GB2184722A

GB2184722A - 7 - hydroxy/amino-cephalosporin antibiotics

Info

Publication number: GB2184722A
Application number: GB08629677A
Authority: GB
Inventors: Dr Colin William Greengrass; Dr Thomas Trefor Howarth; Dr David William Thomas Hoople
Original assignee: Pfizer Ltd
Current assignee: Pfizer Ltd
Priority date: 1985-12-18
Filing date: 1986-12-11
Publication date: 1987-07-01
Also published as: US4684642A; GB8531202D0; GB8629677D0

Description

1

GB2 184 722 A 1

SPECIFICATION

Cephalosporin antibiotics

5 This invention relates to cephalosporin antibiotics, and in particularto cephalosporins have a 7 a-hydroxyamino substituent.

U.S. Patent specification no. 4,297,488 describes cephalosporins of theformula:-

10

15

H

1 '

ET-N-

CH2A

COOH

wherein R1 represents an acyl group, A represents an organic radical or group, and R! represents a radical or. group replacing hydrogen, and derivatives thereof such as esters, amides and salts. Ri in the above formula can for example be a "nitrogen bonded group", one specific case of which is "-RH3OH" where "R3 repre-20 sents R2 (which is a straight or branched chain C-i—C6alkyi group) or hydrogen". No compounds having Rt as "-NR3OH" are exemplified in this specification.

We have found a group of 7 a-hydroxyamino cephalosporins which have unexpectedly good antibacterial properties, particularly against gram negative organisms, and including activity against (3-lactamase producing strains of bacteria.

Thus the present invention provides cephalosporins of theformula:-

25

30

NKOH

R-CH-CONH ira R1

(I)

10

15

20

25

30

35

35 COOH

andtheirsalts and esters,

where R is an unsubstituted thiazolyl, thiadiazolyl,furyl or pyridyl group; R2is-CH2OCOCH3, -CH2OCONH2ora group of theformula:-

40 , /o v 40

45 or-CH2S.Het, 45

where m is 1 or 2 and each R3 is independently H or C!-C4 alkyl; and Het is an optionally substituted 5- or 6-membered heterocyclic group containing up to 4 heteroatoms selected from 0,Sand N, the heterocyclic group being optionally fused to an optionally substituted benzene ring orto a further 5- or 6-membered 50 heterocyclic group containing up to 4 heteroatoms selected from O, S and N; 50

and R1 is a group of theformula :-

55

60

C=0

I

-N

"N

A4

or c=o

I

-N

55

60

wherein R4is CT-C4 alkyl or-S02(C1-C4 alkyl).

When R2is -CH2SHet, "Het" is preferably an optionally substituted triazolyl,tetrazolyl, thiazolyl, iso-65 thiazolyl, oxazolyl, oxadiazolyl, triazinyl, thiadiazolyl, benzoxazolyl, benzothiazolyl, ortetrazolopyridazinyl

65

2

GB 2 184 722 A

2

group. Preferred substituents are C-i -C4 alkyl, Ci -C4 alkoxy, halo, oxo or a group of the formula ~(CH2)PR5 where p is 0,1,2 or 3 and R5 is -COOH, -0S020H, -S020H, -P03H2 or -OH, with the proviso that p is not zero when Het is tetrazoiyl. With hydroxy and oxo substituents tautomerism may of course occur. "Halo" means F,C(, Br or I.

5 The more preferred "Het" groups are attached to the adjacent S atom by a carbon atom of the heterocyclic ring andare{i)thiadiazolyl optionally substituted by Ct-C4 alkyl or 2-hydroxyethyl (ii) tetrazoiyl optionally substituted by CT-C4 alkyl, carboxymethyl,sulphomethyl,2-hydroxyethyl, hydroxysulphonyloxymethylor 2-(hydroxysu!phonyloxy)ethyl (iii) thiazolyl optionally substituted by 1 or 2 substituents each selected from C1-C4alkyl andcarboxymethyl (iv) isothiazolyl optionally substituted by 1 or 2 substituents each selected 10 from hydroxy and carboxy (v) benzothiazolyl or benzoxazolyl optionally substituted by hydroxy, C-i -C4 alkoxy or halo (vi)tetrazolopyridazinyl optionally substituted by carboxy (vii)triazinyl optionally substituted by C| -C4 alkyl and/or by 1 or 2 oxo or hydroxy groups and (vii) triazolyl optionally substituted by car-boxym ethyl.

The preferred individual groups represented by "Het" are asfollows:-

15

20

25

30

N—N,

A.

'N

(CH.,) „OB £,

C02H

,

/ ^ —N

(tetrazolo/I,5-b7pvridazin-6-yl)

and

N.

\

Typical examples of R are 4-thiazolyl, 1,2,3-thiadiazol-4-yl, 2-furyl, and 3-pyridyl. R4is preferably CH3, C2H5 or —S02CH3.

R1 is preferably

10

15

20

25

30

35

40

c=o

I

N N

so2ch3

or

I

C=0 Nv u

C„H„ 2 D

R2 is preferably either (a) -CH2SHet where Het is an "individual group" as defined above, or (b) pyridiniu-45 mmethyl

The salts and esters (including in vivo hydrolysable esters) of the compounds of theformula (I) are well known to those skilled in the art. These salts include not only salts with -COOH, but also with -0S020H and -S020H. The preferred salts are the sodium and potassium salts, and thetriethylammonium salts. Some of the compounds may of course exist in zwitterionicform. The preferred in vivo hydrolysable esters are those 50 of the formula

35

40

45

50

-CHzOCO^u, -CH2OCOCH3, -CH(CH3)OCOCH3, —CH(CH3)OCOOEt, O.

55

60

-CH2^0,

/

The preferred esters which are useful as intermediates arethef-butyl and benzhydryl esters.

In general, the DL- and D- forms at the starred carbon atom of the compounds of theformula (I) are preferred.

65 The compounds of the formula (I) are preferably prepared by the replacement of - S.CH3 in the 7 a-position

55

60

65

3

GB 2 184 722 A

3

10

15

with -NHOH. This can be done on the 7 a-methylthio-7p-acylamino compounds, i.e., after acylation is carried outto introduce the group RCH(NHR1)CO-, or on compounds not having the acyl group present in the 7p-position. The desired 3-substituent can be introduced before or afterthehydroxyaminosubstituent is present. In addition any 0- or carboxy-protecting groups can be removed before or after the - NHOH group is in position.

Typically, the hydroxyamino substituent is introduced by reacting the appropriate methylthio compound with a mercuric salt, e.g. mercurictrifluoroacetate, mercuric chloride or mercuric acetate, and preferably with mercuric acetate, at low temperature, e.g. - 60°C, in a suitable organic solvent, eg dimethylformamide (DMF). Hydroxylamine hydrochloride again typically in DMF, is added, and the solution is slowly warmed to from -20° to +20°C. Hydroxylamine (preferably generated from hydroxylam'ine hydrochloride/ triethylamine) can be used in place of hydroxylamine hydrochloride. The 7 a-hydroxyamino product can then be isolated conventionally. C2-C4 alkythio, phenylthio or benzylthio derivatives can be used in place of the methylthio starting materials. Similarly metal salts (e.g. acetates) such as silver, thallium, lead orcoppersalts can be used in place of mercuric salts.

The 7 a-methylthio compounds are either known compounds or can be prepared conventionally.

Typical routes to the compounds of theformula (I) are illustrated schematically asfollows: v

(a)

10

15

20

25

30

SMe

Acylation

RCH(NHR )C0NH^£

COO."CBP"

(See U.S.P. 3976641)

-Vs,'

COO.CBP

i) hs2+

ii) NH20H

20

25

30

35

40

NHOH

'tin

RCH(NHR )C0t%=

COOH

Removal of carboxy protecting group

NHOH

RCHCNHR1 )C0NPa = ^

"CBP" = a carboxy protecting group

35

40

45 Modification atthe3-position (e.g. -CH2OActo -CH2SHet)can if desired be carried out before or after introduction of the hydroxyamino substituent.

45

4

GB 2 184 722 A

4

SMe

10

15

20

25

30

(b) RCH(MHR')C0NHa^

OAc

COO.CBP Removal of carboxy-protecting group

SMe

10

15

20

25

30

SCH

35

40

45

50

(c) RCHCMHR1)C0NH

sch3

60

.IK

COOH

NHOH

RCH(NHR1)C0NH|k3 ^S \

COOH

E

/

<0

\

X.

/

COOH

I

0=C=N.

SCH. ■

OCONH

RCH(NHR )C0NH^= r Ha /NH^OH

.OCONH.

COOH

and (d) SCH,

55 H^N1

R

COO.CBP

i) Hg2"7NH,OH . <■ > RCH (NHR ) CONH

NHOH

ii) Acylation ill) Deprotect

COOH

35

40

45

50

55

60

65 (see e.g. GB 1526793)

65

5

GB 2 184 722 A 5

Any hydroxy-protecting groups, if present, can again be removed before or after insertion of the hydroxyamino group.

Apart from the insertion of the hydroxyamino group, all the above steps are conventional (see eg British patent application publication no. 2107307Aor U.S. patent no. 4,297,488).

5 Acylation is typically carried out using an acid chloride or bromide of the acid RCH(NHR1)COOH or an 5

O-protected derivative thereof. Alternatives are of course activated esters, mixed anhydrides. The reaction is typically carried out at low temperature (-10°to 0°C) in a suitable organic solvent, eg dichloromethane.

When an acid halide is used, the presence of an acid binding agent such as pyridine ortriethylamine is preferred.

