DK150515B - METHOD OF ANOLOGY FOR THE PREPARATION OF 6- (2-AMI-NO-2-ARYLACETAMIDO) -2,2-DIMETHYL-3- (5-TETRAZOLYL) PENAME COMPOUNDS OR PHARMACEUTICAL ACCEPTABLE SALTS THEREOF - Google Patents

Description

150515

This invention relates to an analogous process for the preparation of novel 6- (2-amino-2-arylacetamido) -2,2-dimethyl-3- (5-tetrazolyl) penam compounds of the general formula I or pharmaceutically acceptable as set forth in claim 1. salts thereof.

These compounds have broad spectrum antibacterial activity and are particularly effective against Klebsiella pneumoniae. They are therefore of value as therapeutic agents for the control of infectious diseases caused by Gram-positive and Gram-negative bacteria.

Despite the large number of penam derivatives that have been proposed for use as antibacterial agents, there is still a need for new agents.

U.S. Patent Nos. 3,427,302 and 3,468,874 are designated penam derivatives which contain a tetrazolyl group as part of the 6-acyl amino substituent. However, the compounds prepared according to the present invention are unique in having the tetrazolyl group bonded directly to the penam core.

The vast majority of penam compounds disclosed in the prior art have a carboxyl group (or a salt thereof) attached to the 3-position. However, penam compounds with other carboxylic acid derivatives are also known in the 3-position. Penam-3-carboxylic acid esters have been reported by, for example, Kirehner et al.

Journal of Organic Chemistry, Ijl ·, 388 (1949); Carpenter, Journal of the American Chemical Society, 70, 2964 (1948); Johnson, Journal of the American Chemical Society, 75, 3636 (1953); Bamden et al., Journal of the Chemical Society (London), 3733 (1953), and Jansen and Russell, Journal of the Chemical Society (London), 2127 (1965); and penam-3-carboxamides have been reported e.g. by Holysz and Stavely, Journal, of the American Chemical Society, 72, 4760 (1950) and Huang et al., Antimicrobial Agents and Chemotherapy, 493 (1963). Peron et al (Journal of Medicinal Chemistry, T, 483 [1964]) prepared several 6- (substituted-amino) -2,2-dimethyl-penam-3-carboxylic acid azides, which were then converted to the corresponding 3-isocyanates and 3-benzylearbamater. Peron et al. (10c. Cit.) Also reported certain 3- (hydroxymethyl) -penam derivatives. Dehydrogenation of the simple amide of benzylpenicillin gives the corresponding nitrile (Khoskhlov et al., Doklady Akad. Nauk S.S.S.R., 135, 875 (“i960]).

The common view in the art has been that modification in the 3 position would be unlikely to produce improved results.

The process according to the invention is characterized by the characterizing part of claim 1.

Particularly valuable compounds prepared by the process of the invention are: 6- (D-2-amino-2-phenylacetamido) -2,2-dimethyl-3- (5-tetrazolyl) penam and 6- (D-2 -amino-2β-hydroxyphenyl] acetamido) -2,2-dimethyl-3- (5-tetrazolyl) penam.

Intermediates for use as starting compounds in the process of the invention or as precursors thereof are set forth in Danish Patent Application No. 2690/77 and have the formulas:

5- "V_III

Γ Τόη3 -Ν- \ V / 'ν-Ν R2 R1-NHn ^

\ _ /, IV

wherein R is hydrogen, trialkylsilyl having 1-4 carbon atoms in each alkyl moiety or an amino protecting group,

ISLAND

R is hydrogen, trialkylsilyl having 1-4 carbon atoms in each alkyl moiety, alkanoyloxymethyl having 3-8 carbon atoms, 1- (alkanoyloxy) ethyl having 4-9 carbon atoms, phthalidyl or a tetrazolylpenam nitrogen protecting group, the species of which will be defined hereinafter. and hydrogen, trialkylsilyl of 1-4 carbon atoms in each alkyl moiety, alkanoyloxymethyl of 3-8 carbon atoms, 1- (alkanoyloxy) ethyl of 4-9 carbon atoms or phthalidyl, or salts thereof.

2 3 5

The compounds of formulas III and IV in which R, R and R are each independently selected from hydrogen and trialkylsilyl of 1-4 carbon atoms in each alkyl moiety are particularly valuable for preparing the novel penam compounds of formula I.

The term "tetrazolylpenam nitrogen protecting group" shall in the most general sense include those groups which protect the tetrazole ring during or after its formation, and may include a group such as trialkylsilyl or triphenylmethyl which may be attached to the tetrazole ring, for example during acylation of amino However, in the specific and preferred embodiment of the invention, this term should include all groups known or obvious to those skilled in the art who (a) will permit the synthesis of the compounds of formula III wherein R amino protecting group, and R is the said tetrazolylpenam nitrogen protecting group, by the process starting from 6- (protected-amino) -penicillanic acid as described below, and (b) can be removed from a compound of the formula Wherein R is an aeyl group and R is said tetrazolylpenam nitrogen protecting group, or from a compound of formula III wherein R is hydrogen or an amino compound. 2 is the protecting group, and R is the said tetrazolylpenam nitrogen protecting group, using a method where the pen embryo system remains substantially intact. The tetrazolylpenam nitro gene protecting group is required to protect the nitrogen atom, which eventually becomes N-1 in the tetrazole ring of said compounds of formulas I and III, during the conversion of a 6- (protected-amino) -phenylacetic acid to a compound of formula III. It is also the ability of the tetrazolylpenam protecting group to fulfill a particular function, which will be explained in more detail below, more than its precise chemical structure, which is important. Selection and identification of suitable protecting groups can be readily undertaken by those skilled in the art, and examples of several useful groups are given below.

5 150515

Of. For convenience, the compounds of the invention are identified as derivatives of "penam" which have been defined by Sheehan et al. in the Journal of the American Chemical Society, 75, 3293 (1953), to designate the structure: 6-8 s \ 2 ”- * 3

Although the term "penam" does not usually have any stereochemical meanings, the stereochemistry of the penam compounds herein is similar to that found in the naturally occurring penicillins. Using this terminology, the well-known antibiotic penicillin G (benzylpenicillin) is referred to as 6- (2-phenylacetamido) -2,2-dimethylpenam-3-carboxylic acid.

The compounds of formula I according to the invention can be considered as 5-substituted tetrazoles, and such 5-substituted tetrazoles can exist in two isomeric forms, namely:

H

H

S and / N

“(Γ 'I g -c i

5 \ J

which occur together in a dynamic, tautomeric equilibrium mixture.

The general formula I should be understood to include this equilibrium mixture. However, in the case of the starting compound, where this is a tetrazole protected derivative, the two forms represent different chemical units of the previously mentioned formulas III and IV which do not spontaneously convert to each other.

6 150515

As will be appreciated by those skilled in the art, the acyl group R7-CH-CO-nh2 may contain one or more asymmetric centers, and asymmetric centers may exist in one or two forms, the so-called D and L forms. Both forms of each asymmetric center and all combinations of each of the forms are to be considered as encompassed by the invention.

The process of the invention and the preparation of the starting materials therefore from the well-known intermediate 6-amino-penicillanic acid are illustrated in the following reaction scheme: 7 150515 η CH

Mt,, sv χι * Γ jr CH.

(R 2 '- NH Sv 3

WM

N-L

/ Ilia V_N ^ (R2) 'X v c ^ CH-j NHp 8 I 5 (H?)' - NH 3 N-S νΨ— (¾ · N-N-CH3 J-N-1> 4-N-1> ^ N \

Illc \ n__n / IIIb / ik— $ (R2) '^ \ / CH3 Ns ^. CH3

r'-ch-co-nh s nh2 A

fe2 b-fX-®3> - (N-æ3 J-N-L ^ N \ J-N-L ^ N \

<r, N ¢ /> c / N

11. V_N '. IIId HN r / (R2 // // 7 ch3 ^

R -CH-CO-NH. A J

% YYN-®3

I} * N_W

7 R has the meaning given in the preamble of claim 1,

ISLAND

(R) 'is a tetrazolylpenam nitrogen protecting group and (R') * is an amino protecting group.

