DERIVATIVES

BACKGROUND OF THE INVENTION

The present invention relates to novel cepha¬ losporin derivatives and their crystalline form, pro¬ cesses for producing them, and to pharmaceutical compositions containing them for treating and/or pre¬ venting infectious diseases.

Developments of cephalosporin derivatives have been remarkable. Some cephalosporin derivatives have been developed which have excellent antibacterial activity against Gram-negative bacteria including Pseudomonas aeruginosa. However, the antibacterial activity of these cephalosporin derivatives is rather poor against Gram-positive bacteria. Several cepha¬ losporin antibiotics have been used for the treatment of Gram-positive bacteria infections and the increase of Gram-positive bacteria resistant to cephalosporin antibiotics, for example, methicillin-resistant Staphylococcus aureus (MRSA) , has become widely known year by year.

From the foregoing background, it has been desired to develop, cephalosporin derivatives having a strong

antibacterial activity against Gram-positive bacteria while retaining a sufficient antibacterial activity against Gram-negative bacteria including Pseudomonas aeruσinosa.

SUMMARY OF THE INVENTION

An object of the present invention is to provide novel cephalosporin derivatives and salts, solvates, salts of solvates thereof and crystalline form of them.

Another object of the present invention is to pro¬ vide processes for producing novel cephalosporin deri¬ vatives and their crystalline form.

A further object of the present invention is to provide compositions for preventing and/or treating infectious diseases which comprise novel cephalosporin derivatives as active components.

A further object of the present invention is to provide intermediate compounds in the synthesis of cephalosporin derivatives and processes for producing such intermediate compounds.

The present invention is based on the selection of a triazolopyrimidine ring as substituent at the 3-position of the cephem skeleton, and a 2-carboxy- 4,5-dihydroxyphenylmethyloxyimino moiety as substi¬ tuent at the 7-position of the cephem skeleton.

The compounds of the present invention containing these substituents have a wide antibacterial soectrum

against Gram-negative bacteria including Pseudomonas aeruσinosa and Gram-positive bacteria including methicillin-resistant Staphylococcus aureus. These compounds are extremely useful for the treatment of infectious diseases.

The trisodium salt of the compounds of the present invention is generally an amorphous solid and possesses an excellent solubility which is strongly required for parenteral use. But the stability of the salt is not necessarily sufficient for the phar¬ maceutical uses as bulk materials or as parenteral preparations. As a result of extensive studies to improve the stability of the compounds, the present inventors have now discovered that highly pure and stable crystalline form of the compounds of the pre¬ sent invention can advantageously and consistently be prepared and isolated.

DETAILED DESCRIPTION OF THE INVENTION

As a result of extensive investigations concerning development of cephalosporin derivatives having a satisfactory antibacterial activity against Gram- negative bacteria including Pseudomonas aeruσinosa and

also having strong antibacterial activity against Gram-positive bacteria, the present inventors have found that cephalosporin derivatives represented by the general formula (I) as shown below satisfy these requirements and, have accomplished the present inven¬ tion.

The present invention is based on the selection of a triazolopyrimidine ring as substituents at the 3-position of the cephem skeleton, and of a 2-carbo- xy-4,5-dihydroxyphenylmethyloxyimino moiety as substi¬ tuents at the 7-position of the cephem skeleton.

The present invention is directed to cephalosporin derivatives represented by the general formula (I):

and salts, solvates and salts of solvates thereof? wherein R ¹ represents a hydrogen atom or an amino-pro-

tecting group, R ² and R ³ independently represent a hydrogen atom or a carboxy-protecting group, R ¹ * repre¬ sents a hydrogen atom, a hydroxy group, an amino group, a sulfo group, a carboxy group or a protected carboxy group, R ⁵ represents a hydrogen atom, a methyl group, a carboxy group, a protected carboxy group, a carboxymethyl group or a protected carboxymethyl group, and the bond shown with a wavy line represents a bond of anti-form or syn-form.

The present invention is also directed to crystalline form of a cephalosporin derivative repre¬ sented by the formula (I ¹ ):

and salts, solvates especially hydrates and salts of solvates thereof.

The present invention is also directed to the pro—

cesses for preparing above-mentioned cephalosporin derivatives and their crystalline form. The present invention is further directed to pharmaceutical com-' positions for treating and/or preventing infectious diseases characterized by containing these cepha¬ losporin derivatives as active components.