10 Many conventional carboxy protecting groups (CBP's) can be used, eg t-butyl, benzhydryl, benzyl, p- 10

methoxybenzyl and p-nitrobenzyl. These can all be removed by conventional means. The preferred protecting groups are t-butyl, which is typically removed with trifluoroacetic acid, and benzhydryl, which istypically removed with anisole/AICI3 or anisole/trifluoroacetic acid.

Modifications at position 3 of the cephalosporin ring are carried out by conventional methods, e.g. :-15 15

Pyridine,.typically with

(a) —CH20Ac

-7> -°H2-0,O

20 ^ ^ \=/ 20

Sodium or potassium iodide catalysis.

HetSH 25 ■ > -CH2S.Het

25 (b) -CH2OAc

30

-ch2i

-CH2Br

(or a salt [e.g. an alkali metal salt] thereof)

30

(Other suitable leaving groups than -OAc, I or Br can be used.)

The C3 acetoxymethyl group can be reacted with thiol in the presence of a Lewis acid such as boron 35 trifluoride,ifdesired(seeJ55020724). 35

It should be mentioned that derivatives of hydroxylamine protected on oxygen can be used in place of hydroxylamine in theformation of the -NHOH group, such derivatives including H2N-OSiMe3, H2N-0SiMe2tBu,H2N-0SiPh2tBu,H2N-0Si(C1-C4alkyl)3,H2N-0.benzyl,H2N-0.C00.benzyl,

H2N -O.COOtBu, H2N -O.COOCH2CCI3, 40 40

H2N-O.COOCH2CH=CH2,H2N-OCH2CH=CH2,H2N-O.COOCH2h0>-NO2,

H2N-O.COOCH2<Q-OCH3,orH2N-O.COOCH2CH2Si(CH3)3.TheO-

45 protecting groups can be removed conventionally. 45

The same O-protecting groups can be used, if desired, to protectthe hydroxy groups of any hydroxy or hydroxyalkyl substituents. The preferred O-protecting groups are f-butyldiphenylsilyl and t-butyldimethylsilyl, removed with aqueous hydrofluoric acid.

The salts and in vivo hydrolysable esters can be prepared conventionally.

50 An alternative method of introducing hydroxyamino can be represented as follows:- 50

6

GB 2 184 722 A

6

t-Bu

10

15

20

25

(Known)

OAc t-Bu

COO.CBP

H N .OH or O-protectad derivative thereof r 2

NHOH or NEO—protecting, group N = /3

OAc

NHOH

CCQC3P

OAc

COO.CBP

etc.

30

35

40

45

50

55

60

For administration to man in the curative or prophylactictreatment of bacterial infections, parenteral dosages of the compounds will typically be in the range of from 10Omg. to 8g. daily for an average adult patient (70 kg), and, most commonly, from 1 g to 4g daily. Thus for a typical adult patient, individual parenteral formulations will contain from 0.5 to 2g. of active compound, in a suitable pharmaceutically acceptable vehicle. In practice the physician will determine the actual dosage which will be most suitable for an individual patient and it will vary with the age, weight and response of the particular patient. The above dosages are exemplary of the average case but there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

For human use, the compounds of theformula (I) can be administered parenterally in admixture with a pharmaceutical diluent selected with regard to the intended route of administration and standard pharmaceutical practice. They can be injected intravenously, intramuscularly or subcutaneously. They are best used intheform of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to makethe solution isotonic with blood, and the solution may also contain an anaesthetic such as lignocaine.

Thus in a further aspect the invention provides a pharmaceutical composition comprising a compound of theformula (I), or a pharmaceutically acceptable salt thereof, or in vivo hydrolysable esterthereof, together with a pharmaceutically acceptable diluent or carrier.

The compounds may also be administered in combination with other antibiotics and/or (3-lactamase inhibitors such as sulbactam.

The invention also provides a compound of the formula (I), or a pharmaceutically acceptable salt or in vivo hydrolysable esterthereof, for use in medicine, in particularfor use as an antibiotic.

The invention also provides a method of treating a bacterial infection in a human patient, which comprises administering to the patient an effective amount of a compound of the formula (I) or pharmaceutically acceptable salt or in vivo hydrolysable esterthereof.

The compounds of theformula (I) and their pharmaceutically acceptable salts and in vivo hydrolysable esters are antibiotics which have unexpectedly high activity. They are particularly active against gram negative organisms, such as E. coii, Klebsiella pneumoniae Proteus mirabilis, Proteus vulgaris, Proteus morganii, Providentia stuartii, Providentia rettgeri, Haemophilus influenzae and Bacteroides fragilis.

Thefoilowing Examples, in which all temperatures are in °C, illustrate the invention:-

EXAMPLE1

7a-Hydroxyamino-7fi-[DL-2-(2-oxo-3-methylsulphonylimidazolidin-1-ylcarbonylamino)-2-(thiazol-4-yl)acetamido]cephalosporanicacid

10

15

20

25

30

35

40

45

50

55

60

7

GB 2 184 722 A

7

(a) DL-2-[2-Oxo-3-methy/sulphonylimidazolidin-1-ylcarbonylamino)-2-(thiazo/-4-yl)aceticacid DL-2-Amino-2-(thiazol-4-yl)acetic acid (214 mg) [prepared by the method described in J. Med. Chem. 16,

978 (1973)] was taken up in water (10 ml) and the pH was adjusted to 6.5 by the addition of 2N sodium hydroxide solution. 2-Oxo-3-methylsu!phonyl-imidazolidin-1-ylcarbonyl chloride (310 mg) was added to the 5 stirred solution at room temperature in portions; the pH being maintained between 6.0 and 7.0 by the addition of 2N sodium hydroxide solution. When the addition was complete, the mixture was stirred for one hour, filtered, and the filtrate was washed with ethyl acetate (2 x 20 ml), acidified to pH 1.0, and extracted with ethyl acetate (2x20 ml). The combined extracts were washed with saturated sodium chloride solution (20 ml), dried (MgS04), filtered and evaporated to dryness in vacuo. The residue was triturated with methylene 10 chloride and the solid product was separated by filtration to give the title compound as a white solid, (250 mg).

NMR (DMSO-d6), 8 = 3.34 (s, 3H); 3.77 (m, 4H); 5.59 (d, 1H, J = 6Hz); 7.79 (s, 1H); 8.76 (d, 1H, J = 6Hz); 9.09 (s, 1H).

15

(b) Benzhydryl 7u-methylthio-7$-[DL-2-(2-oxo-3-methyl-su/phonyfimidazolidin-1-y/carbonylamino)-2-(thiazo/-4-y/)-acetamidoJcephafosporanate

Asuspensionof DL-2-(2-oxo-3-methylsulphonylimidazolidin-1-ylcarbonylamino)-2-(thiazol-4-yl)acetic acid (600 mg) in methylene chloride (20 cm3) stirred at 0° under nitrogen was treated with N-methylpiperidine 20 (230 (xl) in one portion to give a clear colourless solution. This solution was treated with ethyl chloroformate (200 |aI) and the mixture stirred at 0°forthirty minutes to give a solution of the mixed anhydride. Benzhydryl 7p-amino-7a-methylthiocephalosporanate (1.01 g) in methylene chloride (10 cm3) was added to the mixed anhydride solution at 0°, the solution was allowed to attain room temperature, stirred for 60 hours, washed successively with 0.2N hydrochloric acid (2x20 cm3), saturated sodium hydrogen carbonate solution (2x20 25 cm3) and saturated sodium chloride solution (20 cm3), dried (MgS04), filtered and evaporated to dryness in vacuo. The residue was purified by medium-pressure flash-column chromatography on silica gel ("Merck" [Trade Mark] 230-400 mesh ASTM), eluting the product with 50% ethyl acetate/methylene chloridesolution. The appropriate fractions were combined and evaporated to dryness in vacuo and re-stripped with 50% 1,1,1-trichloroethane/methylene chloride to give the title compound as a white solid, (1.1 g).

30

//?(CH2CI2) 1784cm-1; 1738 cm"1; 1691 cm-1.

/VM?(CDCI3), 8 = 1.92 and 1.94 (2 x s, 3H); 2.05 and 2.23 (2 x s, 3H); 3.31 and 3.35 (2 x s, 3H); 3.77 (m, 4H); 4.7 (m, 2H); 5.09 and 5.10 (2 x s, 1H); 5.84 (m, 1H); 6.87 (s, 1H); 7.30 (m, 10H); 7.71 and 7.72 (2 x s, 1H); 8.85 (d, J = 6 Hz, 1H); 9.07 and 9.08 (2 x s, 1H); 9.63 (s, 1H).

35

(c) 7<x-Methy/thio-7p,-[DL-2-(2-oxo-3-methy/sulphony/-imidazo/idin-1-y/carbony/amino)-2-(thiazo/-4-yijacetamido]-cephaiosporanicacid

To a stirred solution of benzhydryl 7 a-methylthio-7p-[DL-2-(2-oxo-3-methylsulphonylimidazolidin-1-ylcarbonylamino)-2-(thiazol-4-yl)acetamido]cephalosporanate (110 mg) in dichloromethane (15 cm3) at 40 room temperature was added anisole (293 fxl) followed bytrifluoroaceticacid (312 (il) and the mixture was stirred for 3 hours. The mixture was then evaporated to dryness and the residue was triturated with ether (50 ml) to give a pale yellow solid which, after rapid stirring for 2 hours, was separated by filtration, washed with ether and dried in vacuo to give the title compound, (80 mg).

