CS231157B2 - Processing method of klavulan acid and corresponding salts and esters - Google Patents

Abstract

1508977 Antibiotic compositions containing clavulanic add BEECHAM GROUP Ltd 11 April 1975 17410/74 27715/74 43651/74 and 53525/74 Heading A5B [Also in Division C2] Pharmaceutical compositions having antibacterial activity comprise, as active ingredient clavulanic acid of the formula or a pharceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier and optionally other therapeutically active agents such as #-lactam antibiotics, e.g. benzylpenicillin, phenoxymethylpenicillin, carbenicillin, methicillin, propioillin, ampicillin, amoxycillin, epicillin, ticarcillin, cyclacillin, 6- aminopenicillanic acid, 7 - aminocephalosporanic acid, 7 - aminodesacetoxycephalosporanic acid, cephaloridine, cephalothin, cephazolin, cephalexin, cefoxitin, cephacetrile, cephamandole, cephapirin, cephradine, cephaloglycine, hetaoillin, metampicillin, the acetoxymethyl, pivaloyloxymethyl or phthalidyl esters of benzylpenicillin, ampicillin, amoxyoillin or cephaloglycine or the phenyl, tolyl or indanyl α-esters of carbenicillin or ticarcillin. They may be administered orally, topically or parenterally e.g. as tablets, capsules, creams, syrups, suspensions, solutions, reconstitutable powders and sterile forms suitable for injection or infusion.

Description

(54) A method for producing clavulanic acid, corresponding salts and esters

A process for the preparation of clavulanic acid, the corresponding salts and esters of formula (I)

(I) characterized in that the strain of Streptomyces clavuligerua is cultivated in a nutrient medium containing nitrogen and carbon sources, as well as containing the necessary salt multiplication, aerobically at a temperature of 15 ° C. to 40 ° C. usually 20 to 35 ° C; and. preferably 25 to 30 ° C and at a pH of from 5 to 8.5, preferably 6 to 7.5, after which it is isolated from the culture of klevulanic acid or its salt after cell filtration by extracting the filtrate into the solvent after treatment. pH into the acidic region, or by absorbing the product on charcoal, by precipitation, salting-out, or by molecular filtration, whereupon optionally the acid. ktavulanic acid or its salt is converted by esterification of diezoalkme of 1 to 12 carbon atoms or by an alcohol of the formula

R-OH and optionally an ester of a compound of formula I wherein R is an alcohol residue is hydrolyzed.

The present invention relates to a process for the production of clavulenic acid, corresponding to the ester salts, namely the novel Mtibatterlálox acting agent, which has been isolated from Streptomyces clavuligerus. This new compound, which was named clavulaoic acid, has structural formulas I

ČOOH

(AND)

In addition to the broad spectrum and antibiotic activity of moderate moohunoose, clavulanic acid, clavulanic acid, and its salts and esters, also have the ability to enhance the effectiveness of beta-lactam antibiotics against numerous beta-lacaamase producing bacteria.

Strepoomyces claculigerus describes in detail Hggana * spii, in Int. J. System. Bscteriol. £ 21,326. (1971). This streptomyces is interesting because it produces some beta-laeamic antibiotics such as penicillin N, 7- (5- <mino-5-carboxyvaleramido) -3-carbamoyloxymethyl-3-cephem-4-carboxylic acid and 7- (5 " (Mino-5-carboxyvaleramide) -ZC-carbonyloxyethyl-4-methoxy-4-carboxylic acid. Said strepoomycetes are deposited in the Reseerch Service Collection under the designation NRRL 3585, and in the Americen Type Culture Collection under the designation ATXX 27064. Streptomyces tlaviligerus is also disclosed in U.S. Patent 3,770,590, and is referred to as Negacejan aoo. J. Lamp. Soc. 23, 2308 (1971); Branoon and spoo. Antimicrob. Agent Chhemohhe., J, 237 (1972), and £ 247 (1972), dULe H. ggeos and together J.Aibbibbler £ 2, 298 (1974).

Beta-lacbebs are the enzymes that open the beta-acam ring of penicillins and cefbltsporiums to produce products free of alpha-bacterial efficacy. These enzymes are produced in numerous microorganisms, particularly in strains or species of KetacHa, Proteus,

Paeudteono8, Staphyltottol, and in many cases the resistance of some organisms or strains to some penicilli or cephalosporos can be explained. Dllžžtost give betb-lakbmbsy can PoC ^ i ^ oo ^ pit melts before ^ ^ ^ i ei _and i, Ge high proportion of clinically isolated microorganism produces a beta-lakaimasy, see for example M. Wilson IA Pre-Mao BbCeritltа! ^! Proceedings 196 * 9, p. 80. Ibid., In an article entitled PenOiHlio ^ ь ^ -уь ^ о by Gammemnoegbive Bacili, it has been shown that 84% of gram-non-inert microorganisms isolated in aeric nemo-organisms produce betb-lakbaeas1.

In many cases, some penicillins and tefblt8porioy prove to be ineffective! in the treatment of oemoocs attributed to and debited by non-beta-labbaebs-producing eicrttrganies due to co-infections by action. betb-lacbembs1, see, for example, K. May et al., Urit. J. Dis. Cheet. 66, 185 (1972). Co-betaine lactamase-inhibiting agents with penicillin or cefblosptamine may, as expected, lead to the protection of the latter from the breakdown of bacterial beterlactma, thereby enhancing their antibacterial efficacy for 1 infectious trgaoemia.

This method of enhancing antibacterial activity is called synonymous if the antibacterial activity of the combination used is likely to be greater than that of the two separate compounds. The component of the composition which is also known to be a sonically acting component, and such substances are highly appreciated in view of enhancing antibacterial activity. penicillins and tefblosptrioes against ™ 8ΐ81βηΟ ^ trganiemůe. One object of the present invention is to provide a process for the preparation of such a physical active ingredient.

Examples of some pouužtí polysynthetictýth penOcilioů and _{cefblosptrioů>} resisteotoíth against úěinkům. beta-lacobases, both beta-lactam inhibitors and the allergic active ingredients of penicillins and cephalosporins, have been described in the literature, see, for example, Sutherland et al., Nature 201, 868 (1964); Sabsth et al., Nstwre 204. 1066 (1964); O'Callbghbn et al., Alimim et al. Agent and Cheimoherapy # 7, 1968 (1969). None of these known agents have a direct effect on the spectrum of other antibiotics present in the mixture. Some cultures of antimomycetes have been described as producing beta-lacamase-inhibiting agents acting synergistically with penicillins or cephalosporins, for example those described in BriS. 1,363,075 and those described by Hsta et al., J. AnH-biotics 25, 473 (1972) and Urnmzswa et al., J. AnUbiotics 26, 51 (1973).

None of these cases of beta-lactase inhibitors of actinomycetic origin were found to be of clinical use. A particularly noteworthy fact with regard to distinguishing clallulanic acid from other beta-lactamase inhibitors of stinomycetate origin is the moonoot extraction into organic solvents from the culture filtrate, acidified to pH 2, the high stability of clalic acid in human blood and its broad spectrum antibiotic efficacy. as well as the purpose of inhibiting beta-lactamase, low molecular weight,. as well as high Rf values on paper using numerous solvent systems.

It has been found that aerobic Strepoomyces claluliglrus in a conventional nutrient medium at a temperature of about 25 ° C to about 30 ° C approximately in a neutral environment produces a beta-lacamase inhibitor which is also known to be effective in inhibiting beta-lacamase activity. We have called this substance Slalalacetic acid; has the following characteristics:

(a) it is a carboxylic acid, ‘

(b) form the sodium salt of the infrared spectrum as shown in the subclass of FIG. 1.

c) can -οΚ ^^ β! growth of Staphylococcus aureus strains,

d) is graspable and ^ ergiso ^! Serilloactivity of amiociin for 1 beta-lacaamase-producing strains of Escherichis coli, Kiesia aerogenes and Staphylococcus aureus,

(e) the efficacy of serotergic efficacy is counteracted by the anti-tridium strains of Proteus and Staulyl aureus, which confer bett-lactase;

f) forms mHH ^ ^ l (É É ^ 1te) of molecular weight 213.06 (by mass spectrum), which is in accordance with the formula CgHH ^O ^N.

Thus, clalultoleic acid may be regarded as the monovalent carboxylic acid of the above formula, forming the sodium salt, the infrared spectrum of which is appended to the drawing.

A compound produced by Streptomlces cltluliglrus and having the above. it has structural formula II

COOH ch ₂ oh (II)

Thus, cltluronic acid can be named as 3- (Seta-Hydroxyethylideo) -7-txt-4-oxa-1-azabicyclo (3,2,0) -hepten-2-ylbenzylic acid.

Stereochemistry at the carbon atoms at the -5- and -2- positions of clavulamic acid is the same as that of natural penicillins and cephalaporins, so that clavulanic acid can be more precisely characterized by structural formula I

CH ₂ OH (I)

COOH

Thus, the exact designation of clavulanic acid in this sense is Z- (2R, 5R) -3- (beta-hydroxyethylidene) -7-oxo-4-oxa-1-azabicyclo (3.2.0.0) heptane-2-carboxylic acid. ’

The great utility of clavulanic acid can easily be appreciated by the fact that for some Klebbiella aerogenes A strains whose growth is not inhibited by the presence of ampicillin, amxycillin, carbonicillin or benzylpinicil · Ciu in the amount of 125 / cg / ml or by the presence of γ / ml clavulanic acid. growth inhibition occurs in the presence of less than 12.5 µg / ml of any of the foregoing µg / ml if Havranic acid is also present at an amount of 5 µg / ml. - 'j

Similar results were observed for combinations containing numerous different clavulanic acid esters. For example, in strains of Kleeelella aerogenes A whose growth is not inhibited by ampiecin at a level of 125 µg / ml, or β-β-γ-γ-γ-clclic acid in the blood. 10 [mu] g / ml, inhaled at a dose below 12.5 [mu] g / ml in the presence of 5 [mu] g / ml of Havranic acid methyl ester. It has also been found that in the case of strains of Staphyltcticui aureus RjssU whose growth is not CnhCb by the presence of 100 µg / ml of ippicus or three tigers of cyanobacteria, it has been found to be present at about 1 µg / ml. hg / ml Ravenic acid. Tests in female mice have shown that blood and tissue levels of Havulanic acid substantially above 5 µg / ml can be easily achieved by subcutaneous administration of 100 mg / kg of Havulpic acid sodium and that the usable level of clavulanic acid can be achieved by oral administration. sodium clavulanic acid at a rate of 100 mg / kg.

Accordingly, the present invention provides a process for the preparation of Havjlfinic acid, the corresponding salts and esters.

SUMMARY OF THE INVENTION The present invention relates to a process for the preparation of acidic salts, esters and esters of the formula I

(I) ·· * where

R represents a hydrogen atom, such as sodium, potassium, magnesium, aluminum, ammonium or ammonium ammonium, such as trimethylammonium, benzatene, procaine and the like salts which usually form penicillins; furthermore, R represents a C1 -C12 aryl group, optionally substituted with halogen, a phenyl radical, a control group, a C1 -C3 alkoxyphenyl group, a C1 -C3 alkoxyphenyl radical, and a C1 -C3 phenylalkoxyphenyl group. carbon in the alkoxy radical, the naphthyl radical, the naphthyl radical, the naphthyl radical and the optionally substituted methoxyl radical; C,-Cikα až aikik atyikik amin amin amin amin amin amin aipine, optionally further substituted with C 1 -C 4 alkyl or benzyloxycarbonyl, carboxy, optionally in the form of> H or ester, further C 2 -C 5 acyloxy, phenylsulfonyl or nitrile, phthalimido or N -benzoxyzolonyl, then C 1 -C 4 alkoxycarboxnrloxy, wherein R is further lower C 2 -C 4 alkenyl or lower C 2 -C 4 alkynyl, C 3 -C 7 cyclohexyl, phenyl or phthalidyl a group, the latter optionally having a methoxy radical, characterized in that the strain of Straptomrcas clavuligerus is contaminated on a nutrient medium containing nitrogen and carbon sources as well as the necessary mineral salts, aerobically at a temperature 15 ° to 40 ° C, usually 20 to 35 ° C and preferably 25 to 30 ° C and at a pH of 5 to 8.5, preferably 6 to 7.5, whereupon clavulanic acid or a salt thereof is isolated from the culture by filtering the cells by extracting the filtrate of the filter into a solvent after adjusting the pH to acidic acid, or by absorbing the product on charcoal, precipitating, salting, or molar filtration. clavulanic acid or a salt thereof is converted by esterification with a C 1 -C 12 diazoalkarne or an alcohol of formula

R = OH where

R is as defined above, or with a thiol in addition to a coaddition facilitating agent, to a lipid ester, or to a compound of formula I wherein R is any of said cations, a compound of formula wherein:

R is as defined above and

Q is a residue that is readily cleaved and replaced in this reaction, such as a chlorine, bromine or iodine atom or a sulfonic acid ester residue such as a methiamulfonic acid or p-toluenesulfonic acid residue, or an active ester group such as formyl or trifluoroacetyl, and optionally the ester of a compound of formula I wherein R is an alcohol residue is hydrolyzed.

The most suitable salts of clavulanic acid include pharmaceutically acceptable salts, such as sodium, potassium, calcium, magnesium, aluminum, ammonium and amines, such as trimethylamine, benzathine, procaine and the like, as are typically prepared in the case of penicillins and cephalospirins. . Pharmaceutically inadmissible salts of clavulanic acid are also included within the scope of the present invention as long as they are useful as intermediates for the preparation of clavulanic acid esters, i.e., the lithium or silver salts, which can be used in reaction with benzyl bromide to form a useful benzyl ester of clavulanic acid.

If clavulanic acid is clearly more stable than the corresponding free acid as such, it constitutes a preferred object of the process of the invention. Particularly preferred salts of the clavulanic landscape include the sodium and potassium salts, i.e. the compounds of formulas III and IV in which case

COOK

(III)

Crystalline forms of these salts may contain crystalline water, suitable suitable clavulanic acids include those derived from such alcohols as methanol, ethanol, propanol, butanol, 2,2,2-trichloroethanol, 2,2,2-trifluoroethanol , benzyl alcohol, p-nitrobenzyl alcohol, phenol, acetoxymethanol, pivaloyloxymethanol, 2-dimethylaminoethanol and other common alcohols. Numerous different clavulanic acid esters are useful intermediates in the purification of this acid, and numerous clavulanic acid esters are useful synergists. Of course, the activity of these esters can be conditioned by their hydrolysis to give the parent acid.

The term ester as used herein generally includes esters derived from alcohols or thiols of the formula ROH or RSH, wherein R is an organic radical. Suitable values for R include alkyl, alkenyl, alkynyl, aryl, arylalkyl and other similar groups, each of which may be substituted, if appropriate. In view of the unsuitability of increasing the molecular weight above the acceptable level, the group R usually contains up to 16 carbon atoms, preferably up to 12 carbon atoms, and most preferably at most β carbon atoms.

Preferably, R is derived from an alcohol of formula ROH, or less suitably from a thiol of formula RSH, and these are pharmaceutically acceptable.

Suitable substituents in the group R are halogen atoms or lower alkoxy groups, furthermore hydroxyl groups, lower acyloxy groups, lower alkylamino groups, lower dialkylamino groups and the like.

By "lower" is meant groups containing up to 6 carbon atoms, preferably 1 to 4 carbon atoms, and thus may be, for example, R, methyl, ethyl, n-propyl, isopropyl, straight or branched butyl, pentyl, non-propyl, octyl groups. , nonyl, decyl, undecyl, dodecyl, vinyl, allyl, butenyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclohexenyl, cyclohexadienyl, methylcyclopentyl, methylcyclohexyl, naphthyl, benzyl, benzhydryl, phenylethyl, phenylethyl, phenylethyl, phenylethyl, phenyl, , 2-chloroethyl, 2,2,2-trichloroethyl, 2,2,2-trifluoroethyl, acetylmethyl, benzoylmethyl, 2-methoxyethyl, 2-dimethylaminoethyl, 2-diethyleminoethyl, 2-piperidinoethyl, 2-morpholinoethyl, 3-dimethylaminopropyl, p -chlorobenzyl, p-methoxybenzyl, p-nitrobenzyl, p-bromobenzyl, m-chlorobenzyl 6-methoxynaphthyl-2-methyl, p-chlorophenyl, p-methoxyphenyl, or any similar group, as well as those known in the penicillins and cephalosporins for the preparation of esters, which are further known to readily hydrolyze in in vivo to produce parent antibiotics.

The easily hydrolyzing esters include, but are not limited to, the compounds of formulas (V) and (VI).

CO ^X o-CH-Z

I (V) (VI)

Wherein A 1 is hydrogen, alkyl, aryl or aralkyl, Ag is hydrogen or methyl, A 1 is alkyl, aryl or aralkyl, X is oxygen or sulfur, X is swath or sulfur, and Z is a divalent organic group. Esters of formulas V and VI, from which es easily releases clavulan viburnum in the blood upon administration of these esters, are those wherein A 1 is hydrogen, Ag is hydrogen or methyl, and A is methyl, ethyl, propyl, butyl, benzyl or a phenyl group, as well as those wherein X is a tyxy, X is oxygen and Z is a group

-CHg-CHg- or -CH = CH-, hereinafter

When used in connection with the aforementioned general formula, the term alkyl refers to such a radical of up to six carbon atoms, the term aryl refers to a phenyl or naphthyl residue, optionally a phenyl group substituted with an inert substituent such as fluoro or chloro, methyl or methoxy, etc. and the term aralkyl means an alkyl group substituted by an aryl radical.

Especially suitable esters of formulas V and VI are compounds of formulas VII and VIII

(VII) '(VIII) wherein Aa is hydrogen or methyl, A ^ is methyl, tert-butyl or phenyl, and Ag is hydrogen or methoxy.

Many esters of clalic acid differ from similar esters of picicillins and cephalosporins to the extent that they have an increased ability to hydrolyze under mild conditions to form clavulanic acid. For example, simple alkyl esters, such as the meethyester of clavulanic acid, are hydrolyzed to formic acid in water, buffered to pH 7. Among the esters which have the following formula: under mild conditions, they fall within the scope of formula IX

COOR ¹

CH ₂ OH (IX) wherein ^R 'represents a hydrocarbon etopinu and 1-9 carbon atoms, optionally substituted by halogen, lower alkoxy or hydroxyl, or optionally on the basic StopIN ^b ecnýc formula ^{NR 2 R h} \ ^ grapevines ita ^ ^^ ^^ Uy L into salt forms toe ^R2 is hydrogen or lower alkyl, R is hydrogen or lower alkyl, or binds to R ² and ^{R 2} so that ^a to ^p in ^and -kr ^2R2 together form a five-lexrný no ^b-six liters of lively cycle.

When used in formula (IX), the cut-out means a lower group having one to four carbon atoms.

Quick match ^R1 includes ^H ODN ^and a collision with ^lkyl Ove ^ -alkyl groups, substituted by halo ^or farm is considerably gene mmthoxylovou, hydroxyl or group of the formula NR RJ převeditenýfa into a salt form where R ² is methyl or ethyl, and Rd represents ekupinu

1 2 1 methyl or ethyl group, or R ^c and R j are bonded to nitrogen, so that the grouping NR ^J R form a pyrrolidine, piperidine or morrolinový cycle.