8

Considering the reaction scheme, the manner in which the binders of formulas II and III, wherein R is a tetrazolyl-penam nitrogen protecting group, are useful as intermediates for the preparation of the antibacterial compounds of formula I, will be clear. In considering the nature of the tetrazolylpenam nitrogen protecting group, the group must fulfill two functions. First, it must allow the synthesis of compounds of formula c 2

IIa, wherein Br is an amino protecting group and R is said tetrazolylpenam nitrogen protecting group. Second, it must be removable from a compound of formula II wherein R is the tetrazolylpenam nitrogen protecting group or from a compound of formula IIIc wherein R is hydrogen and R is the tetrazolylpenam nitrogen protecting group group, or from a compound 52 of formula IIla, wherein R is an amino protecting group and R is the tetrazolylpenam protecting group, in each case without breaking down the pen numbering system. As will be apparent from the following explanation, not all of the tetrazolylpenamnitrogen-protecting groups useful in the process of the invention need to be removed from each of the compounds of formulas II and III. To be useful in the process of the invention, the tetrazolylpenam nitrogen protecting group need only be removable from at least one of the following three types of compounds: (a) compounds of formula II wherein R is said tetrazolylpenam nitrogen protected group; (b) compounds of formula IIIc wherein R 1 is hydrogen and R is the tetrazolylpenam nitrogen protecting group; and (c) compounds of formula IIa, wherein R 1 is an amino protecting group and R 2 is the tetrazolylpenam nitrogen protecting group. The conditions which it will be necessary to use to remove a given tetrazolylpenam nitrogen protecting group will be known or obvious to one skilled in the art. Furthermore, the reaction conditions which can be used without causing the breakdown of the pen embryo system are also well known and obvious in view of the prior art of penam compounds.

A particular series of tetrazolyl protecting groups is -CH 2 CH 2 X, -C (= O) -O-R 14, SO 2 -R 14 or (R 6) * | Wherein Y represents cyano, alkoxycarbonyl of 2-7 carbon atoms, phenoxycarbonyl, alkylsulfonyl of 1-6 carbon atoms, phenylsulfonyl or -SOg-NR ^R ^ ^ wherein R ^ and R ^ are each selected from hydrogen, alkyl with 1-4 carbon atoms, benzyl and phenyl; 14 R is alkyl of 1-6 carbon atoms, benzyl, phenyl or substituted phenyl having up to two substituents selected from nitro, fluoro, bromo, alkyl of 1-4 carbon atoms, and alkoxy of 1-4 carbon atoms; and (R 2) 'means 7 - (3 (

R17 or -CH_i '\ r20

R19 I I

Wherein R RT and R 1 are respectively hydrogen, hydroxy, nitro, fluorine, chlorine, bromine, iodine, alkyl of 1-6 carbon atoms, alkoxy of 1-6 carbon atoms, alkanoyloxy of 2-7 carbon atoms, formyloxy, alkoxymethoxy of 2-7 carbon atoms, phenyl or benzyloxy;

1R

R is hydrogen, alkyl of 1-4 carbon atoms or phenyl; R 20 and R are hydrogen or methyl; and Z means oxygen or sulfur.

10

An example of a typical tetrazolylpenam nitrogen protecting group is:

/ Y

-CH2 - CH

\ Y wherein Y is an electron-attracting group and Y 'is either hydrogen or another electron-attracting group which may be the same or different from Y. The function of an electron-attracting group is to make a hydrogen atom on the carbon atoms to which Y and Y1 is attached, sufficiently acidic to allow the group to be removed by a reverse Michael reaction. Such a reaction is well known in the art, see, for example, House, "Modem Synthetic Reactions," WA Benjamin, Inc., New York / Amsterdam, 1965, page 207. Typical electron-attracting groups are cyan, alkoxy-carbonyl having 2-7 carbon atoms, phenoxycarbonyl, alkylsufonyl having 15 16 15 1-6 carbon atoms, phenylsulfonyl and SOP-NR 2 R, wherein R and R 0 are each independently hydrogen, alkyl of 1-4 carbon atoms, phenyl or benzyl. protecting group is that in which Y 'is hydrogen, and preferred values of Y are alkoxycarbonyl of 2-7 carbon atoms and phenylsulfonyl, such group can be removed by mild hydrolysis such as mild alkaline hydrolysis or by treatment with a nucleophilic reagent, such as an amine or a thiol or thiolate anion.

Although a wide variety of groups can serve as R 2, particularly suitable values are alkyl of 1-6 carbon atoms, benzyl, phenyl and substituted phenyl, e.g. with up to two substituents individually selected from nitro, fluoro, chloro, bromo, alkyl of 1-4 carbon atoms and alkoxy of 1-4 carbon atoms.

Another tetrazolylpenam nitrogen protecting group which can be used has the formula -SO2-R · Such group can also be formed by hydrolysis or by treatment with a nucleophilic reagent as indicated for the group -C (= O) -0- R \ and appropriate values for R ^ are also alkyl of 1-6 carbon atoms, benzyl, phenyl and substituted phenyl, e.g. with up to two substituents individually selected from nitro, fluoro, chloro, bromo, alkyl of 1-4 carbon atoms and alkoxy of 1-4 carbon atoms.

i, 11 150515

An additional tetrazolylpenam nitrogen protecting group which may be used is

-OH

wherein W is phenyl, substituted phenyl, furyl, substituted furyl, thienyl or substituted thienyl, and W is hydrogen, alkyl, phenyl, substituted phenyl, furyl, substituted furyl, thienyl or substituted thienyl. When ¥ is phenyl or substituted phenyl and W is hydrogen, alkyl, phenyl or substituted phenyl, this group can be removed by hydrogenolysis. This group can also be removed by solvolysis in trifluoroacetic acid when the total effect of W and W is sufficient to provide the necessary degree of stability to the arising carbonium ion. ♦ CH ^ Particularly suitable configurations for this protecting group are ΐ-Cvf r17 and i- , -CH -> - _ in λ Λη wherein F and R are each independently hydrogen, hydroxy, nitro, fluoro, ehloro, bromo, iodo, alkyl of 1-6 carbon atoms, alkoxy of 1-6 carbon atoms, alkanoyloxy of 2 -7 carbon atoms, formyloxy, alkoxymethoxy with 2-7 carbon atoms, phenyl or benzyloxy; 12 ISOS 15 to 18 R "means hydrogen, alkyl of 1-4 carbon atoms or phenyl; 1'q of) R and R each independently represent hydrogen or methyl; and X represents oxygen or sulfur.

As will be appreciated by one of ordinary skill in the art, other groups which will also stabilize the carbonium ion (W-CH-W) + may replace those listed above for ¥ and W.

Another tetrazolylpenam nitrogen protecting group which may be used is phenacyl or substituted phenacyl. Such a group is removed by reaction with a nucleophilic reagent such as thiophene oxide. Typical phenacyl groups which can be used have the formula / = kJl21 -CHp-C

0 V V

Wherein R is hydrogen, nitro, fluoro, ehloro, bromo or phenyl.

Several individual reactions which may be included in the process of the invention will now be discussed and explained in detail. For convenience, they will be designated as Method A, B, C and D.

Methods A and B and D relate to the removal of tetrazolylpenam protecting groups, and method C is an acylation to introduce the group R7-CH-CO-nh2

It will be seen that two or more of these methods can be used to form a particular product. For example, method A can be used to remove a protecting group from an intermediate followed by an acylation according to method C. It is clear that the choice of a particular method or sequence of steps is determined only by the known properties of the compounds and the chemical processes involved. .

Method A comprises catalytic hydrogenolysis of a compound 2 of formula II wherein R is ir 'wherein R 1 and R 1 are each independently hydrogen, hydroxy, nitro, fluoro, chloro, bromo, iodo, alkyl of 1-6 carbon atoms, alkoxy of 1-6 carbon atoms, alkanoyloxy of 2-5 carbon atoms, formyloxy, alkoxymethoxy of 2-7 carbon atoms, phenyl or benzyloxy.

The starting material may be prepared by acylating a suitably similar compound of formula III or a salt thereof wherein R 1 is hydrogen or trialkylsilyl of 1-4 carbon atoms in each

ISLAND

alkyl moiety and R is substituted as indicated above. The aoylation can be carried out as described below under Method 0.

