CEPHALOSPORINS

The present invention relates to a novel class of substituted cephems -3 antimicrobial activity, a process for their preparation and intermediates useful for such preparation. The -3 substituted cephems to antimicrobial activity according to the invention are generally active against gram-positive organisms and many of which are effective against organisms both Gram positive and Gram negative.

The core cephem -3 is represented by the formula (I) below and its positions are, conventionally, numbered as indicated:

The cipher 3 in the ' term "cephem -3" indicates the position of the double bond * If the double bond was between and in the formula (I), the core would have corresponded to that of a cephem -2.

This invention is realized in a 7-beta- acylaminocéphème -3 of formula (IX) or an acid addition salt thereof

wherein "Py" represents a group (2ou 3-pyridyl) or a 3-pyridyl group 2ou quaternized, R is 1

organic acyl and R represents a carboxylic acid group or a salt or ester pharmaceutiquemént acceptable of a carboxylic acid group.

2ou 3pyridyle The presence of the group in the compounds of formula (II) allows the formation of acid addition salts. The salts may comprise, for example, inorganic salts such as sulfate, nitrate, phosphate, borate and the halogénhydrates, e.g hydrochloride, the hydrobromide and 1 'hydroiodide, and organic salts as 1' acetate, 1 'oxalate, tartrate, malate, citrate, succinate, benzoate, 1' ascorbate and methane- suifonate. Quaternised The pyridyl groups "Py" compounds (II) comprise 1' methiodide and metho-trifluoroacetate.

R of compounds of formula (II) is a carboxylic acid group or a salt or ester pharraaceutiquement acceptable of a carboxylic acid group. Exemplary suitable salt, salts include sodium, potassium, calcium, magnesium or aluminium and ammonium salts or substituted ammonium, such as those formed with trialcoylamines such as triethylamine, the procaine, dibenzylamine, triethanolamine, 1- éphénamine, the ethylpiperidine and other amines that have been used to form salts with benzylpenicillin. Exemplary suitable esters, include those which dissociate easily in the human body to produce the parent acid, for example the esters acyloxyalcoyliques, as acétoxyméthylique, pivaloyloxymethyl, alpha- acétoxyéthylique, and alpha-alpha- acétoxybenzylique.

pivaloyloxyéthylique. Other esters include

lactone esters, and dithiolactone thiolactone (that is-

1

the compounds of formula (II) in which R is a group of the formula:

rCO-O-CH Z

I I

X C = Y

wherein X and Y are oxygen or sulfur and Z is a divalent hydrocarbon group in particular the estefs phthalides and substituted phthalides, for example the ester of 3,4- diméthylphtalide.

The R group in the formula (II) has been defined as a organic acyl group. A vast majority of cephems -3 with antibacterial activity which have been mentioned in the literature to date carry: a 7-acylamino group. It has been found, over the years, that by varying the identity of the group 7-acylamino, one can modify the spectrum and (or) the intensity of the antibacterial activity of any given cephem -3. Generally the like, in this case, for a given "Py" group in formula (II), can be introduced a very large number of groups 7-acylamino, resulting in a range of compounds with ' spectra and intensities of very different. However, in general, regardless of the identity of the acyl group R, the compounds of formula (II) have a certain activity and those familiar with the technique of cephalosporins may determine the range of acyl groups R may be introduced.

As a result, generally, R in formula (II) may be any of organic acyl groups are present in the natural penicillins and cephalosporins and semi-synthetic mentioned in the literature.

_AOE280A2AO> Examples, include acyl groups of general formulae (i), (ii) and (iii)

r (i)² (ch₅ ) --ch (ch₂ )-C0-

d ⁿ j

X

2

wherein R is hydrogen, or an alkyl group, cycloalkyl (in particular in to Cg cycloalkyl), cycloalkenyl (particularly cyclohexenyl or cyclohexadienyl), aryl (in particular phenyl or substituted phenyl, for example £-hydroxy-phenyl), heterocyclic (e.g. thienyl, pyridyl, isoxazolyl substituted as the group 3-0-chlorophenyl-5-methyl-isoxazolyl -4, sydnonyle, tetrazolyl); X represents hydrogen, a hydroxy group, a halogen atom (in particular chlorine), a carboxylic acid group or a carboxylic acid ester group (such as an ester or phenyl indanylique ), an azido group, an amino group or a substituted amino group (including the units ureido, ureido substituted, and guanidine substituted guanidino), a triazolyl group, tetrazolyl group, a cyano group, an acyloxy group (for example a Formyloxy or lower alkanoyloxy group) or an esterified hydroxy group, and n and m are each independently 0, 1, 2 or 3.

wherein n is an integer from 1 to 4 and X has the same meaning as in (i) above.

(iii)

R

4 _AOE280A2AO>

R-Z-C-CO-

I⁶

4

wherein R is an alkyl group, aralkyl, aryl (in particular phenyl or substituted phenyl), cycloalkyl (in particular a group cyicloalcoyle in to Cg or substituted cycloalkyl), cycloalkenyl (in particular a group

cyclohexenyl or cyclohexadienyl), or a heterocyclic group

5 S

(in particular a thienyl or pyridyl group) 5 R and R are each hydrogen, a lower alkyl group, phenyl, benzyl or phenylethyl; and Z is oxygen or sulfur-.

A Specific examples of organic acyl groups R which may be present in the compounds according to the invention, groups include 2- thiénylacétyle, phenylacetyl, 2- hydroxyphénylacétyle, 2- aminophénylacétyle, 4- pyridylacétyle, 2-amino-p- hydroxyphénylacétyle and 1-tetrazolylacetyl, but other acyl groups are specifically indicated in the examples below.

The compounds of formula (II) may be prepared by a process which comprises reacting a compound of formula (III) or a salt or dérivé_AOE296A0AO> silylated thereof level i

(Formula III See next page)

H

h₂ n-C

(III)

// N C CH₀ Py Υ -/

C

i ¹

wherein R is a carboxylic acid group or a salt or ΐ ester of a carboxylic acid group and "Py" is 2-or 3pyridyle, with a derivative of acylation N activated on the suitable acid ROH, R is an acyl group wherein any of the free hydroxy or amino groups can be blocked and, using. silylated derivative of the compound (III), by removing the silyl group by hydrolysis or alcoholysis, and converting an amino group optionally protected hydroxy or amino group or hydroxy free, and optionally in the quaternization of the pyridyl group.

As used herein, the term "silylated derivative" of the compound (III) the product of the reaction between the compound (III) and a silylating agent such as a halogénodialcoylsilane, a halogénotrialcoylsilane, or a halo-dialkoxysilane halogénotrialcoxysilane, or, a arylou aralcoyl -corresponding silane, and compounds such as 1' hexamethyldisilazane. The silylated derivatives of the compound (III) are extremely sênsibles to moisture and hydroxylated compounds and, after reaction-with the acylating derivative of the acid on N ROH, the silylated acylated intermediate groups of the compound can be removed by hydrolysis or alcoholysis.

N Since acylating derivatives on suitable acid ROH, include chloride, bromide 1e, anhydride, mixed anhydrides and acid derivatives, and the reactive intermediates formed from the acid and a carbodiimide or carbonyldiimldazole. All the reactive groups, such as amino groups or hydroxy groups, present in the acyl group R may be protected during the acylation on N.

Protecting groups suitable for the amino groups are known in the literature for the synthesis of alpha-aminobenzyl penicillin or alpha-aminobenzyl-cephalosporins. For example, any amino group may be blocked by protonation, by conversion to t- butoxycarbony 1amino or N- méthoxycarbonylpropén -2-ylamino. These protecting groups can then be removed to leave the free amino group. A protection mode useful free hydroxy groups includes providing a dicarboxylic anhydride ' suitable, mandelic and carboxanhydride for example. After _AOE296A0AO> N with the acylation on the reagent, 1e free hydroxy group is formed without having to remove the protection.

Since are intermediates useful for the preparation of compounds of formula (II), 1' invention also relates to the compounds of formula (III), i.e. the following compounds and salts and esters s

3 - (2-pyridylmethyl) 7-beta-amino-cephem-4-carboxylic.

3 - (3-pyridylmethyl) - 7 beta-amino-3-cephem-4-carboxylic.

It is sometimes possible to prepare the compounds of formula (II) parjun method which comprises heating a compound of formula (IV) or (IVA)

R11 (IV)

. C IVA)

wherein R and Py have the same meaning as in formula (II), η is equal to 0 or 1, R " is a carboxylic acid group esterified, R_& , R ^ and R are groups alcoy-

the lower, substituted or unsubstituted aryl or aralkyl,

11

and R_& and are alkoxy groups or substituted or unsubstituted aralkoxy, this stage of heating is performed at a temperature of from 30° to 150 °C C in an inert organic solvent and whether a compound of formula (IV) or (IVA) wherein n ≈ 1 is used, subsequently reduce the resulting in a sulfide sulfoxide, and, if desired, to perform deesterification of the esterified carboxylic acid group R " to produce a free carboxylic acid group or a salt thereof. Preferably, the temperature of the heating step is between 75 °C and 125 °C.

Solvents suitable are those which are inert under the reaction conditions and high between 30° and 150 °C, for example dioxane, toluene and benzene. Solvents high boiling point are difficult to remove after the reaction, and as a result they are not preferable. In order to obtain a net reaction, it may be preferable to carrying out the step of heating under an inert atmosphere, although this is not essential. Furthermore, it is preferable the solvent drying carefully to avoid decomposition. of the starting material.

The radicals R_& , R ^ and R_c the phosphine compound of formula (IV) may be lower alkyl or aryl radicals (of

preferably phenyl) optionally substituted, the radicals and

11

R and R ^ of the compound (IVA) may be optionally substituted lower alkoxy.

If necessary, the sulfoxide compound that is obtained after cyclization of (IV) or (IVA) (n = 1) desired sulfide can be reduced by any of the conventional methods, for example by those described in British Patent

NO. 1.280.693. A method of this kind which has been considered particularly useful is the treatment with triphenylphosphine and acetyl chloride.

The method indicated above for the preparation of the compound (II) by cyclization Witting reagents (IV) and (IVA) is applicable only when the acyl group R is sufficiently stable to support the stage and any deesterifying Heating sometimes extended to conversion of the group R " to a carboxylic acid group.

. (N.B. If R in (IV) or (IVA) is a blocking group such as a triphenylmethyl group which after that the bicyclic ring system (II) has been produced, can be removed by conventional means, the amino compound free (III) can be prepared, and then acylated as previously described).

Course It will be appreciated that the compounds according to the invention show a close relationship structurally with the known 3-cephem of formula (V), whose name is "cephaloridine" admitted.

The compounds according to the invention differ from the cephaloridine in that the group "Py" of the formula (II) is attached to the methylene group through a carbon-carbon bond and not a carbon-nitrogen bond. The compounds of formula (II) are not accessible by the channels normally used to prepare cephaloridin. The compounds according to the invention have advantages over compounds known from the prior art which are the closest in that (by comparing the compound according to the invention with its analog of the prior art the nearest) tend to have a wider activity spectrum and (or) an intensity of activity against clinically important organisms. In many cases, is note from work on the mouse that the compounds according to the invention gives rise to blood levels longer lasting as analogs of the prior art the most closely adjacent.

