluoroacetic acid salt The compound of Example 1 (740mg) was stirred with anisole and trifluoroacetic acid (7.5ml) was added. After one minute, diisopropylether (50ml) was added. The precipitate was collected by filtration, washed with diisopropyl ether and dried in vacuo to give the title compound (450mg); [a]21+102.80 (c 0.88,DMSO); D 1" 1' Xmax (pH6 buffer) 236.8nm (E 0 m335), Xinfl 250.0 (E m 257), 277.4 1 1 icm 1cm (E1 1173) and 296.Onm (E 1 139).

1cm cm .Example 3 Diphenylmethyl (6R,7R Z ,S)-7-[2-(2-formamidothiazol-4-yl)-2-[(3,4- °o dihydroxyphenyl)(diphenylmethoxycarbonyl)methoxyimino]acetamido]ceph- S0oo 3-em-4-carooxylate Intermediate 5 (1.2g) was suspended in methylene chloride (20ml) with stirring under nitrogen and N,N-dimethylacetamide (4ml) was added. The o2"y solution was cooled to -250 and phosphorus oxychloride (0.28ml) was "000o' added. After stirring for one hour at -250 to -20 0 a solution of ao diphenylmethyl (6R,7R)-7-aminoceph-3-em-4-carboxylate (derived from its tosylate salt (1.35g) by the action of sodium bicaroonate solution) in methyl chloride (20ml) containing N,N-dimethylaniline (2ml) was added. The reaction solution was now at -100 and it was then "a surrounded by ice-water and stirred thus for 1.75 hours. The reaction mixture was then washed successively with sodium bicarbonate solution, dilute hydrochloric acid and water three times, each time back extracting the aqueous layers with methylene chloride. The combined organic layers were dried with magnesium sulphate and concentrated.

The crude product in methylene chloride was loaded onto a column of Sorbsil U30 (50g) set up in 40% ethyl acetate in petroleum ether (BP to 600) containing 2% acetic acid. Evaporation of the appropriate eluate gave the title compound (1.56g); 27 21.

1aD+ 36.20 (c 1.05, DNSO); 0 Xmax (EtOH) 269.2nm ([1E 184) Xinfl 232.0 (E 1M 8)y26.

E

1 0 m 1 7 n c 0 8 c m 2 8 5 2 6 (1 cm19,ad266.8nm (E 1 8) Example 4 Diphenylmethyl (6R,7R,2'Z,S)-7-112-(2-aminothiazoI-4-yI)-2-[(3,4dihydroxyphenyl) (diphenylmethoxycarbonyl)methoxyiminolacetamido]ceph- 3-em-4-carboxylate The compound of Example 3 (1.5g) was stirred with methanol 0101(15m1) at 210 and 60%0 perchloric acid (lml)was added. After 2.5 hours, 0 0 the solution was partitioned between water and ethyl acetate and sodiumi bicarbonate solution in excess was cautiously added. The aqueous layer was extracted with more ethyl acetate and the combined o oo organic layers were Washed with brine twice, dried with magnesimi 0 0at and evaporated to give the title compound (1.34g); 15ul...t1 1aD+ 36.50 (c 0.96, DNSO); 0 VMax (Nujol) 3600 to 3100 (broad), 1785,1730 and 1680 cm-.

000 Example (6R,7R,2'Z,S)-7-[2-(2-Aminothiazoi-4-yl)-2-[(carboxy)(3,4- 00 dihydroxyphenyl)methoxyiminolacetamidolceph-3-em-4-Carboxylic acid, sodium salt The compound of Example 2 (11g) was dissolved in ethanol with stirring at 210 and saturated sodium acetate solution (4.8m1) was added. After ten minutes, the mixture was filtered and the filter-cake was washed well with ethanol four times, ether four times and dried in vacuo to give the title compound (5.45g). A further crop (2.73g) was obtained by adding more saturated sodium acetate solution (2m1) to the mother liquors; 21 135.00 (c 0.78, DMSO); 30 100 100t Xmax (p[16 buffer) 234.4 (E 1 m 386); Xinfl 250.4 (E 1 cm 276), 276.8 28 (Ec 184) and 294.4nm (E 1 148).