The reaction can be carried out by a variety of procedures well known in the art for this type of conversion, such as those discussed by Augustine in "Catalytic Hydrogenation", Marcel Dekker, Inc., New York 1965, pages 139-142. However, as will be appreciated by one skilled in the art, conditions which are compatible with the β-lactam portion of the penam core must be selected. A particularly convenient procedure consists of shaking or stirring a solution of the reactant in a reaction inert solvent such as methanol, ethanol, ethyl acetate or water or a mixture of such solvents, in the presence of a catalyst such as 10 $ palladium on charcoal, under an atmosphere of hydrogen. The catalyst is usually present in an amount of from about 10 to about 100% by weight, based on the penam starting material, and the hydrogen pressure can vary from about 1 to about 100 atmospheres. At room temperature, the reaction takes a few hours to complete.

14

Method B consists of treating a compound of formula II wherein 3 is (3C, ✓ 4 17 wherein at least one of R and E is a 2-or 4-position hydroxy group with base. The starting material can be prepared by hydrogenolysis of the corresponding compound wherein any hydroxy group at R 2 or R 2 is protected as its benzyl ether. The procedures described under Method A may conveniently be used. In fact, one can proceed directly from Method A to Method B without isolation or where a hydrogenolysis is carried out. in the presence of a basic agent, method B. may be carried out with the hydrogenolysis, under certain hydrogenolysis conditions, the formation of the penam compound wherein R is a hydroxybenzyl group is followed immediately by hydrogenolytic removal of the hydroxybenzyl group to form the compound of formula I. by dissolving the starting material in a suitable solvent and then adding about 1 molar equivalent of a base at about or slightly below room temperature temperature. The reaction is usually completed in a few minutes. Suitable solvents for this process are those which will serve to dissolve the starting material but are otherwise inert. Examples of solvents which may be used are esters such as ethyl acetate and butyl acetate; lower aliphatic ketones such as acetone and methyl ethyl ketone; chlorinated hydrocarbons such as chloroform, methylene chloride and 1,2-dichloroethane; lower alkanols such as methanol and ethanol; and tetrahydrofuran. A wide variety of basic agents are useful in this process as it is found that the main purpose of the basic agent is to remove the hydrogen from the phenolic hydroxy group of the protecting hydroxybenzyl group. Basic agents which can be used include alkali metal salts of alkanoic acids such as sodium acetate and sodium 2-ethylhexanoate; organic amines such as tributylamine, triethylamine, N, N-dimethylaniline, N, N-ethylpiperidine, pyridine and N-methylmorpholine; alkali metal hydroxides such as sodium hydroxide and potassium hydroxide; alkaline earth metal hydroxides such as barium hydroxide and calcium hydroxide; and alkali metal and alkaline earth metal hydrides such as sodium hydride, potassium hydride and calcium hydride. Although it is customary to use about 1 molar equivalent base in this process, under certain circumstances an excess base may be used. In fact, in cases where there is another acidic function in the starting material, it becomes necessary to add two molar equivalents of the basic agent. However, it will be appreciated by one skilled in the art that the conditions of this reaction must be chosen in view of the sensitivity of the β-lactam moiety of the penamkeme, and the presence in the reaction medium of an excess of a basic agent which will react adversely with the β-lactam must be avoided. A particularly convenient method for carrying out this process is to expose the starting material to an aqueous solution system having a pH in the range of from about 7.5 to about 9.5

Method C comprises acylating a compound of formula III

2 or IV or a salt thereof, wherein R is hydrogen, alkanoyloxymethyl, 1- (alkanoyloxy) ethyl, phthalidyl or trialkylsilyl having 1-4 carbon atoms in each alkyl moiety, if necessary followed by treatment with a protic solvent. The latter treatment is needed when either R 1 or R 3 is trialkylsilyl. The acylation is carried out by contacting the compound of formula III or IV or a salt thereof with an activated derivative of the appropriate carboxylic acid in a suitable solvent system.

An activated derivative normally used is an acid halide such as an acid chloride. In a typical acylation procedure, approximately a molar equivalent of an acid chloride is added to a solution of the compound of formula III or IV or a salt thereof dissolved in a solvent such as a chlorinated hydrocarbon, e.g. chloroform or methylene chloride; an ether, e.g. tetrahydrofuran or 1,2-dimethoxyethane; an ester, e.g. ethyl acetate or butyl acetate a lower aliphatic ketone, e.g. acetone or methyl ethyl ketone; or a tertiary amine, e.g. N, N-dimethylformamide or Ν, Ν-methylpyrrolidone; at a temperature in the range of from about -10 to about 30 ° C, and preferably from about -10 to about 10 ° C, optionally in the presence of about a molar equivalent of an acid-retaining agent, e.g. triethylamine pyridine or sodium bicarbonate. The reaction is completed within a short period of time, i.e. about 1 hour. When R and R are hydrogen, it is convenient to use a tertiary amine salt, e.g. the triethylamine salt, of the compound of formula III or IV. An alternative procedure useful for acylating an O 2 c compound of formula III or IV, wherein R, R 'and Rp are all hydrogen, with acid halides involves the use of an aqueous solution system. In this procedure, which approaches the Schotten-Baumann procedure, the acid halide is added to a solution of the starting material in water or a mixture of water and another inert solvent at about or slightly below room temperature, keeping the pH of the solvent within the range of about 6.0 to about 9.0 before, during and after the addition.

Another activated derivative of the carboxylic acid which may be used in Method C is a mixed anhydride. In this case, a carboxylate salt of the appropriate carboxylic acid is treated with about 1 molar equivalent of a lower alkyl chloroformate in a reaction inert aprotic organic solvent at a temperature in the range of about -20 to about 20 ° C, and preferably at about 0 ° C. salts for this process are alkali metal salts such as sodium and potassium salts and tertiary amine salts such as triethylamine, tributylamine, N-ethylpiperidine, N, N-dimethylamine, N-methylmorpholine and pyridine salts; and suitable solvents are, for example, chloroform, methylene chloride, acetonitrile, tetrahydrofuran, dioxane and Ν, Ν-dimethylformamide. The thus obtained mixed earboxylic acid carboxylic anhydride is usually used in situ to acylate the compound of formula III or IV.

Bets are usually made by mixing solutions of the previously formed mixed anhydride and the compound of formula III or IV. When R and R 2 are hydrogen, it is particularly convenient to use a tertiary amine salt, e.g. the triethylamine salt, of the compound of formula III or IV. The acylation is usually carried out at a temperature in the range of about -30 to about 20 ° C, and preferably at about -10 ° C, and it usually takes a few hours to complete. In most cases, the mixed anhydride and the compound of formula III or IV are contacted in a molar ratio of approximately 1: 1.

150515

Another variation of Method C involves converting the carboxylic acid to an active ester followed by treatment with a compound of formula III or IV or a salt thereof. Active esters which may be used in this connection are, for example, phenyl esters such as p-nitrophenyl and 2,4,5-trichlorophenyl esters, thio esters such as thiophenyl and thiol methyl esters, and N-hydroxy esters such as N-hydroxysuccinimide and N hydroxyphthalimidestere. The esters are prepared by methods well known in the art and the acylation is conveniently carried out by dissolving the active ester and the compound of formula III or IV or a salt thereof in a dipolar aprotic solvent such as Ν, Ν-dimethylformamide, Ν, Ν- dimethylacetamide or N-methylpyrrolidone. The solution is stored at about room temperature for several hours, e.g. overnight, and then the product is isolated by standard methods. In many cases, the active ester used in this process can be replaced by the corresponding acid azide.

Yet another variation of method C useful for the acylation of compounds of formulas III and IV consists in contacting the compound of formula III or IV with a carboxylic acid in the presence of certain agents used in the art to form peptide bonds. . Such agents include carbodiimides, e.g. dicyclohexylcarbodiimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, alkoxyacetylenes, e.g. methoxyacetylene and ethoxyacetylene, and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. The reaction is carried out in a suitable solvent, i.e. one which will serve to dissolve the reactants and not react adversely with the starting materials or product, e.g. acetonitrile, N, N-dimethylformamide and N-methylpyrrolidone.