45 //?(KBrdisc) 1780cm-1; 1729cm-1; 1685cm-1.

/WW/?(DMSC)-d6)8 = 1.99and2.01 (2xs,3H); 2.02and 2.22 (2xs,3H); 3.38 (s,3H); 3.5 (m.2H);3.74(m,4H); 4.79 (m, 2H); 5.02 and 5.03 (2xs,1H); 5.84 (m,1H); 7.70 and7.71 (2 x s, 1H); 8.83 (m, 1H); 9.07 (s, 1H); 9.55 and 9.57 (2xs,1H),

50 (d) 7a-Hydroxyamino-7\i-[DL-2-(2-oxo-3-methylsulphony/-imidazolidin-7-ylcarbonylamino)-2-(thiazol-4-yl)acetamido]-cephaiosporanicacid

Asolutionof7a-methylthio-7p-[DL-2-(2-oxo-3-methyl-sulphonylimidazolidin-1-ylcarbonylamino)-2-(1,3-thiazol-4-yl)acetamido]cephalosporanic acid (100 mg) in dimethylformamide (4 ml) stirred at-60°C under nitrogen was treated successively with mercuric acetate (98 mg) in dimethylformamide (1 ml) and hydroxyl-55 amine hydrochloride (12 mg) in dimethylformamide (750 |xl). The solution was then allowed to attain 0°over approximately one hour and added dropwise to dry diethyl ether (80 ml). Afterfiltration, the ether-damp solid was suspended in methanol (20 ml) and saturated with hydrogen sulphide. The mixture was filtered and evaporated under vacuum. The residue was triturated with dichloromethane (50 ml) to give the title compound as a paie yellow solid, (62 mg).

60

//?(KBr) 1772 cm-1; 1730 cm-1; 1675 cm-1.

5

10

15

20

25

30

35

40

45

50

55

60

NMR (DMSO-d6), 8 = 2.01 (brs, 3H); 3.37 (s,3H);3.79(m,4H); 4.78 (m,2H); 5.07 (s, 1H); 5.89 (d, 1H, J =6Hz); 7.67 (brs, 1H); 8.85 (m, 1H); 9.04 (brs, 1H); 9.19 (s, 1H); 10.32 (br, 1H).

8

GB 2 184 722 A

8

EXAMPLE 2

7a-Hydroxyamino-7fi-[DL-2-(2-oxo-3-methy/sulphonylimidazolidin-1-ylcarbonylamino)-2-(thiazo/-4-yl)acetamido]-3-[(tetrazolo[1,5-b]pyridazin-6-yl)-thiomethylJceph-3-em-4-carboxylicacid

(a) 7a-Methylthio-7$-[DL-2-(2-oxo-3-methylsulphonyl-imidazolidin-1-ylcarbonylamino)-2(thiazol-4-

5 yljacetamido]-3-[(tetrazolo[7,5-b]pyridazin-6-yl)-thiomethyl]ceph-3-em-4-carboxylicacid 5

7a-Methylthio-7p-[DL-2-(2-oxo-3-methylsulphonyl-imidazolidin-1-ylcarbonylamino)-2-(thiazol-4-yl)acetamido]ceph-3-em-4-carboxylicacid [Example 1(c)] (100 mg) was dissolved in water (10 ml) containing sodium hydrogen carbonate (35mg)thentetrazolo[1,5-b]pyridazine-6-thiol (35 mg) was added. The solution was warmed at 65°for 6 hours under a nitrogen atmosphere then cooled to room temperature, filtered,

10 cooled in ice-water and acidified to pH2 with 2N hydrochloric acid. The precipitated solid wasfiltered, 10

washed with water (10 ml) and dried under vacuum to yield the title compound, (40 mg).

//?(KBr) 1776 cm-1; 1730 cm-1; 1682 cm-1.

15 /VM?(DMSO-d6),8 = 2.00 and 2.21 (2xs,3H);3.34(s,3H);3.60(m,2H);3.77(m,4H);4.13and4.60(2xABq, 15 2H); 4.99 and 5.00 (2x s, 1H); 5.82 (m, 1H); 7.72 (m, 2H); 8.57 (d, 1H, J = 6Hz); 8.82 (m, 1H); 9.05 and 9.07 (2 xs, 1H);9.56(s,1H).

(b) 7a-Hydroxyamino-7$-[DL-2-(2-oxo-3-methylsulphonyl-imidazolidin-1-ylcarbony/amino)-2-(thiazol-4-

20 yl)acetamido]-3-[(tetrazolo]1,5-b]pyridazin-6-yl)thiomethyl]ceph-3-em-4-carboxylicacid 20

Thetitre compound (89 mg) was prepared from the 7a-methylthiocephem [Example 2(a)] (130 mg), mercuric acetate (112 mg) and hydroxylamine hydrochloride (15 mg) by the method described in Example 1 (d).

//?(KBr) 1772 cm-1; 1730 cm-1; 1675 cm-1. 25 25

NMR (DMSO-d6), 8 = 3.35 (s, 3H); 3.45 (m, 2H); 3.77 (m, 4H); 4.10 and 4.59 (2 x ABq, 2H); 5.00 and 5.02 (2 xs, 1H); 5.86 (d, 1H, J = 6Hz); 7.73 (m, 2H); 8.57 (d, 1H, J = 6 Hz); 8.82 (m, 2H); 9.03 and 9.06 (2 xs, 1H); 9.39 (brs, 1H); 10.28 (brs,1H).

30 EXAMPLE 3 30

7oL-Hydroxyamino-7fi-[DL-2-(2-oxo-3-methylsu/phonylimidazolidin-7-ylcarbonylamino)-2-(l,2,3-thiadiazol-4-yl)acetamidoJ-cepha/osporanicacid

(a) 2-Amino-2-( 1,2,3-thiadiazol-4-yl)acetic acid hydrochloride

35 Asolutionof2-methoxyimino-2-(1,2,3-thiadiazol-4-yl)acetic acid (mixture ofsyn- and anti-isomers) 500 35

mg) [prepared by the method described in Belgian patent no. 859384 (1978)] in a mixture of methanol (10 cm3) and water (2.3 cm3) was treated with 1N hydrochloric acid (2.7 cm3) and hydrogenated under 50 psi of hydrogen with 5%rhodium-on-charcoal (500 mg) for one hour. Afurther portion of catalyst (500 mg) was added and hydrogenation continued for one hour. After filtration the filtrate was evaporated to dryness in 40 vacuo and the residue triturated with etherto give the title compound as an off-white solid, (490 mg). 40

/VM?(DMSO-d6); 8 = 4.98 (s, 1H); 7.43 (br,4H); 9.12 (s, 1H).

(b) DL-2-[2-oxo-3-methyisulphonylimidazolidin- 1-ylcarbonyfamino]-2-( 1,2,3-thiadiazoi-4-yi)acetic acid

45 The title compound (1.3 g) was prepared from the amino acid of Example 3(a) (900 mg) and 2-oxo-3- 45

methylsulphonylimidazolidin-1-ylcarbonyl chloride (1.04 g) by the method of Example 1(a).

NMR( DMSO-d6) 8 = 3.34 (s, 3H); 3.76 (m, 4H); 6.02 (d, 1H, J = 6Hz); 8.96 (d, 1H, J = 6Hz); 9.26 (s, 1H).

50 (c) Benzhydryl 7a-Methylthio-7fi-[DL-2-(2-oxo-3-methylsulphony/-imidazolidin-1-ylcarbonylamino)-2-(1,2,3- 50 thiadiazol-4-yl)acetamido]cephalosporanate

The title compound (900 mg) was prepared from DL-2-[2-oxo-3-methylsulphonylimidazolidin-1-ylcarbonylamino)-2-(1,2,3-thiadiazol-4-yl)aceticacid (1.2 g), N-methylpiperidine (417 jxl), ethyl chloroformate (327 jxl) and benzhydryl 7(3-amino-7a-methylthiocephalosporanate (1.66 g) by the method of Example 1 (b). 55 55

//? (CH2CI2) 1785 cm-1; 1735 cm-1; 1690 cm-1.

NMR (CDCI3) 8 = 2.04 (s, 3H); 2.27 and 2.30 (2 x s, 3H); 3.34 (s, 3H); 3.35 (m, 2H); 3.94 (m, 4H); 4.91 and 4.94 (2x s, 1H); 4.93 and 5.12 (2 x ABq, 2H); 6.36 (m, 1H); 6.91 (s, 1H); 7.40 (m, 10H); 7.76 and 7.86 (2 x s, 1H); 8.72 (s, 1H); 9.21 (m,1H).

60 60

(d) 7a-Methylthio-7fi-[DL-2-(2-oxa-3-methyisulphonyl-imidazolidin-1-ylcarbonylamino)-2-(1,2,3-thiadiazol-4-yl)-acetamido]cephaiosporanicacid

The title compound (603 mg) was prepared from its benzhydryl ester [Example 3(c)] (850 mg), tri-fluoroacetrc acid (2 ml) and anisole (1 ml) by the method of Example 1 (c).

9

GB 2 184 722 A

9

//?(KBr) 1779 cm"1; 1730 cm"1; 1683 cm"1.

/VM/?(DMSO-d6)8 = 1.99 and 2.01 (2xs,3H); 2.03 and 2.23 (2xs,3H); 3.35 (s,3H); 3.42 (m,2H); 3.72 (m,4H); 4.68 and 4.90 (2 x ABq, 2H); 5.05 and 5.06(2xs, 1H); 6.18(d, 1H, J = 6Hz); 9.02 (m, 1H); 9.14and9.16(2xs, 5 1H); 9.80 and 9.83 (2xs, 1H).