Examples below, given as non-limiting, will further include .1 ' invention.

EXAMPLE 1

(A) Preparation of 3 - (3-pyridylmethyl) - 7-beta-(2- thiénylacétamido ) - 3-cephem-4- carboxylatë of t.-butyl

Are added to a solution under stirring of the crude free base (VIII) (75 mg) in methylene chloride anhydrous, to

-10 °, triethylamine (65 mg) and 2- thiénylacétyle (freshly distilled, 39 mg). The mixture was stirred to

-10° during 15 minutes, is diluted with methylene chloride (10 ml) and washed with brine. Evaporated, the organic layer dried (SO ^ Mg), resulting in a crude gum.

Are the raw gum chromatography on a silica gel, elution with ensuring the mixtures ethyl acetate/petroleum ether, to give the desired cephem (IX) in the form of a solid foam (34 mg, 45%).

Cm "" V¹ ( CHC1₀ ) 1780, 1715, 1685.

max 3' ¹

Molecular ion measured at ^ 471.1269 23^H 25^N 3° 4^2^{: calcu} -ⁱ ® 471.1286-, = 3.6 PPM Error). Fragmentation corresponds to the structure.

λ 237.5 nm (ethanol) (13,500): 263 (10,650):

max ''

oC² p = 83.9 ° (CHC1 C = 1% in₃ )

4T PPM ( CDCl ^) :=

1,52 (S, 9:00), 2.96 and 3.42 (nibble AB, 2:00,18:00 J * z), 3.41 and 4.09 (nibble AB, 2:00, ≈ 15:00 J z), 3.85 (s, 2Η ), 4.98 (d, 1:00, J = 5:00 z), 5.85 (dd, 1:00, J ≈ 5:00 z, J ' = 9:00 z), 6.8-7.9 (byte aromatic + NH), 8.2-8.8 (wide, aromatic).

(B) Preparation of trifluoroacetate salt of the acid 3 - (3-pyridylmethyl) - 7 beta-(- 2- thiénylacétamido ) ~ -3 cephem-4-carboxylic The by dissolving the cephem (XX) (33 mg) in trifluoroacetic acid (0.5 ml) and the solution is held. at room temperature for 5 minutes " The solution is evaporated and evaporating the residual gum from anhydrous toluene (4 x 1 ml). Gum In order to triturate the residue with anhydrous ether, to give the desired carboxylic acid trifluoroacetate (X) as a solid (25 mg, 68%)

cm "* ^ (Nujol film) 1780,1670 (wide).

m^aX

The minimum inhibitory concentration (MIC) of the compound necessary to inhibit the growth of gram-positive bacteria five types are indicated in the following table:

(A) Preparation of the 1 - (l-benzyloxycarbonyl-2-methyl-1-propenyl) - 3 - (triphenylmethylamino) - 4 - /" 3 - (3-pyridyl) prop-2- ynylthio 7azétidinone -2.

Are of 6-beta-(triphenylmethylamino) benzyl pénicillanate (1.1 g) in a nitrogen atmosphere in anhydrous tetrahydrofuran (40 ml) of the hydrobromide containing L-bromo-3 - (3-pyridyl)-propyne 2 (0.61 g, 1.1 equivalent, obtained from 3-ethynyl-pyridine by successive reaction with ethyl-magnesium, formaldehyde and finally phosphorus tribromide.) Is added at 30 minutes a solution 0,778 Μ t-potassium butoxide in tert-butanol (5.7 ml, diluted with 10 ml of tetrahydrofuran) and is continued stirring for 2 hours. Evaporated, the reaction mixture (vacuum) at a low-volume and is added ethyl acetate. The washing of the organic phase with water and brine, dried ( Na2S0 ^) and is evaporated to dryness. Are chromatography of the residue on silica, the elution with ethyl acetate/light naphtha (1: 9.2 litres) which, provides an amorphous product (0,187 g) 14%.

V_x 1755 cm^- ^ (beta- làctame ), 1715 cnT ^ (ester). If PPM ✓

( CDC13₎ 2,02 (s, 3Η ), 2.18 (s, 3:00), 2.8-3.1 (m, 3:00 changes with D^O 1:00), 4.8-5.25 (m, 2:00), 5.3 (s, 2:00), 6.9-8.8 (byte aromatic).

(B) Preparation of the ~ 3/4 - (3-pyridyl)-prop-2- ynylthio 7-3-triphenyl-methylamino-azetidinone -2.

The by dissolving the beta-lactam (I) (^ 3 50 g) in a mixture of pyridine (50 ml) and water (5 ml). The mixture is cooled under stirring in an ice bath and salt and is treated with potassium permanganate to the finely divided form (1.25 g). Cooled The mixture was stirred for another one hour. The mixture is diluted with ethyl acetate (300 ml) and brine (100 ml) and the mixture is filtered on the Kieselguhr catalysts. The organic layer is separated and washed with brine. Evaporated, the organic layer dried (MgSO ^), resulting in a raw gum (3.1 g).

The raw gum The chromatography on silica gel eluting with mixtures of ethyl acetate/petroleum ether, to give the desired beta-lactam (II) in the form of a solid foam (0,751 g, 30%)

1

V cm (CHC1 -.) 3380,1765.

d max⁷

% pPM ( CDCl ^) 3.25 (s, 3Η, 1:00 changed with D^O ), 4.60 (broad s, 2:00), 6.61 (s, 1:00 changes with D^O ); 7.0-8.8 (aromatic).

Preparation of the (C) l-(L-hydroxy-l-t- butoxycarbonyltaéthyl ) - 4 - /" 3 - (3-pyridyl)-prop-2 - -- triphénylméthylaminoazetidinone ynylthio_7 3-2.

Is heated with reflux of tertiary butyl glyoxalic monohydrate (1.93 g) and anhydrous benzene (20 ml) under a nitrogen atmosphere, in a Dean and Stark, until all of the water has been removed. Adding the beta-lactam (II) (620 mg) and the mixture is heated at reflux under a nitrogen atmosphere for another 3 hours. The reaction mixture is cooled and washed with water (5 x 10 ml). Evaporated, the organic layer dried ( MgSC >₄ ), resulting in a raw gum (1.45 g).

Are the raw gum chromatography on silica gel, eluting with mixtures of ethyl acetate/petroleum ether, to give the desired beta-lactam (III), which is a mixture of stereoisomers in the form of a solid foam (510 mg, 65%).

V cm^-1 (CHCl,) 1775,1740

max 3'

& pPM ( CDCl ^) 1.48 (s, 9:00); 3.17 (s) and 3.37 (s) superimposed on a broad signal (integration together to 3Η, 1 ℮ broad signal changes with D₂ 0 to leave two singlet); 3.95 (broad s, 1:00 " changes with D₂ 0); 4.85-4.40 (m, 2:00); 5.13 and 5.25 (2 singlet integrating together to 1:00); 7.0-8.7 (aromatic).

Preparation of the (D) l-(l-chloro-l-t- butoxvcarbonvlméthyl )/ "" -4-3 - (3-pyridyl)-prop-2- ynylthio_7 -3-triphenylmethylamino-azetidinone -2.

(III) (IV)

The by dissolving the beta-lactam (III) (515 mg) in a mixture of anhydrous tetrahydrofuran (5 ml) and anhydrous dïoxanne (5 ml) and resultant mixture is cooled between -5 ° and

-10 °C. Is added anhydrous pyridine (202 mg), followed by thionyl chloride (304 mg) purified, dropwise in 3 minutes. The resultant mixture is stirred and between 0 -5 " C for 15 minutes. The laye the mixture and the residue with anhydrous toluene. The combined filtrates evaporated, and removing the residual gum with anhydrous toluene (4 x 10 ml). Combined extracts are filtered and is evaporated, resulting in a gum. The new evaporation of the gum from ether gives the desired anhydrous chloride (IV) in the form of a foam. solid (436 mg, 82%).

Are the chloride (IV) (436 mg), triphenylphosphine (368 mg) and 2,6-dimethylpyridine to 50 °C (90 mg) in anhydrous dioxane (10 ml), under nitrogen, during 5 hours. The mixture is cooled, is diluted with ethyl acetate (200 ml) and washed with brine. Evaporated, the organic layer dried (MgSO ^) ^ resulting in a brown gum (727 mg).

the gum is chromatography on silica gel, eluting with mixtures éthylê acetate/petroleum ether, to give the desired phosphorane (V) in the form of a solid foam (262 mg, 44%).

V max™"¹ C^chc1 ₃ ) 1755,1640.

(P)/ 4-Preparation of the ~ 3 - (3-pyridyl) - 2- oxopropylthio_7 -l (1-t-butoxycarbonyl-l-triphenylphosphoranylidenemethyl) - 3-triphenylmethyl-amino-azetidinone -2.

(See formula V and VI next page)

The phosphorane is heated at reflux (V) (250 mg) in piperidine (5 ml), under nitrogen, for 3 hours. Evaporated, the mixture and the residual gum is dissolved in ethyl acetate. The solution is mixed with HCl 0.1 N

(20 ml) during 5 minutes, οη makes basic with a saturated solution of NaHCOg and the organic layer is separated. Is extracted again the aqueous layer with ethyl acetate. Combined extracts are washed with brine, dried (MgSO ^) and is evaporated, resulting in a raw foam (250 mg).

The raw foam The chromatography on a silica gel, by performing 1' elution with mixtures of ethyl acetate/petroleum ether, to give the desired keto-phosphorane (VI) in the form of a solid foam (197 mg, 77%).

V cm"¹ (CHCl-j) 1750,1630

max i¹

(G) Preparation of 3 - (3- pyridvlméthyl ) - 7-triphenylmethylamino- 3-cephem-4- carboxylata t-butyl.

The keto-phosphorane (VI) (197 mg) at reflux in anhydrous dioxane (5 ml) under nitrogen for 8 hours. Evaporated mixture, resulting in a crude gum.

The raw gum The chromatography on a silica gel by performing 1' elution with mixtures of ethyl acetate/petroleum ether, to give the desired cephem (VII) in the form of a solid foam (95 mg, 71%).

Vma_X ^cm_1 (^chc1 ₃ ) 1775,1715

S pPM ( CDCl ^) 1.52 (s, 9:00); 3.03 (center of the byte, 3:00, collapsing in a nibble AB, 2:00, jr = 18 Hz with D₂ 0); 3.70 (center of the nibble AB, 2:00, J = 15:00 z); 4.31 (d, 1:00, J = 5:00 z);

4,75 (center of the byte 1:00, collapsing in a doublet, J = 5:00 z with D₂ 0); 7.0-8.7 (aromatic, 19 H).

After crystallization from methanol, the product has a melting point of 169-171®, oC ^ = 13.8® (C = 1% in CHCl ^),

X (ethanol) 263 nm (£= 10,500).

max

(H) 7-Preparation of beta-amino-3 - (3- pyridyIméthyl ) - 3-cephem- 4-carboxylate t-butyl .