In the cephalosporin derivatives of the present invention represented by the general formula (I), it is known that the aminothiazole moiety as the substi¬ tuent at the 7-position thereof exhibits tautomerism as shown below:

wherein R ¹ , R ² and the bond shown with a wavy line have the same significance as defined above. In the

present invention, the aminothiazole moiety is expressed as including both isomers since both are generally deemed to 'be the same substance. Accordingly, the compounds of the present invention represented by the general formula (I) also include both of these tautomeric isomers.

The compounds represented by the general formula (I) may form base or acid addition salts. Typical examples of base salts of the compounds represented by the general formula (I) include pharmaceutically acceptable salts such as alkali metal salts (sodium salts/ potassium salts, etc.), alkaline earth metal salts (calcium salts, etc.), salts of organic bases (ammonium salts, benzylamine salts, diethylamine salts, etc.), and salts of amino acids (arginine salts, lysine salts, etc.). These salts of the com¬ pounds may be mono-salts, di-salts or tri-salts. In the case of mono-salts or di-salts, the salts may be salts of the carboxy group at the 2-position and/or salts of the carboxy or sulfo group contained in the substituents at the 3-position, and/or salts of the carboxy group in the acyl group at the 7-position, of the cephem skeleton.

Typical examples of acid addition salts of the com¬ pounds represented by the general formula (I) include pharmaceutically acceptable salts, such as salts of inorganic acids (hydrochlorides, hydrobromides, sulfa- tes, phosphates, etc.), salts of organic acids (acetates, citrates, maleates, tartarates, benzoates, ascorbates, ethanesul onates, toluenesulfonates , etc.), and salts of amino acids (aspartates, glutama- tes, etc.). These salts of the compounds may be mono- salts or di-salts. In the case of mono-salts, the salts may form in the aminothiazole ring as- substi¬ tuent at the 7-position of the cephem skeleton or in the triazolopyrimidine ring as substituent at the 3-position of the cephem skeleton.

The compounds represented by the general formula (I) may form solvates especially formic acid solvates or hydrates.

The compounds of the present invention represented by the general formula (I) may be present as a syn- isomer shown below:

wherein R ¹ and R ² have the same significance as defined above; or as an anti-isomer shown below:

wherein R ¹ and R ² have the same significance as defined above; or as a mixture of these isomers. Among them, the syn-isomer is particularly preferred and, mixtures mainly composed of the syn-isomer are also preferred.

In the compounds of the present invention repre¬ sented by the general formula (I), the amino-pro- tecting groups may be selected from acyl groups such as for yl, acetyl, chloroacetyl, t-butoxycarbonyl, benzyloxycarbonyl, etc.; or aralkyl groups such as benzyl, diphenylmethyl, triphenylmethyl, etc. Tri ethylsilyl group may also be used as an amino- protecting group. The carboxy-protecting groups may be selected from alkyl esters such as methyl ester, ethyl ester, t-butyl ester, etc.; or aralkyl esters such as benzyl ester, diphenylmethyl ester, ^' triphe¬ nylmethyl ester, etc.; or trialkylsilyl esters ^" such as trimethylsilyl ester, etc. Inorganic or organic bases may also be used as carboxy-protecting groups. Collectively taking account of various operations, synthesis of thus protected products, and conditions for the removal of protecting groups, it is preferred to use a triphenylmethyl group as the amino-protecting group and a diphenylmethyl group as the carboxy- protecting group.

The compounds of the present invention represented by the general formula (I) can be produced as follows. Namely;

Process A

The compounds of the present invention represented by the general formula (I) can be produced by reacting compounds represented by the general formula (II):

wherein R ³ , R ¹ * and R ⁵ have the same s ignificance as def ined above , with compounds represented , by ' the general formula ( III ) :

wherein R ¹ , R ² and the bond shown with a wavy line have the same significance as defined above, and R ^s

and R ⁷ are the same or different and independently represent a hydrogen atom or a hydroxy-protecting group or together represent a isopropylidene group.

If necessary and desired, the compounds repre¬ sented by the general formula (II) may be converted into reactive derivatives at the amino group thereof.