It is apparent from the above description of Method C that hydrogen substituents at R, R or R can be successfully replaced by trialkylsilyl substituents by a process for acylating a compound of formula III or IV. These trialkylsilyl substituents are then removed and replaced with hydrogen at the end of the acylation, simply by brief exposure of the product to a protic solution system such as water or a lower alkanol, e.g. methanol or ethanol. Due to the easy availability of the starting materials, the trimethylsilyl group is a preferred substituent. It can be introduced into the starting penam derivative of formula 150515 18 III or IY by well known methods such as the use of trimethylchlorosilane or N-trimethylsilylacetamide, as disclosed by Birkofer and Ritter in Angewandte Chemie (International Edition in English), 4, 417-418 and 426 (1965). However, conditions that are compatible with the β-lactam group in the penamkeme must be selected. Also useful in Method C are the silylated derivatives formed by reacting said compounds of formulas III and IY with dichloro-di- (lower-alkyl) silanes. The silylation step is performed by methods well known in the art (for example, as disclosed in DE patent specification No. 1,933,187). After the acylation reaction, the silyl group is removed by treatment with a protective solvent such as water or a lower alkanol, e.g. methanol or ethanol.

In addition, if desired, the tetrazole ring of a compound of formula III or IV wherein R, RJ and Έτ are all hydrogen may be protected by various other groups before aeylation by method C. The protective group is then removed after the acylation to release the desired antibacterial agent of formula I. A wide variety of protecting groups can be used for this purpose, e.g. triphenylmethyl, substituted triphenylmethyl, alkoxymethyl, benzyl oxymethyl, substituted benzyloxymethyl and cyanmethyl. However, a particularly convenient group is the triphenylmethyl group.

It will be appreciated by those skilled in the art that not all of the variations discussed under Method C are equally effective or appropriate in all cases for aeylating a compound of formula III or IY. The relative efficiency of a particular variant will depend on several factors, such as the precise structure of the compound of formula III or IY, the availability of the starting materials, the scale of the reaction, and in particular the structure and reactivity of the acyl group introduced. In practice, one skilled in the art will select the most appropriate variant in each case, taking full account of the relevant factors. Furthermore, certain additional measures and modifications become necessary or desirable. Eg. it is necessary to protect the amino group of the starting carboxylic acid before activating the carboxy group in the acid. After the amino group has been protected, the carboxy group is activated, the acylation is carried out by one of the methods described under method C, and then the antibacterial penam compound of formula I or II is obtained.

150515 19 by removing the protective group. A wide variety of protecting groups known in the art for protecting amino groups during peptide synthesis can be used for this purpose. Groups which have proved particularly suitable are the benzyloxycarbonyl group, the use of which is described by Doyle et al. in the Journal of the Chemical Society (London), 1440 (1962), and the enamines formed by reaction of the starting amino acid with a β-dicarbonyl compound as disclosed by Dane and Dockner in Angewandte Chemie (International Edition in English) 3, 439 ( 1964) and in the Chemische Berichte der Deutschen Chemischen Gesellschaft, 98, 789 (1965). For the use of other protecting groups, reference may be made to Greenstein and Winitz, "Chemistry of the Amino Acids", John Wiley &

Sons, Inc., New lork / London, 1961, pages 882-922. In some cases, a particularly valuable acylation procedure consists in using the acid chloride hydrochloride of the acid precursor. The acid chloride hydrochlorides are prepared and the acylation is carried out by the methods described for the preparation of 2-amino-2-phenylacetyl chloride hydrochloride and the subsequent acylation of 6-aminopenieillanic acid in the disclosure of U.S. Patent No. 3,140 282nd

Method D consists in hydrolysis of the corresponding compound of formula II wherein R 1 is a group of formula -C (= O) -O-R 2 or 14 14 '.

SO2-R wherein R is alkyl of 1-6 carbon atoms, benzyl, phenyl or substituted phenyl having up to two substituents selected from nitro, fluoro, chloro, bromo, alkyl of 1-4 carbon atoms and alkoxy of 1-4 carbon atoms. The hydrolysis is usually carried out by contacting the starting material with an aqueous or partially aqueous solution system at a temperature usually in the range of about -5 to about 30 ° C, and preferably from about 10 to about 25 ° C, at a pH value in the range of from about 7.5 to about 9.5, and usually about 8.5 until the hydrolysis is substantially complete. The reaction usually takes about 1 hour to complete. It is usual, but not necessary, to use an auxiliary solvent in this process. Auxiliary solvents which may be used are those which are miscible with water and will serve to dissolve the starting compound. lypical examples of useful auxiliary solvents are acetone, lower alkanols such as methanol and ethanol, ethylene glycol, mono- and di (lower-alkyl) ethers of ethylene glycol such as 2-ethoxyethanol and 1,2-dimethoxyethane, tetrahydrofuran, dioxane and acetonitrile. The product is isolated by well known methods.

2 3

The starting penam compounds of formulas III and IT, wherein E, R and 5

Are all 'are hydrogen used in Method 0 can be prepared from the appropriate compound of formula III or an acid addition salt thereof, wherein Ir is hydrogen and R is (r4)' or -OH-τ / ΧνΊ r 2 °; ΉυΓ wherein (R 2) 'and (R 1') 'respectively represent hydrogen, hydroxy, fluoro, chloro, bromo, iodo, alkyl of 1-6 carbon atoms, alkoxy of 1-6 carbon atoms, alkanoyloxy of 2-7 carbon atoms, formyloxy, alkoxy-

JO

methoxy of 2-7 carbon atoms, phenyl or benzyloxy; R is hydrogen, alkyl of 1-4 carbon atoms or phenyl; and R 2 each independently means hydrogen or methyl; and X is oxygen or sulfur; wherein at least one of (R 1 -) 1 and (R 2) 'must be 2- or 4-hydroxy, 2- or 4-alkoxy of 1-6 carbon atoms, 2- or 4-alkanoyl-oxy of 2- 7 carbon atoms, 2- or 4-formyloxy, 2- or 4-alkoxy-methoxy with 2-7 carbon atoms or 2- or 4-benzyloxy when R is hydrogen; by treatment with trifluoroacetic acid. While this is not necessary, it is usually advantageous to add the reaction to an alkoxybenzene such as anisole, phenetol or veratrol. The reaction is conveniently carried out by dissolving the starting material in a small volume of fluoroacetic acid containing anisole, keeping the solution at a temperature in the range of about 20 to about 70 ° C, preferably at 30 - 40 ° C, for a suitable period of time and then precipitating the product by adding a non-solvent. The product can then be recovered by filtration.

21

Alternatively, it is sometimes appropriate, especially when working on a small scale, to stop the reaction by rapid evaporation of the trifluoroacetic acid at about room temperature in vacuo.

The amount of trifluoroacetic acid used in this process is not critical, provided there is enough present to effectively dissolve the starting material and is usually used from about 10 molar equivalents to about 100 molar equivalents calculated on the penam compound. Usually 1 molar equivalent of anisole is used, but larger amounts, even as large as 10 molar equivalents, can be added if desired. A starting material which is particularly effective in this process is a sulfonate salt, e.g. the methanesulfonate or p-toluenesulfonate of the compound of formula III. The reaction time varies depending on a number of factors, such as the reaction temperature, the structure of the starting material and the concentration of the solution. However, an appropriate mode of operation involves control of pilot reactions using magnetic nuclear resonance spectroscopy so that the time leading to the optimal conversion to product for a given set of reaction conditions can be determined. When operating at about 35 ° C, reaction times in the range of from about 0.1 to about 1.5 hours are usually used.

2 3

The starting penam compounds of formulas III and IV wherein R, R and R 2 are all hydrogen used in Method 0 may also be obtained by hydrogenolysis of the appropriate compound of formula III or an acid addition salt thereof wherein R 1 is hydrogen and R is 4. 17 wherein R and R 1 are each independently hydrogen, hydroxy, nitro, fluoro, chloro, bromo, iodo, alkyl of 1-6 carbon atoms, alkoxy of 1-6 carbon atoms, alkanoyloxy of 2-7 carbon atoms, phenyl or benzyloxy The same technique and conditions described earlier under Method A are applicable to this process.