The by dissolving the cephem (VII) (1.0 g) in acetone (10 ml), is cooled to 0 C and® acid is treated with para-toluene-tallow onic, monohydrate {710 mg). The mixture is allowed to return to room temperature and held at this temperature for 2 hours. Evaporated, the mixture is shaken and the resultant gum with ethyl acetate (25 ml) and a saturated solution of sodium bicarbonate (10 ml). Organic coated are separated and is re-extracted the aqueous layer with ethyl acetate (2 x 10 ml). Combined extracts are washed with brine, dried (MgSO ^) and is evaporated, resulting in a raw gum (0.96 g). The raw gum The chromatography on silica gel, eluting with mixtures of ethyl acetate/petroleum ether, which gives the desired free amine (VIII) as a solid foam (0,423 g, 12%).

V_max (CHCl₃ >1775 cm, 1715 cm, measured at 347.1291 molecular ion ( C^I^^N^O^S, calculated 347.1303. = 3.2 PPM Error).

S pPM (CDC1₃ )

1,55 (s, 9:00); 2.7 (wide, 2:00, changes with D2O); 3.02 and 3.48 (nibble AB, 2:00, J = 18:00 z); and 3.44 4.08 (nibble AB, 2:00, J = 15:00 z); 4.76 (d, 1:00, J = 5:00 z); 4.99 (d, 1:00, J = 5:00 z); 7.0-8.8 (byte aromatic).

EXAMPLE 2

Preparation of 3 - (3-pyridylmethyl) - 7-beta-(2- thlényla - cétamido ) - 3-cephem-4-carboxylic .

Can also be obtained the isolated product in the example 1 as trifluoroacetate salt as hermaphroditic ion as follows

(XI)

Dissolve the ester is cephem 4IX) (179 mg) in trifluoroacetic acid (2 ml) and maintained at room temperature for 5 minutes. The solution is evaporated and evaporating the residual gum from anhydrous toluene (4x1 ml). Gum In order to triturate the residue with anhydrous ether, to give the carboxylic acid trifluoroacetate (X) as a solid (188 mg)*

Dissolving the salt is (X) (188 mg) in water (40 ml) and by adjusting the pH of the resultant solution has 5 with triethylamine. The solution is evaporated to dryness and the resulting solid is triturated with water (5 ml). The solid is collected and dried under vacuum, to yield the desired cephem (XI) as a white solid (55 mg, 35%)

Fp = 168 -171°C V (' Nuj61 ) 1765 cm **¹ , 1665 cm"¹ 238 nm max. > = ' max

(ethanol), -£ = 13,200 and 264 nm,. € = 10,150

¹ max ¹ max

The antibacterial activity of the product is substantially similar to that of trifluoroacetate salt described in the example 1.

. EXAMPLE 3 !

Preparation of 7-beta (D-alpha- hvdroxyphénylacétamido ) - 3 - (3-pyridyl-methyl) - 3-cephem-4-carboxylate t-butyl.

(See formulas VIII and XII next page)

Dissolving 1*amine is free (VIII) (347 mg) in anhydrous methylene chloride (20 ml) is cooled to and -20°C.

Are added to the agitated solution cold carboxyanhydride D-mandelic acid (196 mg), by fractions, in 5 minutes. To -20°C The mixture was stirred for another 2 hours and is evaporated, resulting in a crude gum. The raw gum The chromatography on silica gel eluting with mixtures of ethyl acetate/petroleum ether, to give the desired cephem (XII) in the form of a solid foam (318 mg, 66 $ iLa crystallization of the product in it ' ethyl/petroleum ether

gives a white crystalline solid, Fp 157-9°C. V (CHC1_)

' max 3

1780 cm15 cm " \ 1680 ατΤ^. λ = 264 nm (ethanol).

' max

* £11,400

max

°C

23

- 133.1 ° (C. = 1% in chloroform).

Molecular ion measured at 481.1701(^C 25^H 27^N 3° 5^S calculated 481.1671, error ppmj ^ ^ = 6.2 ppm ( CDCl ^)

l,57 (s, 9:00) ; 2,99 and 3.45 (nibble ΑΒ, 2Η, J = 18:00 z); and 3.37 4.18 (nibble AB, 2:00, J = 15 Hz); 4.5-5.4 (wide, 1:00, changes with D₂ 0); 5.05 (d, 1:00, ≈ 5:00 J z); 5.20 (s, 1:00); 5.80 (dd, 1:00, J = 5:00 z, J ' = 10:00 z collapses to d, J = 5:00 z with;

7,0-8.8 (m, 10:00,1:00, changes with D₂ 0).

B Preparation of trifluoracëtate salt of the acid 7-beta-(D- aIpha - hydroxyphénylacétamido ) - 3 - (3-pyridylmethyl) - 3-cephem-4- carboxylic .

(See formulae XII and XIII page ' following) PhCHCONH

I

OH

O ^ Su

. PhCHCONH

t\

_AOE296A0AO> Oh

^ HAS

Ni

C0₂ H

CF₃ ^c 2 θ°

(XII)

(XIII)

Dissolve the ester is céphèrae (XII) (150 mg) in trifluoroacetic acid (2 ml) and maintained at room temperature for 5 minutes. The solution is evaporated and evaporating the residual gum from anhydrous toluene (4x1 ml). Gum In order to triturate the residue with anhydrous ether, resulting in the trifluoroacetate salt cephem

desired (XIII) as a solid (163 mg, 100%) V (KBr)^{3 7} max

1770 cm"¹ ,

1670 cm

-1

^ = 264 nm (ethanol),

max'

10.350.

oC² £= - 83.5

(wide)

(C = 1% in ethanol).

max

The minimal inhibitory concentration (MIC) of the compound required to inhibit the development of various bacteria on agar nutrient is indicated in the following table

EXAMPLE 4

A-Preparation of 7-beta-(D-alpha-t- butQxycarbonylamlnôphénylacétamldo ) - 3 - (3-pyridylmethyl) - 3-cephem-4-carboxylate b-butyl

Is added dropwise while stirring, in 5 minutes, to a solution of methyl chloroformate (60 mg) in anhydrous tetrahydrofuran to -10°C, a solution containing N (t.-butoxycarbonyl)-D-alpha-phenylglycine (159 mg), triethylamine (64 mg) and anhydrous N, N-dimethylbenzylamine (1 drop) in anhydrous tetrahydrofuran (5 ml). The mixture was stirred for another 25 minutes to -10°C. Is added dropwise the free amine (VIII) (200 mg) in anhydrous tetrahydrofuran (3 ml) in 5 minutes and the resulting mixture is stirred for another 2 hours at -10°C. Andthe Filtering the mixture evaporates the filtrate, resulting in a gum, 0n the gum is dissolved in ethyl acetate and washed with a saturated solution of sodium bicarbonate, followed by brine. Evaporated, the organic layer dried (MgSO ^), resulting in a raw foam. The raw foam The chromatography on silica gel, eluting with mixtures of ethyl acetate/petroleum ether, giving the desired ester cephem (XIV) in the form of a solid foam (162 mg, 50%).

max (CHCl ^) 1780 cm ^, 1710 cm ' ^ (wide). ^_max ⁼ 264 nm

£(ethanol)_m = 10,500 oC = - 83.6^e (C= 1% in the ' chlomax D

roforme ).

Molecular ion measured at 580.2358 (Ch ^ H-._c No.0, -S calculated 580.2355, error < Ippm ).

Æ > pPM (CDC12) :-

1,40 (s) and 1.52 Cs) (become integrated together to 18:00);

2,82 and 3.32 (nibble AB, 2:00, J = 18:00 z); 3.29 and 4.07 (nibble AB, 2:00, J = 15:00 z); 4.94 (d, 1:00, J = 5:00 z); 5.30 (d, 1:00, J = 7:00 z);

5,65-6.10 (m, 2:00); 7.0-8.9 (aromatic m + NH).

B-Preparation of di-trifluoroacetate acid salt 7-beta-(D-alpha- aminophénylacétamido ) - 3 - (3-pyridylmethyl) - 3-cephem- 4- carboxyligue.

Dissolve the ester is cephem (XIV) (80 mg) in trifluoroacetic acid (2 ml) and maintained at room temperature for 5 minutes. The solution is evaporated and evaporating the residual gum from anhydrous toluene (4x1 ml). Gum In order to triturate the residue with anhydrous ether, to give the di-trifluoroacetate salt desired cephem (XV) as a solid (76 mg, 84%).

Ax V ^ ^ KBr) 1775 cm "" \ 1670 cm^-1 (wide) λ_max = 264 nm

94

£(ethanol)_{&x m} = 11,050. Oc" = - 18.5^e . (C = 1% in ethanol)

The minimal inhibitory concentration (MIC) of the compound required to inhibit the development of various bacteria on agar nutrient is indicated in the following table-level i CMI (jug/ml) Bacteria Gram-positive

B. subtilis 0.25

Staph, 0.25 aureus Oxford

Staph, Russell 2.5 aureus

Strep, - Hémolytique CN10 0,25

Strep, pneumoniae CN33 0,5

NCTC 10418 EéColi

Salmonella typhi

Shigella sonnei

Klebsiella aerogenes A

Proteus mirabilis 0977

- EXAMPLE 5

A -1 preparation ' methiodide of 7-beta-(P-alpha-hydroxy-phenylacetamido) - 3 - (3-pyridylmethyl) - 3-cephem-4-carboxylate t. of-butyl.

CMI (pq/ml)

2,5

1,25

2,5

2,5

5,0

Dissolve the ester is cephem (XII) (150 mg) in anhydrous tetrahydrofuran (1 ml) and is treated with methyl iodide (0.2 ml)..This maintains the mixture at room temperature for 2 hours. The diluting the mixture with anhydrous ether, resulting in a gum that, by trituration with anhydrous ether, gives 1' desired methiodide (XVI) as a yellow amorphous solid (160 mg, 82%).

^ ^ (CHCl..) 1780 cm^-1 , 1705 cm"¹ , 1680 cm"¹ , max λ = 266 nm 3'⁷ max ' (ethanol)£_raax ⁼ 11-600, oC ^ = - 139.0 ° (0=1% in chloroform).

B" Preparation of metho-trifluoroacetate acid 7-beta-(D- alpha- hydroxyphénylacétamido ) - 3 - (3-pyridylmethyl) - 3-cephem- 4-carboxylic .

(See formulae XVI and XVII next page)

(XVI) (XVII)

Dissolving the is methiodide (90 mg) in trifluoroacetic acid (2 ml) and maintained at room temperature for 5 minutes. The solution is evaporated and evaporating the residual gum from anhydrous toluene (4 x 1 ml). Gum In order to triturate the residue with anhydrous ether, to give the desired metho-trifluoroacetate (XVII) as a solid (80 mg, 100%). V (KBr) ~ 1770 cm\

1670 cm^-1 (wide). A = 265 nm (ethanol) £= 9.550.