(e) 7 a.-Hydroxyamino-7fi-[DL-2-(2-oxo-3-methy/su/phonyl-imidazolidin- 1-ylcarbonylamino)-2-( 1,2,3-thiadiazol-4-yl)-acetamido]cephalosporanicacid The title compound (44 mg) was prepared from the 7a-methylthiocephem [Example 3(d)] (67 mg), mercuric 10 acetate (66 mg) and hydroxylamine hydrochloride (8 mg) by the method of Example 1 (d).

//? (KB r) 1780 cm"1; 1728 cm"1; 1680 cm"1.

/VM/?(DMSO-d6) 8 = 1.99 and 2.00 (2xs,3H); 2.01 and 2.21 (2xs,3H); 3.34(s,3H); 3.41 (m,2H);3.79(m,4H); 15 4.61 and4.91 (2xABq,2H);5.01 and5.03(2xs, 1H);5.84(m, 1H);7.71 (brs,1H);8.84(m,1H);9.03and9.04(2 xs, 1H); 9.39 and 9.60 (2 x s, 1H); 10.24 (s, 1H).

EXAMPLE 4 -

3-[5,6-Dioxo-4-mehyl-1,4,5,6-tetrahydro-1,2,4-triazin-3-y/)-thiomethyl]-7a-hydroxyamino-7fi-[DL-2-(2-oxo-3-20 methylsulphonyl-imidazolidin- 1-ylcarbonylamino)-2-(1,2,3-thiadiazol-4-yl)acetamido]ceph-3-em-4-carboxylicacid

(a)3-[5,6-Dioxo-4-methyl-1,4,5,6-tetrahydro-1,2,4-triazin-3-yl)thiomethyl]-7a-methylthio-7fi-[DL-2-(2-oxo-3-methylsulphony/-imidazolidin-7-y/carbonylamino)-2-(1,2,3-thiadiazo/-4-yl)acetamido[ceph-3-em-4-

25 carboxylicacid

A solution of7a-methylthio-7(3-[DL-2-(2-oxo-3-methyl-sulphonylimidazolidin-1-ylcarbonylamino)-2-(1,2,3-thiadiazol-4-yl)acetamido]cephalosporanic acid [Example3(d)] (220mg)and5,6-dioxo-4-methyl-1,4,5,6-tetrahydro-1,2,4-triazinyl-3-thiol (81 mg) in 1,2-dichloroethane (70 cm3) was refluxed under nitrogen for 14 hours. The mixture was cooled to room temperature and the title compound filtered and dried in vacuo,(W0 30 mg).

//? (KBr) 1777 cm"1,1732 cm"1,1680 cm"1.

NMR (DMSO-d6), 8 = 2.03 and 2.23 (2 x s, 3H); 3.24 and 3.26 (2x s, 3H); 3.35 (s, 3H); 3.52 (m, 2H); 3.75 (m,4H); 35 3.82and4.12(2xABq,2H); 5.02and5.03(2xs,1H);6.18(d,1H,J = 6Hz);9.02(m,1H); 9.13and9.16(2xs, 1H);9.80and9.83(2xs,1H).

(b)3-[(5,6-Dioxo-4-methyl-1,4,5,6-tetrahydro-1,2,4-triazin-3-yl)thiomethyl]-7a-hydroxyamino-7fi-[DL-2-(2-oxo-3-methyl-sulphony/imidazolidin-1-ylcarbonylamino)-2-(1,2,3-thiadiazol-4-yl)acetamido]ceph-3-em-4-

40 carboxylicacid

The title compound (96 mg) was prepared from the 7a-methylthiocephem [Example 4(a)] (158 mg), mercuric acetate (135 mg) and hydroxylamine hydrochloride (16 mg) by the method of Example 1 (d).

IR( KBr) 1772 cm"1; 1727 cm"1; 1700 cm"1.

45 NMR(DMSO-d6),8 = 3.24and3.26(2xs,3H);3.35(s,3H);3.51 (m,2H);3.76(m,4H);3.90and4.1 (2xABq, 2H); 4.99 and 5.00 (2xs, 1H); 6.21 (m, 1H); 8.97,9.00,9.04and 9.07 (2 xd, 1H, J = 6Hz); 9.14and 9.17 (2xs, 1H); 9.46 and 9.62 (2 x s, 1H); 10.24 (s, 1H).

EXAMPLE 5

50 7a-Hydroxyamino-7p-[DL-2-(2-oxo-3-methylsulphonyiimidazol-1-ylcarbonylamino)-2-(1,2,3-thiadiazol-4-yl)acetamido]-3-(pyridiniummethy/)ceph-3-em-4-carboxy/ate

(a) 7a.-Methy/thio-7$-[DL-2-(2-oxo-3-methyisu/phonyl-imidazolidin-1-ylcarbonylamino)-2-(1,2,3-thiadiazol-4-yi)-acetamido]-3-(pyridiniummethyl)ceph-3-em-4-carboxylate 7a-Methylthio-7(3-[DL-2-(2-oxo-3-methylsulphonyl-imidazolidin-1-ylcarbonylamino)-2-(1,2,3-thiadiazol-4-55 yl)acetamido]cephalosporanicacid [Example 3(d)] (500 mg) was dissolved in water (20 ml) containing sodium hydrogen carbonate (71 mg) then potassium iodide (1.92 g) and pyridine (374 ^,1) were added. The solution was heated at 70° for 4 hours, cooled, and concentrated in vacuoto 5 ml. This concentrate was diluted with acetone (50 ml) and chromatographed over silica (75g),eluting with acetone:water (3:1) to afford the crude title compound. This material was dissolved iln water (5 ml), filtered through "Hyflo" [Trade 60 Mark] and freeze dried to afford the pure title compound (110 mg).

IR (KBr) 1780 cm"1; 1730 cm"1; 1656cm"1.

NMR (DMSO-d6), 8 = 1.96 and 2.25 (2 x s, 3H); 2.88 and 3.43 (2 x m, 2H); 3.33 and3.34 (2 x s, 3H); 3.76 (m,4H); 4.92 and4.96(2xs,1H); 5.08 and 5.58 (2 x ABq, 2H); 6.15 (m, 1H); 8.14 (m, 2H); 8.56 (m, 1H); 9.00 (m, 1H); 9.09 65 and 9.14 (2xs, 1H); 9.35 and 9.43 (2xd. 1H, J = 6Hz); 9.74 and 9.75 (2xs, 1H).

5

10

15

20

25

30

35

40

45

50

55

60

65

10

GB 2 184 722 A

10

(b) 7a-Hydroxyamino-7fi-[DL-2-(2-oxo-3-methylsulphonyl-imidazolidin-1-ylcarbonylamino)-2-( 1,2,3-thiadiazol-4-yl)-acetamido]-3-(pyridiniummethyl)ceph-3-em-4-carboxylate

The title compound (67 mg) was prepared from the 7a-methylthiocephem [Example 5(a)] (78 mg), mercuric acetate (75 mg) and hydroxylamine hydrochloride (9 mg) by the method of Example 1 (d). 5 5

//? (KBr) 1777 cm-1; 1735 cm-1; 1657 cm-1.

NMR (DMSO-d6) 8 = 3.35 and 3.36 (2 x s, 3H); 3.40 (m, 2H); 3.79 (m, 4H); 5.08 (s, 1H); 5.09 and 5.47 (2 x ABq, 2H); 6.21 (d, 1H,J = 6Hz);8.21 (m,2H)

8.63 (m, 2H); 9.05 (m, 3H); 9.17 and 9.22 (2x s, 1H); 9.54and 9.66 (2 x s, 1H). 10 10

EXAMPLES

7fi-[DL-2-(4-Ethyl-2,3-dioxopiperazin-1-ylcarbonylamino)-2-(1,2,3-thiadiazol-4-yl)acetamido]-7-a-hydroxyamino-3-[(1-[2-hydroxyethyl\-1H-tetrazol-5-yl)thiomethyl]ceph-3-em-4-carboxylicacid

(a) DL-2-(4-Ethyl-2,3-dioxopiperazin- 1-ylcarbonyiamino)-2-{ 1,2,3-thiadiazol-4-yl)acetic acid

15 Thetitle compound (1.24g) was prepared from 2-amino-2-(1,2,3-thiadiazol-4-yl)acetic acid hydrochloride 15 [Example 3(a)] (912 mg) and 4-ethyl-2,3-dioxopiperazin-1-ylcarbonyl chloride (953 mg) by the method of Example 1(a).

NMR (DMSO-d6) 8 = 1.06 (t,3H, J = 7Hz); 3.37 (q, 2H, J = 7Hz); 3.56 (m, 2H); 3.94 (m, 2H); 6.04 (d, 2H, J = 6Hz); 20 9.26 (s,1H); 10.01 (d, 1H,J = 6Hz). 20

(b) Benzhydryl 7$-[DL-2-(4-ethyl- 1,2,3-dioxopiperazin- 1-ylcarbonylamino)-2-( 1,2,3-thiadiazoi-4-yl)acetamido]-7u-methylthiocephalosporanate

Thetitle compound (130 g) was prepared from DL-2-(4-ethyl-2,3-dioxopiperazin-1-ylcarbonylamino)-2-25 (1,2,3thiadiazol-4-yl)acetic acid [Example 6(a)] (1.20 g), N-methylpiperidine (445 (il) ethyl chloroformate (384 25 (xl) and benzhydryl 7|3-amino-7 a-methylthiocephalosporanate (1.95 g) by the method of Example 1 (b).