It consists Nejvhod ^ alkyl StopIN pHmý řettaec ^r1 and ^F into Seet carbon atoms ^and straight ^beta. ADN su ^ t ^ l ^^ ned mmH StopIN ^^ OH ^ ^b not on six tions ^{P N} R ² R ^{2 p} řevediteným in salt form , chlorine, bromine or iodine, or a trichromomethyl or trifluoromethyl group.

Clavulanic acid esters which are particularly useful as synergists are those which are hydrolyzed in mammalian tissues, particularly in human blood, to form clavulanic acid or a salt thereof, since clavulanic acid is believed to be or its salts are somewhat more suitable synergists than esters per se. Numerous esters of formulas V to IX are suitable for this purpose.

Another group of particularly suitable esters of the present invention are those which are useful intermediates readily converting clavulanic acid or its salts by chemical or biochemical processes known to those of penicillin or cefslosporins to be mild enough to prevent degradation beta-aakaamyl ring, reactive with acid labile.

It is particularly convenient if the ester can be cleaved by hydrogen. Useful esters in such a process include bennyl esters, substituted benzo esters, benzene diesters, substituted benzyl esters, trityl esters and the like. Particularly suitable for this purpose are, as has been shown, the a and z of the ery ters.

Generally speaking, the nature of any substituent in the ester moiety is unimportant unless the substituent of the hydrogen moiety.

Since clavulanic acid and its salts are useful intermediates in the preparation of the suitable artibacterial esters of the present invention, the present invention also relates to clavulanic acid and precipitating salts when used as chemical mesiproducts.

As noted herein, clavulaminic acid and its salts have valuable therapeutic properties, so that according to another aspect of the present invention, pharmaceutical compositions comprising clavulanic acid, a salt thereof or an ester thereof together with a pharmaceutically acceptable carrier are described.

Meei's compositions of the present invention include those suitable for oral, topical or pariaiteral administration, and can be used in the treatment of mammalian infections, including humans.

Suitable forms of the formulation and compositions of the present invention include tablets, tablets, creams, syrups, suspensions, solutions, suspensions, and sterile forms suitable for injection or injection. Such compositions may contain pharmaceutically acceptable excipients such as diluents, binders, colorants, flavors, preservatives, disintegrants, and the like, always in accordance with conventional pharmaceutical practice using techniques well known to those skilled in the art of formulating dosage forms. antibiotics.

Injectable compositions of clavulanic acid or corresponding salts, as well as infusion composites, are particularly suitable because of the high concentration of clavulanic acid in the tissue after injection or infusion. Thus, preferred compositions of the invention include clavulanic acid or a salt thereof in sterile form.

Co-administration in a unitary dosage form containing Havuonic acid or a salt or ester thereof suitable for oral administration constitutes another preferred variation of the invention.

In certain circumstances, the efficacy of an oral clavulanic salt, salt or ester thereof may be improved if such a combo contains a buffering agent or a coating for initial initiation, so that the compounds of the invention are not exposed to the lasting effect of very acid gastric glands for longer. Such buffered or coated compoice can be prepared in accordance with conventional pharmaceutical practice.

The clavulanic acid, its strength or ester may be present in the como as the only therapeutically effective agent, or it may be contained therein together with other therapeutically active agents, such as beta-hams, antibiotics. Suitable beta-aecamic antibiotics which can be added to the synergistic compounds include not only those antibiotics known to be readily attacked by beta-lactamasomes, but also those that exhibit a good degree of intrinsic resistance to beta-acams. Also suitable beta-acamma antibiotics for addition to the compositions of the present invention include benzylspin, phenoxymethylpenicciin, carbeeitin, eethiin, pmpcd, βpiperidinoamoyl, epiectin, ticarcillin, cyclocadinic acid, aminoacetin acid. aminocephalosporanic acid, 7-aminodesacetoxygalosporanic acid, cephaloridine, cephalothine, cephaloline, cephalexin, cepoxitin, cepaceeril, cephamendol, cetamirine, ceparadine, cephaloglycine, and other well-known penicillins and their cephalosporosporins, or their cephalosporosporins hetacillin, peeaIImicdio, further αcetoУsmitthSlsters, pílalosloxspethlS.e8tвrs. a. benzyl phthalidyl esters (C 1 -C 3, applinyl, amoyydine or cephaloglycine), or phenyl, tolylesters or indanyl- or f-esters with carbenicide or ticarcillin and the like.

It is clear that when a substance from the group of oeoicillin or cephalosporins that is present in the colic or suitable for oral administration, the co-administration for oarenteral is administered. When the acid, its salt or ester in the pharmaceutical composition together with the beta-lactoic antibiotic, the ratio of clavulanic acid, its salt or ester to the beta-lactoic antibiotic may be from 20: 1 to 1: 12, usually from 10: 1 to 1: 10 and preferably is from 3: 1 to 1: 3.

Total multiply! Antibacterial agents in any dosage form are generally 50 to 50%. 500 mg, and is generally in the range 100 to 1000 mg.

The compositions of the present invention can be used to treat infections, including other respiratory tract, urinary tract and soft tissues in humans.

The composition of the invention can also be used in the treatment of infections of domestic animals, such as in the case of cattle, udder inflammation.

Usually, 50-6000 mg of a coli of the present invention are administered daily for treatment, typically between 500-3000 mg of a composition of the present invention per day. However, in the treatment of more severe infections or infections caused by particularly resistant organisms, higher doses may be used in accordance with conventional clinical practice.

The exact form of the composition of the invention will depend to some extent on the microorganism to which the treatment relates. In the treatment of the greatest number of infections, the kommpoice of the invention is usually adjusted so as to achieve a blood level peak of at least 0.1 µg / ml, preferably at least 0.25 µg / ml, and preferably at least 1 synergistic component, for example up to / µg / ml synergistically active ingredient.

The content of pencc-Hnu or phospholosporin in the synergistic compositions of the present invention is generally up to the content typically used in the case of pencHin or crphalosporin as yeast when used as the sole ingredient in the treatment of infection.

Particularly preferred compositions of the present invention comprise from about 150 mg to about 1000 mg of acocyclyl, ampicillin or the starting material from which such a prime derivative is derived, and from about 50 to about 500 mg of clavulanic acid, its salt, Si ester hydrolyzable in vivo, and more preferably about 200 mg. up to 500 mg of allyl H 2 O, an appellant or the like starting material and 50 to 250 mg of alanulic acid, salt or ester, hydrolyzable in vitro

The matsials in such a composition may be hydrated if desired. The antibiotic's homogeneity in such a composition is expressed on the basis of an antibiotic which is theoretically available from the poposc and not on the basis of the hpoOn of the drug.

The present invention provides a process for the production of clavulanic acid, comprising salts and esters, comprising the step of filtering the Streptomycas trichloride and isolating clavulanic acid or a salt thereof from the culture medium, where appropriate, if desired. prepare the free acid, salt or ester thereof using otherwise conventional and known procedures.

Preferably, Strepoonyce and olives with ATCC 27064 or mullion are used, producing a high yield of active ingredient.

By cultivation is meant here the aerobic growth of a clavulanic acid-producing microorganism in the presence of assimilated carbon and nitrogen sources as well as inorganic salts. Aerobic growth can occur on a solid or oolorous medium or in a liquid medium where the nutrients are dissolved or suspended. The cone may be carried out on an aerobic surface or in a culture. The culture medium may be composed of a nutrient complex, or may be chemically defined. It has been found to be particularly suitable for the nutrient-containing environment, such as yeast extract, soybean meal and the like.

Nutrient media that can be used to cultivate Strrptopyr claviligerus may contain between 0.1 and 10% organic nitrogen source, such as quartz extract, corn broth, rostlix proteins, seed proteins, hydrolysates of such proteins, hydro milk protein lysetes, fish and meat extracts, and hydrolysates such as peptones. Other sources of nitrogen, such as olefin, amides, single amino acids or mixtures of conventional amino acids such as valine, asparagine, glutamic acid, proline, and Alenin bitches may be used.

It is possible to add some sugars to the culture medium in a quantity of 0.1 to 5%, but glucose has an undesirable effect in some environments, since the yield of clavulanic acid decreases. Starches or starch hydrolysis products such as dexyrin, sucrose, lactose or other sugars, glycerin or glycerin esters can be used. The carbon sources may also be based on animal oils and fats. As a source of carbon for the growth of the microorganism and the production of beta-lacamase inhibitors, they can be used in carboxylic acid and salt environments. An environment which contains soya meal (Arkasoy) and dried malt distillates from Scotssol sd ^ t ^ nem.

The addition of antifoam agents (P &lt; ronic D31) may and may be necessary to manage the loading of some environments in feraimation devices.

Inorganic salts such as sodium chloride, potassium chloride, magnesium chloride, zinc chloride, iron (III) chloride may be added to the medium as described above, and in particular when chemically defined. * sodium sulphate, iron sulphate, magnesium sulphate, or sodium or potassium salts of phosphoric acid; Calcium carbonate may also be added as a source of calcium ions, optionally for buffering purposes. Also salts of trace elements such as nickel, cobalt or manganese may be added. And, if necessary, vitamins can be added.

As used herein, the term &quot; mutant &quot; includes any mutant strain that is produced spontaneously or by the action of an external agent, whether used or otherwise. Suitable methods for obtaining mucoing strains are described in Η. I. Adler in the Tectmiques for the Development of Microorgtoisms in Raadatioo and from phaDdoisotopes for IoduutritL Mc ^ roi ^ irgani ^ ms, Procudings of a. Sy ^ f ^ C ^ osium, Vienna, 1973, p. 241, ^'Atomic Iotlroαtiooαl Eoergy N ut ^ c ^ o ^ i ^ -The, and includes:

(i) ionizing radiation (such as X-rays and gamma-rays) ultraviolet light, ultraviolet radiation together with photosulfonizing agents (such as 8-methoxysatraene), nitrous acid, hydroxylamio, pyrimidine base analogs (such as S-bromo and o); acrides, alkylating agents (such as mustard gas, nitrile sulfonates), hydrogen peroxide, phenols, forteplo and

Li) genetic Ulcenikα, such as recombination, transformation, UrtoLsdukcl, lysonehail, ^ logeno! conversion, and selective procedures for spontaneous use.

А ^ О ^^ по! Stigeptouycles ilululiglrus is usually carried out at a temperature of 15 to 40 ° C, preferably 20 to 35 ° C, and preferably at a temperature of 25 to 30 ° C, the pH being usually 5 to 8.5, preferably 6 to 7.5.

Streptococcus iluliglrus can be cultured in the above mentioned environments using conical glass flasks for aeration and shaking on a rotary shaker, or it can be done in stainless steel feeder equipped with baffles, mixed by disc disks for aeration with air intake. Comments can also be made continuously.

The initial pH of the ferrocyanate is typically 7.0, and the highest yield of clavulanic acid is achieved in 2 to 10 days at 20 to 35 ° C. In feruentαčoíih stainless steel equipment for pouuití environment Arkatoo ^of lSčitαaot / deltrio how. it is mentioned above, the most preferred temperature is 26 ° C, and the maximum formation of clavulsic acid occurs in 5 days.

Klalulanol tyselia can be extracted from the culture medium, ie the culture filtrate in a number of ways. Particularly useful processes include solvent extraction from the cooled culture filtrate after adjusting the pH to acidic, as well as processes based on.

An internal nature such as desert and ion exchange resins. Streptomyces ilululiglrus cells are usually first removed after fermentation is complete by filtration by centrifugation, and then the extraction procedure is performed.

In the solvent extraction procedure, the filtrate from the culture is cooled, the pH is reduced to acidic by 2-3 by acid addition, followed by vigorous stirring of the acidified filtrate with a water-impregnated solvent such as acetic acid o-butyl ester. or ethyl ethyl acetic acid. Typically, monohydric acid such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid and the like are used in the acidification of the culture filtrate. When the above solvents are used, a particularly preferred substance is--Ь.

After separation of the two phases by centrifugation, the eluting SLT-lactate is extracted again from the solvent into an aqueous solution of sodium bicarbonate or potassium phosphate buffer, further into a suspension of calcium carbonate in water, or even into water of maintenance. pH approximately neutral, i.e. around 7.0. The aqueous extract, after separation of the two phases, can be concentrated under reduced pressure, whereupon the lyophilizate is obtained as a crude product of the claotlanic acid salt. This product is stable when stored in a solid state from -20 ° C.

In the anion exchange resin process, the clarified filtrate from the culture at a neutral or slightly acidic pH, for example in the range of 6-7, is percolated down through a column of weak or strong basic anion exchange resin such as Amberllt IR4B or Zerrlit FFIF until saturation and mtteral beta-lactimase, starts to flow from the bottom. The column is washed with water, eluted with aqueous sodium chloride solution, the fractions containing the beta-lacamase inhibitor are collected, pooled, dehydrated and lyophilized to give crude clavulanine tyrosine sodium.

Aimer II IR 4B is an example of a weakly basic anion exchange resin with polyamine active groups and a polystyrene and divinylbenzene matrix. Zeerlit FFIP is a strongly basic anion exchange resin with active quaternary ammonium groups and crosslinking of vinyl benzene and divinebemeen. and Similar resins are Isopor FFIP and DiAcquete FFIP SRA 64, manufactured by BDH Ciemic OLs Ltd, PpoIc, Dor set, UK.

Another form of extraction is by contacting the culture filtrate, usually at neutral pH, with a salt of clavulanic acid, in contact with an organic phase in which am.n is dissolved, conductive insoluble. water such as merculisobutylketone, trichlorethylene and the like. Suitable amines include secondary and tertiary amines in which one of the substituent groups is an aliphatic long chain containing, for example, 12 to 16 atoms, and another substituent on the nitrogen is a tertiary alkyl group so that the molecule is lipophilic in nature. A suitable amine for this purpose is Ammerlit LA2, and the amine is usually used in the form of an acid addition salt.

Following this method of extraction, clavulamic acid is found in the organic phase in the form of an amine salt; the organic phase is separated from the original culture filtrate, and the clavulanic acid is extracted back into the aqueous phase with brine, preferably using a concentrated solution of sodium chloride, sodium nitrate and the like. The crude clavulanic acid salt can then be obtained by lyophilization or similar methods.

Other essential separation processes such as carbon absorption, salting-out and molecular filtration can also be used, but neither of these processes can usually be used as successfully as the two methods described above, which can rightly be said to be advantageous. Further purification of the crude product as obtained by the above-described processes can be carried out in a number of ways, but ion exchange columns are particularly suitable, particularly when using Idopor, De-Acidite FFIP SRA64 or DIEA-cellulose. The acid is to be eluted with a gradient of an aqueous salt solution such as sodium chloride in a concentration of 0 to 0.5 M). A column of 0.01 M phosphate buffer at pH 7 should be eluted with a saline solution, typically a sodium chloride solution of 0-0.2 M in 0.01 M phosphate buffer at pH 7.

The active fractions can be determined by the inhibitory activity on Srtt-lactabase, or by the contribution of the antibiotic activity against Klebstellt arrtgenes in agar dilution testing. The fractions of major activity activity are then pooled, all shrunk in vacuo to a mild volume, and the crude product obtained, clavulturic acid salt, is desalted by purging with a Bio Gel P2 column downstream.

Bio Gel P2 is an example of a very lipophilic resin to which organic substances can be absorbed but which does not retain inorganic salts. It is a polyacrylamide gel manufactured by Bio Rad, Richmond, USA.

The active material is desalted, mixed with ethanol, and further cellulose column chromatography is carried out using 4: 1: 5 / ethanol / water (4: 1: 5) as the upper solvent phase.

Fractions containing the Escherichia cooi beta-lacaarnase-inhibiting material were combined, evaporated to dryness by evaporation in vacuo, redissolved in water, and lyophilized to yield sodium clavulanic acid as a white solid.

The most useful and convenient methods for detecting the presence of clavulanic acid in culture filtrates include biochemistry on paper. The presence of clavulanic acid can be demonstrated by utilizing its efficacy in inhibiting beta-lactamase. Thin-layered chrommftgrfir can be used to clavulanic acid in solid formulations. Such methods of proof, and test procedures are described hereinbelow.

A variation of the method of isolating the pure form of clavulanic acid or of the corresponding salts depends on the isolation of the impure form of clavulanic acid or the corresponding salt, followed by the preparation of the clavulanic acid ester which is then recovered using clavulanic acid or its salt from conventional methods. of the pure ester.

The impure clavulanic acid or salt used in this process usually comprises at least 1% by weight of the antibiotic.

Suitable esters which can be used in such a process are those which can be cleaved by hydrogenolysis, enzymatic processes or hydrplytically under very mild conditions.

One suitable group of esters for use in this process is the compounds of formula (X)

(X) where A? represents hydrogen or a phenyl group optionally substituted, and Aθ represents an optionally substituted phenyl group.

It is the most important, even if A,? hydrogen or a phenyl, tslyl network, chlorophenyl or methaphthyl group; and Αθ represents a phenyl group, i.e., a chlorophenyl or methoxyphenyl group.

Preferably A is? hydrogen and Αθ are phenyl. The esters of formula (X) may be cleaved by hydrogenolysis to form clavulanic acid or a salt thereof.

The other groups of esters which can be used in this process are those of the above-mentioned formulas (V) and (VI), and such esters can be converted to clavulanic acid salts by mild alkaline hydrolysis, e.g. at pH 7.5.

The non-crude form of clavulanic acid or its spliers to be purified by this process may be in the form of a solid or solution, which usually also contains substantial amounts of organic and inorganic impurities.

The clavulanic acid or its corresponding salt can be converted to the ester by an esterification reaction, as discussed hereinafter. A preferred reaction for forming the ilavulanic acid ester is the reaction of the clavuloic acid salt with an esterifying agent such as a reactive halide, a sulfonic acid ester, or the like, as will be discussed. these reactions are usually carried out in an organic solvent medium having a high dielectric constant such as dimethylforaamide, a mixture of acetone, dimethylaxide, N-methacetamide, hexalneethyltrimium phosphoric acid and the like.

If desired, the clavulanic acid salt may be dissolved in the solvent by a conventional method, or it may be bound to a polymeric support. MEEI pouužtí suitable course of this process the anion exchange class ^a1 iontomtoičové resins, strongly basic, especially those which are characterized makoreeikilární structure allowing pouužtí rrzpouštědal nonaqueous system. As found, Amberlit A26 is suitable for these purposes. The clavulanic acid salt can be absorbed on the propyl chloride. from the culture iiirrate, whereupon the resin is suspended in dimethylformamide containing sodium iodide or otherwise eluted with a solution of sodium iodide in dimethyiformamide, or in a mixture of methylremlamium and acetone.

The impure clavulanic acid aster is usually purified by chromatography, and in such procedures, the ester is usually spanned in an organic solvent, such as acetic acid ethyl ether, meyl ether, acetic acid, The solid phase used in the chromatography process is usually an inert material, such as silicagel, or silicon tetrachloride ^/ mbteeal.

The fractions exiting the column can be tested for the presence of oxalic acid by discharging the aforementioned synthetic properties. The other fractions are usually combined, and. the organic solvent under reduced pressure.