The compounds represented by the general formula (II) may be reacted with the compounds represented by the general formula (III) using suitable condensing agents, such as N,N'-dicyclohexylcarbodiimide, N-ethyl-5-phenylisoxazolium-3'-sulfonate, etc. Alter¬ natively, the compounds represented by the general formula (III) may be converted into appropriate reac¬ tive derivatives prior to the reaction with the com¬ pounds represented by the general formula (II). The appropriate reactive derivatives may be, for example, acid halides (acid chlorides, etc.), azides, acid anhydrides, particularly mixed acid anhydride with strong acids, active esters (N-hydroxysuccinimide ester, etc.) or active amides ( i idazolide, triazo- lide, etc. ) .

The reaction between the compounds represented by the general formula (II) and the compounds represented by the general formula (III) may be carried out

by the general formula (III) may be carried out generally in an inert organic solvent such as dioxane, tetrahydrofuran, 'acetonitrile, chloroform, methylene chloride, ethyl acetate, dimethylformamide, etc., if necessary and desired, in the presence of deacidifying agents. The reaction may also be carried out in an aqueous solution, preferably in the presence of deaci¬ difying agents. As the deacidifying -agents, triethy- lamine, diethylaniline, and the like may be used in the organic solvent system, and aqueous alkalis, pre¬ ferably sodium hydroxide, sodium hydrogen carbonate, potassium carbonate, and the like may be used in the aqueous system.

The reaction may be carried out at temperatures ranging from about -30°C to room temperature, and pre¬ ferably from -10°C to 10 ^β C. Under the condition described above, the bond represented by a wavy line in the general formula (III) is retained.

The compounds represented by the general formula (II) used in the process of the present invention can be prepared by the method described in the Japanese Patent okai 142987 (1985). The compounds represented fay the general formula (III) can generally be prepared

by the methods D, E and F described below.

If necessary and desired, the protecting groups may be removed from thus obtained cephalosporin deri¬ vatives represented by the general formula (I).

Process B

The compounds represented by the general formula (I) can be produced by reacting compounds represented by the general formula (IV):

wherein R ¹ , R ³ , R , R ⁵ and the bond shown with a wavy line have the same significance as defined above, with compounds represented by the general formula (V) :

wherein R ² , R ^s and R ⁷ have the same significance as defined above, and X represents a halogen atom or a hydroxy group. When the compounds represented by the general formula (V) are alcohols, they may be either reacted directly with the compounds represented by the general formula (IV) in the presence of appropriate condensing agents such as triphenylphosphine or diethyl azodicarboxylate, or converted into appropriate reactive derivatives such as tosylate and then reacted with the compounds represented by the general formula (IV). However, with regard to reac¬ tivity and operability, halides are preferred for the compounds represented by the general formula (V) to be reacted with the compounds represented ^" by the general formula (IV) .

The reaction between the compounds represented by the general formula (IV) and the compounds represented by the general formula (V) may be carried out in an inert organic solvent such as dioxane, tetrahydro- furan, acetonitrile, chloroform, methylene chloride, ethyl acetate, dimethylformamide, etc. or mixture thereof, or, if necessary and desired, in water or mixture of water and organic solvents, preferably in

the presence of deacidifying agents. As the deaci¬ difying agents, triethyla ine, diethylaniline and the like may tie used in organic solvents, and aqueous alkalis, preferably sodium hydroxide, sodium hydrogen carbonate, potassium carbonate and the like may be used in aqueous solvents.

The reaction between the compounds represented by the general formula (IV) and the compounds represented by the general formula (V) may be carried out at tem¬ peratures ranging from about -30°C to room tem¬ perature, and preferably from -10°C to 10°C. Under the condition described above, the bond represented by a wavy line in the general formula (IV) is retained.

The compounds represented by the general formula (IV) can be prepared by the method described in the Japanese Patent Application 249193 (1984).

The compounds represented by the general formula (V) , in the case of halide form, can be prepared from 4-methylcatechol by the following procedure; firstly, protection of the hydroxy groups, then halogenation of the 5-position by a conventional method, then replace¬ ment of the halogen atom with a carboxyl group, then protection of the carboxyl group by esterification.

and finally halogenation of the benzyl terminus by a conventional method. Then, if necessary and desired, the resultant halides can be hydrolyzed to give hydroxy form of the compounds represented by the general formula (V) .

If necessary and desired, the protecting groups may be removed from thus obtained cephalosporin deri¬ vatives represented by the general formula (I).

