NO844091L - PROCEDURE FOR PREPARING 2-DIOXACYCYLCOYLTYLO-2-PENEM-3-CARBOXYLIC ACID DERIVATIVES - Google Patents

Abstract

Certain 2-dioxycycloalkylthio-2-penem-3-carboxylic acid compounds are useful as antibacterial agents for the treatment of mammals and have the formula. or a pharmaceutically acceptable salt thereof, wherein: R is. A is carbonyl, methylene or thiocarbonyl ,. B is alkylene having 2-5 carbon atoms ,. alk is alkylene of 1-6 carbon atoms ,. is hydrogen or a group resulting in an ester which is hydrolyzable in vivo and n is zero or a. Preparation of the compounds is described.

Description

The present invention is directed to a method for producing a family of antibacterial agents comprising a 2-azetidinone (betalactam) ring. Chemically, the antibacterial agents produced according to the present invention are identified as 6-alpha-1-hydroxyethyl-2-substituted-2-penem-3-carboxylic acid compounds.

Although certain 2-substituted-2-penem-3-carboxylic acid compounds have been previously described, there is a continuing need for new compounds with desirable antibacterial therapeutic properties.

The present invention is directed towards the production of

a connection with the formula

or a pharmaceutically acceptable salt thereof, where: R is

A is carbonyl, methylene or thiocarbonyl,

B is the alkylene with 2-5 carbon atoms,

alk is the alkylene with 1-6 carbon atoms,

is hydrogen or a group resulting in an ester that is hydrolyzable in vivo and

n is 0 or 1.

Included within the scope of the present invention is the preparation of a compound of the formula

or a pharmaceutically acceptable salt thereof, wherein:

R and R^ are as defined above for compounds of formula I.

Preferred compounds of formula I or II include those where R 1 is hydrogen, B is ethylene, A is methylene and n is 1, especially when R is 1,3-dioxacyclopent-4-ylmethyl or 1,3-dioxacyclopent-2-ylmethyl .

Also preferred are compounds of formula I or II where R is hydrogen, B is ethylene, A is carbonyl, n is 1 and alk is methylene, especially when R is 2-oxo-1,3-dioxacyclopent-4-ylmethyl.

Furthermore, compounds of formula I or II are preferred where R is hydrogen, B is propylene, A is methylene and n is zero, especially when R is 1,3-dioxacyclohex-5-yl.

In addition, compounds of formula I or II are preferred where R is hydrogen, B is propylene, A is carbonyl and n is zero, especially when R is 2-oxo-1,3-dioxacyclohex-5-yl.

A pharmaceutical composition may comprise a compound of formula I or II and a pharmaceutically acceptable diluent or carrier, and a method of treating a bacterial infection in a mammal comprising administering an antibacterially effective amount of a compound of formula I or II .

The compounds of the formulas I and II are useful as antibacterial agents, and are derivatives of the bicyclic nucleus of the formula:

In the present specification, the nucleus of formula III is identified by the name "2-penem," and ring atoms are numbered as shown. The carbon atom attached to ring carbon atom no. 6 is given the number 8. In the present description, the abbreviation "PNB" is also used for the p-nitrobenzyl group.

The relationship between the hydrogen atom on the bridgehead carbon atom 5 and the remaining hydrogen atom on carbon atom 6 in connection with formula I can be either cis or trans. The present invention includes methods for the production of both isomers as well as mixtures thereof. The trans isomer is generally preferred in pharmaceutical applications and the cis isomer can easily be converted to the trans isomer.

In general, carbon atom 5 will have the absolute stereochemistry designated R using the Prelog-Ingold R,S stereochemical designation, which is used in the present application. Thus, for example, a compound of formula II where R is 1,3-dioxacyclohex-5-yl and R^ is hydrogen is called (5R,6S)-6-[(R)-1-hydroxyethyl]-2-(1,3- dioxacyclohex-5-yl)thio-3-carboxyl-2-penem.

As will be appreciated, it is possible to prepare different optically active isomers of the new compounds. The present invention includes a method for producing such optically active isomers as well as mixtures thereof.

The present invention is directed towards the production of penems which are substituted in the 2-position with a group of the general formula R-S-.

The present invention comprises the preparation of the penems in which the 3-carboxyl group is esterified with a non-toxic ester group which is hydrolysed in vivo. These esters are rapidly cleaved in mammalian blood or tissue to release the corresponding penem-3-carboxylic acid. Typical examples of such easily hydrolyzable ester-forming residues are alkanoyloxymethyl with from 3 to 8 carbon atoms, 1-(alkanoyloxy)ethyl with from 4 to 9 carbon atoms, 1-methyl-1-(alkanoyloxy)ethyl with from 5 to 10 carbon atoms, al -Coxycarbonyloxymethyl with from 3 to 6 carbon atoms, 1-(Alkoxycarbonyloxy)ethyl with from 4 to 7 carbon atoms, 1-Methyl-1-(Alkoxycarbonyloxy)ethyl with from 5 to 8 carbon atoms, N-(Alkoxycarbonyl) aminomethyl with from 3 to 9 carbon atoms, l-(N-[alkoxycarbonyl]amino)ethyl with from 4 to 10 carbon atoms, 3-phthalidyl, 4-crotonolactonyl, gamma-butyrolacton-4-yl, carboxyalkylcarbonyloxymethyl with from 4 to 12 carbon atoms or 5-methyl-2-oxo-1,3-dioxolen-4-ylmethyl.