³ max max

The minimal inhibitory concentration (MIC) of the compound required for inhibiting the development of 5 gram-positive bacteria types on nutrient agar is indicated. in the following table :-

B. subtilis

Staph, aureus Oxford

Staph, Russell aureus

Strep. Hémolytique CN10-

Strep, pneumoniae CN33

CMI( jnq / rnl )

0,25

0,12

5,0

0,02

0,12

E. coli NCTC10418

Salmonella typhi

Shigella sonnei

Klebsiella aerogenes A

Proteus mirabilis C977

CMI (yuq/ml)

50

50

50

- 50

125

EXAMPLE 6

A-Preparation of 7-beta-(alpha- phénoxycarbonylphénylacétamido )

-3 (3-pyridylmethyl) - 3-cephem-4-carboxylate t.-butyl

Is dissolving the free amine (VIII) (200 mg) in anhydrous methylene chloride (5 ml). The solution is cooled to -10°C and is treated with anhydrous triethylamine (116 mg), and then with alpha-phenoxy-carbonyl-phenylacetyl (238 mg, obtained by treating phénylmalonate mono with thionyl chloride) in anhydrous methylene chloride (2 ml), drop has drop and stirring, in 3 minutes. Are the mixture a -10°C during 45 minutes. The mixture is diluted with methylene chloride, washed with brine, dried, (MgSO ^) and is evaporated, thereby given a raw gum (410 mg).

The raw gum The chromatography on silica gel, eluting with mixtures of ethyl acetate/petroleum ether, thereby giving the cephem desired ester (XVIII) in the form of a solid foam (224 mg, 66%).

V_max (CHCl₃ ) 1785 cm^-1 , 1740 cm"¹ , 1715 cm"¹ , 1685 cm^-1

S pPM ( CDCl ^) :-

1,52 (s, 9:00); and 3.40 2.95 (nibble AB, 2:00, J = 18:00 z); 3.31 and 4.09 (nibble AB, 2:00, J = 15:00 z) f 4.87 and 4.89 (2 singlet integrating together to 1:00); 5.00 (d, 1:00, J = 5:00 z); 5.88 (dd, 1:00, J = 5:00 z, J ' = 9Ηζ ) ; 6,9-8.9 (byte aromatic + NH)

" X_max =²⁶³ nm (ethanol). £= 11,200, ^ ≈ - 66.9 ° (C = 1% in chloroform 23.)

Dissolve the ester is cephem (XVIII) (100 mg) in the acid trifluoracetique and is maintained (2 ml) at room temperature for 5 minutes. The solution is evaporated and evaporating the residual gum from anhydrous toluene (4 x 1 ml). Gum In order to triturate the residue with anhydrous ether, to give the desired trifluoroacetate (XIX) as a solid (98 mg, 89%). V " _ (KBr) 1770 cm^-1

11.300

the compobactéries below:

1670 cm^-1 (wide). λ =263,5 nm (ethanol). £=

max '.

OC² q =- 1,8 ° (C = 1% in ethanol).

The minimum inhibitory concentrations (MIC) of

fabric required for inhibiting the development of various

on nutrient agar are indicated in the table Bacteria Gram-Positive

B. subtilis

Staph, aureus Oxford

Staph, Russell aureus

Strep jS- Hémolytique CN10.

E. coli NCTC 10418

Salmonella typhi

Shigella sonnei

Proteus mirabilis C977

CMI (yng/ml)

12,5

5

25

12,5

CMI( jiq / ml)

50

125

12,5

25

EXAMPLE 7

A- Préparatioh of the 7-beta-(D-alpha-t.- butoxycarbonylaminophénylacétamido ) -3 (2-pyridylmethyl) - 3-cephem-4-carboxylate t.-butyl

The ajoutç dropwise in 5 minutes stirring, to a solution of methyl chloroformate (60 mg) in anhydrous tetrahydrofuran (5 ml), cooled to -10°C, a solution containing N-(t.-butoxycarbonyl)-D-alpha-phenyl glycine (159 mg), triethylamine (64 mg) and N, N-dimethylbenzylamine (1 drop) in anhydrous tetrahydrofuran (5 ml). After 25 minutes, is added dropwise to the free amine (XXVIII) (200 mg) in anhydrous tetrahydrofuran (3 ml) in 5 minutes. To -10°C The mixture was stirred for another 2 hours. Filtering the mixture and evaporating the filtrate, resulting in a gum is dissolved in ethyl acetate and washed with a saturated solution NaHCO ^ and brine. Evaporated, the organic layer dried ( MgS0₄ ), resulting in a raw foam (380 mg).

The raw foam The chromatography on silica gel, eluting with mixtures of ethyl acetate/petroleum ether, thereby giving the cephem desired ester (XXIX) in the form of a solid foam (325 mg, 97%)._max (CHCl ^) 1780 cm "\

1710 cm^-1 , 1690 cm^-1 (bearing)

JS pPM ( CDClg )

1,40 (s, 9 H); 1.50 (s, 9:00); 3.39 (s, 2:00); 3.69 and 4.20 (nibble AB, 2:00, J= 15 Hz); 4.83 (d, 1:00, J= 5:00z); 5.24 (d, lH, J = 7:00 z); 5.60-6.00 (m, 2:00); 6.80-8.70 (byte aromatic + NH > Ion measured molecular to 580.2370

error = 2.6 PPM).

<£ = 14,000.

λ_max = 264.5 nm (ethanol)

(C,_o H_qc No.0, calculated 580.2355 30,36 4,6 S*

λ = 270 nm.

Πΐ3ΐΧ

Oi ≈-- 86.7

£= 13,800 and

(C = 1% in chloroform)

B-di- trlfluoracétate Preparation of acid 7-beta-D-alpha- aminophénylacétamido ) -3 (2-pyridylmethyl) - 3-cephem-4-because carboxylic.

Dissolve the ester is cephem (XXIX) (100 mg) in trifluoroacetic acid to and held at-room temperature for 5 minutes. The solution is evaporated and evaporating the residual gum from anhydrous toluene (4 x 1 ml). Gum In order to triturate the residue with anhydrous ether, to yield the trifluoroacetate salt of the desired cephem (XXX) as a solid (98 mg, 88%). )) (KBr)

1775 cm "^, 1670 cm" (wide) ^. λ. = 264 nm (ethanol), £ =12.300⁷ max⁷

λ and = 270 nm, £ = 12,300. OC ° = -9.0 (C= 1% in

max D⁷

ethanol).

The minimum inhibitory concentrations (MIC) of the compound required to inhibit the development of various bacteria on nutrient agar are indicated in the following table CMI (pq/ml)

1,25

1,25

12,5

1,25

CMI (juq/ml)

50

12,5

25

125

B. subtilis

Staph, aureus Oxford

Staph, Russell aureus

Strep, β) Hémolytique CN10-

E. coli NCTC 10418

Salmonella typhi

Shigella sonnei

Proteus mirabilis C977

Preparation of the starting material of the example 7

(A.) Preparation of the 1 (l-benzyloxycarbonyl-2-methyl-l-propenyl) -3 (triphenylmethylamino) - 4 - /" 3 (2- pYridyl ) prop-2- ynYlthio 7

Are of 6-beta-(triphenylmethylamino) benzyl pénicillanate (146 g) in anhydrous tetrahydrofuran (1500 ml) of the hydrobromide containing L-bromo -3 (2-pyridyl) - 2-propyne (81.4 g, obtained from 2- éthynylpyridine by successive reaction with ethyl-magnesium, formaldehyde and finally phosphorus tribromide) and soda in powder form (23.4 g) during 24 hours, at room temperature.

Filtering the mixture and evaporating the filtrate, resulting in a residual gum. The the residual gum is dissolved in ethyl acetate and washed with brine. Evaporated, the layer dried prganique ( MgS0₄ ), resulting in a crude gum.

The raw gum The chromatography on silica gel, eluting with mixtures of ethyl acetate/petroleum ether, to give the compound (XX) seco as desired

of a solid foam (81,4g, 46%). V (CHC1-) 1760 cm^-1 ,

' ¹ max 3 ¹

1720 cm^-1

*/ S Ppm ( CDCl, J)

2,02 (s, 3:00); 2.18 (s, 3:00); 3.04 (center of the nibble AB, J = 15:00 z superimposed on a broad signal, integrating together to 3:00, the signal changed with wide D^O ); 4.3-4.8 (m, 2:00, collapsing in two doublets, J = 5:00 z with^D 2°) > 4.90 (s, 2:00); 7.0-8.7 (byte aromatic).

Preparation of the (B)/ 4^- 3- (2-pyridyl) prop-2-3 -- ynylthio_7 phénylméthylamino -azetidinone -2.

Seco is dissolving the compound (XX) (105 g) in a mixture of pyridine (1000 ml) and water (100 ml). Agitated mixture is cooled in an ice bath and. salt and is treated with potassium permanganate to the finely divided form (37,6g) by fractions, in 1 hour. Cooled The mixture was stirred for another 1 hour. The. mixture is diluted with ethyl acetate (1000 ml) and the brine (200 ml) and the mixture is filtered on the Kieselguhr catalysts. The organic layer is separated and washed with brine. Evaporated, the organic layer dried (MgSO ^), resulting in a crude gum.

The raw gum The chromatography on silica gel, eluting with mixtures of ethyl acetate/petroleum ether, to give the beta-lactam (XXI) in the form of a solid foam (14.0 g, 18%). V ( CHC1₀ ) 1760 cm"¹ .-⁷ max 3

' S * ppm ( CDCl2 ): 3,05 (d wide, 1:00, J = 8:00 z changes with DgOjjSjSl (s, 2:00); 4,50-4.83 (m, 2:00); 7.0-8.7 (byte aromatic + NH).

Preparation of the (C) l-(l-hydroxy-l-t.-butoxycarbonylmethyl)

4 - / - " 3 (2-pyridyl) prop-2- ynylthio 7-3-triphenylmethylamino-azetidinone -2^r

(Formulae XXI XXI and See. Following Dpage

and the mixture is heated at reflux under nitrogen for another 2 hours. The mixture is cooled and washed with water (5 x 200 ml). Evaporated, the organic layer dried (MgSO ^), resulting in a crude oil (68 g).

Crude oil The chromatography on silica gel, eluting with mixtures of ethyl acetate/petroleum ether, to give the desired beta-lactam (XXII) as a mixture of stereoisomers, having the appearance of a solid foam (14.7 g, 43%). V ( CHC1₃ ) -1765 cm"¹ , 1735 cm"¹

Preparation of the (D) Kl-chloro-l-t.- butoxvcarbonylméthyl )/ ~ 3-4 - (2-pyridyl)-prop-2- ynylthio_7 -3-triphenylmethylamino-azetidinone -2.'

The by dissolving the beta-lactam (XXII) (13,7g) in a mixture of anhydrous tetrahydrofuran (100 ml) and anhydrous dioxane (100 ml) is cooled and the solution between -5 and -10°C.

Is added 2,6-dimethylpyridine redistillée, (7,26g) and thionyl chloride purified (8,0g), dropwise, in 10 minutes. The mixture was stirred between O and -5°C for another 20 minutes. The diluting the mixture with anhydrous toluene (lOO ml) and is filtered. Evaporated filtrate is evaporated and again the residual gum from anhydrous toluene

(2 x 100 ml). A new evaporation of the gum from the ether gives the desired anhydrous chloride (XXIII), contaminated with a small amount of 2,6-dimethylpyridine in the form of a solid foam (16.0 g).