Process C

The compounds represented by the general formula (I) can be produced by reacting compounds represented by the general formula (VI)

wherein R ¹ , R ² , R ³ , R ⁶ , R ⁷ and the bond shown with a wavy line have 'the same significance as de ^' fined above.

and Y represents an acetoxy group or a halogen atom, with compounds represented by the general formula

wherein R ¹ * and R ⁵ have the same significance as defined above. The reaction can be carried out by reacting the compounds represented by the general for¬ mula (VI) with the compounds represented by the general formula (VII) in an organic solvent such as alcohols, dimethylformamide, acetonitrile, etc. or mixture thereof, or in an aqueous system. The reac¬ tion of the compounds represented by the general for¬ mula (IV) and the compounds represented by the general formula (V) may be carried out in an organic solvent, preferably in the presence of Lewis acid, such as boron trifluoride-ether complex and the like, or in an aqueous system in the presence of an appropriate amount of aqueous alkali, such as sodium hydrogen car¬ bonate or potassium carbonate, preferably in a buffer solution at a pH in the range of 6.0 to 7.8, at tem¬ peratures in the range of about 40°C to about 80°C,

preferably at from 55 to 65°C. Under the condition described above, the bond represented by a wavy line in the general formula (VI) is retained. The com¬ pounds represented by the general formula (VI) can be prepared from the compounds represented by the general formula (III) and known 7-aminocephalosporanic acids or derivatives thereof by conventional condensation reaction. The compounds represented by the general formula (VII) can be prepared by the method described in the Japanese Patent Kokai 142987 (1985).

<

If necessary and desired, the protecting groups may be removed from thus ^' obtained cephalosporin deri¬ vatives represented by the general formula (I).

The compounds represented by the general formula (III):

wherein R ¹ , R ² , R ^s , R ⁷ and the bond shown with a wavy line have the same significance as defined above, can conveniently be prepared from the compounds repre¬ sented by the general formula (VIII)

wherein R ¹ , R ² and the bond shown with a wavy line

have the same significance as defined above, and R ⁸ represents a hydrogen atom or a carboxy-protecting group.

The compounds represented by the general formula (VIII) can generally prepared by the methods described below.

Process D

The compounds represented by the general formula (VIII) can be produced by reacting the known compounds represented by the general formula (IX):

wherein R ¹ , R ^a and the bond shown with a wavy line have the same significance as defined above, with the compounds represented by the general formula (V ) :

wherein R ² and X have the same significance as defined above. When the compounds represented by the general formula (V) are alcohols, they may be either reacted directly with the compounds represented by the general formula (IX) in the presence of appropriate condensing agents such as triphenylphosphine or ethyl azodicar- boxylate, or converted into appropriate reactive deri¬ vatives such as tosylate and then reacted with the compounds represented by the general formula (IX). However, with regard to reactivity and operability, halides are preferred for the compounds represented by the general formula (V ) to be reacted with the com¬ pounds represented by the general formula (IX).

The reaction between the compounds represented by the general formula (IX) and the compounds represented by the general formula (V ) may be carried out in an inert organic solvent such as dioxane, tetrahydro-

furan, acetonitrile, chloroform, methylene chloride, ethyl acetate, dimethylformamide, etc. or mixture thereof, or, if necessary and desired, in water or mixture of water and organic solvents, preferably in the presence of deacidifying agents. As the deaci¬ difying agents, triethyla ine, diethylaniline and the like may be used in organic solvents, and aqueous alkalis, preferably sodium hydroxide, sodium hydrogen carbonate, potassium carbonate and the like may be used in aqueous solvents.

The reaction between the compounds represented by the general formula (IX) and the compounds represented by the general formula (V ) may be carried out at tem¬ peratures ranging from about -30°C to room tem¬ perature, and preferably from -10°C to 10°C. Under the condition described above, the bond represented by a wavy line in the general formula (IX) is retained.

The compounds represented by the general formula (V) can be prepared from 2,2,6-trimethylbenzodioxol by the method described above for the compounds repre¬ sented by the general formula (V) .

If necessary and desired, the protecting groups may be removed from thus obtained compounds repre-

sented by the general formula (VIII).