22

Another one. process for preparing the starting materials of formulas III and IV, wherein R 2, R 2 and R 2 are all hydrogen, are removed from the protective triphenylmethyl group from a precursor of formula III wherein R is triphenylmethyl and R is hydrogen . Pure protective triphenylmethyl group is removed by treating the compound of formula III with acid, and a variety of acidic reagents and conditions known in the art for removing the triphenylmethyl group can be used in this process. For example, it is possible to use a sulfonic acid such as methanesulfonic acid, benzenesulfonic acid or p-toluenesulfonic acid; anhydrous hydrogen halide acid such as hydrogen chloride or hydrogen bromide; or an alkanoic acid such as acetic acid, propionic acid, chloroacetic acid, trifluoroacetic acid and the like. The reaction is usually carried out by dissolving the starting material in a suitable solvent and adding about two molar equivalents of the acidic reagent at about room temperature. The reaction is completed within approx. 1 hour and the product is present in the reaction medium in the form of the acid addition salt corresponding to the acidic reagent used. Rer must be selected a solvent which will dissolve the starting penam compound, and examples of useful solvents are: ethers such as diethyl ether, tetrahydrofuran, dioxane and 1,2-dimethoxyethane; chlorinated hydrocarbons such as chloroform, methylene chloride and 1,2-dichloroethane; lower aliphatic ketones such as acetone, methyl ethyl ketone and methyl isobutyl ketone; esters such as ethyl acetate and butyl acetate; hydrocarbons such as hexane, cyclohexane and benzene; and lower alkanols such as methanol, ethanol and butanol. Although it is common to use about two molar equivalents of acid in this process, only one molar equivalent is necessary when either reaction; is administered in the presence of a molar equivalent of water or the acid is introduced as a monohydrate. However, it will be appreciated by one skilled in the art that the product of this reaction should not be exposed to an excess of acid for prolonged periods, as in this case there is a danger of destroying the β-lactam system. A particularly convenient method of operation in this process is to choose such an acid-solvent system that the starting material is soluble, but the acid addition salt formed during the reaction precipitates as it is formed. Yield can then be recovered by filtration after completion of the reaction. When the combination of p-toluenesulfonic acid in acetone is used, the p-toluenesulfonate of the product is precipitated frequently.

Similarly, the starting materials of formula III, C π -ia 14 in which Ir is hydrogen and R * 1 are -C (= O) -O-R, -SOO-R and (R) ', wherein 14 R R means alkyl of 1-6 carbon atoms, benzyl, phenyl and substituted phenyl having up to two substituents selected from nitro, fluoro, chloro, bromo, alkyl of 1-4 carbon atoms and alkoxy of 1-4

C

carbon atoms; and (R) 'is or -oh- / xv- R20; wherein R71 and R ^ are each hydrogen, hydroxy, nitrofluoro, chloro, bromo, iodo, alkyl of 1-6 carbon atoms, alkoxy of 1 -6 carbon atoms, alkanoyloxy of 2-7 carbon atoms, formyloxy, alkoxymethoxy 18 of 2-7 carbon atoms, phenyl or benzyloxy, R is hydrogen, alkyl of 1-4 carbon atoms or phenyl; R and R are each hydrogen or methyl and X is oxygen or sulfur; from the corresponding compound wherein R 1 is triphenylmethyl. The triphenyl methyl group is removed by treatment with acid exactly as described above.

The starting materials of formula III wherein R is triphenylmethyl and 2R are hydrogen are prepared by a reverse Michael reaction to a 2 corresponding compound wherein R is -CHgCHgY wherein Y is cyano, alkoxycarbonyl of 2-7 carbon atoms, phenoxycarbonyl, alkylsulfo 15 16 nyl having 1-4 carbon atoms, phenylsulfonyl or SO 9-NR 2 R, wherein 15 16

Rog R individually means hydrogen, alkyl of 1-4 carbon atoms, phenyl or benzyl. The reverse Michael reaction consists in treating the compound with about an equivalent base under conditions known in the art of reverse Michael reactions but compatible with the pen embryo system. lypically, the compound of formula IV is treated with about one equivalent of a relatively non-nucleophilic base in a nonhydroxylic solvent at a temperature in the range of about 0 to about 25 ° C for a period of from about 10 minutes to about two hours.

(See further Journal of the Chemical Society [London], Part B, 5867 [1970].

Preparation of the starting materials for method C which are joined 5 2 3

compounds of formulas III and IT in which R is hydrogen and R and R

each is alkanoyloxymethyl of 3-8 carbon atoms, 1- (alkanoyloxy) ethyl of 4-9 carbon atoms or phthalidyl is obtained by alkylating a tetrazolate salt such as the triethylamine salt of the corresponding compound of formula III or 17, wherein R and R are both hydrogen, using the appropriate alkanoyloxyalkyl or phthalidyl halide. The method of Method D is used in this method except that it is common to use at least two molar equivalents, and preferably about three molar equivalents, of the alkylating agent.

The final starting materials for the preparation of the antibacterial compounds of the invention are the novel compound compounds of formula III wherein R is an amino protecting group 2, e.g. triphenylmethyl, and R is a tetrazolylpenam protecting group, e.g. selected from -0Ηο0Η9Υ, -C (= 0) -0-R1 ^ and (R) *, wherein Y, R * and (R) 'have the meaning previously defined. The compounds can be prepared by a novel three-step reaction sequence described in Danish Patent Application No. 2690/77.

As previously stated, the synthesis of the antibacterial agents of the invention involves the use of two kinds of protective groups, and these groups have been identified for their ability to function in a particular way.

The tetrazolylpenam nitrogen protecting group has been identified for its ability to fulfill two functions. The first of these, its ability to be removed under certain specified conditions, has already been discussed. The second of these is its ability to allow the synthesis of a compound of formula III, wherein R is an amino protecting group and R is said tetrazolylpenam nitrogen protecting group.

From the foregoing, it will be clear that the tetrazolyl-penam nitrogen protecting group must be a group which will allow for the execution of the above-described three-step reaction sequence.

That is, it must be such that the amide of formula VI can be prepared, that the amide can be converted to an imidoyl halide and that the imidoyl halide can be converted to the said 1-protected 5-tetrazolylpenam compound of formula IV, in essentially as described.

Similarly, the amino protecting group must fulfill two functions. The first of these is that, following association with the 6-amino function of 6-aminopenicillanic acid, it must allow the above-described three-step reaction sequence to be carried out. That is, it must protect the pen embryo system during the formation of the amide of formula VI, during the conversion to an imidoyl halide and during the conversion to the 1-protected 5-tetrazolylpenam compounds of formula III. The other function of the amino protecting group in the process of the invention is that it must be removable under conditions which do not degrade the pen numbering system, from: (a) a compound of formula III wherein Ir is said amino-protecting group; and R is a tetrazolylpenam nitrogen protecting group; (b) a compound of formula III wherein said amino protecting group is and R is hydrogen; or (c) a compound of formula III wherein R 1 is alkanoyloxymethyl, 1- (alkanoyl-oxy) ethyl or phthalidyl.

Selection of appropriate amino protecting groups will be readily appreciated by one skilled in the art. In particular, all such groups known in the art of peptide synthesis are useful. However, particularly suitable protecting groups are triphenylmethyl, substituted triphenylmethyl and β, β, β-trihaloethoxycarbonyl such as β, β, β-tribromethoxycarbonyl and β, β, β-trichloroethoxycarbonyl. Examples of substituted triphenylmethyl groups which are particularly valuable are those of the formula: wherein R22, R25 and R24 each represent hydrogen, fluoro, chloro, hromo, alkyl of 1-4 carbon atoms, alkoxy of 1-4 carbon atoms or phenyl. Due to its easy accessibility, the triphenylmethyl group is particularly valuable.

A characteristic feature of the compounds of formula I and those of formula II and IV wherein R and R are hydrogen is their ability to form salts. Due to the acidic nature of a 5-monosubstituted tetrazole, said compounds have the ability to form salts with basic agents, and these salts, commonly referred to as "tetrazolate" salts in this specification, should be considered to be within the scope of the invention. scope of the invention. The salts can be prepared by standard techniques such as contacting the acidic and basic components, usually in the 1: 1 mole ratio, in an aqueous, non-aqueous or partially 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. The basic agents suitable for salt formation belong to both the organic and inorganic types, and they include ammonia, organic amines and alkali metal hydroxides, carbonates, hydrogen carbonates, hydrides and alkoxides, as well as alkaline earth metal hydroxides, carbonates, hydrides and alkoxides. Representative examples of such bases are primary amines such as n-propylamine, n-butylamine, aniline, cyclohexylamine, benzylamine, p-toluidine and octylamine secondary amines such as diethylamine, N-methylaniline, morpholine, pyrrolidine and piperidine; tertiary amines such as triethylamine, N, N-dimethylaniline, N-ethylpiperidine, N-methylmorpholine and 1,5-diazabicyclo [4,3,0] non-5-ene; hydroxides, 27 tsosis such as sodium hydroxide, potassium hydroxide, ammonium hydroxide and barium hydroxide; alkoxides such as sodium ethoxide and potassium ethoxide; hydrides such as calcium hydride and sodium hydride; carbonates such as potassium carbonate and sodium carbonate; and hydrogen carbonates, such as sodium bicarbonate and potassium hydrogen carbonate.