To prepare compounds of formula I or II where is a group that forms an ester that is hydrolyzed in vivo, the acid of formula I or II (R^ is hydrogen) is reacted with a base to form the corresponding anion. Suitable cations include sodium, potassium, calcium, tetraalkylammonium and the like. The anion can be prepared by lyophilizing an aqueous solution of

I or II, e.g. an aqueous solution containing tetrahydrofuran, and sodium bicarbonate or tetrabutylammonium hydroxide.

The resulting anion of I or II is reacted with the corresponding chloride or bromide of R-^ in a reaction-inert solvent such as e.g. acetone or dimethylformamide at approx. 20 to approx. 50°C, preferably 25°C.

The compounds of formula II can be synthesized according to schemes A-C.

As shown in Scheme A, a compound of formula II can be prepared according to the method of Yoshida et al., Chem. Pharm. Bull., 29, 2899 - 2909 (1981), from the known dibromopenam of formula IV. The dibrompename of formula IV. The dibromopenamate (IV) undergoes an exchange reaction with t-butylmagnesium chloride at a temperature of between -90 and -40°C, preferably approx. -78°C in a reaction-inert solvent such as tetrahydrofuran, diethyl ether or toluene, preferably tetrahydrofuran. Other organometallic reagents can also be used. The resulting reaction mixture is treated in situ with the appropriate aldehyde, e.g. acetaldehyde for the 1-hydroxyethyl derivative. The aldehyde is added at between approx. -80 and -60°C, preferably approx. -78°C for acetaldehyde.

The resulting bromohydroxypenam V is hydrogenated to remove the 6-bromo substituent. A suitable hydrogenation catalyst is a noble metal catalyst, e.g. palladium. The reaction is carried out in a protic solvent such as e.g. 1:1 methanol-water or 1:1 tetrahydrofuran-water, preferably 1:1 methanol-water, at a pressure of approx. 1-4 atmospheres, preferably 4 atmospheres and a temperature of between approx. 0 and 30°C, preferably approx. 25°C.

The resulting alcohol of formula VI can be protected with a trialkylhalosilane of the formula

where Rg in each occurrence is an alkyl group with 1-6 carbon atoms and Q is chlorine, bromine or iodine. Dimethyl-t-butylchlorosilane thus forms in the presence of an amine proton acceptor such as e.g. imidazole in a polar, aprotic solvent such as e.g. N,N-dimethylformamide in a temperature range between approx. 5 and 4 0°C, preferably approx. 25°C, a trialkylsilyl-hydroxyl protecting group as shown in formula VII.

The treatment of VII with mercury(II) acetate in acetic acid at a temperature of approx. 9 0°C gives the olefin VIII.

To obtain the desired azetidinone IX, the olefin VIII is ozonized in a reaction-inert solvent such as e.g. dichloromethane at a temperature of between approx. -80 and -40, preferably approx. -78°C. The reaction product is treated with an alkanol such as e.g. methanol, to give the acetidine IX.

As shown in scheme B, a compound of formula IX is treated with a trithiocarbonate salt of the formula M+R^q-S-C(S)-S where R^q is alkyl with 1-4 carbon atoms, preferably ethyl, and M is a metal such as . sodium or potassium, to obtain a compound of formula X. This conversion of IX to X is carried out in an organic solvent which is reaction inert, water or a mixture thereof, preferably a mixture of water and dichloromethane in a temperature range of about 0 - 35°C, preferably approx. 25°C.

The compound of formula X is condensed with p-nitrobenzyl-chloro-oxalate in the presence of a tertiary alkylamine where each alkyl group e.g. 1-4 carbon atoms, such as e.g. ethyldiisopropylamine, to obtain the compound of formula XI. This condensation reaction is carried out in a reaction-inert solvent, preferably dichloromethane, in a temperature range of approx. 5 - 25°C, preferably approx. 10°C.

The resulting compound of formula XI is ring-closed using a trialkyl phosphite where the alkyl group has 1-4 carbon atoms, such as e.g. trialkyl phosphite, in a reaction-inert solvent, such as e.g. trichloromethane, in a temperature range of approx.

4 0 - 80°C, preferably approx. 60°C, to obtain the penem with formula

XII.

The thio group in compound XII is oxidized to the corresponding sulfoxide XIII with an oxidizing agent such as e.g. m-chloroperbenzoic acid, in a reaction-inert solvent such as e.g. dichloromethane, in a temperature range of approx. -10 to -30°C, preferably -20°C.

The sulfoxide XIII is substituted with the mercaptide with the formula R-S by, for example, using the sodium or potassium salt, which is reacted with the sulfoxide XIII in a polar, organic solvent such as e.g. ethanol or acetonitrile, in a temperature range

OE OE

at approx. -50 to -10 C, preferably approx. -35 C.

Starting mercaptans with the formula R-SH or starting thioacetates

with the formula R-S-C(0)CH3 is known for many of the values of R

and those that are not known can be produced using analogous methods that are known in the field. For an overview see J.L. Wardell, "Preparation of Thiols", in The Chemistry of the Thiol Group, S. Patai, publisher, John Wiley & Sons, London, 1974, chapter 4. See also Volante, Tetrahedron Letters, 22, 3119 -

3122 (1981) for the conversion of alcohols to thiols and thiol esters using triphenylphosphine and a dialkyl azodicarboxylate in the presence of the alcohol and an appropriate thiolic acid.

For compounds of formula XIV, the trialkylsilyl group is preferably removed prior to hydrogenolysis to remove the acid-protecting group (PNB) to obtain a compound of formula XV. The trialkylsilyl group is removed with a tetraalkylammonium fluoride in an ether solvent such as tetrahydrofuran in a temperature range of approx. 15 to 40°C, preferably approx. 25°C.