Process E

The compounds represented by the general formula (VIII) can be prepared by reacting the known compounds represented by the general formula (X):

wherein R ¹ and R ⁸ have the same significance as defined above, with the compounds represented by the formula (XI) :

wherein R ² has the same significance as defined above, in an organic solvents, such as methanol, ethanol.

dioxane, tetrahydrofuran, methylene chloride and ethyl acetate, and if necessary and desired, in the presence of dehydrating agents such as molecular sieve, at tem¬ peratures in the range of from about -30°C to about 100°C, preferably from -10°C to 30°C.

The compounds represented by the general formula (XI) can be prepared from a halide form of the above- mentioned compounds represented by the general formula (V ) by phthalimidoxylation followed by dephthaloyla- tion, both of which can be carried out conventional methods.

If necessary and desired, the protecting groups may be removed from thus obtained compounds repre¬ sented by the general formula (VIII).

Process F

The compounds represented by the general formula (VIII) can be prepared by reacting the known compounds represented by the general formula (XII):

OH r

N

II ( XII )

2-CH ₂ C C C00R ⁹ O

wherein R ⁹ represents a hydrogen atom or a carboxy- protecting group, and Z represents a halogen atom, with the compounds represented by the general formula (V) :

wherein R ² and X have the same significance as defined above, by the method same as above-mentioned process D, then by condensing the product with the thiourea derivatives represented by the general formula (XIII):

wherein R ¹ have the same significance as defined above. Under the condition described above, the bond represented by a wavy line in the general formula (XII) is retained. With regard to reactivity and operability, it is desirable to react an alcohol form of the compounds represented by the general formula (V ) with the compounds represented by the general formula (XII) .

In the present process, the condensation with the thiourea-derivatives can be carried out in an organic solvent such as methanol, ethanol, dioxane, tetra¬ hydrofuran, methylene chloride and ethyl acetate, or mixture thereof, and preferably in the presence of deacidifying agent such as triethylamine, diethylani- line, sodium ,hydrogen carbonate and potassium car¬ bonate, at temperatures in the range of from about -30°C to about 100°C, preferably from -10°C to 30°C.

If necessary and desired, the protecting groups may be removed from thus obtained compounds repre¬ sented by the general formula (VIII).

The trisodium salt of the compound of the present invention represented by the formula (I ¹ ) is generally an amorphous solid. The salt possesses an excellent solubility which is strongly required for parenteral use, but the stability of the salt is not necessarily sufficient for the pharmaceutical use as bulk materials or as parenteral preparations. As a result of extensive studies to improve the stability of the compound, the present inventors have now discovered that highly pure crystalline compound of the present invention represented by the formula (I ¹ ), (6R,7R)-7- [2-(2-amino-4-thiazolyl)-2-[Z-[(2-carboxy-4,5-di- hydroxyphenyl)methyl] oxyimino] acetamido] -3-[(2- carboxy-5-methyl-s- riazolo[1,5-a] pyrimidin-7-yl)- thiomethyl]-8-oxo-5-thia-l-azabicyclo[4.2.0]oct-2- ene-2-carboxylic acid, can advantageously and con¬ sistently be prepared and isolated. In particular, the present inventors have prepared and isolated crystalline hydrated ( 6R,7R)-7- [2-(2-amino-4-thiazol- y1)-2-[Z-[(2-carboxy-4,5-dihydroxyphenyl)methyl] oxy-

iminolacetamido]-3-[(2-carboxy-5-methyl-s-triazolo- [1,5-a]pyrimidin-7-yl)thiome hyl]-8-oxo-5-thia-l-aza- bicyclo[4.2.0Ioct-2-ene-2-carboxylic acid. This new crystalline hydrated compound is superior not only in its crystallinity and increased purity but is also obtained in high yields and has increased stability on storage. These properties of the crystalline material render it of particular value in pharmaceutical use.

The above hydrated crystalline material was characterized by its infrared spectrum and for X-ray powder diffraction pattern.