V ( CHC1₀ ) 1770 cm^-1 , 1740 cm^-1 max i.⁷

Preparation (E) 4 / ~ 3 (2-pyridyl) prop-2- ynylthio 7Kl-t. butoxy-carbonyl-l-triphenylphosphoranylidenemethyl) - 3-triphenylmethylamino-azetidinone -2.

(XXIII) (XXIV)

Are the chloride (XXIII) (16.0 g), triphenylphosphine (11.9 g) and of the 2,6-dimethyl-pyridine (2,6g) to 50 °C in anhydrous dioxane (150 ml) under nitrogen anhydrous, during 10 hours. The mixture is cooled, is diluted with ethyl acetate (250 ml) and washed with brine. Evaporated, the organic layer dried ( MgS0 ^), resulting in a raw gum (28.5 g).

The raw gum The chromatography on silica gel, eluting with mixtures of ethyl acetate/petroleum ether, to give the desired phpsphorane (XXIV) as a brown solid foam (7.82 g, 41%). V (CHC1 -) 1755 cm^-1,7 ~ ^ max 3,1625 cm.

Is heated the phosphorane (XXIV) < -7.22 g) at reflux in piperidine (100 ml) under nitrogen for 2 hours. Evaporated, the mixture and the residual gum is dissolved in ethyl acetate. The washing of the solution with HCl 0.1 N (3 x 20 ml), then with brine. Evaporated, the organic layer dried ( MgS0 ^), resulting in a brown raw foam.

The raw foam The chromatography on silica gel, eluting with mixtures of ethyl acetate/petroleum ether, to give the keto-phosphorane (XXV) as

a dark yellow solid foam (4.90 g, 62%). V ( CHC1₀ ) max 3,1750 ch¹ , 1635 cm"¹ .

(G) Preparation of 3 (2- pyridylméthvl ) - 7-beta- triphénylméthyI - aroino -3-cephem-4-carboxylate t.-butyl.

The keto-phosphorane (XXV) (4.90 g) at reflux in anhydrous dioxane (50 ml) under nitrogen, during 20 hours. Evaporated mixture, resulting in a crude gum.

The raw gum The chromatography on silica gel, by performing 1' elution with ethyl acetate/petroleum ether, to give the desired cephem (XXVI) as a solid foam (2.51 g, 75%)" y (CHCl ^) 1770 cm^- '¹ , 1715 cm^- '*' ,

= 263 nm (ethanol). £= 10,500. oi 39.6 = + ° (C=l υ % ^ max max

in chloroform).

_AOE280A2AO> < Tppm ( CDCl2 ) :-

1,50 (s, 9:00); 2.95 (d, 1:00, J = 10:00 z, changes with D₂ 0); 3.21 10 (s, 2:00); 3.61 and 4.16 (nibble AB, 2:00, J = 15:00 z); 4.30 (d, 1:00,

J = 5:00 z); 4.50-5.00 (m, 1:00); 7.0-8.7 (byte aromatic).

(H) 7-Preparation of beta-amino -3 (2-pyridylmethyl) - 3-cephem-4-carboxylate t.-butyl.

The by dissolving the cephem (XXVI) (1,0g) in acetone (10 ml), is cooled to 0 °C and is treated p- toluènesuif aldonic acid, monohydrate (710 mg). The mixture is allowed to return to 2^room temperature and held at this temperature for 2 hours.

The solid is filtered, washed thoroughly with acetone and are dried under vacuum, to yield the salt of the di- ptoluène -sulphonic acid (XXVII), in the form of a white crystalline solid (519 mg, 44%), Fp 165-7°C. l) - (Nujol film) 1800 cm^- '*' ,

1715 cm λ. = 264 nm (ethanol), SL = 11,800, and 2,70 nm, max 'max''^£ max^{= 11} -⁸⁰⁰ * ° < ² d ="²⁶ "⁹ °^{(C= 1% in} ethanol)

The acid salt are di-p-tolunesulfonic ( XXVÏI ) (465 mg) with ethyl acetate (10 ml) and a saturated solution of sodium bicarbonate (10 ml). The organic layer is separated and is re-extracted the aqueous layer with ethyl acetate (2 x 5 ml). Combined extracts are washed with brine, dried (MgSO ^) and is evaporated, thereby giving the desired free amine (XXVIII), in the form of a solid foam (224 mg, 96% from the salt).^v _max (CHCl-J.) 1775 cm"¹ , 1715 cm"¹ .

S pPM ( CDCl ^)

1,52 (s, 9:00); 1.89 (s, 2:00, changes with D2O); 3.47 (s, 2:00);

3,69 and 4.22 (nibble AB, 2:00, J= 15 Hz); 4.75 (d, 1:00, J = 5:00 z);

4,99 (d, 1:00, J = 5:00 z); 7.0-8.7 (byte aromatic). Molecular ion measured at 347.1321 (C ^ Hg ^ N- ^O^S calculated 347.1304, error = 4.9 PPM).

EXAMPLE 8

A" Preparation of 7-beta- /~ D-2- (1- lmidazolidonecarbonylamino ) phenylacetamido 7-3 (3-pyridylmethyl) - 3-cephem-4-carboxylate t. butyl.

Are added to a mixture of the acid monohydrate (XXXI) (162 mg) and of the free amine (VIII) (200 mg) in the anhydrous tëtrahydrofuranne (20 ml), while stirring, a solution of dicyclohexylcarbodiimide (119 mg) in anhydrous tetrahydrofuran (5 ml), dropwise, in 3 minutes. The mixture was stirred at room temperature for 3 hours. Filtering the mixture and carefully the solid is washed with ethyl acetate. The combined filtrates evaporated, resulting in a raw foam (371 mg).

Chromatography is the crude product on silica gel, eluting with chloroform/methanol mixtures, thereby giving the desired estef cephem (XXXII) in the form of a so-

dangero us (240 mg, 70%). V (CHC1 ") 1780 cnf¹ , 1720 cm"¹ , 1670 cm"¹ ^ 'max 3' ' (bearing).

4Tppm (CDC1₃ ) 1.53 (s, 9 H); 2.9-4.3 (m, 8Η ) ; 4,91 (d, lH, J = 5:00 z) 5.6-6.3 (m, 3Η, 1:00 changes with O^O ); 7,1-8,2 (m, 8:00); 8.51 (broad s, 2:00): 9.15-9.45 (m, 1:00). λ = 263.5 nm (ethanol),

£= 11,060.

θζ = ^^ -64.2 ° (C = 1% in chloroform).

B- Preparation of the salt of 7-beta/ " D-2- (L- imidazolidonecarbonylamino ) phénylacétami do 7 -3 (3-pyridylmethyl) - 3-cephem-4- carboxy1ique.

Dissolve the ester is cephem (XXXII) (100 mg) in trifluoroacetic acid (2 ml) and maintained at room temperature for 5 minutes. The solution is evaporated and evaporating the residual gum from anhydrous toluene (4 x 1 ml). Gum In order to triturate the residue with anhydrous ether, to give the desired trifluoroacetic acid salt (XXXIII) as a solid (93 mg, 85%).

V_max (^KBr ) 1770 cm"¹ , 1715 cm"¹ (wide), 1665 cm"¹ (wide).

Λ_max = 263.5 nm (ethanol), £ = 10,800. O= -2a, 4°

EXAMPLE 9

A-Preparation of 7-beta-phenoxyacet-4 - (3-pyridylmethyl)- 4-carboxylate t.-butyl .

Is dissolving the free amine (200 mg) in anhydrous methylene chloride (5 ml) is cooled and the solution to -10°C. Are added to the cooled, stirred solution of triethylamine (116 mg), followed by phenoxyacetyl chloride (148 mg), dropwise in three minutes. The mixture was stirred for another 30 minutes to -10°C. The mixture is diluted with methylene chloride, (20 ml), washed with brine, dried (MgSO ^) and is evaporated, resulting in a foam (328 mg).

Chromatography is the crude product on silica gel, in 39

y

2214163

performing 1' elution with mixtures of ethyl acetate/petroleum ether, giving the desired ester-cephem-(XXXIV), in the form of a solid foam (173 mg, 63%).

V^m ax^{( chc1} 3^{) 1780} c™"¹ "¹⁷¹⁰ cm*¹ (bearing), 1690 cm^-1 .

- Sppm ( CDCl ^) 1.58 (s, 9:00); 3.05 and 3.50 (nibble AB, 2:00, J = 18:00 z); 3.45 and 4.18 (nibble AB, 2:00, J = 14:00 z); 4.60 (s, 2:00); 5.09 (d, 1:00, J = 5:00 z); 5.96 (dd, 1:00, J = 5:00 z, J * = 9:00 z);

6,8-7,9 (m, 8:00); 8.6 (broad signal, 2' H) _AOE280A2AO>

Molecular ion measured at 481.1673^^C 25^H 27^N 3° 5^{S counta} 481,1671,

error = 0.4 PPM). X = 263.5 nm (ethanol), £. Max = 13,000 λ and^{274 nrn} " £=¹³ -¹⁰ °. Û( ² p = -90.5 ° (C = 1% in chloroform).

B-Preparation of trifluoroacetic acid salt of the acid 7-beta-phenoxyacet -3 (3-pyridylmethyl) - 3-cephem-4-carbo xylol .

Dissolve the ester is cephem (XXXIV) (100 mg) in trifluoroacetic acid (2 ml) and maintained at room temperature for 5 minutes. The solution is evaporated is evaporated and the resultant gum from anhydrous toluene (4 x 1 ml). The resultant gum In order to triturate with anhydrous ether, resulting in the die-trifluoroacetic acid salt

siré (XXXV), as a solid (107 mg, 96%). 1 )^ Max (KBr)

1775 cm^- '¹ , 1675 cm ~ ^ (wide). =\ 263 nm (ethanol),

' max ¹

£= 12,100 and Λ _max = 274 nra, " =£ 11.900. o <:² ^=-35,9⁰ (C = 1% in ethanol).

The minimal inhibitory concentration. (MIC) of the compound required to inhibit the development of various bacteria on agar nutrient is indicated below :-

EXAMPLE 10

A- Preparation of 7-beta- éthylthioacétamido -3 (3- pyrldylméthyl ) - 3-cephem-4-carboxylate t. butyl-"

(VIII) (XXXVI)

Dissolving 1 is' free amine (VIII) (240 mg) in anhydrous methylene chloride (5 ml), is cooled to -10°C and is treated with triethylamine (140 mg), followed by, chloride ethyl- thioacétyle (144 mg), dropwise and stirring, in 3 minutes. The mixture was stirred for another 30 minutes to -10°C " The diluting the mixture with ethyl acetate, washed with brine, dried (SO M ^ ^) and is evaporated, resulting in a crude gum.