Conversion of a compound of formula XV to a compound of formula II is carried out using a conventional hydrogenolysis reaction, and it is carried out in a conventional manner for this type of conversion. Thus, a solution of a compound of formula XV is stirred or shaken under a hydrogen atmosphere, or hydrogen is mixed with an inert diluent such as e.g. nitrogen or argon, in the presence of a catalytic amount of a noble metal hydrogenolysis catalyst, such as a palladium-on-calcium carbonate or a palladium-on-Celite (a diatomaceous earth) catalyst. Suitable solvents for this hydrogenolysis are lower alkanols, such as e.g. methanol, ethers, such as tetrahydrofuran and dioxane, low molecular weight esters, such as ethyl acetate and butyl acetate, water, and mixtures of these solvents. However, it is common to choose conditions under which the starting material is soluble, such as e.g. aqueous ethers, e.g. aqueous tetrahydrofuran, at a pH of approx. 7

to 8. The hydrogenolysis is usually carried out at room temperature and a pressure of approx. 0.5 to approx. 5 kg/cm 2. The catalyst is usually present in an amount from approx. 10% by weight based on the starting material up to an amount equal in weight to the starting material, although larger amounts may be used. The reaction usually takes approx. 1 hour, after which the compound of formula II is easily recovered by filtration followed by removal of the solvent in vacuo. If palladium-on-calcium carbonate is used as catalyst, the product is isolated as the calcium salt and if palladium-on-Celite

is used, the product is isolated as the sodium salt.

The compound of formulas I or II can be purified using conventional methods for beta-lactam compounds. The compound with formula I can, for example, be purified by gel filtration on "Sephadex", or by recrystallization.

An alternative synthesis procedure is shown in scheme C. The aze-tidine of formula IX is reacted with a trithiocarbonate of the formula M<+>R-S-C(S)-S~, where M is a metal such as e.g. sodium or potassium, using the method previously described to prepare X.

The resulting trithiocarbonate XVA is treated with (prnitrobenzyloxycarbonyl)(dihydroxy)methane in an aprotic solvent such as e.g. benzene, toluene or dimethylformamide, preferably benzene, in a temperature range of approx. 25 - 110°C, preferably approx. 80°C, to give the alcohol of formula XVI.

The corresponding chloride XVII is prepared from the alcohol XVI by treatment with thionyl chloride in a reaction-inert organic solvent, such as e.g. dichloromethane, in the presence of a hindered amine which serves as an acid acceptor such as 2,6-lutidine, in a temperature range of approx. -10 to 75°C, preferably 0°C.

The chloride XVII is reacted with a triarylphosphine such as triphenylphosphine, in a reaction-inert solvent such as e.g. tetrahydrofuran in the presence of a tertiary amine such as e.g. 2,6-lutidine at a temperature of approx. 2 5°C, to obtain the compound of formula XVIII, which is cyclized by reflux treatment in an aromatic solvent, such as e.g. toluene, to give the penem of formula XIV.

Trithiocarbonate salts of the formula M+R-S-(C=S)-S are prepared from the appropriate mercaptan of the formula R-SH or by treating a thioacetate of the formula RSC(0)CH^ with an alkali metal alkoxide followed by carbon disulfide.

Using the aforementioned method of Yoshida et al., the stereochemistry at carbon atom 6 of the penem as well as the hydroxyethyl group attached to carbon atom 6 is that shown in formula II. The main stereochemistry of the product from the cyclization using schemes B or C is that where the hydrogen atom in pene-ring position 5 is trans to the hydrogen atom on carbon 6 and in the alpha configuration. Alternatively, the stereochemistry can be described as 5R, 6S, 6-(R)-1-hydroxyethyl.

The compounds of formulas I or II are acidic and will form salts with basic agents. The production of such salts is considered to be within the scope of the present invention. These salts can be prepared using standard techniques, such as e.g. bringing the acidic and basic substances, usually in a stoichiometric ratio, into contact with each other in an aqueous, non-aqueous or semi-aqueous medium, as appropriate. They are then recovered by filtration, by precipitation with a non-solvent followed by filtration, by evaporation of the solvent or, in the case of aqueous solutions, by lyophilization, as appropriate. Basic agents suitably used in salt formation are of both the organic and inorganic types and include ammonia, organic amines, alkali metal hydroxides, carbonates, bicarbonates, hydrides and alkoxides, as well as alkaline earth metal hydroxides, carbonates, hydrides and -alkoxides. Representative examples of such bases are primary amines, such as e.g. n-propylamine, n-butylamine, aniline, cyclohexylamine, benzylamine and octylamine, secondary amines, such as diethylamine, morpholine, pyrrolidine and piperidine, tertiary amines, such as triethylamine, N-ethylpiperidine, N-methylmorpholine and 1,5-diazabicyclo-[4,3,0]non-5-ene, hydroxides, such as e.g. sodium hydroxide, potassium hydroxide, ammonium hydroxide and barium hydroxide, alkoxides, such as e.g. sodium ethoxide and potassium ethoxide, hydrides, such as calcium hydride and sodium hydride, carbonates such as potassium carbonate and sodium carbonate, bicarbonates, such as sodium bicarbonate and potassium bicarbonate and alkali metal salts of long-chain fatty acids, such as e.g. sodium 2-ethyl hexanoate.

Preferred salts of the compounds of formula I or II are sodium, potassium and calcium salts.

The pharmaceutically acceptable salts of formula I or II are those which are free from significant harmful side effects in ordinary amounts of use and include, e.g. the sodium, potassium or calcium salts thereof.