IR spectrum (Nujol): v _maχ 3600 - 2200,- 3275, 1769, 1652, 1596, 1542, 1521, 1519, 1307, 1272, 1190, 1160, 1103, 1064, 1026, 964, 901, 854, 795, 770 cm" ¹ . X-ray diffraction pattern [given as d spacings in Angstrom units and percentage intensities] :

d ( A) I ( ) d ( A) I ( % )

19.11 55 3.79 74

14.34 11 3.66 44

9.48 48 3.56 73

8.73 13 3.36 47

7.19 37 3.22 22

6.27 82 3.15 13

5.73 58 3.11 26

5.34 20 2.85 28

5.21 28 2.75 12

4.78 77 2.62 13

4.53 42 2.49 10

4.26 25 2.12 13

3.97 100 1.98 16

The crystalline hydrated compound represented by the formula (I ¹ ) can be prepared conveniently from a solution of a solvate or a salt of the said compound. Thus, according to a further embodiment of the inven¬ tion we provide a process to prepare crystalline hydrated (6R,7R)-7-[2-( 2-amino-4-thiazolyl)-2-[Z-[(2- carboxy-4,5-dihydroxyphenyl)methyl] oxyimino] acet- amido]-3-[(2-carboxy-5-methyl-s-triazolo[1,5-a]- pyrimidin-7-yl)thiomethyl]-8-oxo-5-thia-l-azabicyclo- [4.2.0]oct-2-ene-2-carboxylic acid by adjusting the pH of a solvate (acetic acid solvate, formic acid solvate, etc.), an acid addition salt (trifluoroacetic acid salt, etc.) or a base salt (trisodium salt, etc.) of the said acid in an appropriate aqueous medium to a pH in the range of from 1.0 to 4.0, preferably from

1.5 to 2.5, and at a temperature in the range of from -40°C to 80°C, preferably from 15°C to 50°C. The adjustment of pH may be performed by the use of an inorganic acid, such as hydrochloric acid, sulfuric acid or phosphoric acid, or a mixture thereof. If necessary and desired, crystallization may be per¬ formed using a seed crystal, and recrystallization may be carried out in an appropriate acidic solvent in the conventional way. The solvent may contain water, lower aliphatic alcohols such as methanol, ethanol, isopropanol, aqueous carbonic acid, lower aliphatic carboxylic acids such as formic acid, acetic acid, organic amides such as N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, ethers such as monoglyme, diglyme, tetrahydrofuran, or a mix¬ ture thereof. Mixed solvents such as 50% acetone- water or 30% methanol-water may also be used. Non-toxic salts at the carboxyl groups of the com¬ pounds represented by the formula (I ¹ ) may include inorganic salts such as alkali metal salts (sodium salts, potassium salts, etc.), alkaline earth metal salts (calcium salts, etc.), amino acid salts (lysine salts, arginine salts, etc.) and organic base salts

(procaine salts, phenylethylbenzylamine salts, diben- zylethylenediamine salts, diethanolamine salts, N- methylglucosamine salts, etc.), most preferably sodium salts. Other non- oxic salts may include acid addition salts formed with, for example, hydrochloric acid, hydrobro ic acid, sulfuric acid, nitric acid, formic acid and trifluoroacetic acid.

In the preparation of crystalline hydrated form of compound represented by the formula (I'), (6R,7R)-7- [2-( 2-amino-4-thiazolyl)-2-[Z-[(2-carboxy-4,5-di- hydroxyphenyDmethyl]oxyimino] acetamido]-3-[( 2- carboxy-5-methyl-s- riazolo[1,5-a]pyrimidin-7-yl)- thiomethyl] -8-oxo-5-thia-l-azabicyclo[4.2.0] oct-2- ene-2-carboxylic acid, its formic acid solvate is especially preferred as the starting material. The formic acid solvate itself can also be crystallized, and the crystallized solvate as well as the method to prepare the said crystallized solvate are also included in the present invention.

The crystallized formic acid solvate of the com¬ pound represented by the formula (I ¹ ) was charac¬ terized by its infrared spectrum (and for X-ray powder diffraction pattern) .

IR spectrum (Nujol) : v-. _ax 3600 - 2200, 3269, 1770, 1654, 1596, 1517, 1509, 1303, 1188, 1159, 1101- 1061, 1025, 962, 904, 853, 794, 770 cm" ¹ .