IR (CH2CI2) 1780 cm-1,1710 cm-1,1670 cm-1.

NMR (DMSO-de), 8 = 1.23 (t, 3H, J = 6Hz); 2.04 (s, 3H); 2.27 and 2.28 (2 x s, 3H); 3.34 (s, 2H); 3.56 (m, 4H); 4.10 30 (m,2H);4.91 and4.95(2xs, 1H);4.93and5.11 (2xABq,2H); 6.38(m, 1H); 6.89(s, 1H);7.40(m, 10H); 8.06and 30 8.68 (2 xs, 1H); 8.81 and 8.82 (22 x s, 1H); 10.16 (m, 1H).

(c) 7B-[DL-2-(4-Ethyl-2,3-dioxopiperazin-1-ylcarbonylamino)-2-(1,2,3-thiadiazol-4-yl)acetamido]-7a-methylthiocepha/osporanicacid

35 The title compound (594 mg) was prepared from the benzyhydryl ester of Part (b) (1.25 g) by the method of 35 Example 1(c).

//?(KBr) 1780 cm-1,1710cm-1,1678 cm-1.

NMR (DMSO-d6), 8 = 1.07 (t, 3H, J = 6Hz); 1.99 and 2.01 (2 x s, 3H); 2.03 and 2.24 (2 x s, 3H); 3.30 (m, 4H); 3.55 40 (m,2H);3.90(m,2H);4.64and4.91 (2xABq,2H);5.06and5.07(2xs,1H);6.21 (m,2H);9.13and9.15(2xs, 40 1H); 9.84 (brs, 1H); 10.05 (m, 1H).

(d) 7fi-[DL-2-(4-Ethyl-2,3-dioxopiperazin- 1-ylcarbonylamino)-2-( 1,2,3-thiadiazol-4-yl)acetamidoJ-3-[( 1-\2-hydroxyethyl\-1H-tetrazol-5-yl)thiomethyl]-7a-methylthiocephalosporanicacid

45 The title compound (100 mg) was prepared from 7p-[DL-2-(4-ethyl-2,3-dioxopiperazin-1-ylcarbonylamino)- 45 2-(1,2,3-thiadiazol-4-yl)acetamido]-7a-methylthiocephalosporanicacid [Example 6(c)](100 mg) and 1-(2-hydroxyethyl)-1H-tetrazole-5-thiol (46 mg) by the method of Example 4(a).

//?(KBr) 1778 cm-1,1711 cm-1,1672 cm-1.

50 NMR (DMSO-ds) 8 = 1.06 (t, 3H, J = 6Hz); 2.01 and 2.23 (2 x s, 3H); 3.37 (m, 4H); 3.56 (m, 2H); 3.78 (m, 4H); 4.15 50 and 4.44 (2x ABq, 2H); 4.24 (m, 2H); 5.00 and 5.01 (2 xs, 1H); 5.02 (br, 1H); 6.21 (d, 1H, J = 6Hz); 9.12 and 9.15 (2xs,1H); 9.82 and 9.84(2xs,1H); 10.07 (m,1H).

(e) 7fi-[DL-2-(4-Ethyl-2,3-dioxopiperazin-1-ylcarbonylamino)-2-(1,2,3-thiadiazol-4-yl)acetamido]-7a-

55 hydroxyamino-3-[(1-(2-hydroxyethyl)- 1H-tetrazol-5-yl)thiomethyl]ceph-3-em-4-carboxylic acid 55

The title compound (55 mg) was prepared from the 7a-methylthiocephem [Example 6(d)] (75 mg), mercuric acetate (67 mg) and hydroxylamine hydrochloride (11 mg) by the method of Example 1 (d).

//?(KBr) 1775 cm-1,1710cm-1,1670 cm-1.

60 NMR (DMSO-de), 8 = 1.06 (t, 3H, J = 6Hz); 3.54 (m, 10H); 4.10 and 4.36 (2 x ABq, 2H); 4.21 (m, 2H); 4.96 and 60 4.97 (2xs, 1H);6.24(m,1H);9.14and 9.16 (2xs,1H); 9.47 and9.62 (2xs,1H);10.04(m,1H); 10.25 (s,1H).

Thefollowing compounds of theformula (I) were prepared from the appropriate starting materials using 65 the methods described above. The side chains all haveDL-stereochemistry; 65

11

GB 2 184 722 A

11

•CH CONH

I

NH

li

COOH

10

15

20

25

30

35

40

45

Example No. .

Preparation as for Example No in (KBr) -X

HNIt (l)MSU-d , ) 0

2(u)-(b)

a

C><

1776 1729 1685

3.34 (B, 3H); 3.35 (m, 2H) ; 3.76 (iu( 4H); 4.15 and 4.56 (2 x AU<|. 2H); 5.00 and 5.0] (2 x a, 1H); 6.21 (m, )H); 7.73 (in. IH); a. 98 (d, 1H. .1 " 6 Hz); 8.(IB aiul 9.05 (2 x d. 111, .1 - 6Hz) j 9.14 and 9.16 (2 x a, 1H); 9.44 and 9.62 (2 x a, 1H).

4(a)—(b)

(X

cx

^ N^o

N—N CII,S-^ \=o N

1776 1708 1676

1.06 (c, 311, J - 7Hz); 3.25 aiu! 3.26 (2 x a, 3H); 3.36 (in, 410; 3.55 (in, 2H): 3.89 (ro. 2H); 3.90 and 4.11 (2 x ABq, 2H); 4.99 and 5.02 (2 x e, lH)i 6.24 (in. 1H); 9.14 and 9.16 (2 x a, 1H); 9.49 and 9.63 (2 x a, 1H)J 10.03 (m, 1H); 10.24 (a, 1H).

4(a)-(b)

xX

c

Vo

1780 1727 1660

3.35 (a, 3H); 3.74 (m, 6H); (2 x Atirj» 2H) ;

3.63 (w, 2H); 4.11 and 4.37 4.30 (m. 211)

N'

B-/ II \.--N

(c»2) 2oh

4.96 (a, 1H); 4.97 (br. 111) 6.22 (m, lH)i 8.99 and 9.05

(2 x d. 1H. J and 9.17 (2 x and 9.61 (2 x (a. 1H).

6IIz); 9.14 s. 1H); 9.44 s, 1H); 10.24

10

2(a)-(b)

•1

N \

N

C )=o

1777 1731 1690

3.36 (B, 3H): 3.50 (m, 6H)i 4.41 and 4.62 (2 x All*), 2H); 4.99 and 5.01 (2 x u, 1H); 6.23 (iu, 1H); 7.89 (a. 1H); 8.10 and 8.18 (2 x s, 1H); 8.98 and 9.06 (2 x d. )H, J » 6Hz)i 9.14 and 9.17 (2 x a, 1H); 9.46 and 9.62 (2. x a, 1H).

11

2(a)-(b)

Ol cr

N -%0

1775 1710 1675 1640

1.06 (in. 311); 3.50 (in, 8H);

4.11 and 4.58 (2 x AB<|. 2H)

5.01 and 5.03 (2 :: 8. 1H);

6.23 (ill, 2H); 7.95 (a. 1H); 9.00 (a. 1H); 9.1H and 9.J9 (2 x a. 1H); 9.51 and 9.61 (2 x a, 1H); 10.06 (in. 1H).

50 EXAMPLE 12 50

3-Acetoxymethy/-7fi-[DL-2-furyl-2-(3-methylsu/phonyl-2-oxo-imidazolidin- 1-ylcarbonylamino)-acetamidoJ-7-a-hydroxyaminoceph-3-em-4-carboxylicacid

(a)DL-2-furyl-2-(3-methy/sulphonyl-2-oxoimidazolidin-1-ylcarbony/amino)aceticacid

55 Thetitle compound was prepared from DL-2-amino-2-(2-furyl)aceticacid [J. Antibiotics 3 7 (6), 546(1978)] 55 by the method of Example 1(a).

NMR (CDCIa), 8 = 3.36 (s, 3H); 3.82-4.05 (m, 4H); 5.71 (d, J = 6,1H); 6.39 (m, 1H); 6.45 (m, 1H); 7.42 (s, 1H); 8.69 (d, J = 6,1H);

60 60

(b)Benzhydryl-3-acetoxymethyl-7$-[DL-2-furyl-2-(3-methyl-sulphorryl-2-oxoimidazo/idin-1-ylcarbony/amino)acetamidoJ-7a-methylthioceph-3-em-4-carboxylate

Asuspension of DL-2-furyl-2-(3-methylsulphonyl-2-oxoimidazolidin-1-ylcarbonylamino)aceticacid (0.97 g) in dichloromethane (25 ml) was treated with pyridine (240 fil). After stirring at room temperature for 5 65 minutes the almost clear solution was cooled to-30° and trichloroacetyl chloride (330 |xl) was added. The 65

12

GB 2 184 722 A

12

solution was stirred at-30°for 1 hour. A solution of benzhydryl 7p-amino-7a-methylthiocephalosporante (1.60 g) in dichloro- methane (20 ml) was added and the reaction mixture warmed to room temperature over 1 hour. Ethyl acetate (100 ml) was added and the mixture washed with 1N hydrochloric acid (2 x 50 ml) and brine (50 ml). Drying, followed by evaporation of the solvent in vacuo gave the crude product which after 5 chromatography on silica gel (eluting with a dichloromethane-ethyl acetate gradient) afforded thetitle 5

compound as a yellow solid, (0.45 g).