The in vitro activity of the compounds of formula I or II and salts thereof can be demonstrated by measuring their minimal inhibitory concentrations (MIC) in mkg/ml against various micro-organisms. The procedure followed is that recommended by the International Collaborative Study on Antibiotic Sensitivity Testing (Ericcson and Sherris, Acta Pathologica et Microbilogia Scandinav, Supp. 217, Section B: 64 - 68. (1971)), and uses brain-heart infusion (BHI) agar as a medium for repeated plating. Tubes grown overnight are diluted 100-fold for use as standard inoculum (20,000 - 10,000 cells in approximately 0.002 ml are placed on the agar surface, 20 ml of BHI agar/dish). Twelve 2-fold dilutions of the test compound are used, with a starting concentration of the test drug of 200 mkg/ml. Single colonies are not taken into account when the plates are read after 18 hours at 37°C. The sensitivity (MIC) of the test organism is received as the lowest concentration of compound capable of producing complete inhibition of growth by assessment with the naked eye.

The compounds of formula I or II, and the pharmaceutically acceptable salts thereof, are suitable for combating bacterial infections in mammals, including humans, e.g. infections caused by sensitive strains of Staphylococcus aureus.

The compounds produced according to the present invention can be administered orally or parenterally, i.e. intramuscularly, subcutaneously, intraperitoneally or intravenously, alone or combined with a pharmaceutically acceptable carrier. The ratio between the active ingredient and the carrier will depend on the chemical nature, solubility and stability of the active ingredient, as well as the intended dosage. The ratio between the pharmaceutically acceptable carrier and the penem compound will normally be in the range of 1:10 to 4:1. For oral administration, the compounds produced according to the present invention can be used in the form of tablets, capsules, lozenges, dragees, powders, syrups, elixirs, aqueous solutions and suspensions and the like. In the case of tablets, carriers that can be used include lactose, sodium citrate and salts of phosphoric acid. Various disintegrants such as e.g. starch, and lubricants, such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets. Useful diluents for capsules are lactose and high molecular weight polyethylene glycols. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. Sweetening and/or flavoring substances can be added. For parenteral administration, sterile solutions of the active ingredient are usually prepared, and the pH of the solutions is adjusted and buffered appropriately. For intravenous use, the total concentration of dissolved substances must be regulated so that the preparation becomes isotonic.

The prescribing physician will determine the appropriate dose for a given patient, and this can be expected to vary depending on the age, weight and response of the individual patient, as well as the nature and severity of the patient's symptoms. The compounds of formula I or II will normally be used orally in doses in the range from approx. 10 to approx. 200 mg/kg body weight per day, and parenterally in doses from approx. 10 to approx. 400 mg/kg body weight per day. In some cases, it may be necessary to use dosages outside these limits. .The following examples and representations shall further illustrate the invention. Infrared (IR) spectra were measured either as potassium bromide discs (KBr disc), Nujol "mull" or as solutions in chloroform (CHCl 2 ), methylene chloride (CH 2 Cl 2 ) or dimethyl sulfoxide (DMSO), and diagnostic absorption bands are indicated either in ym or wavenumber (cm ^). Nuclear magnetic resonance (NMR) spectra were measured for solutions in deutrochloroform (CDCl3), perdeuterowater (D2O) or perdeuterodimethylsulfoxide (DMSO-dg), or mixtures thereof, and the peak positions are expressed in parts per million downstream from tetramethylsilane. The following abbreviations for top forms are used: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; b, wide; w, weak; c, complex. The abbreviations "ss" and "sss" denote that a particular proton occurred as two or three singlets respectively, due to presence of diastereoisomers. In the examples and in the preparations, the abbreviation "PNB" represents the p-nitrobenzyl group. EXAMPLE 1 Sodium-(5R,6S)-6-[(R)-1-hydroxyethyl]-2-(1,3-dioxacyclohex-5-yl)thio-2-penem-3-carboxylate The pH of a suspension of 41 mg of 10% palladium-on-diatomaceous earth in 8 ml of distilled water + 8 ml of tetrahydrofuran was adjusted to approx. 7.5 with 0.02M aqueous sodium bicarbonate solution. A solution of 41 mg of p-nitrobenzyl(5R,6S)-6[(R)-1-hydroxyethyl]-2-(1,3-dioxacyclohex-5-yl)thio-2-penem-3-carboxylate in 4 ml of tetrahydrofuran and 4 ml of distilled water were added and the resulting mixture was hydrogenated at 3.9 kg/cm 2 of hydrogen for 75 minutes. 40 mg more of 10% palladium on diatomaceous earth was added to the reaction mixture and the pH of the suspension was adjusted to approx. 7.0 with 0.02M aqueous sodium bicarbonate solution. The mixture was hydrogenated at 3.9 kg/cm<2>hydrogen for 75 minutes, then the catalyst was removed by filtration and the filtrate was concentrated in vacuo to remove tetrahydrofuran. The pH of the resulting aqueous solution was adjusted to 7.0 and the solution was extracted with two 20 ml portions of ethyl acetate. The aqueous phase was then lyophilized to give 30 mg (96% yield) of the title product as an amorphous solid. The infrared spectrum of the title compound as a Nujol "mull" had an absorption at 5.67 µm.

The procedures were repeated to obtain the title compound in .85% yield. The spectral data are as follows: NMR(D 2 O,25MH 2 ): 1.29 [3H, d, J = 6.5 Hz]; 3.57 [1H, m]; 3.90 [2H, m] ; 3:92 [1H, dd, J = 6.0, 1.4 Hz]; 4.24 [1H, qd, J = 6.5,

6.0Hz]; 4.29 [2H, m]; 4.86 & 4.94 [2H, both d, JAO=6.5 Hz]

and 5.68 [1H, d, J = 1.4Hz] ppm. IR(KBr disc): 3040(b), 2966(b), 2846(w), 1770(s), 1593, 1378, 1295, 1169, 1136, 1053, 1020 and 924 cm-1. UV(H20) (extinction coefficient in parentheses) : 254 (4470) and 320(5240) nanometers. [ct]D (H 2 O) : + 139 , 9°.