The compounds of formulas I, III and IV which contain a basic group have the ability to form acid addition salts. These acid addition salts are also to be considered within the scope of the invention. Examples of acid addition salts which are particularly valuable are: hydrochlorides, hydrobromides, phosphates, perchlorates, citrates, tartrates, pamoates, glutarates, benzoates, sulfates, lactates and arylsulfonates.

Of course, when contemplating therapeutic use in mammals of a salt of a compound of the invention, it is necessary to use a pharmaceutically acceptable salt. However, other salts are useful for a variety of other purposes, e.g. isolation and purification of individual compounds, alteration of the solubility properties of an individual compound, and mutual conversion of pharmaceutically acceptable salts and their non-salt forms.

The antibacterial penam compounds of the invention exhibit activity over a wide variety of Gram-positive and Gram-negative bacteria. In vitro activity can be demonstrated by the conventional double-row dilution technique in the cardiac-heart infusion broth (Difco). The broth is seeded with the bacterial culture and with the sample biotic and then incubated overnight. The next day the test is visually read. The minimum inhibitory concentration (MIC) is the lowest concentration of antibiotic which prevents turbidity, ie. which prevents growth of the microorganism.

Minimum inhibitory concentrations (MIC) in yag / ml were determined for compounds A - F of the invention and ampicillin gains the use of a standard dilution method in brain-cardiac infusion agar and that of Steers et al. described multiple-time inoculator (Steers et al., Antibiotics and Chemotherapy, 2, p. 307 (1959); Ericsson et al., Acta Pathol. Microbiol.

Scand., Sect. B, 217 (Suppl.), Page 64 (1971)). The standard graft material contained about 20,000 organisms. The incubation occurred for 18 h at 37 ° C.

The following Table I lists the results of a broad first in vitro assay for a variety of microorganisms, and the following Table II lists the results of a test against various strains of Klebsiella pneumoniae, a common widespread pathogenic Gram-negative bacterium .

Compounds examined: “3 S-CB-CO-SH. _ S / <Β3

J- * —L

°. > # A: R7 is / ~ \

Bs R7 is H2N · - ^ "" "O-. C B: R7 is

N S

E: r7 is H0-cix F: R7 is' HO -o- CH3

0 X C00H

Ampicillin 29

1505 1S

ti • no

H

Η 1Λ VD U3 C \ l u

• Η Η H CM in an H CV

• H OOVQHrinOOOOOOOOOOintn

p, V 'OOOinOOOOOOCMCM

s CM CM CM CM CM H CM Η H

, · Λ Λ Λ Λ oo cn <η cm m in mm

IS Ν '* fO H CM in CM CM CM

**** · »*» «* ft ft ft fc OOmOfOVOCMOOOOOVOOinOVOVO.

CM ΗΟΟΙΠΟΟ O CM o

CM CM CM CM CM H

_Λ_A_

O O vo CM CM CM in CM CM

cm cm in m η h in h cm in η h ft fs (\ · »* k O ft ** *« k H ooHOfnmcMominoofnococMmtn

Η O CM CM O O O H

CM H CM CM

__Λ_Λ_

VO VO VO CM CM

^ m in in rH rH m fjn i — i λ ^ λ λ Λ · »ti a fi HHinHmfninooooocMOOomin

• Η \ CM CMOOOOOHOOOCMCM

d bO CMHHHCMHiHr- (

> SL Λ Λ Λ Λ A

a ο-- ρ ο σν cm cm vo in in p, + cm cm m fn η h in cm cm 03 IV fv A · «η M ft ft ft +5 h o ooojomtnoooooooor-iovovo bo g H ooooooino o

• r | HCMHHCMH CM H

ω A A A - --- ω cn in go in in incMinin

> ri IO CM S ΙΊ CM CM, H CM CM

M ° pq oovoovovoinooooovoofcsinvovo

HO CMOOinOO OCM

HP CM Η H CM CM

pq -P A Λ A

<ς · Η ------------------ - EH> cn CO CTi 00 h to in is irv ιλ m in ts-m

• H <J OOCMOCMCMOOOOOOOOCMmOCM

\ j / H HHinOinOOO Or-1 CM H

φ v / CM CM CM CM CM

-P A A A

as' P ______—- <s>

H

Φ incMOvoinvocMov4KvovinovsovHic \ H

a · OinOOHvOOCMOISCrvOOlSISOHO

g P 00 <t0CMCM0HHHin00'0 0000 old «aj <iJ <j <jJ« a! «aJ <i! <iJ« aJ <i; <! pq <J <J <!! 00 <a! • P HHHCMHHHHCMCMCMCMinmtniS-OOrn w ooooininininininininininininvo

m cd cd cd S

cqcqcq-h ω ω ω φ φ φ p pppH ooo cdcdcd ή cd <d α> co a SS · η · η · η ρω ρρρο -H-ri-Hcdddd pa p Ρ Ρ Φ bO b0 bO Ό Ο S O Sd) cd cd cd Cd · Η -Η · Η · Η P pp · Η S ia · Η o 'ηΗγΗΗΗ PP Ρ -PPP 3 Ή d ω cd ωωω οοοοφφφρφφφρΡιΦ a ppprooooocdcdcdP) dP5iHffl >> o ωο η οοοοοΰιΰΦΐΰωωωω p cd d οοοο-Ητ-1-H-Hcdcdcdcdcdcdcdocda cd bo OOOOOOOOOOOOr — 1HH H Cd ρ ΗΗΗθ · Η · Ρ · Ρ · Η888βΦ3> ωωω · Η> +3 o iji | Baii) (i) ii) ii), d, d, iij »iB» ii) Oci Ρ ρ, ρ, ρ, ΦΠΛΛΛ ^ ΡΡΡ, Ω, Ω, Ω-ΡθΡ, ϋ) ΦΦΦΡΟΟΟΟΦΦΦΦΦΦΦΟΗΡ • η · ρ · Ρ · ρ · ΡωωωωωωωωΗΗΗΡΦΦ g cocococoHWHHPHpHpHPuHWWfccoco 150515 30 fl • Η Η Η • Η ΙΛ ΙΛ Ο * λ • Η (Μ ΙΑ ΙΓ \ CM Ο ft Η CM CM Η Ο S Η S , VO CM CM ΙΑ Ο Η Η CM Ο%%%% CM CM CM CM ΙΑ ΙΑ Ο Η Ο CM CM Ο Η - - - "~ 3- Ω ΙΑ ΙΑ ΙΑ ΙΑ Ο 4- CM CM CM CM Ο Ο Η Η s-- ΙΑ ΙΑ tm • tm, Ο CM CM ΙΑ CM Ο Η H CM Η ΙΑ -Ρ d - ω W ΙΑ Ti .ρ ΙΑ ΙΑ ΙΑ CM Η C - * «·« ο κ £ 0 Ο fQ CM CM Od VD Ο <Ο Η Η Η Η ιΗ Η · Η __- Ρ Η cm ω Ω ΙΑ ΙΑ ΙΑ Λ Λ »> I — j CD c CM CM CM ΚΛ ΙΑ d

<| Η H i — l CM

£ 3 -H

_____ I A

ISLAND

H

0 <i- σν fA is σ »p g. O O O O O d a? H ooooo> vs ΰ c <jrøm <;