1cm I(Ec m Example 6 (6R,7R,2'Z,S)-7-[2-(2-Aminothiazol-4-yl)-2-[(carboxy) (3,4dihydroxyphenyl)methoxyimino]acetamido]ceph-3-em-4-carboxylic acid The compound of Example 5 (1.09) was mixed with ,ater (4ml) and sodium acetate (41mq) was added when a solution formed. This solution was filtered and scratching initiated crystallisation. The mixture was stored at 210 for 14.5 hours and then the crystals were collected by filtration, washed with water and dried in vacuo at 210 to give the e title compound (250mg). Addition of 2N hydrochloric acid (0.9ml) qave .n further crystalline product (360mq); 21 136.270 (c 0.54, DMSO); X° kmax 234.4nm (E 1 408), Xnfl 249.6 (E 1 o 295), 277.8 (E 192) and S0, 1cm 1cm 1cm 295.0nm (E 'cm 156).

1cm Example 7 (6R,7R,2'Z,S)-7-[2-(2-Aminothiazol-4-yl)-2-[(carboxy)(3,4dihydroxyphenyl)methoxyimino]acetamido]ceph-3-em-4-carboxylic acid, hydrochloric acid salt.

The compound of Example 4 (27.4g) was dissolved in formic acid (110ml) with stirring at 200 and concentrated hydrochloric acid (8.2mi), 3 equivalents was added. The mixture was stirred for a further fifteen minutes and then added dropwise over six minutes to 2" diisopropyl ether (1.1 litres) with stirring at 200. The precipitate was collected by filtration, washed with diisopropyl ether (500ml) and dried in vacuo to give the title compound (18.02g); v (Nujol) 3600-2800 (broad) and 1780 cm-1; max 6 3.3-3.7 5.05(d, 5.4(s, CHON), 5.8(m, 6.46(m, H-3) and 9.6(d, NHCO).

29 Example 8 (6R ,7R,2' Z,S)-7-[2-(2-Aminothiazol-4-yl)-2-[(carboxy)(3,4dihydroxyphenyl)methoxyimino]acetamido]ceph-3-em-4-carboxylic acid.

The compound of Example 7 (2.65g) was added, with stirring, to distilled water (10.6ml) at 20 0 and the mixture was stirred for thirty minutes, during which time crystallisation occurred. The stirred suspension was chilled in ice/water for fifteen minutes and then the crystals were collected by filtration, washed with chilled distilled water (10 ml) dried in an air oven at 30 0 C to give the title compound (2.09 g); S0 ,Water content 12% (Karl Fischer); IR Spectrum (Nujol) x v 3700-2100 (broad), 1760, 1720, 1660, 1555, 1520, 1350, 1300, max 0 0 1290, 1240, 1215, 1170, 1155, 1120, 1030, 1000, 920, 860 and 755cm- 1 Iso X-ray Diffraction Pattern (given as d spacings in Angstrom units and o percentage intensities I).

12.5 9.97 9.51 6.79 6.33 5.29 4.76 4.63 (100); 4.39 4.32 4.18 4.09 3.91 0 3.78 3.69 3.61 3.52 3.48 3.42 3.33 3.15 3.07 3.02 2.96 2.85 2.78 2.75 2.65 2.59 2.50 2.44 2.38 2.31 2.27 2.20 2.16 2.10 2.05 2.01 Example 9 tert-Butyl (6R,7R,2'Z,S)-7-[2-(2-formamidothiazol-4-yl)-2-[(3,4- 0 dihydroxyphenyl)(diphenylmethoxycarbonyl)methoxyimino]acetamido ceph- 3-em-4-carboxylate.

Intermediate 5 (1.53 g) was dissolved, with stirring, in methylene chloride (15 ml) and N,N-dimethylacetamide (2.9 ml) and the solution was cooled to -35 0 and phosphorus oxychloride (0.41 ml) added The mixture was stirred at -25 0 to -20 0 for one hour, and then a solution of tert-butyl (6R,7R)-7-aminoceph-3-em-4-carboxylate [derived from its tosylate salt (1.0 g) by the action of aqueous sodium bicarbonate solution as described in Example 1 for the diphenylmethyl ester] in methylene chloride (15 ml) containing N,N-dimethylaniline (2.9 ml) was added. The reaction mixture was kept at ca. 0 0 for 16 hours, then washed successively with water, dilute hydrochloric acid (twice) and water; each time the aqueous layer was back-extracted with 30 methylene chloride. The combined organic layers were concentrated to a foam, which was redissolved in methylene chloride (15 ml) and passed down a column of "Sorbsil" U30 silica (5 g) eluting with methylene chloride (30 ml). The eluate was concentrated to low volume and treated with diisopropyl ether (15 ml) to precipitate the title compound as a white solid (1.2 g); 6 (CDC13) 1.57 (CH 3 3 5.74 OCHCO), 6.15 6.49 (d, 8.4-8.7 NHCO and -OH).