The ester of this process is usually of satisfactory purity, but the resulting material can be rechromatographed again, if desired.

The clavulanic acid ester thus purified can be converted to the crowd acid! «New or a salt thereof using procedures already discussed herein.

A particularly suitable process for obtaining clavulanic acid or a salt thereof is lyophilizing the compound of formula (X) as described herein. Reactions of this type are usually carried out in the presence of a transition metal allyl ether and with low or medium hydrogen overpressure. The reaction may be carried out at high temperature, room temperature or lower, for example in the range of from 0 to 100 ° C. Particularly preferred conditions for carrying out such hydrogenation include the use of a slightly higher hydrogen pressure at a temperature of 12-20 ° C. The reaction is carried out in conventional solvents such as lower alkanoic acid. for example ethanol. Palladium-on-carbon is, in our view, a particularly suitable calatate.

When the hydrogenation is carried out in the presence of a base, a clavulanic acid salt such as sodium or potassium is formed using sodium or potassium acidic carbonate.

The claryacid, or its addition salt, as obtained from similar reactions, is typically characterized by good purity.

The esters of the first acid may be prepared by clavulonic acid or its salts using conventional procedures.

Suitable methods for preparing esters include:

a) reacting a clavulanic acid salt with a compound of formula Q-R, wherein Q is an easily substituted moiety and a displaceable group, and R is an organic group,

b) reaction of clavulanic acid with diazoalkin,

c) reacting the klay Mandeic acid with an alcohol of formula ROH in the presence of a condensation or condensation-promoting agent such as a carbodiimide or the like. .

Suitable salts of clavulanic acid which may be used in the reaction with compounds of formula R-Q include alkali metal salts such as sodium or potassium salts, or other suitable salts such as silver salts.

Suitable quaternary groups Q include those known and known to be substituted by carloxylate anions, including, for example, chloro, bromo and iodo, sulfonic acid esters such as -o esters. -SD ₂ 'CH 3 or -O-W 8 -C 8 H 8 CH 3 for longer term active ester groups, such as -O-CO-H or -O-CO-CF 3, and other conventional groups that can be easily replaced by occlusive reagents.

The foregoing reaction is usually carried out in an organic solvent of high di-dialectic content, such as dimethylforaamide, acetone, dloxane, trahydroxybenzoate or the like, operating in a temperature range from -5 ° C to 100 ° C, more usually. in the range from * 5 ° C to 30 ° C, for example at a temperature of about 1 ° C to about 1 ° C.

The reaction of klayulanic acid and diazo compounds is a mild reaction for the preparation of alkyl esters, aralkyl esters or the like esters. This reaction with diazo compounds can be carried out under conventional reaction conditions, for example in the usual temperature range and in common solvents. Thus, such a reaction is carried out at a temperature of from about -5 ° C to 100 ° C, for example at a temperature of between 0 ° C and between suitable. solvents for this reaction are varied to lower bullying such as methane! and ethanol, and among the solvents that may be used are _tetrahydrate and dioxane and the like. Ethanol is a particularly suitable solvent for this reaction.

The reaction of clavulanic acid with an alcohol or thiol with a condensation facilitating agent is usually carried out in an inert organic solvent with a relatively high concentration, such as scetor. The reaction is usually carried out at a temperature lower than Si, for example in the range of -10 ° C. C up to * 22 ° C, usually! at temperatures from -5 ° C to +18 ° C, for example by first cooling to 0 ° C with gradual heating to about 15 ° C. As a reagent for promoting and facilitating the reaction, a material which is generally used to obtain water from the reaction mixture, and suitable reagents of this type include those from the group of carbodiimides, carbodiimidazoles or the like. The reagent is (xyllarlodiimide) In order to minimize the self-condensation of Halanic acid, this reaction is usually carried out in a large excess of alcohol or thiol.

Another suitable process for preparing esters is:

d) cleavage of carbon dioxide from compounds of formula XI

(XI) wherein R * is an inert organic group, and

e) reacting a compound of formula XI with an alcohol of formula ROH, or less preferably with a thiol of formula RSH.

831157

The carbon dioxide can be eliminated from the compounds of formula XI spontaneously during preparation, or otherwise by heating the compound of formula XI in an inert solvent. Suitable inert solvents include ethers such as diethyl ether, tetrahydrofuran, dioxane and the like. In many cases, the compound of formula XI decomposes spontaneously even at reduced temperature, for example at -5 ° C, to form an ester of formula

where ^{r /} is inert within the definition of R.

If the reaction of a compound of formula XI is to be carried out with an alcohol, or less preferably with a thiol, this reaction is usually carried out in an inert solvent such as an ether, in the presence of an excess of alcohol or thiol to avoid self-condensation. a clavulanic acid derivative.

Similarly, esterification procedures are generally not as suitable as those using a clavulanic acid salt and its reaction with a compound of formula R-Q, as discussed herein.

Compounds of formula XI may be prepared by reacting the salt of clavulanic acid with the compound - of ^- the formula Cl-CO-OR ^ or '' ^{Odpovíddjící:} chertLckou analogy · - typically ^- the reaction is conducted at a reduced temperature, for example at most 5 ° C, and in an inert solvent such as tetralylprofuran, dioxane and the like. Most preferably, the clavulanic acid salt is used in this reaction - a lipophilic salt, so that it dissolves in the solvent, although - if desired - a less suitable sodium salt can be used to provide a suspension of the salt in the reaction medium.

Description 1

Tests suitable for the detection of clavulanic acid

Test subtypes

Solutions containing clavulanic acid clavulanic acid, samples from isocyanate processes and the like) are incubated for 15 minutes - with a beta-actaanns preparation at pH 7 in - 0.05 M phosphate buffer at 37 ° C. During this time, enzyme inhibition or inhibition also occurs, followed by addition of the substrate as a substrate, and the incubation at 37 ° C is continued for 30 minutes. The enzymatic degradation of the substrate to βnicilovic acid is determined by a hydroxylactic assay with a pH value. The molar content of the beta-lactams used is so large that it causes 75% of the hydrolysis of the banozypeptide at 37 ° C in 30 minutes.

The extent of hydrolysis is a measure of the amount of enzyme that remains uninhibited. The results are expressed as% inhibition of enzyme activity by a dilution of clavulanic acid, i.e. a solution containing clavulanic acid (for example, with a cultured fittate) or a concentration of clavulanic acid in pg / ml, when 50% of the enzyme inhibits. for the above four conditions (I _5Q).

Beta-lactase enzyme *

The enzyme used is beta-lactase produced by Ketichia coli JT4. This culture is resistant to ampiiClin, and its resistance is conditioned by the production of beta-lactasease controlling the R-factor. They can be used, if desired, other similar beta-lakaamases controlling the R-factor.

The Kuutura maintained on nutrient agar slices is inoculated into 400 ml of sterile tryptone medium contained in a 2-liter conical flask. The environment has the following composition:

trypton ((ooid) 32 g / liter, yeast extract: (oxoid) 20 g / liter, sodium chloride 5 g / liter, and chloride hexahydrate in addition to 2.2 g / liter.

The final pH was adjusted to 7.4 with dilute sodium hydroxide solution. The flask was shaken for 20 hours at 25 ° C on a 240-rocker rotary shaker.

The microcell cells were collected by centrifugation, washed with 0.05 M buffered saline at pH 7 (with suspension and centrifugation) and resuspended in water to a cell concentration that was 25 times higher than in the culture medium. the cell suspension is disrupted in an MSE-ultrasonic disintegrator at 4 DEG C. The cell debris is removed by centrifugation, and aliquots of supernatant are stored under deep supercooling.To use the assay procedure, supernatant is diluted with 0.005 M phosphate buffer, This results in about 75% hydrolysis of the 1 mg / ml solution of yenpeenidine in 30 minutes at 37 ° C.

Test procedure

Appropriate dilutions of the blood preparation and the ietaulactpmase solution are mixed and incubated for 15 minutes at 37 ° C (test). The Koonrole assay with the buffer buffer of the preparation is also incubated. A solution of benzzepeatcillin (s) is then added to the test and control mixtures, and incubation is continued for an additional 30 minutes at 37 ° C. The residual benzylpenin-C-lin in each mixture is then determined using a lydiuxyl amine assay as described by Beckel et al., Proc. Roy. Soc., 15154. 498 (1961).

6 ml of the reagent hydrocyanide reagent is added to all tests, controls and blank, and the reaction is allowed to proceed at room temperature for 10 minutes before adding 2 ml of iron (II) sulfate solution as another reagent. The absorption of the final solutions is measured on an E. E. L. coloripetrl or spectrophotometer at 490 nm against the blank reagent solution. The composition of the solutions, tests and blank tests before lydruxy.

Folders

(all dissolved or diluted by adding 0.005 M phosphate buffer pH 7) Test ientzjpeatkClin, blank test ml kontrola ml blank reagent ml Ietaulaktapásy solution of E8Chlaichia coli 1.9 0.0 1.9 1.9 Inhibitor solution 0.1 0.0 0.0 0.0 BeatzjieatkClin 5 mg / ml 0.5 0.5 0.5 0.0 0.005 M phosphate buffer, pH 7 0.0 2,0 0.1 0.6

results

The percentage inhibition of beta-lsktiraaay is calculated as follows:

(beazylpenicilia uptake) - (control uptake) uninhibited reaction) - x (test uptake, ie, inhibited reaction) - coatrol uptake (ie, uninhibited reaction) = y

У% inhibition = -. 100 x

To achieve an Iθθ value, the inhibitor preparation is diluted until 50% inhibition of bennylpeeiiClinC beta-lactase is achieved in the above procedure.

Description 2

Proof of clavulanic acid by chrommtography on paper

The culture filtrate and reference, clavulanic acid solution (250 µg / ml, partially purified preparation) are applied in a trace (20 µl) onto a 1 cm wide Whstman No. 1 filter paper. The chromatogram was run descending for 16 hours at 5 ° C using n-butyltoluyl-isopropylpylalkechyl 7: 7: 6 (v / v) as a system. The strips are dried at 40 ° C and plated on agar plates containing the plates. 6 .mu.l / ml beniylpeeillin, and vaccinated with Klebsiella aerogenes, extending the be-tactamtsu (synergistic system). The plates are kept overnight at 30 ° C, and klavulanic acid appears as a zone of inhibited growth. The Rf value is 0.46. Mn ^ sv! 6 .mu.g / ml of bennyl picillin as such is subject to the need to kill K-obese aerogeaes, but with the beta-tactamase inhibitor this concentration is already toxic, i.e. synergism appears.

Using the above synergistic system allows clavulanic acid to be shown to be less than ever antibacterial.

Description 3

Evidence of clavulanic acid sodium salt by thin layer chromatography

Solutions of the clavulanic acid sodium salt formulations are applied in feet (5 µl / 1 µg / ml solution) to glass plates (F254, Merck) with a thickness of 0.25 mn. The chromatogram is run at 22 ° C using the upper phase of a 4: 5: 1 mixture of n-butyltol-ethyl alcohol-water (v / v). The chromatographic plates are dried at 40 ° C, and sodium clavulanic acid is detected by agar plates. containing 6 [mu] g / ml of protein and inoculated with K-obese aerogenes (synergistic system, see section on pepper chromatography for an explanation above).

The agar surface is covered with a fine filter cloth before the thin-layer plate is placed on the agar surface. After an additional 15 to 30 minutes required for diffusion, the thin-layered plate is removed with a coated filter, and the agar plate is incubated overnight at 30 ° C to expose inhibited growth zones. . The Rf of the clavulanic acid sodium salt in the above solvent system is about 0.37. It is also possible to react two spray-applied reactions, i.e., Ehlich's and Orifeyl-tertazium aluminum, to detect the clavulanic acid sodium salt band. The first of these contained 300 mg of p-dimethylbenzaldehyde in 9 ml of ethanol, 54 ml of n-butylolaltic chloride and 9 ml of concentrated hydrochloric acid.

By heating the β sprayed plate for 1 to 2 minutes at 120 ° C, a pink spot appears in place of clavulum acid sodium salt. The triphenyltetraoolium chloride reagent is a mixture of 1 volume of a 4% solution of this compound in methanol and 1 volume of methanolic sodium hydroxide solution. After spraying, the plate is heated to 80 ° C and a red spot appears on the white background in place of clavulanic acid sodium salt.

Example 1

Cultivation of Streptomyces clavuligerus

Streptomyces clergigus was cultured at 26 ° C on agar slices containing 1% Yeatex (yeast extract), 1% glucose and 2% Oxoid # 3 agar, pH 6.8. A sterile scraper is used to transfer the microtiter and spore from the slice to a 100 ml liquid medium in a 500 ml Erlenmayar flask. The liquid medium has the following composition:

malt extract Oxoid bacteriological peptone Oxoid 'glycerin tap water g / liter g / liter 20 g / liter liter

The pH of the hoodiota is adjusted to 7.0 by the addition of sodium hydroxide solution, and 100 ml volumes are dispensed into flasks which are closed with a foam stopper. before sterilizing for 20 minutes under a pressure of 0.1-0.5 MPa. The seeded bottle was shaken for 3 days from a temperature of 26 ° C on a 5 cm rotary shaker at 240 rpm. Production flasks containing the above liquid medium were inoculated with a vegetative inoculum and grown under the same conditions as before. Culture filtrate samples were tested for inhibitory activity against betl-laclauls from Escheeichia coli JT4. The highest and most potent efficacy is achieved in 3 days. The results are shown in Table 1. The clavularic acid band of Rf 0.46 is evident when the culture filtrate is tested on paper by paper using the procedures described herein. The increase in bandwidth is directly proportional to the increase in beta-lactamase inhibition.

Streptomyces clavuligerus is also cultured in two-liter shake flasks containing 400 ml of medium from the production stage, using the same medium and culture conditions as previously described in this example. In these larger vessels, the growth of the organism is more subdued and the optimum inhibitory activity of the beta-lactuase is reached 7 to 9 days after inoculation with the vegetative stage. The results are also shown in Table 1.

Table 1: Binding activity of beta-lacamase for Streptomyces cllvuligetus in 500 ml and 2 000-ml flasks

Number of days fer meding % Inhibition of la-cherichia coli beta-lactamase by final dilution of the culture filtrate 1/2 500 flask 500 ml flask 2000 ml

15 Dec

30

Example 2

Cultivation of Streptomyces clavuligerus

The seed flask - see Preparation of Example 1 - is used to inoculate 500 ml conical flasks containing 100 ml of the medium listed below in 100 ml deionized water:

% (weight by volume)

Soluble Starch 2% glycerin 0.3% Scotasol 0.1% Arkassoy 1% ferrous sulfate heptahydrate 0.01%

The medium is sterilized by autoclaving at 0.1-0.5 MPa for 20 minutes, and seeded with 5% of the vegetative grade stitches. The flasks were shaken at 26 ° C on a rotary shaker as in Example 1. The optimum titer of clavulanic acid was reached after 3 to 5 days. Dilution of the tartrate filtrate in a ratio of 1/200 gives 60% inhibition in the beta-lactase inhibition assay. The clavulamic acid band with an Rf value of 0.46 can be determined using paper chromatography (bioautographically) as previously described. This bandwidth increases in size in proportion to the increase in beta-lacamase inhibition activity.

Comment:

Soluble Starch is a product of Bitish Drug Houues, Poole, UK

Scotasol is a soluble fraction of ethanol distillation, doddvaael Thomas Brthwich Ltd, Glasgow, Great Bitanie, Arkasoy is a soy meal, supplier of Arcade Co., Mancheeser, UK.

Example 3

Rolling a Streptomyces clavuligerus flask according to Example 1 is used to inoculate 500 ml conical flasks containing 100 ml of the medium described below in deionized water under sterilization as described herein. The inoculum fluid is used at a rate of 5%.

% (weight by volume) dextrin 2% Arkasoy 1% Scotasol · 0.1% Ferrous sulphate heptahydrate 0.01%

The seeded flasks were shaken at 26 ° C. The optimum inhibitory activity of beobalactamts was reached in 3 to 5 parts. The activity was similar to that observed in Example 2.

Note: De ^ tt ^: Ln is a product of C P C, ManchheSer, UK.

Example 4

KHtvanie Stxaptomycea clavullgeius

The inoculation stage as in Example 1 ae is used to inoculate 500 m conical flasks containing the medium as described below, prepared using deionized water:

% (weight by volume) glucose 1 »soya meal 1% Scotaaol 0.05 5» calcium carbonate 1 ·

Flasks are treated and processed exactly as in the previous examples, and the killing is performed exactly under the same subpages. Beta-lactamase inhibiting activity is generated at 3 and 5%. The tailturate filtrate at a final dilution of 1/200 is found to be 35 to 45% inhibition in the beta-lactamase inhibition assay.

Example 5

Streutomyces clavuligerus

The β-lactimase inhibitory activity attributable to clavulanic acid is generated under the following conditions and using the same inoculation stage and culture conditions as in Example 1:

% (weight by volume)

glycerine 2 % soya meal 1.5 % magnesium sulfate O' % sec. potassium phosphate KgHPO4 0 (, 1 -%

The medium is prepared in deionized water. Inhibitory activity of beta-lacaamase reaches a maximum level at 3 and 5 Λιϊ, and is of the same order of magnitude as in Example 4.

Example 6

Streutomyces clavuligerus

Using the medium described below, clavulanic acid is formed under the same conditions and using the same inoculum from the vegetative step as in Example 1.

glucose. 1% Lab Lam Co (Oxoid). 1% Yeast (Oxoid) 0.3% calcium carbonate 0.3%

The medium is prepared in deionized water.

The optimum titer ae is obtained in 3 to 5 Da, and at a dilution of the culture filtrate of 1/2 500, 35 to 45% inhibition is also obtained in the bets-SakSm-enzyme enzyme inhibition assay.

Cultivation of Streptomyces clavuligerus

As in Examples 4, 5 and 6, 35-45% inhibition (at a dilution of 1/2500) in beta-lactammas assays at the optimum titer reached 3 to 5 days after vaccination was carried out using the medium below. All conditions are the same as previously described.

% (weight by volume) glucose 2%

Arkasoy 1% calcium carbonate 0, ½% hexobutyrate 0.000 1%

The medium is prepared in deionized water.

Example 8

Cultivation of Streptomyces clavuligerus

Using the following production medium under standard culture conditions, as described in the previous examples, 20 to 30% inhibition occurs at a dilution of be500 in the bela-culture test at 3 and 5 days after seeding. x ^ í. And, using paper-on-paper as described herein, a clavulanic acid band of Rf 0.46 can be detected when testing the culture filtrate.

Scotasol 2%

Extract Yeast (Oxoid) 0%

The medium is prepared using tap water, pH 7.0.