-P N N IS O N S

m co vo vo is- ο- ω

_ bD

--j-j CQ M -rl 0 0 t3 s s o g 0 0 d · Η> »

faO b £> ο -Η -P

ο O d tJ P

2 Ρ o 2 o

0 0 H 0 'H

0 d d ο ρ 0

S * H H

a fi ft fi d

• H 0 0 0 p d -H

fl -P -P -P 0 -Η P

d OOO -PO 0 bo dddos -pp ο ο od 0 doooo, q tJ ao UUO -H 0 P 0 0 0 Sh> Tj M -P +> +> -P on rl fl GS -rl P. 04 smw η o 04 *

TABLE II

31515 31

In vitro Activity over Klebsiella pneumoniae Strain I MIC * No. * A Ampicillin Trihydrate 53A009 6.25 25 53A015 3.12 50 53A022 6.25 50 53A021 12.5 50 53A028 6.25 25 53A042 6.25 100 53A044 3 , 12 1.56 53A047 6.25 50 53A056 12.5 50 • 53A063 25> 200 53A064 3.12 25 53A067> 200> 200 53A068 6.25 50 53A069 12.5> 200 53A076 12.5 50 53A077> 200> 200 53A079 25 50 53A082 25 100 Φ -2

The inoculation dilution was 10 in all cases. ττ continued

Tribe | MC * frg / Bl)

nr 'B C D E F G

53A015 12.5 12.5 6.25 12.5 6.25 50 53A028 12.5 25 25 12.5 6.25 50 53A068 25 25 25 12.5 6.25 50 53A069 100 50 25 12.5 12, 5> 200 53A070> 200> 200> 200> 200> 200> 200 53A72 6.25 12.5 12.5 25 6.25 50 53A073 25 50 50 12.5 25 50 53A075 100> 200> 200 200 100 100 53A076 25 50 100 12.5 25 25 53A078 6.25 12.5 100 50 6.25 50 53A080 50 50 50 12.5 25 100 53A081 25 50 50 6.25 25 50 53A086 12.5 50 50 6.25 25 50 53A088> 200> 200> 200> 200> 200> 200 53A089 25 25 25 25 12.5 50 53A090 6.25 3.12 3.12 6.25 3.12 25 53A091 25 50 12.5 6.25 25 50 53A093 6.25 25 25 3.12 3.12 50 * -2

The inoculation dilution was 10 in all cases

Table I shows that all the compounds of the invention have substantially the same broad spectrum of activity and that they have useful activity against all the microorganisms against which ampicillin has useful activity. Table XI shows that the compounds of the invention have substantially greater activity against a large number of strains of Klebsiella pneumoniae than ampicillin. This is an important benefit as Klebsiella pneumoniae often occurs in mixed infections with other Gram-negative organisms, such as Proteus, Pseudomonas and Enterobacter, especially in urinary tract infections. Mixed infections are a common problem in clinical practice today, and the use of an anti-biotic against which one of the organisms is resistant leads to an even greater problem, such as over-infection. Therefore, it is an important technical advance that the compounds of the invention, in addition to having the same broad-spectrum activity as ampicillin, have significantly greater activity against a troublesome microorganism such as Klebsiella pneumoniae.

The in vitro activity of the antibacterial compounds of the invention makes them particularly suitable for topical application, e.g. in the form of creams and ointments.

The antibacterial penam compounds prepared according to the invention are also active in vivo. To determine this activity, the sample antibiotic is administered to infected mice using a multiple dose regimen. The severity of the infection ranges from about 1 to 40 times that required to kill 10090 of the mice under the conditions of the test. At the end of the test, the activity of a compound is judged by counting the number of survivors among the exposed animals. Both the subcutaneous (sc) and oral (po) dosing route are used. Results are listed in Table III for two compounds of the invention. The ability of the compounds to protect mice from systemic infections caused by a lethal intraperitoneal inoculation of Staphylococcus aureus or of Escherichia coli is indicated.

TABLE III

34 1S0515

Dosage Dose- * protection

Formation (mg / kg) of ring pathway S. aureus E.coli 6- (D-2-amino-2- [5-thienyl] -acetamido) -2,2-dimethyl-3- (5-tetrazazolyl) -penam 50 sc 40 20 "25 sc 60 20» 12 sc 50 "6 sc 50" 200 po 0 6- (D-2-amino-2- [p-hydrosyphenyl] acetamido) -2,2-dimethyl-3 - (5-tetrazolyl) -penam 50 sc 80 100 "25 sc 70 80" 12 sc 50 "6 sc 50" 200 po 100

The in vivo activity of the anti-bacterial compounds of the invention makes them suitable for controlling bacterial infections in mammals, including humans, in both the oral and parenteral modes of administration. The compounds will find widespread use in controlling infections caused by sensitive Gram-positive and Gram-negative bacteria in humans.

For therapeutic use of a compound made according to the invention, or a salt thereof in a mammal, especially the human, the compound can be administered alone or it can be mixed with other antibiotic agents and / or pharmaceutically acceptable carriers or diluents. The carrier or diluent is selected on the basis of the intended mode of administration. For example, the penam compound by the oral route may be administered in the form of tablets, capsules, lozenges, trocans, powders, syrups, elixirs, aqueous solutions and suspensions and the like in accordance with standard pharmaceutical practice.

The amount of active ingredient-carrier ratio will, of course, depend on the chemical nature, solubility and stability of the active ingredient, as well as the intended dosage. In the case of tablets for oral use, commonly used carriers include lactose, sodium citrate and salts of phosphoric acid. Various degradation agents such as starch and lubricants such as magnesium stearate, sodium lauryl sulfate and talcum are commonly used in tablets. For oral administration in capsule form, useful diluents 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. If desired, certain sweetening and / or flavoring agents may be added. For parenteral administration which includes intramuscular, intraperitoneal, subcutaneous and intravenous use, sterile solutions of the active ingredient are usually prepared and the pH of the solutions is adjusted and appropriately buffered. For intravenous use, the total concentration of solutes should be controlled so that the preparation is isotonic.

As previously stated, the antibacterial penam compounds prepared according to the invention can be used in humans, and the daily dosages to be used will not differ significantly from other clinically used penam antibiotics. The physician will ultimately determine the appropriate dose for a given individual, and this may be expected to vary according to the individual patient's age, weight and reaction, as well as the nature and severity of the patient's symptoms. The compounds of the invention will normally be used orally at doses of from about 10 to about 200 mg / kg body weight per day. per day and parenterally in doses of about 5 to about 100 mg / kg body weight per day. day. These figures, however, are merely illustrative, and in some cases may be necessary; -to use dosages outside these limits.

Certain of the compounds of the invention have the ability to form solvates (eg hydrates) and all such hydrates are to be considered within the scope of the invention.

The following examples are provided for further illumination purposes only. Infrared (IR) spectra are measured as potassium bromide slices (EBr slices) or as Nujol slurries, and diagnostic absorption bands are reported in wave numbers (cm). Magnetic nuclear resonance spectra (MR) are measured at 60 MHz for solutions in deuterochloroform (ODCl 2), perdeuterodimethylsulfoxide (DMSO-dg) or deuterium oxide (D20), and peak positions are expressed in parts per minute. million (ppm) down from tetramethylsilane or sodium 2,2-dimethyl-2-silapentane-5-sulfonate. The following abbreviations for tip forms are used: s, singlet; d, duhiet; t, triplet; g, quartet; m, multiplied.

EXAMPLE 1 6- (1β-2-Amino-2-pyrenylacetamido) -2,2-dimethyl-3- (5-tetrazolyl) penam

To a stirred solution of 23.8 ml of ethyl chloroformate in 600 ml of acetone is added 25 ml of a 3 $ solution of N-methylmorphiline in acetone. The resulting solution is cooled to -40 ° C and then 75.2 g of sodium N- (2-methoxycarhonyl-1-methylvinyl) -D-2-amino-2-phenylacetate is added. The temperature is set to -20 ° C and stirring is continued for 28 minutes. The solution is again cooled to -40 ° C and an ice-cold solution prepared by suspending 60.0 g of 6-amino-2,2-dimethyl-3- (5-tetrazolyl) penam in 250 ml of water and then adjusting the pH value to 7.0. The resulting solution is stirred for 30 minutes without further cooling, and then the acetone is removed by evaporation in vacuo. To the aqueous residue is added an equal volume of tetrahydrofuran and then the pH is adjusted at 5 ° C to 1.5 with dilute hydrochloric acid. The mixture is kept at this temperature and pH for 30 minutes and then the tetrahydro uranium is removed by evaporation in vacuum.