Chromatography is the crude product on silica gel, eluting with mixtures of ethyl acetate/petroleum ether, giving the desired ester cephem (XXXVI) in the form of a solid foam (184 mg, 59%). V ' ^ (CHCl ^). 1780 cm^- '³ ', 1715 cm^-1 , 1675 cm"¹ .

J) Ppm ( CDC1₃ ) 1.26 (t, 3:00, J7Hz); 1.85 (s, 9:00); 2.61 (q, 2:00, J = 7:00 z); 3.10 and 3.51 (nibble AB, 2:00, J = 18:00 z); 3.97 (s, 2:00);

3,49 and 4.15 (nibble AB, 2:00, J =15Ηζ); 5.07 (d, lH, J = 5Ηζ ) ; 5,87 (dd, 1:00, J = 5:00 z, J ' = 9:00 z); 7.10-8.8 (byte aromatic + NH, 5Η ). λ = 263.5 nm (ethanol), £ = 12,000. OC² ! , ° = -88.7

(C = 1% in chloroform).

Molecular ion measured at 449.1435 (Cg ^ Hg^N^O^Sg calculated 449.1443, error = 1.8 PPM).

B-Preparation of trifluoroacetic acid salt of the acid 7- beta- éthylthioacétamido 3 - (3-pyridylmethyl) - 3-cephem-4-because carboxylic.

Dissolve the ester is cephem (XXXVI) (100 mg) in trifluoroacetic acid (2 ml) and maintained at room temperature for 5 minutes. The solution is evaporated is evaporated and the resultant gum from anhydrous toluene (4 x 1 ml). The resultant gum In order to triturate with anhydrous ether, to give the desired trifluoroacetic acid salt (XXXVII) as a solid (96 mg, 85%).

V (KBr) 1775 cm^-1 , 1670 cm"¹ (wide), λ ≈ 263 nm

(ethanol), £ = 11,700. ot² ^ ≈ - 22.8 ° (C = 1% in ethanol). The minimal inhibitory concentration (MIC) of the compound required to inhibit the development of various bacteria on agar nutrient is indicated in the following table CMI (yuq/ml) Bacteria Gram-positive

B. subtilis 0.5

Staph, 0.05 Staph aureus Oxford, Russell 1.25 aureus

Strep. j3 CN- Hémolytique 10,0.12 Strep, CN 33,0.25 pneumoniae

IJC (yuq/ml) Bacteria Gram-negative

E. coli NCTC 10418,50 Shigella sonnei Salmonella typhi 50 50 50 Klebsiella aerogenes A

C 977,25 Proteus mirabilis

EXAMPLE 11

A-Preparation of trifluoroacetic acid salt of the 7-beta-(D-alpha- quanidinophénylacétamido ) -3 (3-pyridylmethyl) - 3- cephem-4-carboxylate t.-butyl

(VIII)

Is dissolving the free amine (VIII) (250 mg) and the salt of trifluoroacetic acid D-alpha- guanidinophénylacétique (244 mg) in a mixture of methylene chloride and anhydrous dimethylformamide (2 ml) of anhydrous (level i ml). The resultant mixture is stirred, cooled in a cold water bath and is treated with a solution of dicyclohexylcarbadiimide (163 mg) in anhydrous methylene chloride (2 ml). The mixture was stirred for 30 minutes, and then for another 30 minutes in an ice bath. Filtering the mixture and lë solid is washed with chloroform. Combined filtrates are washed with water, dried (MgSO ^) and is evaporated, resulting in a gum that, by trituration with anhydrous ether, ester gives the desired cephem (XXXVIII) as a yellow solid (100 mg, 22%)

V_v (CHCl,) 1775 cm^-1 , 1710 cm^-1 , 1670 cm^-1 max. λ =263 nm 3 '' max (ethanol), £ = 10,000. oC = - 39.6 ° (C = 1% in ethanol).

B- Preparation of di-trifluoroacetic acid salt of ^ The acid 7-beta-(D-alpha- quanidinophénylacétamido ) -3 (3-pyridylmethyl)

- 3-cephem-4- carboxyligue

4

(XXXIX)

Dissolve the ester is cephem (XXXVIII) (60 mg) in trifluoroacetic acid (1 ml) and maintained at room temperature for 5 minutes. The solution is evaporated is evaporated and the resultant gum from anhydrous toluene (3 x 0,5 ml). In order to triturate the resultant gum avecde i-her anhydrous, to give the di-trifluoroacetic acid salt desired (XXXIX) as a solid (51 mg, 79%).

V (KBr) ~ 1775 cm \ 1670 cm " '*' (wide), max ⁷ /³

The minimal inhibitory concentration (MIC) of the compound required to inhibit the development of various bacteria on agar nutrient is indicated below

A -1 preparation ' methiodide 7-beta (D-alpha-N-t, butoxycarbonylaminophénylacétamido ) -3 (3-pyridylmethyl) - 3- cephem-4-carboxylate t.-butyl

NH PhCHCONH CO ^ u^i O- ΛC 0₂ Bu Λ Mel

(XIV)

Dissolve the ester is cephem (XIV) (150 mg) in anhydrous tetrahydrofuran (1 ml) and is treated with methyl iodide (0.2 ml). The mixture is maintained at room temperature for 16 hours. The diluting the mixture with anhydrous ether are pulverized and the resultant gum with anhydrous ether, resulting in 1 * desired methiodide (XL) as a solid. (178 mg, 9-3%). V ( CHC1₃ ) 1785 cm^-1 , 1710 cm^-1 ,

1690 cm^-1 (bearing). iTppm (CDC1₃ ) 1.39 (s, 9 H), * 1.50 (s, 9:00);

3.2-4.3 (broad m, 4:00); 4.55 (s, 3:00); 5.02 (d, 1:00, J = 5:00 z);

5,34 (d, 1:00, J = 7:00 z); 5.6-6.0 (m, 2:00); 7.40 (s, 5:00); 7.7-8.2 (m, 2:00); 8.35-8.75 (m, 1:00); 8.8-9.35 (m, 2:00).

λ = 265 nm (ethanol), < £= 14,300. oC² \ ° = -114.0

max¹ D

(C = 1% in chloroform).

B-Preparation of trifluoroacetic acid salt of the metho-trifluoroacetate of 1' 7-beta (D-alpha- aminophénylacétamido )

-3 (3-pyridylmethyl) - 3-cephem-4-carboxylic .

Dissolving 1 is' methiodide (XL) (100 mg) in 1' trifluoroacetic acid (2 ml) and held at the room temperature for (10 minutes). The solution is evaporated, resulting in a gum that is made to evaporate again from anhydrous toluene (4x1 ml). The resultant gum In order to triturate with anhydrous ether, to give the desired trifluoroacetic acid salt (XLI) as a solid (93 mg, 100%). V (KBr) 1775 cm '"'¹ ', 1670 crrT ^ darge ). A = 265 nm (ethanol), £ = 11,500. θ( ° = -27.7 (C= 1% in ethanol).

The minimal inhibitory concentration (MIC) of the compound required to inhibit the development of various bacteria on agar nutrient is indicated below

IJC Bacteria Gram-positive (^ / qq/ml)

B. subtilis 0.5

Staph, 0.25 aureus Oxford

Staph, Russell 12.5 aureus

Strep. CN- Hémolytique 10,0.05

Strep, CN 33,0.25 pneumoniae

E. coli NCTC 10418

Salmonella typhi

Shigella sonnei

Klebsiella aerogenes A

C 977 Proteus mirabilis

CMI (^ Aiq /ml)

50

50

50

50

125

EXAMPLE 13

4-A t.- Butoxycarbonyl -7 - (2-t-butoxycarbonylamino-2-p-hydroxy- phenyl-acetamido) - 3 - (3-pyridylmethyl)-cephem 3

Is dissolving N-t-butoxycarbonyl-D-alpha- phydroxyphénylglycine (169 mg), triethylamine (64 mg) and dimethylbenzylamine (1 drop) in anhydrous tetrahydrofuran (5 ml). Aj. ny The dropwise the solution, in 10 minutes, to a solution of methyl chloroformate (60 mg) in anhydrous tetrahydrofuran (5 ml), which has been agitated in a bath of C0₂ solid. After 20 minutes, is added dropwise to the 7-amino-4-tert-butoxycarbonyl-3 - (3-pyridylmethyl)-cephem 3 (VIII) (200 mg) in anhydrous THF (5 ml) and the reaction mixture is stirred at -10°C during 2 hours. The product is filtered and the filtrate is concentrated to a yellow syrup is chromatography on silica gel 60. (particle size < screen № 230), eluting with _AOE296A0AO> chloroform/methanol 19:1. The main product is isolated (XLII) as a pale yellow glass (41 140 mg. %).

^ max^{( CHC1} 3 >³⁴⁰⁰ >2970, 1780, 1720, 1620, and 1600 1500 cm^-1 ;

ifppm (CDC1₃ ) 1.41 (9:00, s, Bu^fc ); 1.51 (9:00, s, Bu*¹ ); 2.87 and 3.33 ( 2Η, nibble AB, J=I8Hz, S-CH₂ ~); 3.33 and 4.06 (2:00, quarteb AB, J = 15:00 z, CH₂ pyridyl); 4.92 ( 1Η, d, J=4,5:00 z, C6 of the

i

lactam); 5.17 ( 1Η, d, J = 6:00 z, CO-CH-N); 5.79 ( 1Η, m, C7 lactam-); 5.87 ( 1Η, exchangeable, wide s, OH); 6.73 ( 2Η, d, J = 9:00 z, phenyl); 7.19 ( 2Η, d, J = 9:00 z, phenyl); about 7.4 ( 1Η, m, C5 pyridyl); 7.75 ( 1Η, d, 7Ηζ, C4 of pyridyl) and 8.37 ( 4Η, wide, ΝΗ, s and C2 and C6 of pyridyl).

Β-Salt of bis- trifluoracétlque of the 7 - (2-amino-2- phydroxyphénylacétamido ) - 4-carboxy-3 - (3-pyridylmethyl)- céphènte 3

Dissolving the is 4-t-butoxycarbonyl-7 - (2-t- butoxycarbo - nylamino -2-p- hydroxyphénylacétamido ) -3 (3-pyridylmethyl)-cephem 3 ( XLIlj (135 mg) in trifluoroacetic acid (2 ml) and is allowed to stand at room temperature for 15 minutes. Is removed excess trifluoroacetic acid by distillation under reduced pressure is removed and the final traces by evaporation with anhydrous toluene. Several times In order to triturate the residue with anhydrous ether, which gives the product (XLIII) in

form of a whitish powder (115 mg, 76%). (KBr) 3410,7 max⁷

3180, 3040, 2600, 1770, 1670 and 1515 cm^-1 , λ (EtOH) 229 nm⁷⁷⁷⁷ max

(£= 15,100), 259 nm. (£= 10,400), 264 nm (= £11,100) and 269 nm (£= 10,700). A biochromatogramme in butanol/ethanol/water 4:1:5 indicates a zone of inhibition to R ^. 0.28.

The minimal inhibitory concentration (MIC) of the compound required to inhibit the development of various bacteria on agar nutrient is indicated below

EXAMPLE 14

A-4-t- Butoxycarbonyl -7- carboxyméthylthioacétamido -3 - (3-pyridyl methyl) cephem -3 .