Example 9

Cultivation of Streptomyces clavuligerus

Using standardized culture conditions, the medium below produces 3 to 5 days after inoculation with the vegetative step. At a dilution of 1/500 of the culture, 20 to 30% inhibition in the bela-lactate activity assay was obtained.

g / lltr glycerine 15 Dec sucrose 20 May proline 2.5 glutamic acid monosodium salt 1.5 sodium chloride 5.0 potassium phosphate 2,0 calcium chloride 0.4 Meltin tetrachloride tetrahydrate 0.1 ferric chloride hexatyyditeCt 0.1 zinc chloride 0.05 magnesium sulfate heptahydrate 1.0

The medium is prepared in deionized water; final pH = 7.1 ·

Example 10

Cultivation of Streptomyces clavuligerus

A Yeatex and glucose stock agar slice was used to inoculate a Yeatex glucose slice in a Roux flask to prepare a mycelial suspension and a spore in sterile water. The sliced Roux flask was incubated at 26 ° C for 10 days. After adding 100! sterile water is prepared by a mycelial suspension, and this suspension is used to inoculate a 50 liter steam sterilized inoculum medium of the following composition (using tap water):

% (weight by volume) malt extract (Oxoid) 1% bacteriological peptone (Oxoid) 1% glycerine 1% 10% Pluronic L81 as reagent against foam formation (in soybean oil) 0.05%

Pluronic is supplied by Jacobs snd Van den Berg UK Ltd, London, and contains a block polymer of polypropylene and polyethylene, and soybean oil supplied by British Oil and Cake Mills Ltd., Hulli, United Kingdom.

A 90 liter stainless steel fermenter equipped with baffles agitated by a 13 cm diameter disc mixer at 240 rpm is filled with this medium. Sterile air is admitted at 50 liters per minute, and the tank is incubated at 26 ° C.

After 72 hours, the inoculation feraentation device was used to inoculate 150 liters of the same medium using 5% (by volume) addition under sterile conditions. The production medium is contained in a 300-liter stainless steel fermenter equipped with baffles and a 20 cm stirred disc stirrer at 210 rpm, adding 150 liters per minute sterile air to the reaction mixture and fermenting at a rate of 150 liters per minute. temperature 26 ° C. 10 ml of soybean oil containing 10% '' Pluronic L81 '' is added as a reagent if necessary. Samples were tested at regular time intervals to inhibit beta-lactamase. The highest level of beta-lactamase inhibitory activity is achieved in 4-5 days, see Table 2.

Table 2

Beta-lactamase inhibitory activity in samples taken from culture filtrate after fermentation of 300 liters of mash with Streptomyces clavuligerus

Fermentation time (days) % inhibition of beta-1actamase. Inhibition test with final dilution 1/2 500 1.0 1.5 2,0 2.5 3.0 3.5 4.0 4.5 5.0 12 20 May 31 36 50 54 51 56 55

23OO 57

Example OO

Kujti. Streptomyces davjl-gerus

The inoculation fermenter works exactly the same as in Example 10 using the same medium.

After 72 hours, the inoculum medium was used as a 5% (by volume) inoculum for 150 liters of steam medium contained in a 300 liter stainless steel filter apparatus. with a 20 cm diameter disc stirrer at 210 rpm and aeration with sterile water at a rate of 150 nm / min. The melt temperature is maintained at 26 ° C, and an anti-gentle agent is added in portions of 10 ml, i.e., soybean oil containing 10% Ponicone L8, as necessary.

The medium used in the production step is identical to that of Example 3, and is added prior to sterile 0.05% (by volume) soybean oil containing 10% Pluronic L81.

The β-lactase activity of the fermentation samples was similar to that of Example 10, see Table 2. Chromatography on paper can be used to determine the Havulanic acid band at Rf 0.46 using bioPutographic procedures. (syne ^ -smus) as described herein. The size of the clauulant acid bandwidth increases evenly with the rise of the iltP-lacPapais inhibition assay.

Example 12

Activating Streptomyces davjl-gerus

100 ml of sterile water is added to a sporoculture culture which has been grown on bitter agar in R 2 flasks for 10 days at 26 ° C. A suspension of myccHa and spores is used to inoculate 75 liters of steam with the following composition (using tap water):

dextrin 2%) motnoattoblpm

Arkasos 50 1% P ^ motnoit0bblpи

0% Priconic L8O in soybean oil 0.03%, by volume

The pH of the medium is maintained at 7.0.

The medium is placed in a 100 O stainless steel fermenter with a 100 O volume and mixed with a 17 cm disc mixer at 140 rpm. In роЖу! 75 liters per minute of air is permitted, and n ^{c l} u ^b Ouan will burst Todina BC ^{72 - 26} ° C.

The contents of the inoculation fermenter are used to inoculate 500 liters of steam in a medium of the following composition (using tap water):

Ark _Pso with 50 1.5% potmotio by volume glycerine 1.0% potmotio by volume prim potassium phosphate 0.1% potmotio by volume soybean oil containing 10%

Pluronic L8O 0.2% ppdle from the environment The pH of the environment is maintained at 7.0. The environment is in a 2000 liter feeder, constructed of stainless steel, containing bulkheads, while the environment is agitated by a disc mixer with a diameter. 45 - cm at 106 rpm.

1,200 liters of sterile air are introduced into the environment per minute and, if necessary, 25 ml of a foam-forming agent, i.e. soybean oil containing _10% Pluronic LB1, is added in portions of 25 ml. The fermentation temperature is maintained at 26 ° C, and the maximum yield of clavulanic acid is reached in 3 to 5 days; the yield corresponds to 200-300 [mu] g / ml clavulanic acid.

Example 13

Cultivation of Streptomyces clavuligerus

The inoculum was prepared in seed flasks as described herein but using the medium described in Example 3 (pH of the medium was adjusted to 7.0). This medium is used to inoculate 100 µl aliquots of the following medium prepared in deionized water and stearate. Said subdl. 100 ml is always contained in a 500 ml conical flask. The stitch content is 5%.

Incoming P 224 1% weight by volume

Arkasoy 50 1.5% by weight ojeem prim, potassium phosphate O11% t weight by volume pH of the medium is adjusted to 7.0.

The inoculated flasks are grazed at 26 ° C, and the optimum inhibitory activity on beta-lacammas is reached in 3-5 days. A clavulanic acid content of 300 to 500 µg / ml is reached.

The belly of P 224 is a triglyceride (Prices Limited, Wirral, Chhehhre, UK). It contains 65% oleic acid, 11% palmitic acid and other similar acids.

Example 14

Isolation of crude clavularic acid in the form of the sodium salt

The culture drip obtained by the procedure of Example 10 is clarified by continuous centrifugation, whereby separate celite is unnecessary. With 150 liters of fermentation liquid, 120 liters of clarified culture liquid are obtained, and this filtrate shows a 58% inhibition in the beta-lakaimase inhibition test at 1 / 2.500 dilution. - The filtrate is cooled to 5 ° C, added 40 liters of n-butanol, the reaction mixture is further stirred with the addition of 25% sulfuric acid solution until pH 2.0 is reached. The acidic mixture is stirred for an additional 10 minutes before the two phases are separated by centrifugation. The aqueous portion is not needed. 0.5% Nooit GJSX activated charcoal is added to the n-butamol extract, the mixture is stirred for 15 minutes, then the charcoal is filtered off (unnecessary) using infiltrated clay as filter aid.

Ion-free water is added to the n-butanol phase in a melt volume of 0.25 volumes, and the mixture is stirred with the addition of 20% sodium hydroxide solution until the pH has stabilized at 7.0. The phases are separated by centrifugation, and the n-butanol phase is given to a tree (not needed). The aqueous fraction was bent under reduced pressure to a volume of 800 ml, followed by isolation, to give 35 g of a crude solid product of clavulanic acid ^X 50 of ^1.3 µg / ml in the beta-lactase inhibition test. is stored dry with cooling to -20 ° C before further cleaning.

Example 15

Isolation of the crude sodium salt of k-avuoic acid

Liter of culture filtrate of 53% inhibition at a dilution of 1/2500 in the test for. The beta-lalimase inhibition obtained according to Example 12 was percolated by downward flow through a 2.5 cm by 15 cm diameter column packed with Penmutt Isopore resin FF IP (SRI 62) in a chlorine cycle (Permuuit Co product, Isleworth) , Middlesex, United Kingdom). The column is then washed with 300 ml of distilled water, and the active beta-lacamase inhibitor is eluted with 0.2 M sodium chloride solution. Fractions of 20 ml are collected and beta-lacamase inhibition assays are performed with a final dilution of 1 / 2,500. The active fractions were pooled, concentrated in vacuo to a volume of 20 < -1 &gt;, and the solution obtained was desalted using a 3.6 cm diameter Biorad Blogel P2 column and a 40 cm gel bed eluting with 1% n- butanol in water <6. Biogel P2 is a product of Bio Rad Laboratories, Richmond, Calif., USA. The active fractions were pooled as evaluated by the beta-lactamase inhibition assay, followed by sodium chloride eluting after clavulanic acid, following the elution with a silver nitrate solution. The combined active fractions are concentrated and then lyophilized.

0.45 g of a crude solid preparation of clavuamic acid having a pore size of 0.92 pg / ml is obtained from a liter of culture filtrate. _

The solid product is stored with cooling to -20 ° C before further purification.

Example 16

Isolation of the crude sodium salt of k-avuanoic acid

The filtrate of the culture containing 300 .mu.g / ml of 1-alanic acid is stirred under stirring, after which it is extracted with n-butanes, and from this solvent the halanic acid is extracted again into water at neutral temperature. pH.

The cooled filter filtrate (5-10 ° C) is pumped through a built-in mixer, adding a sufficient quantity of nitric acid (by volume) nitric acid at the inlet to maintain the pH at the outlet in the range of 2.0 - 0.1. The octylated filtrate is passed through a heat exchanger whose plates are cooled in an amount (A.Ρ. V. Ltd). 4 liters per minute to maintain the temperature in the range of 2 to 5 °. The pH ae follows the flow rate before the flow is fed to a three-stage countercurrent separator (Weatffia Seppaator Ltd, Model EG 1006).

Water-saturated N-butyl alcohol (at a temperature of about 5 ° C) is pumped at a rate of 3 liters per minute to a countercurrent separator. A water outlet from said manifold and discharges to waste; from the butamone outlet, the entrained water is removed using a centrifugal separator for separating the liquids (Alfa Laval Ltd, Moolel 3024X-G), and the buccane fraction is collected in a stainless steel vessel equipped with a cooling jacket where it is stored at a temperature about 5 ° C.

40 liters are removed from this machine and are carefully cleaned. with 2 'liters of chilled water, saturated with n-butyl alcohol. The pH was adjusted to 6.8-1.1 using 20% sodium hydroxide solution.

The resulting mixture of aqueous extract and β-cot beech is fed to a centrifugal separator for liquid separation (Shu ^lung Cenogil Ltd., Moilel Μ35ΡΪ-5 PH) at a pumping rate of 2 liters per minute.

From 1,800 liters of the capsule, 90 liters of the aqueous phase containing 39% of clavulanic acid from the pot contained in the filter filtrate are obtained.

In 15 liters of aqueous extract, ee adjusts the total solids content from 2% to 8% by adding 60 g of sodium chloride per liter, and all is spray-dried (Aclh-Drug, Copenhagen, Type Lab S). Operating conditions used: feed rate of 2 liters per hour, 170 V sprayer feed, 6 to 7 settling in heated equipment, 150 ° C inlet temperature, 80 ° C outlet temperature.

The dried product with a total weight of 1 kg contains 62% of the clavulanic acid originally contained in the feed.

The remaining 75 liters of the aqueous extract was concentrated (De Dbnske

Sukkkefabrikker, Laboratory MoUuC, Mernmx type 900). The operating procedure is to recirculate the retained residue from the stainless steel reservoir, which reservoir is equipped with a cooling device, by adjusting the outlet valve so that a differential pressure of 2.5 MPa over 40 m @ 2 is achieved. The temperature is maintained at 2 to 5 ° C, and the pH is 6.8 - 0.1 by adding 2 N hydrochloric acid solution, if necessary. This reduces the volume to 34 liters containing 72% of the original supply of clavulanic acid.

The aqueous concentrate is stored at about 5 ° C, adjusted to 8% solids, and spray dried as described above. The dried batte contains 75% of the clavulanic acid content of the spray-drying feed.

All spray drying product obtained from 90 liters of aqueous extract contains

69.4 g of clavulanic acid, corresponding to 72% of the clavulanic acid content of the spray-drying line and 21% of the original amount of clavulanic acid contained in 1800 liters of culture.

Example 17

Partial purification of crude clavulanic acid

The crude preparation of clavulanic acid, as described in Example 15, is purified by ion exchange chromatography. 18 g maaeeiál, prepared. according to the procedure described in Example 15 and an Iθθ value of 1.3 µg / ml (final concentration) is dissolved in 25 ml of distilled water, and the solution is applied to a bed of 3.6 cm on a 40 cm PerrnuUit FF IP (SRA 62). ) in the chloride cycle. The column is eluted with a sodium chloride gradient, resulting in a spontaneous drop in the feed of 0.5 M sodium chloride solution to the mixing tank containing a liter of distilled water, and from there the feed is fed ^{to the} African column. 10 ml aliquots are collected, and beta-lacamase inhibition activity is tested using 1/2 500 fraction dilutions. The activity is eluted along the main strip of color between the fractions. 24 and 30 · The active fractions are q> draw and concentrated to 30 ml ·

The solution obtained is separated using a 5 cm bed. to 45 cm Biorad Biogel P2, and eluting with 1% n-butyl alcohol in water. The 20 ml fractions were tested for clavulanic acid content using the beta-lactamase inhibition test. The fractions were also applied in traces to a strip of paper sprayed with either Eirlich's reagent or triple-methyl-tetrol spray as described in description 3. The inhibitory activity of beialacaimase is comparable to the pink or red spots that are caused by the agents. The chan- ges of activity exhibiting activity are combined after cutting, separating those containing sodium chloride and concentrating in vacuo to dryness to give 520. mg of purified clavulanic acid sodium salt, with an Iθθ value of 0,2 pg / ml when in use. Standardized Test for Inhibition of Beas-Lactamis ·

TLC on silica gel reveals the following Rf values for clavulanic acid: using an α-butyl alcohol / ethanol / water 4: 1: 5 phase, R _f 0.37;

using .alpha.-butyl alcohol, acetic acid and water 12: 3: 5, Rf 0.44; and using a 7: 3 mixture of isopropyl alcohol and water R 0,7 0.78 (all mixtures by volume).

The bands are detected by spraying with Ehrlich's reagent. 6-Aminopenicillanic acid is used as a marker under detection by the same spray, having R _f values in which case the system is 0.30, 0.39 and 0.77.

Example 18

Partial purification of clavulanic acid sodium salt

The culture filtrate as obtained in Example 12 was extracted with the solvent as described in Example 14 to give a solid preparation which was further purified by ion exchange chromatography using Whstman's diethylaminomethylcellulose DE 52. The resulting solids in the vase Dissolve 10 g in 20 ml of distilled water and apply the solution to a column of 3,6 cm per 50 cm of DE-52-cellulose previously equilibrated using 0,01 M phosphate buffer pH 7,5 The column is eluted using a sodium chloride gradient. A 0.1 M solution of sodium chloride in a 0.01 M sodium phosphate buffer solution of pH 7.5 is fed into a mixing chamber containing a liter of 0.01 M phosphate buffer at pH 7.5, said mixing chamber being further combined and column.

Fractions of 10 ml each were collected and tested for beta-lactamase inhibiting activity at a dilution of 1/2500. Fractions were also tested for antibacterial activity by the needling procedure using Klebaiella aerogenes nutrient agar procedure. The fractions showing the highest beta-lactamase inhibiting activity and characterized by inhibition bands in the plate puncture assay were pooled, concentrated and desalted on a Biorad Biogel P2 column. These fractions contain clavulanic acid, as evidenced by paper chromatography and thin layer chromatography.

Example 19

Isolation of solid clavulanic acid sodium salt

The partially purified 500 mg clavulanic acid solid preparation, prepared as described in Example 17, is applied to a 2.5 cm / 50 cm bed of Whstman microcrystalline CC31 cellulose. The upper phase of the α-butyl alcohol mixture is used as the chromatographic solvent. ethyl alcohol / water 4: 1: 5 (v / v), then 4 ml fractions are collected from the column in a 4 ° C procedure. These fractions were tested for the presence of clavulanic acid by applying traces on filter paper sprayed with Ehrlich's reagent (pink spot) or triphenyltetrazolium (red spot). These trace assays have been shown to correspond to beta-lactamase inhibition assays at a dilution of 1/1 250. The active fractions are combined, and the combined fraction is dried in a rotary evaporator under vacuum. The solid was dissolved in a small volume of distilled water and a freeze-dried to give a solid white product sodium clavulanate to 40 mg of a value ^{T 0} 50, 08 .mu.g / ml in the assay for inhibition of beta-lactamases.

Example 20

Isolation of solid clavulanic acid sodium salt

The concentrated reverse extract (6 liters) of ultracentrifugation according to Example 16 and containing 10 g of clavulanic acid as determined using the beta-lactamase inhibition test of description 1 was percolated per liter to a column of 5 cm per 60 cm ion exchange resin Permutit Zerolit FF 1 P SRI 62 in the chloride cycle · Wash the column using two liters of deionized water before eluting with a sodium chloride gradient.

The gradient is formed in a container containing 4 liters of 1.4 M sodium chloride and from there a stirred container containing 4 liters of 0.7 M sodium chloride solution is in turn connected to a stirred container containing 4 liters of deionized water, and it is connected to the column by a pump. The column was eluted at a rate of 2.5 ml per minute and 25 ml fractions were collected. Fractions were assayed using a beta-lacamase inhibition assay.

The active fractions (No. 140 and 230) were combined and concentrated in vacuo to near dryness. After the addition of 500 ml of ethanol, the solids are filtered off with vigorous stirring, and the ethomol extract is filtered in vacuo to dryness on a rotary evaporator, then redissolved in 40 ml of deionized water. This solution is applied to a 10 cm by 60 cm Biorad Biogel P% column, and the column is eluted with 1% n-butanol solution. Fractions of 25 ml are collected and tested for beta-lskamase inhibiting activity at a final dilution of 1/2500. Tests for sodium chloride content of 1/25 fractions were utilized using silver nitrate solution. These fractions, containing sodium chloride-free clavulsic acid, are pooled, bent by evaporation of the solvent to 20 ml under reduced printing, and lyorilized. It is obtained

4.6 g clavulanic acid sodium salt (^-θ) is about 0.06 pg / ml).

Example 21

Preparation of clavulanic acid ester (methyl ester)

COONa

CH ₃ I *

O

CH2OH

COOCH ₃

19.6 g of clavulanic acid sodium salt are dissolved in 0.5 ml of dry dimethylformamide, and 0.25 ml of methyl iodide are added. The reaction mixture was allowed to stand for 1.5 hours at room temperature in anhydrous medium, after which the solvents were distilled off in vacuo. The residue was purified by thin layer chromatography (6OF 254, Merck, Dearmtadt) eluting with ethyl acetate to give clavulanic acid methyl ester as a colorless oil of Rf 0.38; the red color is induced by spraying (spray) (Sioculoal spray). The isolated ester has the following composition:

Analysis:

P ^ro -H ^- Calculated: 50.49% C 5.43% H 6.29% N Found: 50.70% C, 5.20% H, 6.57% N λ ___ (mod EEE bsoorcevadd DNM 21 .

max <

vmax 3,300-3,600 (Wide Belt), 1,800, 1,750, 1,695 cm.