//?(KBr) 1785 cm"1.

NMR (CDCI3), 8 = 2.08 and 2.12 (2 x s, 3H); 2.24 and 2.32 (2 x s,3H); 3.24-3.58 (m, 2H); 3.82-4.06 (m, 4H); 4.92 10 and5.16,4.94and5.16(2xABq,J = 14,2H);4.96and4.98(2xs, 1H); 5.78and5.81 (2xd,J = 6,1H);6.41 (m, 10 1H); 6.55 (m,1H); 6.95and6.97 (2xs,1H); 7.19-7.57 (m,11H); 8.81 and 8.82 (2xd, J = 6,1H);.

(c)3-Acetoxymethyl-7fi-[DL-2-furyl-2-(3-methylsulphonyl-2-oxoimidazolidin-1-y/carbony/amino)-acetamido]-7a-methylthioceph-3-em-4-carboxylicacid

15 To a solution of benzhydryl 3-acetoxymethyl-7p-[DL-2-furyl-2-(3-methylsulphonyl-2-oxoimidazolidin-1- 15 ylcarbonylamino)-acetamido]-7a-methylthioceph-3-em-4-carboxylate (0.45 g) in dichloromethane (25 ml) at -65° under nitrogen was added anisol (0.36 g) followed by a solution of aluminium chloride (0.224 g) in nitromethane (2 ml) with stirring. After 5 minutes, ethyl acetate (10 ml) and 1N hydrochloric acid (15 ml) was added and stirring continued for 15 minutes to room temperature. The ethyl acetate layer was separated and 20 dried. Evaporation gave a yellow residue which on trituration with ether gave the product as a yellowsolid, 20 (0.315 g).

//? (KBr) 1785 cm-1.

NMR (DMSO-d6), 8 = 2.02 and 2.04 (2xs, 3H); 2.12 and 2.23 (2x s, 3H); 3.38 (s, 3H); 3.38-3.67 (m, 2H); 3.69-3.92 25 (m,4H);4.67and4.96,4.69and5.01 (2xABq,J = 14,2H);5.10and5.12(2xs,1H); 5.80(d,J = 6);6.43(m,2H); 25 7.66 (m, 1H); 8.59 and 8.63 (2 x d, J = 6,1H); 9.68 (d, J = 6,1H).

(d) 3-Acetoxymethyl-7$-[DL-2-furyI-2-(3-methylsulphonyl-2-oxoimidazolidin-1-y/carbonylamino)acetamido]-7a-hydroxyamino-ceph-3-em-4-carboxyiic acid

30 The title compound was prepared from the product of part (c) by the method of Example 1(d). 30

IR (KBr) 1785 cm""1.

NMR (DMSO-de), S = 2.01 and 2.03 (2 xs, 3H); 3.38 (s, 3H); 3.37-3.62 (m, 2H); 3.70-3.91 (m, 4H); 4.61 and 4.92, 4.63 and 4.96 (2 x ABq, J = 14,2H); 5.06 (s, 1H); 5.81 (d, J = 6,1H); 6.36-6.59 (m, 3H); 7.61 (m, 1H); 8.16and 35 8.18(2xs, 1H); 8.58and8.62(2xd,J = 6,1H); 9.37and9.46(2xs, 1H). 35

EXAMPLE 13

3-Acetoxymethyl-7fi-[DL-2-(2-oxo-3-methylsulphonylimidazolidin-1-ylcarbonylamino)-2-(3-pyridyf)acetamido]-7a-hydroxyaminoceph-3-em-4-carboxylicacid

40

(a) DL-2-(2-oxo-3-methy/sulphonyiimidazolidin)-1-ylcarbonyl-amino)-2-(3-pyridy/)aceticacid The title compound was prepared from DKL-3-pyridylglycine [prepared similarly to the method of Davis ef a/for4-pyridylglycine. Arch. Biochem. Biophys. 87,88 (1960)]. by the method of Example 1(a).

NMR (DMSO-de)B = 3.34 (s, 3H); 3.70 (m, 4H); 4.78 (d, J = 6Hz, 1H); 7.27 (m, 1H); 7.62 (d, J = 6Hz, 1H); 8.35 (d, 45 J = 3Hz, 1H); 8.49 (s, 1H); 9.00 (d, J = 6Hz, 1H).

(b) Benzhydryl 3-acetoxymethyl-7$-[DL-2-(2-oxo-3-methylsulphonylimidazolidin-1-ylcarbonylamino)-2-(3-pyridyl)acetamido]-7a-methylthioceph-3-em-4-carboxylate

The pyridylacetic acid (0.55 g) from Example 13(a) was sonicated with pyridine (0.13 ml) in acetonitrile (10 50 ml) for 0.5 hours. The suspension was cooled to -40°C, trichloroacetyl chloride (0.215 ml) was added and the mixture was stirred for 2 hours at -40°C. Benzhydryl 3-acetoxymethyl-7p-amino-7a-methylthio-ceph-3-em-4-carboxylate (0.935 g) in acetonitrile (10 ml) was then added, and the reaction mixture was warmed to 20°C and stirred overnight. The solvent was removed in vacuo, the residue dissolved in methylene chloride (20 ml) and washed with aqueous sodium bicarbonate (2 x 20 ml). The organic phase was dried over 55 magnesium sulphate and the solvent removed in vacuo. The residue was chromatographed (silica gel, ethyl acetate:isopropanol, 95:5) to give the title compound (0.26 g).

IR (KBr), 1785 cm"1.

NMR (DMSO-de) 8 = 1.91,1.94,1.97 and 2.21 (4xs, 6H); 3.27 (s, 3H); 3.48 and 3.60 (ABq, J = 14 Hz, 2H); 3.70 60 (m,4H); 4.60,4.67 and 4.48,4.90 (2xABq,J = 12Hz,2H); 5.14and5.15(2xs, 1H);4.63(m, 1H); 6.87 (s, 1H); 60

7.36 (m, 12H); 7.77 (m, 1H); 8.50 (m, 1H); 8.65 (s, 1H); 8.83(2xd, J = 6Hz,1H); 9.82 and 9.87 (2xs,1H).

40

45

r

50

55

(c) 3-Acetoxymethyl-7fi-[DL-2-(2-oxo-3-methylsulphonylimidazolidin-l~ylcarbonylamino)-2-(3-pyridyl)-acetamido]-7oL-methy/thioceph-3-em-4-carboxylicacid

13

GB 2 184 722 A

13

The benzhydryl ester (0.25 g) from Example 13(b) above in methylene chloride (10 ml) at -20°C was treated with anisole (0.27 ml) followed bytrifluoroaceticacid (1 ml). The reaction mixture was warmed to 20°Cand stirred for 1 hour. The solvent was then removed in vacuo and the residue dissolved in methanol (15 ml). Isopropyl alcohol (10 ml) was added and the methanol was removed in vacuo. The solid was filtered off, 5 washed with ether (2 x 20 ml) and dried/'/? vacuo, yielding the title compound as an off white solid, (0.16g).

/fl(KBr) 1780 cm-1.

NMR (DMSO-de) § = 1 -95,1.98,2.01 and 2.21 (4x s, 6H); 3.34 (s, 3H); 3.50 (m, 2H); 3.70 (m, 4H); 4.60,4.66 and 4.92,5.04 (2 x ABq, J = 12Hz, 2H); 5.07 and 5.08 (2 xs, 1H); 5.64 (d, J = 6Hz, 1H); 7.47 (m, 1H); 7.84 (m, 1H); 8.55 10 (m, 1H); 8.67 (s, 1H); 8.82,8.86 (2 x d, J = 6Hz, 1H); 9.77 and 9.82 (2 xs, 1H).

(d) 3-Acetoxymethyl-7$-[DL-2-(2-oxo-3-methylsulphony/imiclazolidin-1-ylcarbonylamino)-2-(3-pyridyll-acetamido]-7<x-hydroxyamino-ceph-3-em-4-carboxyiicacid

(i)The7a-methylthio cephem (0.14g)from Example 13(c) in dimethylformamide (3 ml) was treated with

15 mercuric acetate (0.076 g) in DMF (1 ml) at -60°C followed by O-fert-butyl-diphenylsilylhydroxylamine (0.71 g) in dimethylformamide (1 ml). The reaction mixture was warmed to -5°C and stirred at -5°Cfor 1 hour. The reaction mixture was diluted with ethyl acetate:THF (2:1,90 ml) and washed with brine (3x50 ml). The organic phase was dried over magnesium sulphate and the solvents removed in vacuo yielding 3-acetoxymethyl-7(3-[DL-2-(2-oxo-3-methyl-sulphonylimidazolidin-1-ylcarbonylamino)-20 2-(3-pyridyl)acetamido]-7a-0-fe/t-butyldiphenylsilylhydroxyamino-ceph-3-em-4-carboxylate, (0.145 g).

(ii)The7a-tert-butyldiphenylsilylhydroxyamino-cephalosporanate(0.14g)from Example 13(d)(i) in acetonitrile/tetrahydrofuran (1:1) (4 ml) was treated with 40% hydrofluoric acid at 0°C for 0.5 hours. The solvents were removed under high vacuum and the residue dissolved in methanol (10 ml), filtered, isopropyl

25 alcohol (10 ml) was added and the methanol removed in vacuo. The solid was filtered and washed with ether (2x20 ml) yielding the title compound (0.08 g) as the hydrofluoride salt.

IR (KBr), 1785 cm-1.