EXAMPLE 2

The procedures of Example 1 were applied using compounds of formula XV to obtain the corresponding compounds of formula II as the sodium salt whose R and infrared spectral properties are as shown in Table 1, with the medium in parentheses.

MANUFACTURE A

p- nitrobenzyl( 5R, 6S)- 6-[(R)- 1- hydroxyethyl]- 2-( 1, 3- dioxacyclohex- 5- yl) thio- 2- penem- 3- carboxylate

To a solution of 70 mg (0.12 mmol) p-nitrobenzyl-(5R, 6S)-6-[(R)-1-t-butyldimethylsilyloxyethyl]-2-(1,3-dioxacyclohex-5-yl)thio -2-penem-3-carboxylate in 4 ml of anhydrous tetrahydrofuran, 0.07 ml (1.2 mmol) of acetic acid and 0.36 ml (0.36 mmol) of a 1M solution of tetrabutylammonium fluoride in tetrahydrofuran were added. After stirring for 40 hours at room temperature under nitrogen, 40 ml of ethyl acetate was added and the resulting solution was washed with 25 ml of saturated aqueous sodium bicarbonate solution, 25 ml of water and 25 ml of saturated aqueous sodium chloride solution. The ethyl acetate solution was then dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product (70 mg) was chromatographed on silica gel (35 g), eluting with 2:1 chloroform-ethyl acetate, to give 46 mg (82% yield) of the title product.

The infrared spectrum of a dichloromethane solution of the title compound had absorptions at 5.58, 5.91 and 6.58 µm. 2 The 50 MHz NMR spectrum of a perdeuterodimethylsulfoxide solution of the title compound had peaks at 1.17 (D,3H); 3.54 (m, 1H); 3.76-3.95 (c,3H); 4.02 (m,1H); 4.2 (m, 2H); 4.82 (q, 2H), 5.25 (d, 1H); 5.38 (q, 2H); 5.78 (d,1H); 7.7 (d,2H) and 8.25 (d,2H) ppm.

The procedure was repeated to obtain the title compound

in 94% yield, mp: 214-215°C (from tetrahydrofuran). The spectral data are as follows: NMR(DMSO-dg, 250 MHz): 1.17 [3H, d, J = 6 Hz]; 3.53 [1H, m] ;

3.82 [2H, m]; 3.90 [1H, dd, J = 6.1 Hz]; 4.01 [1H, m]; 4.20 [2H, m]; 4.79 & 4.83 [2H, both d, J__ = 6 Hz]; 5.26 [1H, d, J = Hz];

To ride

5.31&5.45 [2H, both d, J = 14 Hz]; 5.78 [1H, d, J = 1 Hz]; 7.69 [2H, d, J = 9 Hz] and 8.24 [2H, d, J = 9 Hz] ppm. IR(KBr disk) : 3452, 2965, 2851 (w), 1776(S), 1693 (S) , 1609 (w) , 1520, 1502, 1376, 1220, 1194, 1176, 1135, 1119, 1047 and 1023 cm."<1>

MANUFACTURE B

The procedures from preparation A were applied using compounds of formula XIV to obtain the corresponding compounds of formula XV whose R, yield and spectral properties, with solvent in parentheses, are shown in Table 2.

MANUFACTURE C

p- nitrobenzyl-(5R, 6S)- 6-[(R)- 1- t- butyldimethylsilyloxyethyl]-2-( 1, 3- dioxacyclohex- 5- yl) thio- 2- penem- 3- carboxylate

Sodium methoxide (27 mg, 0.5 mmol) was added to a solution of 1,3-dioxacyclohex-5-yl-thioacetate (81 mg, 0.5 mmol) in 5 mL of anhydrous ethanol cooled to -35° C under nitrogen. After

45 minutes at -35°C, a solution of 300 mg (approx. 0.5 mmol) of crude p-nitrobenzyl-(5R,6S)-6-[(R)-1-t-butyldimethylsilyloxyethyl]-2- ethylsulfinyl-2-penem-3-carboxylate in 5 mL of tetrahydrofuran that had been cooled to -50°C.. The resulting solution was stirred at -35°C for 60 min, then 0.029 mL (0.5 mmol) acetic acid added and the solution concentrated in vacuo. The residue was dissolved in 50 ml of ethyl acetate and the resulting solution was washed sequentially with 25 ml of saturated aqueous sodium bicarbonate solution, 25 ml of water and 25 ml of saturated aqueous sodium chloride solution. The ethyl acetate layer was dried over anhydrous sodium sulfate and concentrated in vacuo. Chromatography of the crude product (360 mg) on silica gel (100 g) eluting with chloroform gave 70 mg (24% yield) of the title compound as a viscous gum.

The infrared spectrum of the title compound in a dichloromethane solution had absorptions at 5.59, 5.9 and 6.57 µm. The NRM spectrum of the title compound in a deutrochloroform solution peaked at 0.03 (S,3H); 0.06 (s,3H); 0.8 (s.9H); 1.25 (d, 3H); 3.4 - 3.86 (c,4H), 4.0 - 4.5 (c,3H); 4.63 (d,1H); 4.95 (d, 1H); 5.27 (q, 2H); 5.63 (d,1H); 7.56 (d,2H) and 8.18 (d,2H) ppm.