Approx. M-1-α of first-order CD1 ^ = 2.49 (broad, S, 1, DgO swap), 3.05 (d, 1, J = 17.5 Hz), 3.54 ( dd, 1, J = 17.5 Hz), J g = 2.5 Hz), 3.84 (s, 3), 4.24 (d, 2, J = 7 Hz), 4.93 (dt, 1, J = 7 Hz, J ₂ = 1.5 Hz, 5.07 (d, 1, J = 1.5 Hz), 5.72 (d, 1, J = 2.5 Hz). = 231.06 Calculated for C 14 H 16 NO 3 = = 213.06.

In the case of cholmottlrotil on a thin layer behind oethylene-5ters, a single band appears in each of the following solvent systems:

isopropyl ether / ethyl acetate 4: 1: 5, upper phase, Rf ³ 0.75, isopropylpylalcohol / silica, 7: 3, Rf 0.95, ethyl acetate / ethanol 8: 2, Rf 0.87, all by volume.

The bands were determined iocutoglyphically by using K i (i s) and K i (i s) with addition of cillin (synergism).

Example 22

Preparation of clavulanic acid ρ-nitroenzylsulfate

ch ₂ qh \

COONa

Reaction of the clavulanic acid sodium salt with p-nitrogobenzylroomide in dry dimethylformamide produces a colorless oil after thin-layer chromatography on chloroform-ether, a p-eitrlisnzylester of clavulanic acid, to form fine white powdery needles. Mp 111-112 ° C; recrystallization increases m.p. to 117.5-118 ° C.

Example 23

Preparation of bennylester of clavulanic acid

Crude cellulosic acid, ie 3- (ethoxy-oxyethyl) -7-lxl-4-oxc-1-cicylcyclic acid (3 _l 2.0) lSepCr-22-C-bexylic acid (provided that The amount of solution (55 mg pure) is mixed with 0.18 g of benzyl bromide in 0.64 ml of dry dimethyl bromide, and the reaction solution is allowed to stand at room temperature (about 17-18 [deg.] C.) for 3 hours inaccessible to humans. The reaction mixture was fractionated on silica eluting with ethyl acetate to give essentially the bennyl ester of the above acid as a colorless oil in a yield of 63 mg. The infrared spectrum (in flhu) ^with j ou ataorpční ^Pa.s at ^{1800 s 1 74} 5 ^{1 6} 95 cm ht ~ ^NM -sppktru in euterovaném ^d c ^h lorofoamu the peaks at 2.25 (s, 1, s zaminntsleý DgO), 3.05 (d, 1, J = 17 Hz), 3.51 (dd, 1, J 3 17 Hz, J ₂ 3 2.5 Hz), 4.24 (d, 2, J 37) 5 Hz), 4.92 (dt, 1, J = 7.5 Hz, -1.5 Hz), 5.15 (d, 1, J 3 1.5 Hz, 5m24 (s, 2) 5) 71 (d, 1, J 3 2.5 Hz 7 7.45 delta (s, 5)).

Example 24

Preparation of benzyl ester of clavulanic acid from a crude mixture of extracts of Streptomyces clcauligsrup culture filtrate

A 20 liter culture filtrate, obtained as described in Example 10, is bent under vacuum to a volume of 5 L using film evaporation, then lyon-pressed using an Edward EF 6 shelf freeze drier (manufactured by Edward Ligh) Vacuum Ltd). The solid obtained, weighing 300 g, contains 3 g of sodium claulanic acid, as determined by the enzyme inhibition assay. The solid obtained was suspended in 900 ml of dry dimethylformamide and 150 ml of benzyl bromide was added, after which the reaction mixture was stirred at room temperature for 2 hours, and further diluted with 1 liter of ethyl acetate. After filtration of the reaction mixture, the filtrate is turned to as little volume as possible and the oily residue is extracted into 1 liter of ethyl acetate and the extract is filtered. The filtrate was concentrated again, and the resulting oily residue was loaded on a 7.5 cm by 35 cm silica gel column (Biogel BioesA.1, 100 mesh) in cyclohexane. Elution with cyclohexane removed the benzyl bromide from the column, then ethyl acetate was used as the solvent, and fractions of 20 ml were collected.

The fractions were tested for the presence of clavulanic acid benzyl ester by plating onto thin-layer silicone plates (Merck precoated silica ® 60 F 254) followed by spraying with 2,3,8-trichloro-azrazoliumcyclide rearens. The fractions that give an intense red spot with this rearens are further tested by thin layer chromatography using a chloroform-thyylacetate 8: 2 mixture followed by spray induction of the above rearens.

The benzyl ether of clavulanic acid has an RI of 0.31 at 22 ° C. Fractions containing this ester were combined, all bent to a volume of 15 ml, and this solution was further chromatographed on a Merck silica rel H, type 60, 3.6 x 40 cm column using a mixture of chloroform and ethyl ester. of acetic acid 8: 2 was heated. Fractions of 15 ml are collected and tested for the presence of the benzzlester as described above.

The fractions containing the ester are pooled, bent to 8 ml, and then purified on a 2.5 to 40 cm size silica column (Merc silica gel H, type 60) using a mixture of ethyl acetate and cyclohexane 8: 2 to elute. The properly determined fractions were collected and concentrated in vacuo to give the pure benzene ester as an oil in a yield of 160 mg.

Example 25

Preparation of clavulanic acid ester

3.3 kg of spray dried solids containing 69.4 g of clavulanic acid as determined by the enzyme inhibition assay were obtained as described in Example 26. The solid was slurried in 5.5 liters of dimethylformamide. 500 ml of SenzilStrimide are added, and the reaction mixture is stirred at room temperature for 2 hours; after addition of 12 liters of ethyl acetate, the solids were filtered off, and the filtrate was concentrated in vacuo to give 212% of an oily residue which was applied to a column and a 10 cm bed of 33 cm @ 3 (Hopkkns & Williams MFC) in cyclohexane. The column was eluted using 12 liters of cyclohexane to remove excess Senotilide, then ethyl acetate was used for further elution and 500 ml fractions were collected. These were tested for the cresyl avulanine benzyl ester content by plating onto a thin precoated silica rel 60 F 254 plate followed by spraying with 2,3,5-triphenyltrtruzole silicon rearens.

The intense red spot fraction fractions were further tested on a thin layer of silica gel using a 8: 2 mixture of chloroform and ethyl acetate to elute and spray the developed platelets using the above-mentioned fractions. This product was chromatographed on a 10 cm by 45 cm column of Merck silica rel H (type 60) using a mixture of chloroform and ethyl acetate. 8: 2 as solvents. The aqueous fractions were determined as described hereinbefore and concentrated to give 45.9% of an oil having a purity of 62% as assessed by NiM spectroscopy.

This product is then chromatographed. on a Sephadex LH 20 column of 7 cm by 45 cm in a 1: 1 mixture of cyclohexane and chloroform. After fraction determination and concentration, 27.6 g are obtained.

The colorless oil, which was found to be 95% pure benzyl ester, was clavulanic acid as determined by NMR spectroscopy.

Sephadex LH 20 is a hydroxypropyl derivative of Sephsdex Q25, Pharaacia, London, UK.

Example 26

Preparation of benzyl ester of clavulsic acid

150 liters of culture filtrate at pH 7.0 contained 16.2 sodium clavulanic acid as assessed by enzyme inhibition assays. This filtrate was stirred and 5 kg of Amberlyst A-26 anion exchange resin in a chloride cycle (Rohm & Hase 0 °, Philsdephia, USA), stirred for one hour, then the resin was filtered off, tested with the filtrate to remove The resin was washed with 20 liters of deionized water, followed by 20 liters of acetone and 10 liters of diethylformamide, after which the resin was suspended in 2.3 liters of a mixture of dimethylformamide with a 0.2 M solution after filtration. sodium iodide, and after adding 200 ml of benzyl bromide, the suspension is vigorously mixed thoroughly. After standing at room temperature for 16 hours, 2 liters of ethyl acetate are added, the resin is filtered off, the ethyl acetate is washed with ethyl acetate, and the washings are added to the original filtrate.

The extract obtained is concentrated to a small volume, and the residue is chromatographed on a silica gel column (Merck essential gel H, type 60) of 7.5 by 45 cm size using a 8: 2 mixture of ethyl acetate and cyclohexane as solvent. The fractions containing benzyl ester of clavulsic acid were evaluated by testing on thin-layer plates of silica gel 8 followed by spraying with 2,3,5-triphenyltetrazolium chloride reagent as described previously in Example 24. The correctly determined fractions were pooled, all concentrated to 20 ° C. ml and the residue is chromatographed on a silica gel column (Merck essential gel H, type 60) of 3.6 cm by 45 cm using a mixture of chloroform and ethyl acetate 8: 2 as solvent. Appropriate fractions were combined and concentrated to give 440 mg of a colorless oil which was judged to be 90% benzyl ester ester of clavulsonic acid by NMR spectroscopy.

Example 27

Preparation of clavulsinic acid benzyl ether from crude extracts of culture filtrate from Streptomyces clavuligerua

A portion of the aqueous butanol solution back extract from the culture filtrate as obtained in Example 14 was lyophilized. A 24 g solid contains 0.96 g of clavulsinic acid sodium salt as assessed by the enzyme inhibition assay. This solid was suspended in 75 ml of dry dimethylformamide, 75 ml of benzyl bromide was added, and the reaction mixture was stirred at room temperature for 2 hours. The suspension was diluted with 500 ml of ethyl acetate, and after filtration the filtrate was concentrated to a small volume of oily residue. on a rotary evaporator.

This residue is applied to a 5 cm by 35 cm silica gel column (biogel Biosil A) in cyclohexane, the benzyl bromide is eluted from the column, then the solvent is replaced with ethyl acetate, collected by elution with a 10 ml fraction. Those fractions containing benzyl ester of clavulsinic acid were evaluated as in Example 24. Further purification was also carried out as in Example 24 by column chromatography to give 220 mg of pure benzyl ester.

Example 28

Preparation of clavulanic acid sodium salt

⁴ COON 'cooch ₂ c ₆ h ₅

A solution of 281 mg of substantially pure benzyl ester of clavulanic acid in 25 ml of ethanol was hydrogenated to 90 mg of 10% palladium on carbon for 25 minutes at atmospheric pressure and room temperature in the presence of 82 mg of sodium bicarbonate. After filtering off the catalyst, it is also washed with water and ethanol, the combined filtration and washings are concentrated under reduced pressure, and the remaining semi-solid is triturated with acetone. After filtration and washing with ether, 135 mg of clavulanic acid sodium salt are obtained.

Example 29

Hydrolysis of clavulanic acid methyl ester to clavulanic acid

Dissolve 2.17 mg of methyl clavulanate in 0.1 ml of methanol, and add 0.208 ml of 0.0482 N sodium hydroxide solution. After standing at room temperature for one hour, the reaction mixture contains several products. According to thin-layer chromatography, it is clear that one of the major portions contains a substance equal to the sodium salt of clavulanic acid; color tests and biological evaluation are also consistent with this component being the clavulanic acid sodium salt.

Slow conversion of the clavulanic acid ester to the acid was observed when 1 mg / ml of compound was stored at 37 ° C, in 0.05 M phosphate buffer at pH 7. The reaction was monitored by paper chromatography (bioautographic system). Using a butyl alcohol / ethanol / water system to monitor the reaction over 2 hours, it was found that the methyl ester band of R ^0.79 changed as the clavulanic acid band of 0.12 increased.

Example 30

Antibacterial spectrum of clavulanic acid

Antibacterial activity of sodium clavulanic acid against numerous microorganisms was determined using the microtiter method. Serial dilutions of sodium clavulanic acid on a sensitivity test (Oxoid) in a microtiter plastic crucible were inoculated overnight by culture of each microorganism so that the final inoculum dilution was 0 5 · Ю “ ⁴ . The cultures were incubated overnight, and microorganism growth sites were recorded the next morning by observing the turbidity of the culture. The results, expressed as an approximate minimum inhibitory concentration of µg / ml, are shown in Table 3, and from this it is clearly seen that the compound is characterized by a broad spectrum of antibacterial activity.

Table 3

Antibacterial Spectrum · Sodium salts of clavulanic acid

Strain Minimum inhibitory concentration Hg / ml Staphylococcue sureua (Oxford H), Russeli Bacillus eubtilia Streptococcus faecalis Streptococcus pyogenes CN 10 Escherichia coli NCTC 10418 Klebeiella aerogenes Klebsiella oxytocum Enterobscter aerogenes T 624 Ihterobacter cloacee Acinetobacter anitratus Provideatia stuartii Serratia marceecens Proteus nirabilis C977 Proteus vulgaris W090 Salmonella typhimurium Shigella sonnei Pseudomonas aeruginosa A. 7.5 7.5 62 over 500 125 31 31-62 62 31 62 125 125 125 62 31 31 62 500

Example 31

Examples of inhibition of beta-lactamase by clavulanic acid sodium salt

Clavulanic acid successfully and irreversibly inhibits beta-lactamase from Escherichia coli. The method of description 1 shows. That other beta-lactamases - see Table 4 - are also inhibited by clavulanic acid.

Table 4

Inhibition of beta-lactams of clavulanic acid

Source of beta-lactamase Approximate value taken in to Bscherichia coli JT 4 = 1 Staphylococcus aureus (Russell) Escherichie coli JT4 Escherichia coli BI1 Klebeiella aerogenes Pseudomonas aeruginosa 1622 (R factor) Pseudomonas dalgeish 1.0 1.0 2,0 0.6 5.0 0.1

With peniciline · 0 as eubstrate, the clavulanic acid sodium salt against Staphylococcue aureus (Russeli) beta-lactamase is about 0.06 µg / ml.

Example 32

Examples of activities. meehhyaitar clavulanic acid

It is apparent from the tests of aatibacterial efficacy of the mash that the clavulanic acid is characterized by a broad spectrum of efficacy, but of a lower order compared to clavulanic acid. or the activity of clalanolaic acid is released by slow hydrolysis of the ester in water.

Clavulanic acid is characterized by a clear and bacterial synergism in combination with ampicillin or phthaltiiditis against bacteria resistant to these antibiotics. Also, the peak concentration of sphine versus Staurium (-3 µL) was reduced from 500 ps / ml to less than 0.4 in the presence of 1.0 µg / ml retty. 8 clavulanic acid. The minimum inhibitory concentration of β-oridim is reduced from 1.5 below 0.03 pg / mL per. . presence of 1 pg / ml clavulanic acid beta-ester.

Minimizing the concentration of phosphoridim. decreases from 1.5 µg / m 2. below 0.03 pg / ml per 1 / ug / ml clavulanic acid beta. The minimum inhibitory concentration of ampiiClit against Proteus ρΙγζΙ ^ Ι ^ C889 is reduced from 500 pg / ml to 31 µg / ml. in the presence of 15 µg / ml of methyl clavulanic acid. ,

Example 31

Preparation of Clavulanic Acid Pivaltyltiethylether

0.49 g of clavulanic acid sodium salt was added to a stirred solution of 0.37 g of tert-butyl tert-butyl ester in 5 ml of dry diethyltrimide, and the reaction mixture was kept at 2 ° C for 2 hours. dilute by adding 20 ml. ethyles ^^. acetic acid, ml cyclohexane and 20% water. The reaction mixture was stratified into 2 layers, nonaqueous. Separate the fraction, wash with 20 ml of water and sieve with sodium sulfate. Concentration of the dried filtrate gives the expected product as a pale yellow oil; 500 mg. NM-siktium in di-chloroform: 1.26 (s, 9), 3.13 (d, 1 J = 17 Hzz, 3.62 (dd, 1, J = 17 Hz), J = 2.5 Hz ), 4.3 (d, 2, J = 7.5 Hz), 5.0 (dt, 1, J »7.5 Hz, J = 1.5 te), 5.16 (d, 1, J = 1.5 Hz \, 5.79 (d, 1, J = 2.5 Hz), 5.92 δ (s, 2), infrared (liquid film), C 0 0 ⁸⁰⁰ cm ^-1, ester C = ^{0 1,760} cm ^-1 .

Example 34

Preparation of phthaldo ester. clavulanic acid

0.5 g of clavulanic acid sodium salt is added to a stirred solution of 0.43 3- &lt; 5 &gt; aldehyde in 5 ml of dry dimethylformamide, and the reaction mixture is left at rt _for 2 hours. The solution is then added with 20 ml of ethylene acetic acid, 10 ml of cyclohexane and 30 ml of water and mixed well, and the non-aqueous portion is washed with 20 ml of water, dried over sodium sulfate and concentrated to give a pale yellow gum. The two six-liter ester esters were separated using a high pressure liquid thitmbltlgium column (Merckosorb SI 60.5u) sized 40 by 10 cm, eluting with ethyl acetate at a flow rate of 3 Π per minute.

The first phthaldic ester (retention time 7.15 min) crystallizes from 11ν1 ‰ acetic acid in the form of needles of bt 102 ° and has the following infrared spectrum (in nujol): beta-lacamide C = ⁰ 1790 cm -1 ester C = 0 ¹ 755 cm ¹ . Nlffi-s? Ktrim in r rdede vinго acet acet acet acet acet acet acet acet 3 3: 3 ^, 14 L,

J (17.5 Hs), 3.76 (dd, 1, J = 17.5 Hz, J ₂ 2.5 Hz), 4.25 (d, 2, J = 7.5 Hz), 5.0 (dt, 1, J = 7.5 Hz, J = 1.5 Hz), 5.4 (s, 1, J = 1.5 Hz), 5 * 82 (d, 1, J = 2, 5 te), 7.7 (s, 1), 8.06 δ (m, 4); motetauLtvl weight by mass spectra: 331.07, what corresponds to the formula? 6 ^ 3? °? (calc. 331107). Second ěitstereottober (retention time 8.85 min), this worm infra231157 spectrum (in solution mettylencWortoovép-MTA-iaktim ° 0 1600 cm ^-1, 0 = 0 ester ^1,780 cm roffiRS ktrum ^ v ^ utorovittém cMoroformu): 2.42 ^{(ši.ro to} Y ^{P as,} s, 1-ionisable eclipse with d ₂ O), 3.12 (d, 1, J '16 Hz), 3.60 (dd, 1 = 16 Hz, J = 2.5 Hz), 4.30 (d, 2,

J = 7.5 Hz), 5.0 (dt, 1, J 1 = 7.5 Hz, J ₂ = 1.5 Hz), 5.12 (d, 1, J = 1.5 Hz ^ 5.76 (d, 1, J = 2.5 Hz), 7.52 (S, 1), 7.65 (m, 4)

Example 35

Preparation of clavularic acid

To 44 mg of clavulanic acid sodium salt was added 76 mg of nonyl iodine in 2 ml of dry dimethylformamide, and the reaction mixture was left at room temperature for 2 hours. Thereafter, the solution is concentrated, and the residue is fractionated on a column, eluting with ethyl acetate / hexane (2: 1); the product is obtained as an oil. IR (film): ¹ ° 8 ^{0, 1 745, 1 6} 90 cm ^- '. B nm spectroscopy: molar molecular weight = 325.19, which corresponds to the formula C 18 H 18 N 2 O 3 (calc. 325.19).