Example tert-Butyl (6R,7R,2'Z,S)-7-[2-(2-aminothiazol-4-yl)-2-[(3,4dihydroxyphenyl)(diphenylmethoxycarbonyl)methoxyimino]acetamidolceph -3-em-4-carboxylate.

The compound of Example 9 (6.29 q) was stirred in methanol (64 ml) and 1,4-dioxan (16ml) at 200 and 60% perchloric acid (5.63ml) was added.

o* o After 3.5 hours, the solution was added to rapidly stirred sodium bicarbonate solution (240 ml). The mixture was stirred for a further hour and then chilled. The product was collected by filtration, 0 0 washed with water and dried. It was then dissolved in a mixture of ethyl acetate and methylene chloride and the solution loaded onto a column of "Sorbsil" U30 silica (65 g) which was eluted with further solvent. Evaporation of the appropriate fraction of eluate gave the title compound (4.0 g); 6 1.5 (CH 3 3 5.06 5.61 CHON), 5.8 H-7), 6.41(m, 9.0(broad, 9.5 NHCO).

Example 11 (6R,7R,2'Z,S)-7-[2-(2-Aminothiazol-4-yl)-2-[(carboxy)(3,4dihydroxyphenyl)methoxyimino]acetamido]ceph-3-em-4-carboxylic acid, hydrochloric acid salt.

The compound of Example 10 (1.5 q) was dissolved in formic acid (6 ml) with stirring at 200, and concentrated hydrochloric acid (0.35 ml, 2.1 equivalents) was added. The mixture was stirred for 2 hours then clarified by filtration and then evaporated under reduced pressure.

Ethyl acetate (6 ml) was added to the residue and the mixture was re-evaporated to give a foam, which was triturated with ethyl acetate

I

I

31 (6 ml). The resulting suspension was stirred for 30 minutes at ambient temperature. The solid was collected by filtration, washed twice with ethyl acetate, and dried in vacuo to give the title compound (1.33 g) identical with the product of Example 7.

Pharmacy Example Dry Powder for Injection Per vial .t S0"15 o 0 t 20 (6R,7R,2'Z,S)-7-{2-[2-Aminothiazol -4-yl]-2-[(carboxy)(3,4-dihydroxy phenyl)methoxyimino]acetamido ceph -3-em-4-carboxylic acid.

Sodium carbonate (anhydrous) 500mq 99mg Blend the two sterile components aseptically and fill into sterile vials. Purge the vial headspace with sterile nitrogen; close the vials using rubber plugs and metal overseals (applied by crimping).

The product may be constituted by dissolving in Water for Injections or other suitable sterile vehicle shortly before administration.

Claims (5)

1. Compounds of formula (I) HR 3 OO(') 0I 1 OCHcOORt ^oR' w 1 2 wherein R and R 2 which may be the same or different, each represents a hydrogen atom or a carboxyl blocking group; R represents a hydrogen atom or an amino protecting group; R 4 and R 5 each represents a hydroxy or a formyloxy group or a group of formula ao -OCOR 6 (where R 6 is a Ci- 8 alkyl group), a group -OCO2 R 6 (where R 6 is as defined above), a (C 1 4 alkyl) silyloxy group, or a group [-OB(OR )21 or t C 1 4 alkoxy or oxo substituents, or and R 5 together eresent -OB(OH)O-, -OP(O)(OR 7 (where R 7 is as defined above) or a di(Cl_4 alkyl) silyldioxy group; z is or -SO- or and the dotted line bridging the and