The procedure was repeated to obtain the title compound in 65% yield, m.p.: 133 - 134°C (from diethyl ether/petroleum ether). The spectral data are as follows: NMR(CDCl 3 , 250 MH 2 ): 0.04 (3H, s); 0.07 (3H,s); 0.83 (9H, s); 1.26 (3H, d, J = 6.3 Hz); 3.5 - 3.75 (3H, m); 3.75' (1H, dd, J = 4.0, 1.5 Hz); 4.2 - 4.4 (3H, m); 4.67&4.97 (2H, both d, J^ = 6.2 Hz); 5.21 & 5.42 (2H, both d, JAD= 13.7 Hz); 5.67 (1H,

d, J = 1.5 Hz); 7.61 (2H, d, J = 8.7 Hz) and 8.21 (2H, d, J = 8.7 Hz) ppm. IR(KBr disk) : 2946, 2927, 2850, 1797 (S) , 1697 (S) , 1610, 1512, 1374, 1345, 1320, 1230, 1189, 1168, 1122, 1064, 1025, 982, 931, 835 , 801 and 772 cmT<1>.

MANUFACTURE D

The procedures from preparation C were applied using the appropriate thioacetate to obtain the corresponding compound of formula XIV where R, yield and spectral properties, with solvent in parentheses, are shown in Table 3.

MANUFACTURE E

p-nitrobenzyl-(5R,6S)-6-[(R)-1-t-butyldimethylsilyl-oxyethyl]-2-ethylsulfinyl- 2- penem- 3- carboxylate

A solution of 970 mg (4.78 mmol, 85% pure) of m-chloroperbenzoic acid in 25 ml of methylene chloride was added to a solution of 2.5 g (4.78 mmol) of p-nitrobenzyl-(5R,6S)- 6-[(R)-1-t-butyldimethylsilyloxyethyl]-2-ethylthio-2-penem^3-carboxylate in 125 ml of methylene chloride cooled to -20°C under a nitrogen atmosphere. The mixture was stirred at -20°C for 3 hours, then washed sequentially with two 70 mL portions of saturated aqueous sodium bicarbonate solution, 70 mL water, and 70 mL saturated aqueous sodium chloride solution. The methylene chloride solution was dried with anhydrous sodium sulfate and concentrated in vacuo to a yellow foam of the title compound (2.2 g, 86% yield).

The infrared spectrum of the title compound as a dichloromethane solution had absorptions at 5.54, 5.86 and 6.53 µm. The NMR spectrum of the title compound as a deutrochloroform solution had peaks at 0.06, 0.08, 0.1 and 0.12 (4s, total 6H); 0.8

(p, 9H) ; 1.12-1.58 (m, 6H); 3.1 (m, 2H); 3.86 (m, 1H); 4.3 (m, 1H); 5.3 (m, 2H); 5.67 and 5.78 (2d, total 1H); 7.54 (d, 2H) and 8.18 (d, 2H) ppm.

MANUFACTURE F

p- nitrobenzyl-(5R, 6S)- 6-[(R)- 1- t-butyldimethylsilyl- oxyethyl]- 2-ethylthio- 2- penem- 3- carboxylate

p-nitrobenzyl-oxalyl chloride (5.85 g, 0.024 mol) was added to a mixture of 7.3 g (0.02 mol) of (3-alpha-t-butyldimethylsilyloxyethyl-4-ethylthio(thiocarbonyl)-thio- 2-oxo-azetidine and 4.8 g (0.048 mol) of calcium carbonate in 70 ml of methylene chloride cooled to 10° C. under a nitrogen atmosphere.A solution of 4.17 ml (0.024 mol) of diisopropylethylamine in 20 ml of methylene chloride was added dropwise with at a rate such that the temperature was maintained below 12° C. The mixture was stirred for 60 min at 10° C., then washed with two 50 mL portions of ice-cold water, dried over anhydrous sodium sulfate, and concentrated in vacuo to a viscous oil. The resulting crude p -nitrobenzyl-(3-alpha-t-butyldimethylsilyloxyethyl-2-oxo-azetidinyl)oxoacetate was dissolved in 300 ml of ethanol-free chloroform and then the resulting solution was refluxed under nitrogen while a solution of 6.85 ml (0. 04 mol) of triethyl phosphite in 50 ml of ethanol-free chloroform was added dropwise over 2 hours. The resulting solution was refluxed in 16 hours, and then concentrated in vacuo. The residue was chromatographed on silica gel (800 g) eluting with 95:5 toluene-ethyl acetate to give 5.5 g (53% yield) of the title compound as a yellow foam.

The infrared spectrum of the title compound as a dichloromethane solution had absorptions at 5.56, 5.89 and 6.54 µm. The NMR spectrum of the title compound as a deutrochloroform solution had peaks at 0.07-(s,3H); 0.1 (s,3H); 0.85 (s.9H); 1.12-1.53 (m, 6H); 2.97 (q, 2H); 3.7 (m, 1H); 4.25 (m,1H); 5.3 (q,2H); 5.63 (d, 1H); 7.38 (d,2H) and 8.18 (d,2H) ppm.

The NMR spectrum of the intermediate 4-ethylthio(thiocarbonyl)thio-azetidinone as a deutrochloroform solution had peaks at 0.0 6 (s,6H); 0.8 (s.9H); 1.14-1.62 (m, 6H); 3.14-3.63 (m, 3H); 4.33 (m, 1H); 5.16 (s, 2H); 6.7 (d, 1H); 7.5 (d,2H) and 8.17 (d,2H) ppm.