Example 36

Preparation of clavularic acid

A solution of 100 g of cyanobenzoic acid benzyl ester in 5 ml of ethanol is hydrogenated in the presence of 30 mg of 10% palladium on carbon for 45 at atmospheric pressure and stirring temperature. After filtering off the catalyst, it was also washed with ethanol, and the combined liquid fractions were concentrated in vacuo to give a volatile, experimental oil which was obtained in a yield of 56 mg. NlMR8ppktrum in pardaut (tartaric pyridine): 3.05 (d, 1, J = 16 Hz), 3.60 (dd, 1, J, = 16 Hz% J ₂ = 2.5 Hz), 4.75 (d , 2, J = 7.5 Hz), 5.56 (t, 1, J = 7.5 Hzz, 5.66 (s, 1), 6.0 δ (d, 1, J »2.5 Hz) ).

Example 37

Preparation of peehhУe8terl clavulan

A mixture of 130 mg of clavulanic acid and 10 ml was mixed with an excess of a solution of decumethanol in ether. Depending on the thin layer observation, the reaction is complete in 2 times. Concentration of the solution in vacuo affords a residue which is purified by chromatography on a column of silica gel, eluting with ethyl acetate. The fractions containing meetyypeter of clavulanic acid are pooled and concentrated to give 104 mg of a clear oil.

Example 38

Preparation of methyl ester of clavinic acid

A mixture of 200 mg of clavulanic acid and 5 ml was cooled to 0 ° with stirring, 0.5 ml was added. methanol and then 206 mg of dicyclohexylcarbodiimide, and the reaction mixture is stirred at room temperature overnight. The suspension is filtered, and the filtrate is bent under vacuum to give crude clavulsic acid, which is purified by chromatography on silica gel using ethyl acetate to elute. The product was isolated as a clear oil in a yield of 140 mg.

Example 39

Preparation of clavulanic acid phenyl ester

A mixture of 100 mg of clavularic acid and 5 ml of acetooliril is cooled to 0 ° with stirring, 0.94 g of phenol and 100 m of dicyclohexylcatbodiol are added thereto, followed by stirring at room temperature overnight. The suspension is filtered, the filtrate is concentrated, the residue is fractionated on silica gel, eluting with a 1: 1 mixture of ethyl acetate and hexane; 70 mg of clavulanic acid phenyl ester are obtained. Infrared (film) spectrum: 1,800 ^, 1,770 ^, 1,690 cm -1. NIM spectrum in ^d eutero wine cliloroformu: ^2, 18 ^{(Si to} S ^p гo ^as s, 1), 3.06 (dd, 1, J = 17 Hz, J ₂ »= 0,9 HO, 3.54 ( dd, 1, J = 17 Hz, Jg = 2.6 Hz), 4.29 (d, 2, J = 7.5 Hzz, 5.1 (dt, 1, J = 7.5 Hz), J = 1.5 HO, 5.29 (d, 1, J = 1.5 Hz), 5.76 (dd, 1, J, = 2.6 Hz, Jg - 0.9 Hz), 7.35 δ (m, 5) · Moleloil mass spectrometry = 275 * 08, which corresponds to the formula (calculated 275.08).

Example 40

Preparation of Clavulanic Acid 2,2,2-tetrotlottyl

221 mg of sodium vul kl ^ ^^ n ^ ew is suspended in 5 ml of dry tetra-i yrdrofurlos _s susetnse was stirred and cooled to 0 ° during 20 mime is added to the obtained suspension a solution of 211 mg tricУlotttУyltpttts acid in 1 ml of dry tetrahydrofuran, the temperature of the reaction mixture was then allowed to rise to 20 °, and the reaction mixture was stirred overnight. The pense was then filtered and the filtrate was dried to dryness in vacuo.

The residue is chromatographed eluting with a 2: 1 mixture of ethyl acetate and hexane to give the expected product, which is obtained as an oil. In ioftaačereoдéo ^ ektru (film) are absorbed in the ^{e-1 8 00} 1 760 1 69 ⁰ cm ^{'1. N} ] ^M -8pectrum in touterovirus chloroform: 1.56 (wide band S, 1), 3.07 (dd, 1, J, = 17.5 Hz, J * - 0.7 Hz), 3.56 (dd) 1, J = 17.5 Hz, = 2.5 Hz), 4.24 (d, 2, J = 7.5 Hz), 4.69 (d, 1, J = 12 Hz), 4.92 (d, 1, J = 12 Hz), 5.02 (dt, 1, J = 7.5 Hz, J g = 1.3 Hzz, 5.19 (d, 1, J = 1.3 Hz), 5.735 (dd, 1, J 1 = 2.5 Hz, J g = 0.7 Hz. Mechelear mass by mass spectrum = 328.96, which corresponds to the formula C CH ^N NO ^Cl Cl (calcd 328.96).

ČOONa

CICOOR r

CH ₂ OH

CO-O-COOR

COOR

P HILD 41

Preparation of tysalloy clavulanic soda salt

With 840 mg of lactic acid benzyluterium, 30 ml of ethanol and 5 ml of water, hydrogenated at room temperature under atmospheric pressure for 25 minutes in the presence of 267 mg of palladium on carbon and 244 mg of sodium bicarbonate. After filtering off the catalyst, it is also washed with water and ethanol, and the combined filtrates are heated under vacuum. The product crystallized from a mixture of water and acetone in the form of milk ^{to a} solid of 565 mg. Crystallization from water and acetone gives needles which, after drying over phosphorus pentoxide in vacuo (24 hours), have the following composition:

Found: C, 41.01; H, 3.77; N, 5.68

C, 40.86; H, 3.64; N, 5.51

Infrčervené Spectrum (l-bromo-oaniém rlse u ^{p l} m n ^{s): 1 7 65 1 70} 0 1 ⁶² 0 cm ^-1; Λ max: 3.06 (d, 1, J = 18.5 Hz), 3.57 (dd, 1, J, = 18.5 Hz, J ₂ = 2.5 Hz), 4.15 (d 2, J = 8 (f), 5.3 (HOD), 4.9 (m), 5.71 (d, 1, J = 2.5 Hz).

Example 42

The synergism of nicotinin and clavulanic acid sodium salt

The minimum inhibitory concentration of clavulanic acid sodium and ipppacillin in the presence of 1 µg / mL sodium tyselloylable sodium was determined for a large number of beta-lactamide irradiated microorganisms. The organisms are applied by inoculation to a mash of a sensitive test (Ccoodd, which is placed in a corrugated ripple on a plastic support and contains separate concentration gradients ο ^^ ΙΙο ^ sodium salt ^ sellny clavulanic or ιιπρ ^ ΗΙnu and 1 µg / pl sodium kllvullnvoé ( (Http: / / method ().

_2

The final negative dilution overnight corresponds to a concentration of Ι ^^ Ιι 0.5 · 10. The plate was incubated overnight at 37 ° C, and microbial growth endpoints were recorded the next morning. The minimum concentrations in µg / ml are shown in Table 5, and it can be seen that synergism at a low concentration of 1 µg / ml clearly increases the efficacy of the ppcd versus some of the Gram-positive and grap-negative microspheres. Probably can oyyvOttit this synergism by causing СоНСЬ ^ -п! beta-lactapaps that would disrupt the app of CHO, He cannot exclude the function of other mechanisms.

Similar results to Table 5 were obtained when αβcCllio was replaced by amooycilOnep or phthalic ester lmpiiClCl.

Table 5

AntibblktrtιϋLoí synergiamus α p piiCli ^ ns and sodium salt of claolinic acid

Strain The minimum concentration is / µg / ml sodium salt of clays and ^ ppiClin and? ppcClin in the presence of α 1 sodium salt of clavullinic acid Escheer.cMa coli-NCTC 10481 31 1,8 below 0.4 Echerichia coli B 11 62 over 500 125 and / or aerogenes 31 ' 125 below 0.4

Salla sp. 62

125 below 0.4 strain Minimum inhibitory concentration / µg / µl

clavulanic acid sodium salt ampicillin «Πρ ^ ϋ ^ in the presence of X jug / PL · clavulanic acid sodium salt Ennerobscter cloacae 62 250 62 Serratia εριτ88 ^ μ 125 over 500 62 Staphylococcus aureus 15 Dec 500 below 0.4 Staphylococcus aureus 62 methimicin) 250 7.5

Example 43

AnibaOkeei £ ILE eynergiemus cephaloridine and clavulanic acid sodium salt

The minimum inhibitory constants of cefaloridine, clavulanic acid sodium salt, and cephaloridine in the presence of 5 µg / µl clavulanic acid sodium salt were determined using the procedure of Example 42. The results are shown in Table 6 and show that there is synergy between sodium . salts of clavulanic acid and cephaloridine, especially for strains of Staphylococcus aureus (Russll), producing beta-ascamaa.

Table 6

Α ^ Ь ^ от ^ synergism between cephaloridine and clavulenic acid sodium

Strain Minimal inhibitory concentration / µ &lt; / RTI &gt; clavulanic acid sodium salt cephaloridine With 5 µg / µl sodium clavulanic acid froteus ad 899 na ^d 500 ⁺ * ^// 62 7.5 Staphylococius aureus (Riussn) 15 Dec 3.1 below 0,03 ^{+ /} Staptylococcus aureus (strain Resi-to-covalent ^{¼ '¼} / ⁱ řlsahužíi ^{bo d} ppehimicin) 62 15 Dec 3.7

♦ / the same value was achieved if the synergist was added in an amount of 1 µg / ml instead of 5 µug / pi <Example. 44

ΑΜβΜ ^ Η & ηί synogism between clavulanic acid sodium salt with various penicillins

The results are shown in Table - 7; were obtained using the procedure described in Example 42.

Table 7

Antibacterial synergism between clavulanic acid sodium salt and various penicillins against Klebsiella aerogenes strains

Strain Amoxyciliá Karb.nicilia ^{+ /} Benzylpenicillin alone * 5 Mg / ml synergistic agents alone * 5 Mg / ml synergistic agents alone * 5 pg / ml synergistic agents AND 500 0.97 500 7.6 250 7.8 E 70 500 3.9 500 15 Dec 500 15.6 62 250 15.6 125 7.8 250 15.6 “^ Similar The results were achieved with Use of phenyl-alpha- of an ester carbenicillin or tikareilin place carbenicillin. Example 1 a d 45

Antibacterial synergism of ampicillin and clavulanic acid esters

The results are shown in Table 8; were obtained using the procedure of Example 42.

Table 8

Antibacterial synergism of ampicillin and clavulanic acid esters against Klebsiella aerogenea strains

Strain Ampicillin as such Ampicillin * 5 µg / ml clavulanic acid methyl ester Ampicillin * 5 µg / ml clavulanic acid beazylester AND 500 1.9 1.9 E 70 500 • 3.9 3.9 62 500 3.9 3.9

Neither clavulanic acid methyl ester nor benzyl ester of clavulanic acid inhibits the growth of the test organism at a concentration of 100 µg / ml.

Example 46

Amtibacterial activity of clavulanic acid esters

Using the procedure of Example 30, but using a dilution of 1/100 zapers, the following minimum inhibitory concentration values - see Table 9 - of some clavulanic acid esters against these microorganisms were achieved.

Table 9 Antihakteriální efficiency esters of clavulanic acid Organism Minimal inhibitory concentration Min. inh. k. ^{+ /} esters of clavulanic acid sodium salts benzyl nonyl- pivaloyl- phthalidyl- acid kla- ester ester oxymethyleater ester vulanové Bacillus subtilis A. 250 31 62 125 62 Staphylococcus aureus Oxford 62 31 31 31 15 Dec Staphylococcus aureus Russeli Escherichia 125 31 62 15 Dec 15 Dec coli 10418 125 over 500 125 125 125

^+/- The minimum inhibitory concentration of clavulanic acid sodium salt is added for comparison; high minimum inhibitory concentration values (as compared to those of Example 30) are given using a heavy inoculum.

Example 47

Extraction of clavulanic acid using liquid ion exchange resin

200 ml of the culture filtrate obtained similar to Example 3, but using a medium containing 0.1% by volume of potassium phosphate instead of 0.01% of ferrous sulfate heptahydrate, is extracted for 30 minutes at 5 ° C with Amberlite LA2 *. in a chloride cycle, 15% by volume in methyl isobutyl ketone, 66 ml total. Amberlit LA2 is a product of Rohm and Haas, Croydon, United Kingdom.

The phases were separated by centrifugation (1660 g, 20 minutes) to give 60 ml of the solvent phase and this phase, withdrawn by pipette, was divided into 4 equal portions. Each was extracted with stirring at 5 ° C for 20 minutes using a quarter volume (3.75 mL) of an aqueous extractant as detailed in the following table. The resulting mixture was centrifuged (1660 g, 15 minutes), and 3.6 ml of an aqueous phase was obtained from each extraction.

Sample Volume (ml) Clavulanic acid concentration (pg / mT ¹ ) Clavulanic acid (mg) cleared mash 200 128 25.4 extracted mash 200 15 Dec 3.0 extract M NaCl 3.6 305 1, ’ 2 M NaCl extract 3.6 598 2.5 extract M NaNO 4 3.6 63Θ 3.3 extract 2 M NaNO 4 3.6 758 2.73

The result obtained with a 2 M sodium nitrate solution corresponds to an isoler of 43% of the clarified mash.

Example 48

Extraction of clavulsic acid using liquid ion exchange resin liters of clarified mash as obtained in Example 12 is extruded using Amberlit LA2 (acetate cycle, 15% v / v methylisobutyl ketone, 12.5 liters), with stirring at 17 ° C. After addition of 500 ml of 1-octanol, the phases are separated by continuous centrifugation to give 9.2 liters of the solvent phase, and the mixture is further stirred with cooling at 5 ° C for 90 minutes with 2.3 liters of sodium nitrate solution. The mixture is separated by continuous centrifugation to give 2.4 liters of an aqueous phase (this includes the water used for the make-up). The pH of the aqueous phase (originally 8.0) was adjusted to 7.0 by addition of concentrated hydrochloric acid.

Sample Volume (liters) Concentration Acid acid klavulové klavulové (mg)

tulle g / ml ¹ )

cleared mash 47 146 6 862 extracted mash 47 19 Dec 893 extract in M NaNO4 2.4 1 638 3 931 Extraction efficiency from cleared to extract in nitrate sodium corresponds to 57%.

Example A of p-bromobenzyl clavulanic acid

To 70 mg of clavulanic acid sodium salt in 1.0 ml dimethylformamide, 245 mg of p-bromobenzyl bromide was added, and the reaction mixture was left at room temperature for 2 hours. The solvent is distilled off under vacuum, and the residue is fractionated on a silica gel column eluting with a 3: 1 mixture of ethyl ester and hexane to give the expected product in the form of needles, mp 101-102 ° C. Infrared spectrum (CHCl3): 1,805, 1,750 and 1,695 cm @ -1. HMR-spectrum (CDCl3), 61 * 94 (1H, singlet, OH), 3.12 (1H, doublet of doublets, J = 17.5 and 0.7 Hz, 60-CH), 3.62 (H) , doublet of doublets, J »17.5 and 2.5 Hz, 6 '-CH), 4.32 (2H, doublet, J = 7.0 Hz, 9-CHg), 4.99 (1H, doublet of triplet, J = 7.0 and 1.5 Hz, = CHCH ₂ OH), 5.20 (1H, singlet, 3-CH), 5.28 (2H, ainglet. COOCHp, 5.81 (1H, doublet of doublets, J) = 2.5 and 0.8 Hz, 5-CH), 7.45 (2H, doublet, J ³ 8.5 Hz, aromatic protons), and 7.68 (2H, doublet, J ³ 8.5 Hz, aromatic protons.

EXAMPLE Clavulanic Acid N- (N, N-Dimethyl) -aminoethyl ester

(a) Silver salt of Clavuloic acid (all procedures described below are performed almost completely).

Sodium clavulanic acid tetrahydrate (2.93 g) in deionized water (6 ml) is mixed with a solution of 1.69 g of silver nitrate in deionized water (2 ml), the brownish-yellowish precipitate is filtered off in 5 minutes, washed with 2 ml of deionized water water, followed by acetone (10 ml) and vacuum drying to give 620 mg of clavulanic acid silver salt as a brownish-yellow solid. IR: _m (nujol) 1785, 1695 Unflex), and 5 ^{80 1} (S ^ ^P with mesh) cm ^-1.

b) Formation of ester

500 mg of clavulanic acid silver salt are added to 135 mg (0.6 equivalents of β - (N, N-diethyl) -a-ai-ethyl) -clerif in 3 ml of dimethylformamide, and the reaction mixture is stirred in the dark at room temperature. suspension diluted with 10 ml of ethyl acetate, filtered and the filtrate concentrated to dryness to afford a pale yellow oil which ^p ost ^ u ^ ^{p some} darkens on standing. ^I n ^f r ^No DURATION ^E ^ ktrum ^{(film): 1800,} 75 ^{1 0, 1 6} 95 cm ^-1;

NIM-spectrum (CIl)), δ 1.04 (triplet, J = 7.5 Hz, H2CH3CH3g, 2, 43-3.0 (mltlplet, -CH3CHgCl2), 3.07 (^ k) δ σ, J = 17 Hz, 6? -CH), 3.53 (doublet of doublets, J = 17 and 2.5 Hz, 6? -CH), 3.62 (broad singlet, OH), 4.32 (η 2), 9-CH 3 and COOCH 3 CH 3 N), 4.98 (broad triplet, J = 7 Hz, 8-CH), 5.12 (broad singlet, 3-CH), 5.76 (dubbet, J) = 2.5 Hz, 5-CH 2).

Example i

Clavulanic acid amino-2-ylmethyl ester

A mixture of 0.5 clavulanic acid sodium salt and 1.0 g of 9-chloromethylanthracene was stirred in femethylformamide overnight at room temperature, then the reaction mixture was partitioned between ethyl acetate and water, and the ethyl acetate solution was concentrated under reduced pressure. . Fractionation on silica gel eluting with ethyl acetate / hexane (1: 1) gave a yellow crystalline product in a yield of 0.5 g (57%), mp 120 ° C. Infrared (CHCl3): 1 805, 1 745 and 1 695 cm @ ^-1 . NIM spectrum (CD1 ₃ δ 1.35 (1H, broad band, OH), 2.90 (1H, doublet, J = 47 Hz, 6 / J-CH), 3.35 (1H, doublet of doublets, J = 17 and 2.5 Hz, 6a-CH), 3.96 (2H, doublet, J = 7 Hz, 9'CH 2 g, 4.61 (1H, broad triplet, J = 7 Hz, CH-C 4 OH) 4.98 (1H, singlet, 2-CH), 5.52 (1H, doublet, J = 2.5 Hz, 5-HH), 6.13 (2H, singlet, COOOHh), 7.2- 8.5 (9H, muutiplet, aromatic protons).