The aqueous residue is extracted with ethyl acetate followed by ether and the extracts are discarded. The pH of the remaining aqueous phase is raised to 5.4 and the product begins to crystallize. After 1 hour, filter off and dry. The crude yield is 68.8 g.

The product is suspended in water at 25 ° and the pH is lowered to 1.5. After stirring for a short time, the insoluble material is filtered off and the filtrate is extracted with ether. The aqueous solution is then cooled to 5 ° C and the pH adjusted to 5.2.

The precipitated solid is filtered off to give 62.7 g (58.7 $ yield) of 6- (D-2-amino-2-phenylacetamido) -2,2-dimethyl-3- (5-tetrazolyl) penam trihydrate, m.p. 201-202 ° C; [.alpha.] D @ 20 + 228.2 (1io in CH2 OH). IR (KBr disk): 1780 cm -1 (β-lactam). NMR (in DMSO-dg / DgO); 7.60 ppm (s, 5H), 5.70 ppm (d, 1H), 5.55 ppm (d, 1H), 5.20 ppm (s, 1H), 5.15 ppm (d, 1H), 1.50 ppm (s, 3H), 0.90 ppm (s, 3H).

Analysis "calculated for Ο ^ Η ^ Ο ^^ ε ^ Ε ^ Ο (¢): 0 44.95 - H 5.89 - N 22.94 - S 7.50 Round: C 45.01 - H 5.84 - N 22.81 - S 7.34.

Sodium N- (2-methoxycarbonyl-1-methylvinyl) -P-2-amino-2-phenyl-1-acetate is prepared from methylacetoacetate and D-2-amino-2-phenylacetic acid by the procedure which is used by Long et al. (J.

Chem. Soc., London, Part C, 1920 [1971]) for the corresponding β-hydroxy compound.

The MIC of the compound prepared against a strain of Streptococcus pyogenes is <0.1 µg / ml.

The compounds of the following table are prepared by coupling 6-amino-2,2-dimethyl-3- (5-tetrazolyl) penam with the appropriate reagent according to the above procedure. Also, minimal inhibitory concentrations (MICs) of the compounds against a strain of Streptococcus pyogenes are indicated. The structure of the products is confirmed by magnetic nuclear resonance spectroscopy.

150515 38 τ CH3 R -NEL_ .SX / dH3 oJ-l — vs,

\ = N *

IT

ΤΤΛ, Ι ++ 0 IR MIC

r1_ud ^ te <™ -h W *) Footnote D 3-H0C6H4-CH-C0- 5 1776, 1686 <0.1 1, 3 nh2 L 4-H0CgH4-CH-C0- 7 <0.1 3 nh2 P j J 28 1770 0.1 3 ^ CH-CO- nh2 p 3-Cl-4-H0C6H3-CH-C0- 44 1783 0.1 1, 3 NH2 DL 4-ClC6H4-CH-C0- 7 1785, 1695 <0 , 1 1, 3, 4 nh2 DL 3-ClC6H4-CH-CO- 14 1780, 1700 0.79 1, 3, 4 nh2 □ —CH-CO- ^ 38 1770, 1680 <0.1. 2, 3p 4-NH2C6H4-CH-CO 4 1770 0.039 1, 3 nh2 p 3-NH2C6H4-CH-CO 13 1375, 1650 <0.01 3, 5 nh2, Ω 150515 39

Footnotes 1. Isobutyl chloroformate is used to form mixed anhydride.

2. The MIC value of this compound is measured against a strain of Staphylococcus aureus.

3 The starting enamines are prepared by condensing the appropriate glycine with methyl acetoacetate according to the procedure described by long et al. (Journal of the Chemical Society [London], Part C, 1920 [1971]). These α-amino acids, described in the literature, are prepared by the published procedures. The novel α-amino acids are prepared from the above aldehydes via a Strecker synthesis, for which the technique is discussed by Greenstein and Winitz in "Chemistry of the Amino Acids," John Wiley & Sons, Inc., New York / London, 1961 , pages 698-700 and the references therein. The strecker synthesis produces DL amino acids which are separated into their optical isomers by conventional means (see Greenstein and Winitz, loc. Cit., Pages 715-755; Nishimura, et al., Nippon Kagaku Zasshi, 82, 1688 [1961] [Chemical Abstracts, 58, 11464 (1963)]; and Belgian Patent No. 795,874). See also British Patent No. 1,221,227. 5- (3-Pyridyl) hydantoin is prepared by the method described by Henze and Knowles, J. Org. Chem., IJJ, 1127 (1954), and it is hydrolyzed to 2-amino-2- (3-pyridyl) acetic acid by the method described by Davis et al. (Archives Biochem. And Biophys. 87,88 [i960]) for the corresponding 4-isomer.

4. The compound is isolated as its triethylamine salt.

5. The amino groups in the starting material are protected by benzyloxy-carbonyl groups during coupling and they are removed by hydrogenolysis after coupling.

EXAMPLE 2 2- (D-2-Amino-2- [4-hydroxyphenyl] acetamido) -2,2-dimethyl-3- (5-te-azolol) -1amine

Strain a stirred solution of 0.19 ml of ethyl chloroformate in 15 ml of dry acetone, cooled to 0 ° C, add 1 drop of N-methylmorpholine followed by 576 mg of sodium N- (2-methoxycarbonyl-1-methylvinyl) -D-2- amino-2- (4-hydroxyphenyl) acetate (Long et al., Journal of the Chemical Society [London], Part C, 1920 [1971]). The mixture is stirred for a further 30 minutes and then cooled to about -35 ° C. Then an ice-cold solution of the sodium salt of 6-amino-2,2-dimethyl-3- (5-tetrazolyl) penam is added, prepared by adding 10 $ sodium hydroxide solution. to a suspension of 436 mg of 6-amino-2,2-dimethyl-3- (5-tetrazolyl) penam in 5 ml of water (to obtain a pH of 7.8), followed by dilution with 25 ml of acetone. The cold bath is removed and the reaction product is further stirred for 30 minutes. At this point, the acetone is removed by evaporation under reduced pressure and then 20 ml of methyl isobutyl ketone is added to the aqueous residue. The iDofase system is cooled to 10 ° C, acidified to pH 0.9 with dilute hydrochloric acid and then stirred at 10 ° C for 1 hour. The methyl isobutyl ketone is removed and thrown away. The pH of the aqueous phase is raised to 6.6 and then stored in the refrigerator for 3 hours. The precipitate formed is filtered off to give 320 mg of 6- (D-2-amino-2- [4-hydroxyphenyl] acetamido-2,2-dimethyl-3- (5-tetrazolyl) penam. Infrared spectrum (KBr disc) ) shows absorptions at 1775 cm (β-lactam carbonyl) and 1680 cm cm (amide I band). The NMR spectrum (in DMSO-d , aromatic hydrogen atoms), 5.60 ppm (quartet, C-5 and 0-6 hydrogen atoms), 5.10 ppm (multiplet, benzyl hydrogen atom and C-3 hydrogen atom), 1.45 ppm (singlet, C-2 methyl hydrogen atoms) and 0.95 ppm (singlet, C-2 methyl hydrogen atoms).

The MIC of the above compound against a strain of Streptococcus pyogenes is <0.1 µg / ml.

Claims (2)

150515 o p a_t_e_n_t_k_r_a_v__: 1. Analogous Process for Preparation of 6- (2-Amino-2-aryl-acetamido) -2,2-Dimethyl-3- (5-tetrazolyl) Penam Compounds of the General Formula 7-ch-c-nh ch NB , NS '"" fsT 2. CH3! J-N- o C ΊΪ \ I HN-N wherein R7 means 2-thienyl, 3-thienyl or (R7), ^ y · (R7) "^ K = J where (R7)" and (R7) "each hydrogen, hydroxy, chloro or amino, or pharmaceutically acceptable salts thereof, characterized in that a penam compound of the formula RX-NH \ CH3 f ^ CH3 \ II HN-N wherein R1 represents hydrogen or a trialkylsilyl or dialkyl chlorosilyl group, or a tetrazolyl protected derivative thereof, is reacted with an acylating agent to introduce the group R7-CH-cI u nh2 o