MANUFACTURE G

3- alpha- t- butyldimethylsilyloxyethyl- 4- ethylthio( thiocarbonyl) thio-2- oxo- azetidine

Ethanethiol (8.5 mL, 0.115 mol) was added to a solution

of 4.18 g (0.104 mol) of sodium hydroxide in 250 ml of water cooled to 0-5°C under a nitrogen atmosphere. After 15 minutes, 7.73 mL, (0.12 mol) of carbon disulfide was added and the mixture was stirred at 0-5°C for 35 minutes. A solution of 15.0 g,

(0.0522 mol) of 4-acetoxy-3-t-butyldimethylsilyloxyethyl-2-azetidinone in 500 ml of methylene chloride was added and the mixture was stirred vigorously at room temperature for 24 hours. The aqueous phase was separated and extracted with two 150 ml portions of methylene chloride. The combined methylene chloride fractions were washed with two 200 mL portions of water and 200 mL saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated in vacuo.

The crude title product (18 g) was chromatographed on silica gel (500 g) eluting with 99:1 chloroform-ethyl acetate to give 9.1 g (48% yield) of the title trithiocarbonate as a yellow foam.

The infrared spectrum of the title compound in dichloromethane solution had absorptions at 5.62 and 9.2 µm. The NMR spectrum of a deutrochloroform solution of the title compound had peaks at 0.08 (s,6H); 0.8 (s.9H); 1.02 - 1.5 (m, 6H); 3.0 - 3.48 (m, 3H); 4.12 (m, 1H); 5.54 (d,lH) and 6.57 (b,lH) ppm.

MANUFACTURE H

1,3-dioxacyclohex-5-yl-p-toluenesulfonate

p-toluenesulfonyl chloride (38.1 g, 0.2 mol) was added to a solution of 20.8 g (0.2 mol) glycerol formal (a mixture consisting of 67% 1,3-dioxane 5-ol and 33% (1,3-dioxolan-4-yl)meta-

noi in 200 ml of pyridine cooled to 0°C under nitrogen. The reaction mixture was stirred at 0°C for half an hour, then at 25°C for 20 hours. The mixture was added to 500 ml of 6N aqueous hydrochloric acid solution and the resulting mixture was extracted with four 200 ml portions of ethyl acetate. The combined ethyl acetate extracts were washed with two 200 ml portions of a 1N aqueous hydrochloric acid solution, two 200 ml portions of water and 200 ml saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate and concentrated in vacuo to an oil. The crude product was dissolved in 500 ml of diisopropyl ether, whereupon the desired 1,3-dioxacyclohex-5-yl-tosylate crystallized. Filtration gave 17.4 g of white crystalline tosylate, m.p. 91 - 92°C. A further 4.3 g of crystalline tosylate was obtained from the mother liquor (total yield 42%). The NMR spectrum of the title compound as a deutrochloroform solution peaked at 2.45 (s,3H); 3.54 - 4.13 (c, 4H); 4.26 - 4.6 (m, 1H); 4.75 (s, 2H); 7.3 (d,2H) and 7.8 (d,2H) ppm.

MANUFACTURE I

1, 3-dioxacyclopent-4-methylmethyl-p-toluenesulfonate

p-toluenesulfonyl chloride (76.2 g, 0.4 mol) was added to a solution of 104.1 g (1 mol) glycerol formal (a mixture consisting of 67% 1,3-dioxacyclohex-5- ol and 33% (1,3-dioxacyclopent-4-yl)methanol) in 1000 ml of pyridine cooled to 0°C under nitrogen. After standing at 0°C for 20 hours, the reaction mixture was allowed to warm to room temperature and was added to 1500 ml of 6N aqueous hydrochloric acid. The resulting mixture was extracted with four 500 mL portions of ethyl acetate. The combined ethyl acetate extracts were washed with two 500 ml portions of 6N aqueous hydrochloric acid solution, two 500 ml portions of water and 500 ml saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate and concentrated in vacuo. The oily product (78.4 g) consisted of approx. a 2:1 mixture of 1,3-dioxacyclopent-4-ylmethyl tosylate and 1,3-dioxacyclohex-5-yl tosylate which was used without purification in a subsequent step (Preparation K).

MANUFACTURE J

1, 3-dioxacyclohex-5-yl-thioacetate

A solution of 28.0 g (0.108 mol) of 1,3-dioxacyclohex-5-yl-p-tosylate and 24.6 g (0.216 mol) of potassium thioacetate in 500 mL of dimethylformamide was heated at 80°C under nitrogen for 20 hours. . The reaction mixture was then cooled to room temperature and diluted with 1000 ml of water. The resulting mixture was extracted with six 300 mL portions of ethyl acetate. The combined ethyl acetate extracts were washed with four 500 mL portions of water and 500 mL saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated in vacuo to an oil. Distillation of the product gave 6.25 g (36% yield) of the title thioacetate, b.p.

70°C (0.4 mm).

The NMR spectrum of the title compound as a deutrochloroform solution peaked at 2.34 (s,3H); 3.4 - 4.36 (c,5H) and 4.8 (q,2h) ppm.

The procedure was repeated to obtain the title compound in 49% yield, bp. 85 - 89°C (0.7mm).

NMR (CDCl 3 #250 MH 2 ): 2.34 (3H, s); 3.7 - 3.85 (3H, m); 4.05 - 4.2 (2H, m); 4.79 (1H, d, Jgem = 6.2 Hz) and 4.89 (1H, d, Jgem = 6.2 Hz) ppm.