The by dissolving the 7-amino-4-tert-butoxycarbonyl-3 - (3-pyridylmethyl)-cephem 3 ( VIXI ) (250 mg) in anhydrous methylene chloride (5 ml) and is cooled in an ice bath. Is treated with l-oxa-4- thiacyclohexane -2,6-dione (95 mg) and the resulting solution is allowed to stand at room temperature for 3 hours. Is separated solvent removal and the residue is purified by chromatography on a short silica gel column (< screen n® 230) 60 ; eluting with chloroform/methanol 9:1. The trituration with ether gives the required acid (XLIV) in the form of a

powder uncrystallised (260 mg, 76%). V ( CHC1 -) 3200

⁷ max 3

(wide), 2980,2500, (wide), 1785, 1720, 1680 and 1515 cm"¹ .

£Ppm (CDC1₃ ) 1.53 (9:00, s, t-butyl); 2.9-3.8 (7:00, m, -S-CH₂ and-CH pyridyl); 4.18 (1:00, d, J = 15:00 z, ÇH -pyridyl. );

5,07 (1:00, d, J=4,5 Hz, C6 of the lake tame by); 5.86 ( 1Η, . dd, J=8 and 4.5 Hz, C7 lactam-); about 7.4 ( 1Η, m, C5 pyridyl);

7,90 (1:00, d, J=7,5:00 z, C4 of pyridyl); 8.45 (1:00 exchangeable, d, J = 8:00 z, NH); 8.58 (2:00, wide, C2 and C6 of pyridyl) and 11.53 (1:00 exchangeable, wide s, CO-H).

.

B-trifluoroacetic acid salt of 4-carboxy-7-carboxymethyl- thloacétamido -3 - (3- pyridvlméthyÎ ) cephem -3.

Dissolving the is 4-t-butoxycarbonyl-7- carboxyméthylthioacétamido -3 - (3-pyridylmethyl) - 3-cephem (XLIV) (250 mg) in trifluoroacetic acid (3 ml) and is allowed to stand at room temperature for 15 minutes. Trifluoroacetic acid is removed under reduced pressure and the last is removed by adding anhydrous toluene and re-evaporation.

The resumes the residue in acetone (1 ml) are discarded and the small amount of insoluble material. The compound of the title is precipitated ( XLVj carefully by addition of acetone to the solution of the anhydrous ether (10 ml). Is achieved uncrystallised whitish powder (150 mg, 54%). V (Nujol film) 3300-2600 (Broadband), 1775, 1710, 1670, 1640 and 1545 cm^_1 . A-(EtOH) 259 nm( ε = 9600), 264 nm (£= 9.800 ) and 269 nm (£= 9.500). ^ ppm (TFA) 3.5-5.0 (8:00, m, S-CH ^ cH and ^ pyridyl); 5.39 (1:00, d, J = 4:00 z, C6 of the lactam); ^ 5.96 (1:00, dd, J=8 and 4:00 z, C7 lactam-); 8.0-8.4 (2:00, m, pyridyl) and 8.6-9.0 (3:00, m, pyridyl and NH). The biochromatogramme in butanol/acetic acid/water 12:3

: 5 reveals a zone of inhibition to R..0,24.

X

The minimal inhibitory concentration (MIC) of the compound required to inhibit the development of various bacteria on agar nutrient is indicated in the following table :-

EXAMPLE 15

A-Preparation of 7-beta-/ * (dl)-alpha-t-butyloxycarbonylamino-2- thiénylacétamido _7 -3 - (3-pyridylmethyl) - 3-cephem-4-carboxylate t.-butyl.

Is added dropwise acid (dl)-alpha-t- butylo - xycarbonylamino -2- thiénylacétique (rag 163), triethylamine (64 mg) and dimethylbenzylamine (1 drop) in tetrahydrofuran (4 ml) to methyl chloroformate (60 mg) in tetrahydrofuran, between -30 and -40°C °. The mixture was stirred to -30^e during 30 minutes, is added to the mixture, to

-30 °C, 7-beta-amino-3 - (3-pyridylmethyl) - 3-cephem-4-carboxylate (VIII) t.-butyl (200 mg) in tetrahydrofuran (3 ml). The mixture is cooled to -70°C and subsequently to warm up to -30^e C in 1 hour. Solvent is then removed and the residue is chromatography on silica gel eluting with dichloromethane, then with a mixture 1:1 of ethyl acetate and petroleum ether (Eb 60-80°), to yield the acylated cephem (206 mg). Is performed thereof new chromatography on silica gel eluting with chloroform, to give the 7-beta-/ * (dl) alpha-t-butyloxycarbonylamino-2 - ± do_7 thiénylacétam -3 ~ (3-pyridylmethyl) - 3-cephem-4-carboxylate t.-butyl (XLVI) (180 mg), in the form of an amorphous solid. Found: M⁺ m/e 586,1925:

^C' 28:00³ 4N⁴ ° 6S^{2 5 ac} - *-^cu -¹ ®-^{K+ m} ^^e 586,1920 (0.85 pPM error).

λ (EtOH) 240 nm (£14,600) and 263 nm (£11,500).

" max max max

(CH₀ C1₀ ) 3420,1785, and 1720 1705 (sh) cm^-1 , ppm ( CDCl,) ^ max 22⁷⁷ (s) - 3,1.47 and 1.58 (s) (total 18Η ); 2.82 and 3.37 (nibble ΑΒ, J = 20:00 z); 3.28 and 4.08 (nibble ΑΒ, J = 14:00 z) (total for nibbles 4Η 2); 5.02 (d enlarged, J = 5:00 z, 1Η ); 5.55-6.20 (m, 3Η );

6,85-7,45 (m, 4Η ); 7.78 (d, J = 7:00 z, 1Η ); 8.48 (broad s, 3Η ).

Β-Preparation the acid salt of di-trifluoroacetic acid? -beta-/ ~ (dl)-alpha-amino-2- thiénylacétamido 7-3 - (3-pyridyl methyl) - 3-cephem-4- carboxyiique .

XLVl

Dissolving is 7-beta-/^- (dl)-alpha-t- butyloxycarbony - lamino thiénylacétamido7 -2 - - 3 - (3-pyridylmethyl) - 3-cephem-4-carboxylate t.-butyl (XLVI) in trifluoroacetic acid (150 mg) during 15-20 minutes at room temperature.

Trifluoroacetic acid is removed under reduced pressure, toluene is added to the residue and is removed by evaporation (2X).

Then In order to triturate the residue with ether, to give the di-trifluoroacetic acid salt of the acid 7-beta-/ " (dl)-alpha-amino-2- thiénylacétamido 7-3 - (3-pyridylmethyl) - 3-cephem-4-carboxylic. (XLVII) as a solid colourless, λ

max

240 nm (EtOH). (£11,600) and 263 nm (£9.200).

max max

^V _m=w (KBr) 3410,1775 and 1765 cm"¹ ,

max'

The minimal inhibitory concentration (MIC) of the compound required to inhibit the development of various bacteria on agar nutrient is indicated ci4 -after :-

B. subtilis

Staph, aureus Oxford

Staph, Russell aureus

S rotational . β Hémolytique CN10-

CMI (judicata/ml )

1,25

0,5

5,0

0,5

CMI (judicata/ml )

E. JT 1 coli

Salmonella typhi

Shigella sonnei

Klebsiella aerogenes A

Proteus mirabilis C977

12,5

2,5

5,0

5,0

12,5

By replacing the starting acid in the part dl A above using the starting acid d corresponding, the process shown gives the D-cephem.

EXAMPLE 16

A-Preparation of 7-beta (4- pyridylthioacétamldo ) - 3 - (3- pyrldyl methyl) - 3-cephem-4-carboxylate t.-butyl .

Are added to a solution of the free base (VIII) (250 mg) in anhydrous methylene chloride, at room temperature, dicyclohexylcarbodiimide (149 mg) in methylene chloride (1 ml), followed by the acid hydrobromide 4- pyridylthioacétique the (180 mg) in dimethylformamide (2 ml), dropwise and stirring, in 2 minutes. After stirring the mixture for 30 minutes by surrounding this mixture by a water bath, and for another 30 minutes in the surrounding by an ice bath, is removed by filtration the white solid precipitated and. the filtrate is diluted with ethyl acetate, washed with an aqueous solution of sodium bicarbonate and brine, dried and evaporating. Chromatography is the residue on silica gel eluting with chloroform/methanol mixtures, thereby giving the desired ester cephem (XLVIII)

(160 mg) as a solid. V ( CHC1₀ ) 1785,1718,

max 3 ''

1685 cm λ. = 263 nm (ethanol), <£. = 14.320. =

max ' max. D

-12.8 ° (C= 1% in chloroform). Molecular ion measured at 498.1377 24° 4^2 26 ^^ ^ac ^-^cu ^-® 498.1395. 3.6 PPM Error). <4>pPM ( CDClg ): -1.53 (s, 9:00); 2.97 and 3.44 (nibble AB, 2:00, J = 19:00 z);

3,41 and 4.09 (nibble AB, 2:00, J = 14:00 z); 3.83 (s, 2:00); 5.00 (d, 1:00, J=4,5 Hz); 5.85 (d, d, lH, J=4,5:00 z, J ' = 9:00 z, collapsing to d, J=4,5:00 z with D₂ 0); 7.16-7.83 (m, 4:00); 8.31 (d, 1:00, J = 9:00 z changing with D₂ 0); 8.40-8.67 (m, 4:00).

B-Preparation of di-trifluoroacetic acid salt of the acid 7-beta (4- pyridylthioacétamido ) -3 (3-pyridylmethyl) - 3-cephem

4-carboxylic- .

(see formulae next page)

Dissolve the ester is cephem (XLVIII) (89 mg)

in trifluoroacetic acid (2 ml) and held at the -20 temperature of ambient temperatures. 5 minutes. The solution is diluted with toluene and anhydrous is evaporated (2 times). Pulping gum residue with anhydrous ether gives the acid salt di ^ irifluoracétique cephem desired (IL) (60 mg), as 2 read 3.X lUcLX ^ of a solid. V (KBr) 1775,1670 (wide) cm^-1 , λ = 263 nm (ethanol), £_max = 13,320. ot = + 12.8 ° (C = 1 % in ethanol).

The minimal inhibitory concentration (MIC) of the compound required to inhibit the development of various bacteria

30 on nutrient agar is indicated below :-

Bacteria Gram-Positive IJC ( yuq /ml )

B. subtilis < 0.02

22 Staph, < 0.02 aureus Oxford

Staph, Russell Strep aureus 1.0. ^- Hémolytique CN10 < 0.02

Strep, CN 33 < 0.02 pneumoniae

Bacteria Gram-negative IJC ( juq /ml )

A-Preparation of 7-beta-(4- pyridylacétamido ) -3 (3- pyridylmé' methyl) - 3-cephem-4-carboxylate t.-butyl.