NMR (DMS0-d6), § = 2.00 and 2.04 (2 x s, 3H); 3.34 (s, 3H); 3.50 (m, 2H); 3.70 (m, 4H); 4.58 and 4.90 (2 x ABq, 30 2H); 4.97 and 4.99 (2x s, 1H); 5.02 (s, 1H); 5.76 (m, 1H); 7.82 (m, 1H); 8.23 (m, 1H); 8.74 (br, 1H); 8.82 (br, 1H); 8.92 (m,1H); 9.64 (m,1H).

EXAMPLE 14

7$-[DL-2-(2-oxo-3-methylsuiphonylimidazolidin- 1-ylcarbonyi-amino)-2-(3-pyridyi)acetamidoJ-7a-hydroxy-35 amino-3-(pyridinium-methyl)ceph-3-em-4-carboxylate

(a) 7^-[DL-2-(2-oxo-3-methylsulphonylimidazolidin-1-ylcarbonyl-amino)-2-(3-pyridylacetamido)]-7a-methyl-thio-3-(pyridinium-methyl)ceph-3-em-4-carboxylate

The title compound was prepared from the product of Example 13(c) by the method of Example 5(a).

40

//?(KBr) 1765 cm-1.

/V/W?(DMSO-d6),8 = 1.86 and 2.23 (2xs, 3H); 3.34 (s,3H); 3.50 (m,2H); 3.70 (m,4H); 4.95 and 4.98 (2xs, 1H); 5.12 (m,1H); 5.60 (m,2H); 7.36 (m,1H); 7.73 (m,1H); 8.13 (m,2H); 8.4-8.62 (m,4H); 8.80 (m, 1H); 9.34and9.42 (2 d, J = 4Hz, 1H); 9.73 (s,1H).

45

(b) 7fi-[DL-2-(2-oxo-3-methylsu/phony/imidazolidin-1-y/carbonyl-amino)-2-(3~pyridyi)acetamido]-7a-hydroxyamino-3-(pyridinium-methyi)ceph-3-em-4-carboxylate

The7a-methylthiocephem (0.12g)from Example 14(a) was dissolved in dimethylformamide (3 ml) and cooled to -60°C. A solution of mercuric acetate (0.064 g) in dimethylformamide (1 ml) followed by 50 hydroxylamine hydrochloride (0.015 g) in dimethylformamide (1 ml) and triethylamine (0.025 ml) were added. The reaction was stirred at -5°C for 2 hours, diluted with acetone (50 ml) and chromatographed over silica gel. Elutionwith acetone: water (3:1) and evaporation of solvents yielded the title compound, (0.052 g).

//?(KBr), 1775cm-1.

55 /V/W/7(DMS0-d6),8 = 3.34(s,3H); 2.52 (m,2H);3.70(m,4H);4.95and5.04(2xs,1H);5.05and5.10(2xd,J = 6Hz, 1H); 5.43 and 5.49 (ABq, J = 12Hz, 1H); 5.63 and 5.65 (2 xs, 1H), 6.36 and 6.42 (2 x s, 1H); 7.33 (m, 1H); 7.73 (m, 1H); 8.12 (m, 3H); 8.40-8.85 (m, 4H); 9.31 and 9.42 (2d, J = 4Hz, 1H); 9.53 and 9.59 (2 xs, 1H).

EXAMPLE 15

60 7$-[DL-2-(2-oxo-3-methylsuiphonyiimidazolidin- 1-ylcarbonylamino)-2-(3-pyridyi)acetamido]-7a-hydroxyamino-3-[tetrazolo[1,5-b]pyridazin-6-yl)thiomethyi]ceph-3-em-4-carboxylate

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(a) 7fi-[DL~2-(2-oxo-3-methy/su/phony/imidazo/idin-1-ylcarbonyl-amino)-2-(3-pyridyi)acetamido]-7a-methyl-thio-3-[(tetrazolo[1,5-b]pyridazin-6-y/)thiomethyl]ceph-3-em-4-carboxyiate

14 GB 2 184 722 A

14

Thetitle compound was prepared from the 7a-methylthio-cephalosporanic acid (0.35 g) of Example 13(c)

by the method of Example 2(a).

IR (KBr), 1775 cm"1.

5 NMR(DMSO-d6), 8 = 1.93 and 2.20 (2 xs, 3H); 3.33 (s, 3H); 3.55 (m, 2H); 3.77 (m,4H); 4.15 and 4.60 (2 xABq,J 5 = 12Hz,2H); 5.04 (s, 1H); 5.63 (d, J = 6Hz, 1H); 7.38 (m,1H); 7.75 (m,2H); 8.56 (m,3H); 8.80 (m, 1H); 9.78 and 9.82 (2xs,1H).

(b) 7fi-[DL-2-(2-oxo-3-methy/su/phonylimidazolidin-1-10 ylcarbonyl-amino)-2-(3-pyridyl)acetamido]-7a-hydroxyamino-3-[(tetrazolo-[ 1,5-b]pyridazin-6-y/)thiomethyl]ceph-3-em-4-carboxylate

Thetitle compound was prepared from the 7a-methylthio-cephem (0.13 g) of Example 15(a) by the method of Example 1(d).

IR{ KBr), 1775 cm-1.

15 NMR(DMSO-d6) 8 = 3.3 (s,3H); 3.52 (m, 2H); 3.72 (m,4H); 4.15 and 4.60 (2xABq,J = 12Hz, 2H); 5.00 and5.01 (2 xs, 1H), 5.70 (m, 1H); 7.52 (m, 1H); 7.71 and 7.74 (2x d, J = 5Hz, 1H); 8.10 (br, 1H), 7.92 (m, 1H); 8.43 and8.46 (2 xd, 1H); 8.56 (m, 2H); 8.69 (m, 1H); 8.82 and 8.87 (2 x d, J = 6Hz, 1H); 9.46 and 9.65 (2 xs, 1H).

EXAMPLE 16

20 3-[(2,5-Dihydro-6-hydroxy-2-methyl-5-oxo- 1,2,4-triazin-3-yl)-thiomethyl]-7a-hydroxy- 20

amino-7fi-[DL-2-(3-methylsulphonyl-2-oxoimidazolidin-1-ylcarbony/amino)-2-(1,2,3-thiadiazol-4-yl}-acetamido]ceph-3-em-4-carboxylicacid

(a) 7fi-Benzy/ideneamino-3-[(2,5-dihydro-6-hydroxy-2-methy/-5-oxo-1r2,4-triazin-3-y/)thiomethy/Jceph-3-em 25 4-carboxy/fc acid

Asuspensionof7p-amino-3-[(2,5-dihydro-6-hydroxy-2-methyl-5-oxo-1,2,4-triazin-3-yl)thiomethyl]ceph-3-em-4-carboxylic acid (see EP 65748) (3.71 g) in dimethylformamide (50 ml) was treated with benzaldehyde (4.24 g) and the mixture was sonicated in an ultrasound bath at 25° for 0.5 hours, by which time a clear solution had resulted. After evaporation to dryness in vacuo the residue was triturated with ethertogivethe 30 title compound,

(4.28 g).

I.R. (KBr), 1770 cm-1.

N.M.R. (DMSO-d6), 8 = 3.53 and 3.73 (ABq, J = 18,2H); 3.58 (s,3H); 4.08 and4.35 (ABq, J = 13,2H); 5.28 (d, J 35 = 4.5,1H); 5.62 (d, J = 4.5,1H); 7.46-7.77 (m,5H); 8.56 (s,1H). 35

(b) Benzhydryl 3-[(6-benzhydryloxy-2,5-dihydro-2-methyi-5-oxo-1,2,4-triazin-3-yi)thiomethyl]-7$-benzylideneaminoceph-3-em-4-carboxylate

The productfrom Example 16(a) (4.02 g) in dichloromethane (50 ml) was treated dropwise over 0.5 hours 40 with a solution of diphenyidiazomethane (3.74 g) in dichloromethane (50 mi). After stirring for a further 18 40 hours the solution was evaporated to dryness and the residue was triturated with ether (150 ml) to afford the title compound as a pale cream solid, (5.45 g).

I.R. (KBr), 1780 cm-1.

45 N.M.R. (CDCI3),8 = 3.53 (s,3H); 3.54 and 3.68 (ABq, J = 19,2H); 4.06 and 4.43 (ABq, J = 13,2H); 5.17(d, J = 5, 45 1H); 5.43 (d, J = 5,1H); 6.75 (s, 1H); 6.96 (s, 1H); 7.20-7.79 (m, 25 H); 8.62 (s, 1H).

(c) Benzhydryl 7fi-amino-3-[(6-benzhydryloxy-2,5-dihydro-2-methyl-5-oxo- 1,2,4-triazin-3-yl)thiomethyl]-7a-methylthioceph-3-em-4-carboxylate

50 Thedibenzhydryl cephem (Example 16(b)) (3.02 g) intetrahydrofuran (20 ml) was treated at-65° with a 50

solution of potassium fe/t-butoxide (0.43 g) in tetrahydrofuran (10 ml), added dropwise over 1 minute. The resulting dark red solution was stirred for a further 2 minutes at -65°then methyl methane-thiolsulphonate (0.48 g) was added and the mixture stirred to —30° over 1 hour. The reaction mixture was added to water (50 ml) and dichloromethane (50 ml) and the dichloromethane layerseparated, dried (Na2S04) and evaporated in 55 vacuo giving a foam (3.3 g). 55

The crude 7 a-sulphenylated imine thus obtained was dissolved in methanol (10 ml) and dichloromethane (10 ml) and treated with Girard Reagent-T. After stirring for 4 hours at 25° the solution was evaporated//7 vacuo and the residue dissolved in dichloromethane (20 ml) and water (20 ml). The dichloromethane layer was separated, dried (Na2S04) and evaporated in vacuo giving a foam. The product was purified by silica gel 60 chromatography (dichloromethane-ethyl acetate gradient) to afford thetitle compound, (0.96 g). 60

I.R. (KBr), 1775 cm-1.