X-ray diffraction pattern [given as d spacings in Angstrom units and percentage intensities] :

d (A) I (%) d (A) I (%)

18.87 39 3.63 52

10.01 22 3.51 88

9.17 22 3.32 60

8.25 20 3.12 48

7.54 23 3.00 28

6.18 40 2.76 32

5.71 22 2.67 27

5.05 34 2.52 32

4.77 41 2.49 31

4.53 55 2.47 29

4.24 42 2.31 26

3.97 57 2.23 25

3.77 100 2.03 21

Crystalline formic acid solvate of (6R,7R)-7-[2- (2-amino-4-thiazolyl)-2-[Z-[(2-carboxy-4,5-dihydroxy- phenyDmethyl]oxyimino] acetamido]-3-[(2-carboxy-5- methyl-s-triazolo[l,5-a] pyrimidin-7-yl)thiomethyl]-8- oxo-5-thia-l-azabicyclo[4.2.0] oct-2-ene-2-carboxylic acid can generally be prepared from its amorphous base salts, such as trisodium salts, or from acid addition salts, such as hydrochloric acid salts or trifluoroacetic acid salts. Thus, corresponding

cephalosporin derivative or its salt can efficiently be crystallized from formic acid at a temperature in the range of from 0°C to 60°C, preferably from 15°C to 50°C, and if necessary and desired, using acids such as hydrochloric acid. The crystallization may also be facilitated by adding water to the solution, or the use of seed crystals.

The compounds of the present invention show a potent antibacterial activity against a wide range of bacteria including Gram-positive and Gram-negative bacteria, especially against methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa, and are quite useful as therapeutic agents for infectious diseases.

To demonstrate the utility of the compounds of the present invention, data on antibacterial activity of a representative compound (referred to as Compound 1) are shown below. A compound described in another invention of the present inventors, Japanese Patent Application 147359 (1985), is used as a reference com¬ pound.

Compound 1: (6R,7R)-7-[2-(2-amino-4-thiazolyl)-2-[Z- [(2-carboxy-4,5-dihydroxyphenyl)methyl]- oxyimino]acetamido] -3-[(2-carboxy-5-meth- yl-s-triazolo[1,5-a] yrimidin-7-yl)thio- ethyl]-8-oxo-5-thia-l-azabicyclo[4.2.0]- oct-2-ene-2-carboxylic acid Reference compound: (6R,7R)-7-[2-(2-amino-4-thiazolyl)-2-[Z- [(4-acetoxy-2-carboxy-5-hydroxypheny1)- methy]oxyimino] acetamido]-3-[(2-caboxy-5- methyl-s-triazolo[1,5-a] pyrimidin-7-yl)- thiomethyl]-8-oxo-5-thia-l-azabicyclo- [4.2.0] oct-2-ene-2-carboxylic acid

Experimental Example 1

Antibacterial activity _in_ vitro was determined in accordance with the agar plate dilution method.

A platinum loop each of test bacteria (10 ⁶ cells/ml) , cultured in Mueller Hinton broth, was ino¬ culated on Mueller Hinton agar plates which contained test compounds at various concentrations. After cultivating at 37°C for 20 hours, the minimum inhibi¬ tory concentration (MIC μg/ml) was determined.

Table 1-a

MIC (μg/ml)

Staphylococcus Escherichia Serratia compound aureus coli marcescens

Smith NIHJ JC 2 IFO 3759

Compound 1 1.56 0.39 0.10 Reference compound 1.56 0.39 <0.05

CAZ ** N.D. 0.20 <0.05

N.D.; Not determined. **; Ceftazidime

Table 1-b

Proteus Klebsiella Pseudomonas compound morganii pneumoniae aeruginosa IFO 3848 IFO 3317 13

Compound 1 0 .39 <0 . 05 0.10 Reference compound 0 . 20 <0 . 05 1.56

CAZ ** 0. 20 <0 . 05 1.56

N.D.; Not determined. **; Ceftazidime

As shown in Tables 1-a through 1-b, Compound 1 was about equipotent to the reference compound against the bacterial strains except Pseudomonas aeruginosa, against which Compound 1 was more than 10 times as potent as the reference compound and Ceftazidime.

Experimental Example 2

Protection ability against systemic infection was determined as follows. An aqueous suspension of test bacteria was intraperitoneally injected into 10 four week old ICR mice. One hour after the infection, test compounds were intravenously administered. The number of surviving mice was counted 1 week after injection to determine the dose at which 50% of the test animals were alive (EDs.: mg/kg) .