Example E

N-Benzoxxzooonnymethhyeeser clavulanic acid

To a solution of 0.6 g of clavulanic acid sodium salt (in the form of tetrahydrate) in 6 ml of N, N-diethylforeaeide is added at room temperature and stirring with 0.4 N-chloromethylbenzone xlonlonone; After 4 hours, the solvent was distilled off in vacuo and the residue was partitioned between 25 ml of ethyl acetate and 25 ml of water. The ethyl acetate layer was dried over anhydrous sodium sulfate and concentrated to dryness to give an oil which was purified by column chromatography on silica gel using cyclohexane and ethyl acetate to elute. After separation of the solvents, 0.5 g of product is obtained in a foamed form. Infrared (film), 3. 400 (Si ^ ^ y says a belt, OH), 1790 (br, fly-lactam C = 0 both ester), ^{1695 cm} @ ^-1 (C = CC. NJM-S ^ ktrum: ^{1.6 (1} H , Greek sin ^g years, interchanged with ^D 2 O ^, CH 2 H) ^{, 3.0} (1H, doublet, u = 17 Hz, 6- / 7-CH), 3.44 (1H, doublet of doublets, J = 17 Hz, J = 3 Hz, 6- and -CH, 4.08 (2H, doublet, J = 7 Hz, CH3 OH, 4.79 (1H, triplet, J = 7 Hz, CH), 5.02 ( 1H, singlet, 3 &apos; CH), 5.60 (1H, doublet, J = 3 Hz, 5-CH), 5.86 (2H, singlet, CH2 --N), 7.12 (4H, broad singlet). C ^ H),

The other sample, prepared non-swellable, solidifies on standing and then has a melting point of 95 ° C. Infrared SPE ^to trum (Nujol ^ ^{3 5 6 ^ 3 490 1 70} 5 1 750 and ^{1 690} c ^m '.

Example F

Clavulanic acid 2- (eenzylbenzyl) 22benzzloxylbenzyl

O

\ /

COOCChCC ^

NHCOOCH2C ₆ H ₅

COOCH2C6H ₅

In dry tetrahyerofuran, 199 mg of glacial acid is cooled to 0 DEG C. and 660 mg of N-beneyloxyl-carbonyl-Lenzylester-dicyclohexyl-cyclodilatyl ester is added.

The reaction mixture was allowed to stand when the temperature reached 20 ° C and left to stir overnight, by distilling off the solvent under reduced pressure and fractionating the residue on a silica gel. The residue was eluted with ethyl acetate / hexanes in hexane

108 mg of the product as an oil.

Infrared ktruo s ^ (^ ΟΗ) ^{1 1 760 -1 2} 7 5 cm. NM-S ^ ^ ktiuio CCDCl) δ 2 ² (1Щ br, OH), 2.94 (1H, doublet, J = 17 Hz, 6 C = CH), 3.33. (1H, doublet of doublets, J = 17-2.5 Hz, 6µ-CH), 4.99 (1H, singlet, 3-CH), 5.10 (2H, singlet, SO 2) / δ, 5.18 (2H, sin / flight, COOCHhHcH _5), 5.45 (1) dubbet, J = 2.5 Hz, 5-CH), 5.81 (1H, br dubbet, J = 8Hz, NH), 3.35 (10H, sin / Let, 2 y (m).

The protons appear to be complex in the 4.1 to 4.9S region.

Example G

3-Уyoorcopyee8ter pack acidLL ^ 010vé

NM-ssektrum (CIDCj 2.03 (2H, mžtip0et, CH ₂ C 1 H> CN), 2.50 (2H, triplet, J = 6 Hz, HHgCn, 2.68 (1H, br, OH), 3.05 (1H, doublet, J = 17.5 Hz, 60-CH), 3.55 (1H, doublet, j = 17.5 = 2.5 Hz, 6a-CH), 4.1 to 4.5 ( 4H, muLipbet, ΗΟΟΗ) and CH 3 OH), 4.93 (1H, triplet, J = 7.5Hz, 8-CH), 5.13 (1H, singleton, 3-CH), 5.72 (1H, Dubbet, 2.5 Hz, 5-CH 2).

2.9 g of sodium clavulanic acid are dissolved in 50 [mu] l of dimethylformaoide and 2.2 g of n-bromobutyric acid nitrile are added at room temperature. The reaction mixture is stirred at room temperature for 3 hours, then the sample is tested by thin layer chromatography on silica gel. developing ethyl ester of acetic acid; significant ester formation is observed (Ry = 0.6).

The reaction mixture is worked up in the usual manner, i.e. by extraction into ethyl acetate and acetic acid by wiping the organic layer with water, drying the mixture with silica and then removing the solvent until a yellow oil remains. According to the N1-M-Spray, the product contains an excess of unreacted nitrile, so that the chloroform is spread, and the solution is applied to a column of 20 g of silica 060 followed by elution with CH 2 D 2 (rm 2). The product was taken together with concentration in vacuo and the expected product was isolated as a pale yellow product. The infrared spectrum and the NM spectrum were consistent with the product structure, containing a trace of solvents.

Example H

O-Tritylrthrr benzyl ester of clavulanic acid

Α D 20, zCHCH, OH ^{0 =} Cl x / XOON.

XK ^ CHCH ₂ OH COOCHj ^ - / ^ - OC (C _t H _{s) 3}

From maternal temperature, 17.1 g of clavulanic acid sodium salt tetrahydrate are dissolved in 500 ml of dimethylformamide and then 23 g of p-tritylatharbutylbromide (vsLPL crude product) are added. The solution is stirred for one hour at a temperature of 1 to 10 ml, and further worked up in the usual manner by extraction into ethyl acetate, etc. The product is isolated by column chromatography to 400 g of sL10kagslt G60 using chloroform and methanol. hexane (1: 1).

The product thus obtained is still ultraviolet-sensitive impurities, but not from the acid group, and further chromatographic purification was performed on 40 g of sL10kagslt 060 using acetic acid / n-hexane (1: 1) to elution. Thus, a satisfactory product was obtained. The NMR spectrum is consistent with the indicated structure: in CDCl 3 - δ 2.94 (1H, doublet, J »17 Hz, 6 β -CH), 3.40 (1H, doublet dubbet, H = 17 and 2), 5 Hz, α-CH), 4,16- (2H, doublet, J = 6.5 Hz, CH 2 O 4), 4.83 (1H, triplet, J = 6.5 Hz, 8-CH), . '4.99 (3H, - Wide - sing ^ t С00 <Ш ₂ - and 3-CH), 5.61 (1H, ditbat, J - 2.3 Hs, - 5-CH 6,6- 7.7 (m / m, salmpsic - protons) Infrared spectrum in nujol: 1,805, 1,747, and 1,695 cm ^-1 .

Example I

Е ^ охху ^ Ьу ^ с ^ г О ^ еИлу 01avulsmlve

To a solution of 5.0 g of clavulanic anhydride sodium salt in 50 ml of dry dimethylformamide was added, with stirring, 2.14 g (1.5 ml) of ethyl alcohol and stirred for 30 minutes while stirring at the temperature of the milliotrope. Water and ethyl acetate were added to the residue. The solution in the solvent was dried over anhydrous sodium sulphate, and the filtrate was concentrated to give an oil which was chromatographed on sL10ksgslt using cyclohexam / sthylstsat (1: 1). k elulván. The fractions containing the product by TLC analysis were combined, concentrated under reduced pressure, to give 2.98 g of pale yellow oil. Infrared spectrum (film) ^Vmax * ^'3400 (br), 1800, 1746, 1698 cm. * ¹ OTM spectrum (CDCl3), [delta]: 22 (3H, triplet, J * 7 to, CH2), 2.94 (1H, doublet, J &lt; 17 &gt; Hz, 60-CH), 3.13 (IH, singlet) , OH), 3.50 - (1H, doublet of doublets, J = 17 and 3 Hz, 6 .alpha.-CH, 3.68 (2H, quadrulet, J · 7 Hz, CH2Cl3, 4.16 (2H, doublet) J, 7 Hz, 9-CH3), 4.91 (IH, triplet, J = 7 Hz, 8-CH, 5.06 (IH, elnglet, 3-CH, 5.29, 5.37 (2H)) , ABkovadruplt, J »6 Hz, CH ₂ C ₅ H ₅ ) and 5.68 (1H, J 3 Hz, 5-CH,

Example J

2-bromo-stearate-1-amino acid

To a solution of 2.05 g of the sodium salt of acid in 5 ml of dipethylfordapide was added 1 ml of 1,2-bromoethane, and the reaction mixture was stirred at room temperature for 7 days. Thereafter, most of the solvent was removed under vacuum at 30 ° C, water was added to the residue, and the mixture was extracted into ethyl acetate. The solution obtained in this solvent is washed with water, dried over magnesium sulphate and, after filtration,

The product was isolated by column chromatography on silica gel using ethyl acetate.

Yield 168 mg.

3.49 (2H, triplet, J = 7 Hz), 3.48 (1H, doublet of doublets, J = 3 and 17 Hz), 4.18 (2H, doublet, J = 7 Hz), 4.43 (2H, triplet, J = 7Hz), 4.93 (1H, triplet, J = 7Hz), 5.05 (1H, fine coupling singlet), 5.67 (1H, doublet, J ^with 3Hz).

Example К

Phenacylester of clavulanic acid

OH

COOCH ₂ COC _e H _p

0.34 g of phenscyl bromide is added to a solution of 0.5 g of clavulanic acid sodium salt hydrate in the smallest possible amount of dimethylformamide, and the reaction medium is stirred at room temperature for 3 hours. Then the solvent is distilled off in vacuo and the residue is dissolved in ethyl acetate. The solution in the solvent is washed with water, dried over magnesium sulphate, and the filtrate is concentrated to give 400 mg of phenacyl ester as a solid, mp 89-90 ° C. Infrared (film): 3450, 1800, 1760 and 1700 cm ^-1 . NMB-Spectrum (CDCl3), δ 3,02 (1H, doublet of doublets, J »17 Hz · 1 H2, 6 Д-СН), 3,65 (1H, doublet of doublets, J = 17 and 3 Hz, 6« - CH), 4.20 (2H, doublet, J = 8 Hz, _9-CH2), 5.00 (1H, doublet of triplets, J '8Hz, »CHCH ₂ OH), 5.23 (1H, broad singlet 3-CH), 5.58 (2H, singlet, CH ₂ COOC ₆ H ₅ ), 5.74 (1H, doublet of doublets, J = 17 and 1 Hz, 5-CH), 7.7 (5H, multiplet Molecular weight (mass spectroscopy):

calculated: 317.09 found: 317.09 analysis: calculated: 60.57% C, 4.73% H, 4.42% N found: 60.23% C, 5.00% H, 4.40% N

Example L

Clsvulanic acid 2-pyridylmethyl ester

To a solution of 1.17 g of clavulanic acid sodium hydrate and 1.01 g of 2-pyridylmethyl bromide hydrobromide in 8 ml of dimethylformamide was added 552 mg of potassium carbonate, and the reaction mixture was stirred overnight. After diluting with an equal volume of ethyl acetate, the solution is filtered, the filtrate is concentrated and the residue is extracted into ethyl acetate, this process repeated, and the product is then purified by chromatography on silica gel eluting with ethyl acetate / ethyl acetate.

Yield: 335 mg · IR (film): 3450, 1805, 1750 and 1695 cm ^1st NMR Spectrum (CDCl3): δ 2.48 (1H, broad, interchangeable band), 3.01 (1H, doublet, J = 17 Hz), 3.45 (1H, doublet of oak, flight, J = 17 and 3) Hz), 4.19 (2H, doublet, J = 7 Hz), 4.91 (1H, triplet with fine coupling, J = 7Hz), 5.1 (1H, singlet, with fine coupling), 5.25 (2H, singlet), 5.66 (1H, doublet, J = 3 Hz), 7.1-7.4 (2H, multiplet), 7.67 (1H, triplet doublet, J = 1 and 1 Hz), 8.52 (1H, doublet, J = 6Hz).

Example Μ

C-N-Methyl-C-N-methyl-clavulanic acid ⁴ ΟΟΟΟΗ ₂ ΟΗ ₂ ΟΗ ₂ Ν ^

CH ₃

OOOCH ₂ C _e H ₅ g bennyCesteru clavulanic acid is hydrogenated přítomnnoti 400 mg of 10% palladium on iího uxlí in 30 ml THF until no detectable ester. Then, the solution is filtered and used as described below:

^- the solution, ^3, 71 ^{g of N-rneehyíL-N'-nitroso-N, t cl} OX -dibeoz ^yk Arbon ^{oC -l,} 2-d ^{i h} amiooet AOU ¹⁰ 'mt d ^ t ^ J ^ ^ l.ft The reaction solution was cooled to -30 ° C, 0.71 g of pyrimidine was added, followed by stirring the reaction solution at -25 to -30 ° C for 30 minutes, cooled to -35 ° C. To this reaction mixture is added the above-prepared clavulanic acid solution with stirring so that the temperature of the prepared diazo compound solution does not rise above -30 [deg.] C. The reaction mixture is stirred for a further 15 minutes, 0.5 ml of thorium fluoride atherate is added. After stirring for 5 minutes, an excess of sodium hydrogencarbonate solution is added, the aqueous portion is separated and extracted with methylene chloride. The combined solutions in methylene chloride are washed with water and dried over magnesium sulphate and concentrated. in ethyl acetate, eluting with chlooom and ether gave 180 mg of a colorless gum.

Infra-rvené spe ^ rim (tfLta) 3450 ^{1800 1 7} 45 ¹ 60 cm "'. OT-spectrum (CID &lt; + &gt;): [delta] 2.25 (1H, broad band, single, exchanged ^), 2.93 (3H, single ^, 3.00 (1H, dudee)), 3.3-3.6 (5H, η ^ αρίβ ^, 4.17 (2H, pip, J 37 Hz), 3.85 (1H, triplet, J → 7 Hz with fine k ^ I ^ p ^ ui ^ ai), 4.98 (1H, singlet), 5.08 (2H, single *), 7.28 (5H, single · *) ·

Example N

Clavulanic acid, 1-acetyloxycarbonyl

To 5 ml of dry dimethylformamide, while cooling to 0 ° C, add 1.025 g of clavulanic acid sodium salt hydrate, then, in 10 minutes, 1 ml of 1-bromomethyl acetic acid. The reaction mixture is allowed to stand overnight in the refrigerator, then The solvent was distilled off in vacuo, water was added to the residue, and the reaction mixture was extracted with ethyl acetate. The organic phase was washed with water, dried over magnesium sulphate and concentrated after concentration to give the residue. 60 and acetic acid using ethylene; he laments. thus 176 mg of the compound whose composition is given in otdpPse, R? 3 · 0.49, ^I ofrtčtov · ^s range of 3 ^{1 81} 0 ^{1 770} and ^{1695 cm} ''. @ 1 H NMR spectrum .delta. 1.47 and 1.49 (3H, 2 d, J = 5 Hz, first isomer mixture of ethyl), 2.04 (3H, singlat), 3.01 (1H, doublet, J 17 Hz), 3.45 (1H, doublet of doublets, J 17 and 3 Hz Hz 3.15 (1H, wide and lockable band), 4.17 (2H, duHei, J = 7 Hz), 4, 80 (1 H, muHiplet), 4.98 (1 H, fine puff singles), 5.63 (1 H, bag, δ J 3 3 Hz, 6.78 (1 H, quadruplet, J 3 5 Hz).

Example O 1-EthoxyOttObralooSehlS · stcr acid was calculated for the first time (a) chloimyric acid A mixture of 108.5 g of ethyl chloimrossioate and 0.5 g of alpha-tert-butyl ester was stirred under reflux; a stream of dry chlorine is passed through the solution until a weight gain of 34.5 g (olefolding monachlorohydrate) is obtained, and then the liquid is concentrated under atmospheric pressure to give 33 g of 1-illcoctlslestcot illrxoric acid of b.p. 117-118 ° C. b) Ethyl S-1-Cl-ethylsulfonic acid 4 H, 3 g of the product of step (a) herein above is added dropwise to 50 ml of ethanol; the temperature is then spontaneously raised up to 62 ° C. After the temperature has dropped to 30 ° C, 200 ml of water are added to precipitate a heavy oil which is decanted. The aqueous phase was extracted three times using 50 ml L k The extracts are combined and, after drying with sulphate, the filtrate is concentrated. The residual oil was distilled at e atm. pressure to give 12.1 g of the expected compound at 155-157 ° C. The structure was confirmed by NM-spectrum L elemental analysis results: calculated for C5H9ClO3: 39.4% C, 5.9 iH, 23.2 Cl found: 39.5 Ca 6.2% H and 23.25 Cl c) 1- ^{(Etlcxskιu:,} boonSooχ) eChlSeзter k ^ i ^ i ^ lL ^ ny ^ ew put solution of 3.04 g of the product ee step (b) in 5 ml of dimethylfomamide are added to a stirred solution of 5.86 g of sodium and kltvulancvě 500 mg of potassium potassium in 15 ml of methylformamide. After 21 hours, the reaction mixture was diluted with 250 mL of acetic acid, washed with saturated aqueous sodium bicarbonate, dried over magnesium sulfate, and concentrated to an oil. This was purified by filtration on LH 2 O and scrubbing with chloroform and cyclohexane (1: 1) to give 206 mg of a pale hollow oil. Thin layer chromatography and the use of a 1: 1 chloroform / cyclchexane system gave a single spot. The structure of this compound was confirmed by NM-sppetry. EXAMPLE P Methanol of Klalalanic Acid In 20 m of dimethylrormamide, a span of 2.93 g (0.01 mol) of sodium salt of Klalalanic Acid was spanned. Λ and cooling, stirring, was added 0.8 ml, i.e. 0.86 g of illoilimethyl glycol. After two hours of reaction at room temperature, the limethyl bromide was removed under vacuum, and the residue was dissolved in a mixture of 40 ml of ethyl acetate and 20 ml of cyclolexant. 7 g silL- > of acetic acid gel and 20 ml of cyclolextin. ... ' Add 7 g of sulfuric acid for thin layer chromatography, insoluble materials are filtered off, washed with a small amount of fresh solvent, and set aside (not needed). The filtrate was flooded under reduced pressure to a small volume and the residue was chromatographed on a column using acetic acid and cyclohexane to elute. The product was isolated as a colorless oil výtěiek: 2 g. IR spectrum: ^u _{and x} (film) 1802 (C = 0-lactam), 1755 (C = 0 of ester), 1695 < »(C = C ). O

NMR Spectrum (CDCl3): 2.3 (1H, broad singlet, OH), 3.02 (1H, doublet, J &lt; 17 &gt; Hz, 6- / 9 -CH),

3.42 (3H, singlet, OCH3), 3.47 (1H, doublet of doublets, 17 Hz, 3 Hz, 6-a-CH), 4.16 (2H, doublet,

J 6 Hz, CH ₂ OH), 4.89 (1H, broad triplet, J 6 Hz, CH = CH ₂ ), 5.03 (1H, broad singlet,

3-CH), 5.21, 5.29 (2H, AVquadruplot, J> 5.5 Hz, C 2 OCH 4, 5.65 δ (1H, doublet, J 3 Hz),

5-CH3).