N.M.R. (CDCI3),8 = 2.11 (s,2H); 2.35 (s, 3H); 3.54 (s,3H); 3.57 and 3.68 (ABq, J = 18,2H); 4.14and 4.46 (ABq, J = 13,2H); 4.78 (s, 1H); 6.75 (s, 1H); 6.90 (s, 1H); 7.21-7.49 (m,20H).

10 15

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15

GB 2 184 722 A

15

(d) Benzhydryl 3-[(6-benzhydryloxy-2,5-dihydro-2-methyl-5-oxo-1,2,4-triazin-3-yl)thiomethyl]-7$-[DL-2-(3-methylsulphonyl-2-oxoimidazo/idin-1-ylcarbonylamino)-2-(1,2,3-thiadiazol-4-yl)-acetamido]-7a-methylthioceph-3-em-4-carboxylate

The title compound (210 mg) was prepared by the reaction of the product from Example 16(c) above (400 5 mg)with DL-2-(3-methylsulphonyl-2-oxoimidazolidin-1-ylcarbonylamino)-2-(1,2,3-thiadiazolyl)aceticacid [from Example 3(b)] (167 mg)bythe procedure described for Example 13(b).

I.R. (KBr), 1780 cm-1.

N.M.R. (CDCI3), 8 = 2.18 and 2.23 (2 xs, 3H); 3.21 (s, 3H); 3.33-3.56 (m, 2H); 3.51 and 3.53 (2xs,3H); 3.75-4.08 10 (brm,4H); 4.18-4.62 (m, 2H); 4.84and 4.93 (2xs, 1H); 6.42-6.46 (m,1H); 6.72 and 6.75 (2xs,1H); 6.84and6.87 (2xs, 1H); 7.17-7.50 (m, 20H); 8.92-9.07 (brm, 2H); 9.21 and 9.23 (2xs, 1H).

(e)3-[(2,5-Dihydro-6-hydroxy-2-methyl-5-oxo-l,2,4-triazin-3-yl)-thiomethyl]-7$-[DL-2-(3-methylsulphonyl-2-oxoimidazolidin-7-ylcarbonylamino)-2-(1,2,3-thiadiazol-4-y/)acetamido]-7cL-methy/thioceph-3-em-4-

15 carboxylicacid

Asolution of the dibenzyhydryl cephem [from Example 16(d)] (210 mg) in dichloromethane (3 ml) containing anisole (260 mg) was precooled to -65° and treated with a solution of aluminium chloride (160 mg) in nitromethane (1 ml). After 0.5 hours at —65° the reaction mixture was added to saturated aqueous sodium bicarbonate (20 ml) and ethyl acetate (20 ml). The mixture wasfiltered to remove insoluble inorganic 20 salts and the aqueous layer separated. Concentrated hydrochloric acid was added to pH 1.0 and the solution saturated with solid sodium chloride. Extraction with 2 x 20 ml portions of ethyl acetate:tetrahydrofuran (1:1), followed by drying (Na2SC>4) and evaporation of the solvent in vacuo, afforded the title compound, (112 mg).

25 /./?. (KBr), 1780cm-1.

N.M.R. (DMSO-d6),8 = 2.03 and 2.23 (2xs,3H); 3.30-3.83 (m, 12H); 4.00-4.08and4.30-4.37 (2xm,2H);5.03 and 5.04(2xs, 1H); 6.18 (d, J = 8,1H); 8.98-9.03 (m, 1H); 9.14and9.15(2xs, 1H); 9.78and 9.81 (2xs,1H).

(f) 3-[(2,5-Dihydro-6-hydroxy-2-methy/-5-oxo-1,2,4-triazin-3-yl)thiomethyl]-7a-hydroxyamino-7fi-[DL-2-(3-methylsulphonyl-2-oxoimidazo/idin-1-y/carbonylamino)-2-(1,2,3-thiadiazol-4-y/jacetamidoJceph-3-em-4-

30 carboxylicacid

Thetitle compound (60 mg) was prepared by reaction ofthe7 a-methylthiocephem [Example 16(e)] (105 mg) with mercuric acetate and hydroxylamine hydrochloride as described previouslyfor Example 1 (d).

I.R. (KBr), 1775 cm-1.

35 N.M.R. (DMSO-d6), 8 = 3.21 -3.41 ((m, 2H); 3.34 (s, 3H); 3.56 and 3.57 (2 xs, 3H); 3.72-3.83 (m, 4H); 3.97-4.04 and 4.28-4.35 (2 x m, 2H); 4.99 and 5.02 (2 x s, 1H); 6.22 (d, J = 8,1H); 6.51-6.53 (br m, 1H); 8.08 and 8.17 (2 xs, 1H); 8.97 and 9.05 (2xd, J = 8,1H); 9.12 and 9.15 (2xs,1H);9.43and 9.59 (2xs,1H).

Claims

40

1. Acephalosporinoftheformula:-WOH

R-CH-CONH

COOH

or a salt or esterthereof,

where R is an unsubstituted thiazolyl, thiadiazolyl,furyl or pyridyl group;

R2is-CH20C0CH3, -CH2OCONH2 or a group of theformula:-

55

-CH N x)

W

or -CH2S.Het,

where m is 1 or 2 and each R3 is independently H or C1-C4alkyl; and Het is an optionally substituted 5-or 6-membered heterocyclic group containing up to 4 heteroatoms selected from 0,Sand N, the heterocyclic group being optionally fused to an optionally substituted benzene ring or to a further 5- or 6-membered 65 heterocyclic group containing up to 4 heteroatoms selected from O, S and N;

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GB 2 184 722 A

16

and R1 is a group of theformula:-

c=0

or

C=0

t

10

where R4 is 0^4 alkyl or-S02(C1-C4 alkyl).

2. A compound as claimed in claim 1 wherein Rz is either (a) pyridiniummethyl or (b)-CH2SHet where "Het" is a triazolyl, tetrazoiyl, thiazolyl, isothiazolyl,oxazolyl,oxadiazolyl,triazinyl,thiadiazolyl, benzoxazolyl, benzothiazolyl, ortetrazolopyridazinyl group, said groups being optionally substituted by

15 D1-C4 alkyl, C1-C4 alkoxy, halo, oxo or a group oftheformula-(CH2)PR5 where p is 0,1,2or3andR5is-COOH, -0S020H,-S020H,-P03H2 or-OH, with the proviso that p is notzero when Het is tetrazoiyl.

3. A compound as claimed in claim 2 wherein "Het" is attached to the adjacent S atom by a carbon atom of the heterocyclic ring and is (i) thiadiazolyl optionally substituted by C!-C4 alkyl or 2-hydroxy ethyl (ii) tetrazoiyl optionally substituted byC1-C4alkyl,carboxymethyl,sulphomethyl,2-hydroxyethyl,

20 hydroxysulphonyloxymethyl or2-(hydroxysulphonyloxy)ethyl (iii) thiazolyl optionally substituted by 1 or 2 substituents each selected from ^-64 alkyl and carboxymethyl (iv) isothiazolyl optionally substituted by 1 or 2 substituents each selected from hydroxy and carboxy (v) benzothiazolyl or benzoxazolyl optionally substituted by hydroxy, (VC4 alkoxy or halo (vi) tetrazolopyridazinyl optionally substituted by carboxy (vii) triazinyl optionally substituted by Ct-04 alkyl and/or by 1 or 2 oxo or hydroxy groups or (vii) triazolyl

25 optionally substituted by carboxymethyl.

4. Acompound as claimed in claim 3 wherein "Het" is a group selected from

N — N

30

35

40

A.

H

H

(ch2)2oh

CH.

and

(tetrazolo/i,5-b/pyridazin-6-yl)

5. Acompound as claimed in anyone of the preceding claims wherein R is4-thiazolyl, 1,2,3-thiadiazol-4-yl,2-furyl or3-pyridyl.

6. Acompound as claimed in any one of the preceding claims wherein R4is CH3, C2HS or-S02CH3. 45 7. AcompoundasclaimedinclaimewhereinRMs

50

r L

c=o t

N

N

SO^CH.

C-Q N>.

C2H5

8. A pharmaceutical composition comprising (i) a compound of theformula (I) as claimed in any oneof 55 the preceding claims, or a pharmaceutically acceptabfe salt or in vivo hydrolysable esterthereof, (ii) a pharmaceutically acceptable diluent or carrier and, optionally, (iii) a p-lactamase inhibitor.

9. Acompound of the formula (I) as defined in anyone of claims 1 to 7, or a pharmaceutically acceptable saltor//7 vivo hydrolysable esterthereof, for use as a medicament.

10. The use of a compound of theformula (I) as defined in any one of claims 1 to 7, or of a

60 pharmaceutically acceptablesalt or/'/7v/Vo hydrolysable esterthereof, forthe manufacture of a medicament for use as an antibiotic.

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Printed for Her Majesty's Stationery Office by Croydon Printing Company (UK) Ltd, 5/87, D8991685. Published by The Patent Office, 25 Southampton Buildings, London WC2A1 AY, from which copies may be obtained.