4-positions indicates that the compound is a ceph-2-em or ceph-3-em compound; and salts and solvates (including hydrates) thereof. 2. Compoundts claimed in claim 1 of formula (Ia) 6 N -i i i; I; 33 J r" (Ia) O CHc.OOH CooH S wherein R 4 a is a hydroxy or acetoxy group; SZ' R 5 a is a hydroxy or acetoxy group; I I r and the non-toxic salts, solvates (including hydrates) and metabolically labile esters thereof. S 3. Compounds of formula (Ia) as claimed in claim 2 in which the oxime etherifying group has the S-configuration. 4. (6R, 7R, 2'Z, S)-7-[2-(2-Aminothiazol-4-yl)- 2-[(carboxy) (3,4- dihydroxyphenyl)methoxyimino]acetamido]ceph-3-em-4-carboxylic acid and the 10, non-toxic salts, solvates (including hydrates) and metabolically labile esters thereof. The compound of claim 4 in crystalline hydrated form characterised by an infrared spectrum containing absorption bands at 3700-2100 (broad), 1760, 1720, 1660, 1555, 1520, 1350, 1300, 1290, 1240, 1215, 1170, 1155, 1120, 1030, 1000, 920, 860 and 755cm" 1 and characterised by an X-ray diffraction pattern (expressed as O d spacings in Angstrdm units and percentage intensities) as follows:

12.5 9.97 9.51 6.79 6.33 5.29 4.76 4.63 (100); 4.39 4.32 4.18 4.09 3.91 3.78 3.69 (17); 3.61 3.52 3.48 3.42 3.33 3.15 3.07 3.02 2.96 2.85 2.78 2.75 2.65 2.59 2.50 2.44 0- 2.38 2.31 2.27 2.20 2.16 2.10 2.05 2.01 34 6. A process for the preparation of compounds of formula as defined in claim 1 or salts or solvates thereof which comprises acylating a compound of formula (HI) H H COQR' 4C COO ;i*-il -~~:liii .i I t f I I f f I 1 I I II (wherein R 3 R 4 and R 5 are as defined in claim 1, and R 2 is a carboxyl blocking group) or a salt thereof, or with an acylating derivative thereof; whereafter, if necessary and/or desired in each instance, any of the following reactions, in any appropriate sequence, are carried out:- i) conversion of a 2 -isomer into a desiredA 3 -isomer, ii) reduction of a compound wherein Z is -SO- to form a compound wherein Z is iii) conversion of a carboxyl group into a non-toxic metabolically labile ester function, iv) formation of a non-toxic salt or solvate S v) removal of any carboxyl blocking and/or N-protecting groups, and vi) removal of any hydroxy blocking groups. 7. A process for the preparation of the crystalline hydrate of claim which comprises forming an aqueous solution of (6R, 7R, 2'Z, aminothiazol-4-yl)-2-[(carboxy) (3,4-dihydroxyphenyl)methoxyimino]-acetamido]- ceph-3-em-4-carboxylic acid or an acid addition or base salt thereof or a solvate of said salt and crystallising the said hydrate therefrom. 8. A process for the preparation of the crystalline hydrate of claim which comprises forming an aqueous solution of an acid addition or base salt of (6R, 7R, 2'Z, S)-7-[2-(2-aminothiazol-4-yl)-2-[(carboxy) (3,4- dihydroxyphenyl)methoxyimino]-acetamido]-ceph-3-em-4-carboxylic acid or a solvate thereof and adjusting the pH of said solution to between 1.0 and 9. A pharmaceutical composition comprising as active ingredient a compound of formula (Ia) as defined in claim 2, in association with a pharmaceutically acceptable excipient. A pharmaceUi. al composition as claimed in claim 9 wherein the active S, ingredient is a compound as defined in claim 4, in association with a 'I I I A -N I 36- pharmaceutically acceptable excipient. 11. A pharmaceutical composition as claimed in claim 10 wherein the active ingredient is the crystalline hydrated compound of claim 5, in association with a pharmaceutically acceptable excipient. 12. A method of combatting bacterial infections which comprises administering to a subject a compound of formula (Ia) as defined in claim 2.

13. A method as claimed in claim 12 wherein a compound as defined in claim 4 is administered to said subject.

14. A method as claimed in claim 13 wherein the crystalline hydrated 10 compound of claim 5 is administered to said subject. I t 4 4 t 4 4 I 4 i' DATED this 13th day of December 1990 GLAXO GROUP LIMITED By their Patent Attorneys CALLINAN LAWRIE i 4 t 4 t t