Example Q Clavulanic acid p-chlorophenoxyethyl ester

To cold (January and water cooling) and stirred solution of 6 g (0.02 mol) klsvulsonic acid sodium hydrate in 60 µl of diethylfomanide was added 3.5 g (0.02 nol) of p-chlorophonoxymothyl chloride, after cooling 2.5 hours to a temperature below 10 ° C and the diethylfomamide was distilled off at high temperature (20 ° C) under reduced printing under high pressure. 200 ml of ethyl acetate and 20 ml of chilled water are added to the remaining syrup. The solution in ethyl acetate is dried over sodium sulphate after separation, concentrated to a syrup and the residue is chromatographed on a silica gel column using ethyl acetate with cycloxane gradient from 1: 2 to 2: 1 to elute. First, a portion of the clavulanic acid member of p-chloro-phenoxymethyl ester, and then a major portion of 4.7 g of pure c-chlorophenoxymethyl ester of clavulanic acid, is obtained. This product contained a large amount of dlen, detectable by thin layer chromatography in an amount sufficient to be detected in the NMR spectrum.

IR: 3400 (br, OH), 1805 (C = 0 groups of beta-lactam), 1765 (C ^S O ester group), 1695 cm ^-1 (C = C).

NMR Spectrum (CDCl3): 1.5 (OH, 3.00 (1H, doublet, J% 17 Hz, 6- β-CH)), 3.44 (1H, doublet of doublets, D x 17 → 3 Hz, 6-a-CH), 4.14 (2H, doublet, J = 7 Hz, CH 2 OH), 4.77 (1H, broad triplet, J x 7 Hz, N ₂ N ₂ ), 5.02 (1H , broad singlet, 3-CH), 5.60 (1H, doublet, J = 3 Hz, 5-CH), 5.70, 5.77 (2H, AB-quadruplet, J χ 7 Hz, OCH ₂ O), 6 , 89, 7.218 (4H, AVquadruplet, J χ 9 Hz, с ₆ н ₄ ) ·

Example R

Clavulanic acid benzyloxynethyl ester

2.93 g of sodium clavulanic acid tetrahydrate are dissolved in 20 µl of dimethylformamide, and the cloudy solution is filtered. The clear yellow filtrate is cooled in January, and stirred with the dropwise addition of 1.57 g of benzyl chloromethyl ether prepared by Graham and McQuillin. J. Chem. Soc. 1963. 4634) Stirring and cooling is maintained for 2.5 hours, and then the reaction mixture is concentrated to give an orange oil which is extracted several times into ethyl acetate. Concentrating the extract in the solvent yields a yellow oil.

When analyzing the thin-layered oil, silica gel eluting with 1: 1: 2 ethyl acetate / ethyl acetate / methyl ethyl clopentane to induce permanganate spots shows 2 bands of R? 0.21 and 0.55, about the same intensity. The oil is chromatographed on a silica gel column, eluting with a mixture of ethyl acetate and cycloxane (1: 1). The composition of the eluted fractions is analyzed by thin layer chromatography, and the slower moving substance is collected in a pure state, free of any detectable impurities. 0.7 g of an almost colorless oil is collected by concentration.

NMR Spectrum (CSCl 3): $ $ 2.08 (1H, broad singlet), 3.02 (1H doublet, J = 16 Hz), 3.3β (1H, doublet of doublets, J 16 16 Hz, J «3) Hz), 4.15 (2H, doublet, J = 7 Hz), 4.64 (2H, singlet),

4.87 (1H, broad triplet, J = 7 Hz), 4.99 (1H, broad sioglet), 5.34 (2H, sioglet), 5.58 (1H, doublet, J = 2 Hz), 7, 25 (5H, broad sioglet).

Example S '

Preparation of 0 &quot; Ombogyozyloxy teethhlebter1 kebuwontic acid

2.93 g of sodium clavulanic acid tetrahydrate are dissolved in 20 ml of diethylforeaeide and the cloudy solution is filtered. The clear yellow solution was cooled in ice, and 2.02 g of theatree-ethyl ether was added dropwise to the cooled solution with stirring. This reagent was prepared as described by D. N. Kursanov and P. A. Solodkov, Ž. Prikl. Chim. 6, 351 (1943) of the English edition.

The reaction mixture was stirred for 2 hours, and the progress of the reaction was monitored by thin layer chromatography on silicon waste plates. eluting with a 1: 1: 2 mixture of ethyl acetate, ethyl acetate, and ethylcyclopropane (1: 1). At the end of the reaction time, the main product of clavulanic acid β-olefin-teethhleste is Rp = 0.18 with a small proportion of ether esters.

(R p = 0.35). The solvent was separated, and the crude reaction product was purified by chromatography on a silica gel column eluting with a mixture of cyclohexane and ethyl acetate (5: 4). The product is obtained by concentrating the appropriate fractions from flash-boiling, initially forming a yellow oil, crystallizing.

X-ray powder diffraction (CuK), reflection at 20

29.95.

NMRЗSPktгue (CDL ₃ j: ^ £ 1 5 (1H, br sioglet), 3.01 (1H, duet, J = 16Hz), 3.42 (1H, doublet duet, J = 16 Hz, 3 Hz), 4 14 (2H, doublet, J = 7Hz), 4.73 (2H, sioglet), 4.85 (1H, broad triplet, J = 7Hz), 4.97 (1H, sioglet), 5.39 ( 2H, Sioglet), 5.59 (1H,

J = 3 Hz), 7.75 (4H, doublet of doublets, J = 62 Hz, 7 Hz).

Example T

Allyl ester of clavulanic acid

COONa

COOCH2CH-CH2

To a stirred solution of 2.05 g of clavulanic acid sodium tetrahydrate in 9 ml of dry diethylphosphate was added 2.44 ml of ethyl acetate, the reaction mixture was stirred at room temperature for 3 hours, and the solvent was distilled off under vacuum at 30 ° C. Deň: 32 ° C. The residue was partitioned between water and ethyl acetate, the ethyl acetate layer was washed with water, dried over anhydrous magnesium sulfate, and the filtrate was concentrated. The residue is purified by chromatography on a column of 60, eluting with ethyl acetate, Rp = pH = 0.53. This gives 1.31 g of the title compound as an oil.

IR (film): 3 -400, 1805, 1745, 16195th mш-bsoktr1m (CDC., _90ΜΗζ β): 61.98 (1H, zaeeěOtelnУ sioglet), 3.02 (1H, doublet, J = 17 Hz ), 3.46 (1H, doublet of doublets,

J = 17 Hz = 3 Hz), 4.19 (2H, J = 7 Hz), 4.61 (2H, doublet of doublet),

J = 6 Hz), 4.88 (1H, triplicate with triplicate, J = 7 Hz), 5.02 (1H, triplicate with triplicate); J = 6 Hz), 5.64 (1H, J = 3 Hz),

5.7 - 6.1 (1H, mutiplet).

Example U

Acetonnleater of clavulanic acid

O

CH ₂ OH

COONa

O

'COCHjCOCHj

To a stirred solution of 2.05 g of clavulanic acid sodium salt hydrate in 9 ml of dry diethyl laanide is added 2.35 m. Of bromoacetone, the reaction mixture is stirred at room temperature overnight, and then the solvent is removed in vacuo at not more than 30 ° C. The residue was partitioned between water and ethyl acetate. The solution in the organic solvent is washed with water, dried with magnesium sulphate and the filtrate is bent. The ester was isolated by silica gel column chromatography 60, eluting with ethyl ester ester acetic acid. *

This gives 565 mg of the title compound, j = 0.36.

IR (film): 3400, 1600, 1755, 1730

NiR-spectrum (CDCl 3 90 Miz) δ 2.14 (3H, singlet), 2.20 (1H, broad ^ Λ ^ ΜβΙ ^), 3.04 (1H, dubbet, 17 Hz ^ 3.46 (1H, dubbet) doublet, J = 17 Hz and 3 Hz ^ 4.20 (2H, fine copping dubbet, J = 6 Hh), 4.7 (2H, singlet), 4.97 (1H, triplet dubbet, 6 Hz, H ^) u aei 1.5 HO, 5.12 (1H, slng .et e fine), 5.67 (1H, duHet, J * 3 Hz).

Example V

Clavulanic acid cyclohexyl ester

⁴ COOH 'coo

CH 2 OH <3

At 0 [deg.] C., 0.62 [mu] l of pyrrolidine is added to a solution of 1,4-N-ethuxycarbonyl-N-nitro-xocyclohexylemine in 25 ml of dichloromethane. Said material is prepared by reacting D 2 O with N-e-carbonylcarbocyclohexyluminium in carbon tetrachloride from the presence of anhydrous sodium acetate. The reaction mixture was stirred at 0 ° C for 30 minutes, and a solution of the gaseous acid in tetrahydrofroline prepared by hydrogenation of the calytic acid benzyl ester (1 g) and 30 ml was added to this solution over 5 minutes. The price for using palladium bed on charcoal. To the resulting solution was added an excess of sodium bicarbonate solution, the reaction mixture was extracted several times into chloroform, and the solution in chloroform was concentrated after drying with magnesium sulphate. Repeated chromosilicate on silica gel ^л

Two times using 1: 1 ethyl acetate and cyclohexane and once using chloroform, 46 mg of the title compound is obtained. <

^ fratarve ^ spe ^k thum ^{(1 m)} : ^{3 40} 0 1 ⁸⁰ 0 1 ⁷⁴ 0 ^{1 695} cm \ ^NR -spe ^ko rum ^ DC1 ^): δ 1,1-2,0 (11H, тИОрЫ, 1 hydrogen exchangeable), 3.01 (1H, doublet, J = 16 Hz), 3.45 (1H, doublet of doublets, J = 16 Hz = 3 Hz), 4.17 (2H, duWet, J = 7 Hz), 4 , 5-5.0 (1H, trans),

4.87 (1H, triplet, J = 7 Jz), 4.96 (1H, singlet), 5.63 (1H, duWet, J = 3 Hz).

Example W

Clavulanic acid propargyl ester

To a solution of 4.4 g (0.0150 mol) of sodium clavulsinic acid tetrahydrate in 25 L of dry dimethylformamide was added dropwise slowly and with stirring 1.6 ml (2.53 g, 0.021 2 mol) of propargyl bromide. The reaction mixture is stirred at room temperature for 2 hours, then poured into 250 ml of water, and the product is extracted three times into 100 ml of ethyl acetate each time. The solution in this solvent was washed four times with water (100 ml each), dried and magnesium sulfate, and concentrated to a clear and dry solution in ethyl acetate to give a thick oil which was judged to be a thin layer of silica gel using ethyl acetate to develop. 2 ingredients.

The oil was dissolved in 2 mL of chloroform, and the solution was chromatographed on sephadex LH 2 O using a 1: 1 mixture of chloroform and cyclohexane to elute. The 25 ml fractions were tested by thin layer chromatography, and those fractions containing the faster moving component in substantial amounts were combined and concentrated in vacuo to a thick oil which was isolated by 2.2 g. The oil solidified overnight, and only one component when tested by thin layer chromatography. It contains some chloroform by NMR spectrum.

The product was washed with ether to give 700 mg of a solid, m.p. Mp 65-66 ° C. The low yield is affected by the considerable solubility of the product in ether. The results of the various tests are as follows:

Analysis:

H, 4.68; N, 5.91. Found: C, 55.55; H, 4.68; N, 6.12.

NMR Spectrum (CDCl3): 2.01 (ainglet, 1H), 2m6 (triplet, 1H), 2.95-3.75 (multiplet, 2H), 4.29 (doublet, 2H), 4.79-5 .00 (multiplet, 3H), 5.13 (doublet, 1H), 5.75 (doublet, 1H).

Example X

Clavulanic acid 6-methoxynaphth-2-ylmethyl ester

OCOONa

ОСН ₃

A solution of 2.2 g (10 mmol) of clavulanic acid anhydrous sodium and 2.51 g (Ю mmol) of 6-mothoxy-2-bromomethylnaphthalone was stirred in 80 ml of dry dimethylformamide at room temperature in an environment; after 3 hours, thin layer chromatography indicated that the reaction was complete and the mixture did not contain the bromomethylnaphthalene derivative. At 30 ° C, the solvent is distilled off in vacuo, 100 ml of ethyl acetate are added to the residue, the suspension is filtered, and the filtrate is washed with water (2 x 25 ml). After drying and filtering the magnesium sulphate used and distilling off the solvent, 3.2 g of an orange oil are obtained, and chromatographed on 100 g of sephadex LH20, eluting with a 50:50 mixture of chloroform and cyclohexane and collecting 10 ml fractions. ester as determined by thin layer chromatography to induce TTC-stained stains (a solution of 4% triphenyltetrazolium chloride in methanol with the addition of an equal volume of N-sodium hydroxide in methanol).

Concentration of the fractions gave a clear oil which solidified on standing. This material was dissolved in dechlomethane and petroleum ether (b.p. 60-80 ° C) was added until the solution became cloudy. Upon standing in the beaker, a gum was precipitated, the liquid over this material was allowed to evaporate further to give a white crystalline solid, m.p. 89-90 °, yield 2.52 g, i.e. 68 ° C.

Analysis:

pr C _{20 H) 9 6} vypoSteao HO: 65.04% C, 5.15% H, 3.79% Я Alza: 64.91% C, 5.00% H, 3.77% N

NBR ep.ktrua (CDC1 _3): 6 1.45 (IH, broad .iaglet, CHgOH), 2.9 (IH, dubl.t, 6β-H), 3.36 (IH, dubl.t doublets, 6αH), 3.8 (3H, eiag, t, OCH3), 4.03 (2H, doublet, CH3 OH), 4.7 (1H, triplet ==), 4.93 (IH, eiag) t, CHCOO-), 5.27 (2H, eiag, t, -CH ₂ OCO), 5.65 (1H, doublet, 5 -H), 7.37 (6H, multiplet, aromatic hydrogen).

Example X

Clavulanic acid 2-methoxyethyl ester

(a) 800.2 clavulanic acid sodium salt

CH ₃ OCH ₂ OH ₂ OH ---- ► CH ₂ OCH ₂ CH ₂ I as a tetrahydrate

b) MAX I

COOCH ₂ CH ₂ OH

To a solution of 75 g (0.50 mol) of sodium iodide in 500 ml of acetone was added 43 g (0.455 mol)

2-Methoxyethyl chloride (see J. Amer. Chem. Soc. 52, 653, 1930), and the reaction mixture is heated under reflux for 3 to 4 hours. Most of the acetone was distilled off under normal pressure, the residue was cooled and diluted with 200 ml of ether. The ether solution is freed from inorganic impurities by filtration, washed successively with 100 ml of sodium chloride solution, 100 ml of 5% aqueous sodium nitrate solution, drunk using 100 ml of sodium chloride solution and then dried over magnesium sulphate. After filtration, the ether is filtered off at normal pressure, the product is isolated by ethyl acetate collecting a fraction of m.p. 130 to 140 [deg.] At the pressure sto-opere. The product, which is a thick, pale-yellow liquid, is obtained in a yield of 20 g (23%, assuming 100% purity).

To a solution of 6 g (0.020 5 ml) of clavulinsic acid sodium hydrate in 100 ml of ethyltriethylamine at room temperature was added 5.6 g (0.03 mol) of 2-methoxyethyl ether, the reaction mixture was stirred for 5 hours and then detected by TLC. layer, ie d ^ ΐ ^: Lt. to some ester formation. Chlorosolutions were carried out on a thin layer of silica gel with ethyl acetate aspiration, the layers being developed by spraying with TTCC reagent (see J Example X), while the sodium tulsulsic acid remained at the start (R = 0.0), and R i.

g of ester is 0.5.

The reaction mixture is diluted with 200 ml of ethyl acetate and 500 ml of water, the aqueous extract is extracted into 100 ml of ethyl acetate, the combined organic extracts are washed four times with water, 100 ml each, followed by 100 ml of N sodium hydrogencarbonate solution. , and again with water using 3 100 ml slides. The solution was dried over magnesium sulfate, and after filtration, the solvent was removed under reduced pressure. This yields an ester in the form of a pale yellow oil, which is dried under high vacuum, thereby removing the last traces of solvent and unreacted Jtd. According to NM-speetre, the isolated compound is substantially pure. Yield: below 5 6.

NlMR commanded microns (CIDC _3): delta 5.65 (1H, Dubeč), 5.05 (1H, singlet) and 4.9 (1H, triplet), overlap each other, furthermore 4.2 (4H, mutipeet ), 3.6 (2H, mutippet), 3.35 (3H singlet), overlaps

3.3 (1H, multielβ) with 2.9 (1H, singlet).

Claims (2)

SUBJECT OF THE INVENTION A process for the production of clavulanic acid, corresponding salts and esters of the general formula I (I) wherein R represents a hydrogen atom, a cation such as sodium, potassium, magnesium, aluminum, ammonium or substituted ammonium, such as the trimethyl ammonium cation, a cation derived from benzatene, p-kalnene and the like salts which usually form penicillins and phosphetettins; Alcohol or silver, furthermore R represents a C1-12 alkyl group, optionally a halogen atom, a phenylalkyl group, a nitrophazyl group, a C1-3 alkylcarbonyl phenyl group, a C1-4 alkylphenyl group and a C1-3 alkylphenyl group. an alkoxy group, a halophenyl group or a pyridyl group, a naphthyl radical or an optionally substituted methyl group, an anthracenyl group, an alkoxy group having 1 to 4 carbon atoms, an optionally further substituted phenoxy or benzyloxy group, an alkylthio group and 1 to 4 carbon atoms, aminoacid, optionally sub - substituted and (C1 -C4) -alkyl or benzyloxycarbonyl, furthermore carboxyl, optionally in the form of an aoli or ester, furthermore a C2 -C5 acyloxy group, a phenylsulfonyl or nitrile group, a phthalimido group or an N-benzoxyzolonyl group, followed by an alkoxycarbonyloxy group with 1 and wherein R is a lower alkenyl group of from 2 to 4 carbon atoms or a lower alkynyl group of 2 to 4 carbon atoms, a cycloalkyl group and 3 to 7 carbon atoms, a phenyl or phthalidyl group, an optionally or aubstituted methoxy group, by cultivating a strain of Streptomyces clavuligerus n with a nutrient medium containing nitrogen and carbon sources, including the necessary mineral salts, aerobically at a temperature of 15 to 40 ° C, usually 20 to 35 ° C, and preferably 25 to 30 ° C, and at a pH of from 5 to 8, preferably from 6 to 7.5, and then isolated from the culture of the acid to clavulanic or its alo after cell filtration by extracting the culture filtrate into a solvent after adjusting the pH to the acidic region, or adsorbing the product to charcoal, aging, salting out or molecular filtration, whereupon the clavulanic acid or its salt is converted by diesoalkalysis by 1 to 12 carbon atoms or an alcohol of general formula R-OH, where R is as defined above, or with a thiol in the presence of a condensation facilitating agent, to the corresponding ester, or treated with a compound of formula I wherein R is any of said cations R - Q, where R is as defined above and Q is a residue that is readily cleaved and replaced in this reaction, such as a chlorine, bromine or iodine atom, or a sulfonic acid ester residue, such as methanesulfonic acid or p-toluenesulfonic acid residue, or an active ester a group such as a formyl or trifluoroacetyl group; and optionally a ester of a compound of formula I wherein R is an alcohol residue is hydrolysed. 2. The method of claim 1, wherein the Streptomyces clavuligerus strain is cultured ATCC 27064 or the corresponding mutant.