Are added to a solution of the free base (VIII) (250 mg) in anhydrous methylene chloride (1 ml), at room temperature, dicyclohexylcarbodiimide (149 mg) in methylene chloride (1 ml), then the 4- pyridylacétique acid hydrochloride (125 mg) in dimethylformamide (2 ml), dropwise with stirring, in 2 minutes. After stirring the mixture for 30 minutes while it is surrounded by a water bath, and for another 30 minutes while it is surrounded by an ice bath, is removed by filtration the white solid precipitated. The filtrate is diluted with ethyl acetate, washed with an aqueous solution of sodium bicarbonate, then with brine, dried (MgSO ^) and is evaporated. Rédidu The chromatography on silica gel, eluting with chloroform/methanol mixtures, gives the desired ester cephem (L) (76 mg), in the form of a solid foam._max (CHCl^)

1780, 1715, 1685 cm " ~ ^. λ = 263 nm (ethanol), £. = 10,140.

'' max ' max

Molecular ion measured at 466.1671(^C 24^H 26° 4^SN 4 466.1675 calculated. 0.9 PPM Error)

% ppm ( CDCl ^): -1.52 (S, 9:00) ^ and 3.45 3.00 (nibble AB, 2:00, J = 18:00 z); and 3.41 4.09 (nibble AB, 2:00, J = 15:00 z); 3.66 (s, 2:00); 5.01 (d, 1:00, J=4,5:00 z); 5.87 (d, d, 1:00, J=4,5:00 z, J ' = 8,5:00 z collapsing to d, J= 4.5 Hz by shaking with D₂ 0); 7.15 -7.76 (m, 4:00); 7.89 (d, 1:00, J=8,5 Hz, changing with D₂ 0); 8.35-8.65 (m, 4:00).

B-Preparation of di-trifluoroacetic acid salt of the acid 7- bê ta-(4- pyridylacé tamido ) -3 ( pyridylmé 3-methyl) - 3- céphëme - 4-carboxylic .

Dissolve the ester is cephem (L) (63 mg) in trifluoroacetic ^ eide (2 ml) after aging at room temperature for about 10 minutes, the solution is diluted with anhydrous toluene and evaporating to dryness (2 times). Pulping gum residue with anhydrous ether gives the di-trifluoroacetic acid salt cephem -40 die

siré (LI) (60 mg) as a solid. V (KBr) 1775, _AOE296A0AO>

³ ₁ max'

1670 cm (wide) ~. X = 262.5 nm (ethanol), £ = 9.600.

³ max '' max

oL * = - 25 ° (C = 1% in ethanol).

The minimal inhibitory concentration (MIC) of the compound required to inhibit the development of various bacteria on agar nutrient is indicated below.

EXAMPLE 18

A- Preparation of 7-beta (DL-3-cyclohexene / ~ - 3-7-3 t.- butoxycar - bonyl - aminopropionamido ) - 3 - (3-pyridylmethyl) - 3-cephem-4- t.-butyl carboxylate .

Is added dropwise while stirring, in 5 minutes,

to a solution of methyl chloroformate (61 mg) in anhydrous tetrahydrofuran (5 ml), a solution containing 1' DL-3 - (3-cyclohexene)-t.-butoxycarbonyl-3-amino-propionic (172 mg), triethylamine (59 mg) and anhydrous N, N-dimethyl-benzylamine (1 drop) in anhydrous tetrahydrofuran (2.5 ml). After stirring the mixture for another 15 minutes to -10°C, is added dropwise to the free amine (VIII) (204 mg) in anhydrous tetrahydrofuran (2.5 ml) and the resulting mixture is stirred for another 1 hour between and 0 -10^e C.

Filtering the mixture and evaporating the filtrate, resulting in a gum that is dissolved in ethyl acetate and is washed with water, to dilute hydrochloric acid, water, aqueous sodium bicarbonate, then brine, are dried (MgSO ^) and is concentrated. The chromatography of the residue on silica gel, eluting with chloroform, gives the desired ester cephem (Lll) (82 mg) as a solid foam. V (CHC1 .,) 1780,1710 (wide) cm^-1 , = λ

264 nm (ethanol), £, and = 9.250 λ= 269.5 nm (ethanol), max max

£_max ⁼ 9.150. Molecular ion measured at 598.2815

calculated 598.2825,1.7 PPM error).

B-Preparation of di-trifluoroacetic acid salt acid 7-beta-(DL-3 - /* cyclohexene-3- aminoproplonamido 7-3) -3 (3- pyrldylméthyl ) - 3-cephem-4- carboxyllque .

Dissolve the ester is cephem (LU) (70 mg) in trifluoroacetic acid (2 ml). After allowing the solution at room temperature for 10 minutes, diluted with toluene and anhydrous is evaporated to dryness. A further evaporation from toluene and triturating the residual gum with anhydrous ether gives the acid salt ditrifluoracétique desired cephem XLIII) (73 mg) as

of a solid.

^V max^{1775 (KBr)} '^{1670 (sum <} EROISM^{e) cm} '

-1

263,5 nm (ethanol), £.

max

9.550 and λ.

max

max

= 269 nm (ethanol),

max

9.290.

The minimal inhibitory concentration (MIC) of the compound required to inhibit the growth of various bacteria on agar nutrient is indicated in the following table.

B. subtilis

Staph, aureus Oxford

Staph, Russell aureus

Strep. Ji- Hémolytique CN 10

CMI(

ji q/ml

)

1,25

0,5

1,25

0,5

Bacteria Gram-negative IJC (pq/ml )

EXAMPLE 19

A-Preparation of 7-beta-(DL-alpha-t.-butoxycarbonyl- aminocyclo - propylacétamido ) -3 (3-pyridylmethyl) - 3-cephem-4-carboxylate t.-butyl

Is added dropwise while stirring, to a solution of methyl chloroformate (69 mg) in anhydrous tetrahydrofuran (5 ml), ° to -10, vine solution containing 1' N-(t.-butoxycarbonyl)-DL-alpha-amino- cyclopropylacétique (157 mg), triethylamine (74 mg) and anhydrous N, Ndiméthylbenzylamine (1 drop) in anhydrous tetrahydrofuran (2.5 ml). After agitation of the mixture to -10 ° during 15 minutes, is added dropwise to the free amine (VIII) (230 mg) in anhydrous tetrahydrofuran (2.5 ml) and the. the resulting mixture is stirred for another 1 hour at this temperature. Filtering the mixture and evaporating the filtrate, resulting in a gum that is taken up in ethyl acetate, washed with an aqueous solution of sodium bicarbonate, then brine, dried (MgSO ^) and evaporated. The chromatography of the residue on silica gel, eluting with chloroform, gives the desired ester cephem (LIV) (200 mg) as a solid foam. V (CHC1,) 1780, 1710, 1692 (bearing), 1682 (bearing) has ÏH3.X cm. Λ = 263 7 nm (ethanol), £. Max = 10,430 and A =269 nm '' ^ max max (ethanol), £_max _io.230. Molecular ion measured at 544.2331 ( C^H ^ OgSN ^ calculated 544.2355,4.8 PPM error).

B- Preparation of acid salt of the di-trifluoroacetic acid 7-beta (DL-alpha- aminocyclopropylacétamido ) -3 (3-pyridylmethyl)- 3- céphàme -4- carboxyligue.

(see formula next page)

the solution at room temperature for 10 minutes, diluted with toluene and anhydrous is evaporated to dryness.

Again evaporated, the residue from toluene are pulverized and then with anhydrous ether, to give the di-trifluoroacetic acid salt desired cephem (LV) (150 mg),

as a solid. V (KBr) 1775,1675 cm (wide)^- ^.

max

\ = 263,5nm (ethanol), £. _ =9.630, and λ =269,5 nm (ethanol) max^{7 7} max⁷ max⁷

£=9.345.

max

The minimal inhibitory concentration (MIC) of the compound required to inhibit the development of various bacteria on agar nutrient is indicated in the table aorès.

B. subtilis

Staph, aureus Oxford

Staph, Russell aureus

Strep, haemolytic 10-CN

Gram- néqàtives Bacteria

E. JT 1 coli

Salmonella typhi

Shigella sonnei

Klebsiella aerogenes A

C 977 Proteus mirabilis

CMI (q/ml) 12,5

5,0

12,5

2,5

CMI (^ q/ml ) 500

250

250

250

500

EXAMPLE 20

Acid 7-beta- / ~ D-2 - ( cinnamylméthyluréido ) phénylacgtamido 7-3 - (3- pyrldylméthyl ) - 3-cephem-4-carboxylic.

(see formula next page)

Is treated, di-trifluoroacetic acid salt of 1' 7-beta < D-alpha- aminophénylacétamido ) - 3 - (3- pvridylméthyl )

3-cephem-4-carboxylic-{ XV) (300 mg. 1 equivalent) in methylene chloride (10 ml) containing triethylamine

(0.2 g, 4.4 equivalents)-with N-chlorocarbonyl-N- méthylcinnamide (0,113 g, 1.1 equivalent) in methylene chloride (5 ml), at room temperature, during 3 hours. After the removal of the solvent, the total product is a gum which, by trituration with water, provides the compound of the title (LVI) as a brown solid (150 mg) after drying under vacuum.

V _max (KBr disc. ) 3600-3200, 1770, 1660, 1610, cm^-1 .

The minimal inhibitory concentration (MIC) of the compound required to inhibit the development of various bacteria on agar nutrient is indicated below :-

CMI ( jaq /ml )

B. subtilis

Staph, aureus Oxford

Staph, Russell aureus

Strep. J5- Hémolytique CN 10

E. JT 1 coli

Salmonella typhi

Shigella sonnei

Klebsiella aerogenes

C 977 Proteus mirabilis

EXAMPLE 21

32

4

63

0,25

CMI ( Jjq /ml )

125

>125

63

63

>125

7 ~ acid-beta/alpha-(3- benzylidèneimino -3- méthyluréldo ) phenylacetamido 7-3 (3-pyridylmethyl) - 3-cephem-4-carboxylic

(see formulae next page)

The cephem is cooled (XY) (260 mg, calculated as salt di-trifluoroacetic acid) in methylene chloride (10 ml) in an ice bath. Triethylamine (182 mg) is added, then the N-carbonyl chloride (LVII) (88 mg) in methylene chloride (2 ml). The reaction mixture is left at room temperature for 3% hours, and then is evaporated to dryness. After washing with ether and water, the residue (LVIII) gives the product as a solid Desirable buff from chloroform-ether (140 mg).

y (CHC1,) 3400, 3300, 1780, 1680 (b), 1610 cm^-1 , max 3 * '''

EXAMPLE 22

By generally as described in the example 2 for converting the. acid salt of trifluoroacetic 1' 3 acid (3-pyridylmethyl) - 7-beta-(2- thiénylacétamido ) - 3-cephem-4-carboxylic acid free, can be converted into the free acid corrëspondant also each of the trifluoroacetic acid salts examples 3Β, 4Β, 6Β, 7Β, 8Β, 9Β, 10B, 11B, 12Β, 13Β, 14Β, 15B, 16Β, 17B and 18B.

Alterations can be made to the present embodiments, in the field matches ^ techniques without departing from the invention.