MANUFACTURE K

(1, 3-dioxacyclopentyl-4-yl) methyl thioacetate

A mixture of 78 g (0.3 mol) of 1,3-dioxacyclopent-4-yl-tosylate (from Preparation I) and 27.4 g (0.24 mol) of potassium thioacetate in 1500 ml of acetone was refluxed under nitrogen overnight. The reaction mixture was then concentrated in vacuo and the residue was dissolved in 500 ml ethyl acetate + 500 ml water. The aqueous layer was extracted with 500 ml of ethyl acetate. The combined ethyl acetate extracts were washed with two 500 mL portions of water and 500 mL saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue, which consisted of a solid suspended in an oil, was filtered. The filtrate was distilled under reduced pressure, yielding 20.8 g (54%) of 1,3-dioxacyclopent-4-yl-methyl-tosylate, b.p. 65 - 70°C (0.2 mm).

The solid product was washed with ether to give 18.4 g of 1,3-dioxacyclohex-5-yl-tosylate.

The NMR spectrum of the title compound as a deutrochloroform solution showed peaks at 2.37 (s, 3H); 3.1 (d,2H); 3.4 - 4.4 (c,3H); 4.86 (s,H) and 5.02 (s,lH) ppm.

MANUFACTURE L

1, 3- dioxacyclopent- 2- yl) methyl thioacetate

A mixture of 5.0 g (0.03 mol) of 2-bromomethyl-1,3-dioxa-cyclopentane and 5.13 g (0.045 mol) of potassium thioacetate in 60 ml of acetone was refluxed under nitrogen overnight. The mixture was filtered and the filtrate was concentrated in vacuo. The residue was partitioned between 100 ml of ethyl acetate and 60 ml of water. The aqueous layer was extracted with 50 mL ethyl acetate and the combined ethyl acetate portions were washed with 50 mL water and 50 mL saturated aqueous sodium chloride solution. The ethyl acetate solution was dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was chromatographed on silica gel eluting with methylene chloride to give 4.0 g (83% yield) of the desired thioacetate as an oil.

The NMR spectrum of the title compound as a deutrochloroform solution peaked at 2.36 (s,3H); 3.16 (d,2H); 3.94 (c, 4H) and 5.0 (t, 1H) ppm.

MANUFACTURE M

2-oxol, 3-dioxacyclopentyl-4-ylmethylthioacetate

Di-isopropyl-azodicarboxylate (3.9 mL, 0.02 mol) was added to a solution of 5.2 g (0.02 mol) of triphenylphosphine in 50 mL of anhydrous tetrahydrofuran cooled to 0°C under nitrogen. The solution was stirred at 0°C for 30 minutes and during this time a precipitate formed. To this mixture was added dropwise at 0°C a solution of 1.18 g (0.01 mol) glycerol carbonate and 1.4 ml (0.02 mol) thioacetic acid in 20 ml anhydrous tetrahydrofuran. The mixture was stirred at 0°C for 1 hour and then at room temperature for 2 hours. The mixture was concentrated in vacuo and the residue was partitioned between 70 ml of ethyl acetate and 70 ml of water. The ethyl acetate layer was washed with two 50 ml portions of saturated aqueous sodium bicarbonate solution, 50 ml water and 50 ml saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was chromatographed on silica gel and eluted with methylene chloride to give 600 mg of impure material. Further purification of the crude product by chromatography on silica gel and eluting with 3:1 hexane/ethyl acetate gave 220 mg.

(13% yield) of the desired thioacetate as an oil.

The NMR spectrum of the title compound as a deutrochloroform solution peaked at 2.4 (s,3H); 3.25 (d,2H) and 3.94 - 5.06 (c,3H) ppm.

Claims (9)

1. Method for producing a compound with for the flour or a pharmaceutically acceptable salt thereof, wherein R is A is carbonyl, thiocarbonyl or methylene, B is the alkylene with 2-5 carbon atoms. alk is the alkylene with 1-6 carbon atoms, R^ is hydrogen or a group resulting in an ester which is hydrolyzable in vivo and n is zero or one, characterized by the step of converting a compound of the formula to the corresponding compound of formula I by hydrogenolysis, where R 1 is a carboxylic acid protecting group which is removed by hydrogenolysis. 2. Method according to claim 1, characterized by at is hydrogen, B is ethylene, A is methylene, n is one and alk is methylene. 3. Method according to claim 2, characterized in that R is 1,3-dioxacyclopent-4-ylmethyl or 1,3-dioxacyclopent-2-ylmethyl. 4. Method according to claim 1, characterized in that is hydrogen, A is carbonyl, n is one and alk is methylene. 5. Method according to claim 4, characterized in that R is 2-oxo-1,3-dioxacyclopent-4-ylmethyl. 6. Method according to claim 1, characterized in that R^ is hydrogen, B is propylene, A is methylene or carbonyl and n is zero. 7. Method according to claim 6, characterized in that R is 1,3-dioxacyclohex-5-yl or 2-oxo-1,3-dioxacyclohex-5-yl. 8. Method according to claim 1, characterized in that the compound of formula I is or a pharmaceutically acceptable salt thereof, wherein R is A is carbonyl, thiocarbonyl or methylene, B is alkylene with 2-5 carbon atoms, alk is the alkylene with 1-6 carbon atoms, is hydrogen or a group resulting in an ester which is hydrolyzable in v.ivo and n is zero or one. 9. Method according to claim 8, characterized in that R^ is hydrogen and R is 1,3-dioxacyclopent-4-ylmethyl, 1,3-dioxacyclopent-2-ylmethyl, 2-oxo-1,3-dioxacyclopent-4-ylmethyl, 1,3-dioxacyclohex- 5-yl or 2-oxo-1,3-dioxacyclohex-5-yl.