Table 2-a

ED ₅₀ (mg/Kg)

Staphylococcus Escherichia Serratia comDound aureus coli marcescens 242 ^' 67 274

Compound 1 7.68 1.05 0.77 Reference compound 12.4 7.66 2.56

CMD** 8.94 N.D. N.D.

CAZ*** ^• >100 6.48 3.62

N.D.; Not determined.

*; Methicillin-resistant strain **; Cefa andole ***; Ceftazidime

Table 2-b

ED _S o (mg/Kg)

Proteus Klebsiell .a Pseudomonas compound mirabilis pneumoniae aeruginosa

IFO 3849 IFO 3317 13

Compound 1 5.07 0.27 61.8

Reference compound 20.1 3.58 >500

CMD** N.D. N.D. N.D.

CAZ*** 4.36 10.6 145

N.D.; Not determined. **; Cefamandole ***; Ceftazidime

As shown in Tables 2-a- through 2-b, Compound 1 w evidently more potent than the reference compound i protecting the animals from experimental infectio and, especially, was more than 10 times as potent a the reference compound against infections wit Klebsiella pneumoniae and Pseudomonas aeruσinosa.

Next, LD ₅ o of representative examples of the com pounds of the present invention is shown in Table wherein LD ₅₀ was determined in accordance with th Probit method.

Table 3 compound LD ₅₀ (mg/Kg, i.v.)

Compound 1 >4000

CAZ*** >4000

***;Ceftazidime

The compounds of the present invention are active against microorganisms, such as Gram-positive aerobic bacteria such as Staphylococcus aureus, streptococci, etc.. Gram-negative aerobic bacteria such as Escherichia coli, Klebsiella pneumo ^' niae, Proteus mira- bilis, Proteus morganii, Serratia marcescens, Pseudomonas aeruσinosa, Citrobacter, Enterσbacter, Flavobacter, etc. and are useful for the- ^' treatment of infectious diseases caused by these microorganisms. The compounds of the present invention show a thera¬ peutic efficacy against Pseudomonas aeruginosa infec¬ tions which is more potent than those of β-lactam antibiotics ever known; these compounds are also thought to be highly safe. Therefore, the compounds of the present invention are expected to be extremely useful against Pseudomonal infections.

The cephalosporin derivatives provided by the pre¬ sent invention can be employed as pharmaceutical com¬ positions, for example, in the form of pharmaceutical compositions containing cephalosporin derivatives together with appropriate, pharmaceutically acceptable carriers. The pharmaceutical composition may take a solid form, such as tablets, capsules, etc. or a liquid form, such as injections, etc. The com¬ positions may be sterilized and may contain auxiliary agents generally employed in the pharmaceutical art, such as sodium hydrogen carbonate, citric acid, propy- lene glycol, Tween 80, etc.

Further, it is also preferred to use the compounds of the present invention after they are formed into freeze-dried products or powders followed by dissolving them in a conventional solvent, e.g., water or physiological saline, before use. The compounds can be used orally or parenterally. While dose varies depending upon age and conditions of the patient, con¬ ditions and kind of diseases, etc., from about 0.01 to about 10 g, preferably from about 0.1 to about 5 g, can be used as a daily dose for an adult. Parenteral administration of the compounds provided by the pre-

sent invention is particularly preferred.

Hereafter the present invention will be described with reference to the examples below but is not deemed to be limited thereof.

Example 1

Preparation of (6R,7R)-7-[2-(2-amino-4-thiazol- yl)-2-[Z-[(2-carboxy-4,5-dihydroxyphenyl)meth- yl] oxyimino] acetamido]-3-[( 2-carboxy-5-methyl-s- triazolo[1,5-a] yrimidin-7-yl)thiomethyl] -8-oxo- 5-thia-l-azabicyclo [4.2.0] oct-2-ene-2-carboxylic acid (Compound 1).

Step 1

Preparation of 5-bromo-2,2,6-trimethylbenzodi- oxol

To a solution of 2,2,5-benzodioxol (21 g) in dichloromethane (160 ml) was added pyridine (12.1 ml), followed by dropwise addition of a solution of bromine (7.5 ml) under ice cooling, and the solution was stirred at room temperature for 1 hour. After removing the solvent under reduced pressure, the resi¬ due was extracted with ether (200 ml). The solution was washed thrice with water (100 ml each), twice with aqueous solution of citric acid (IN, 100 ml each), once with saturated aqueous solution of sodium hydro¬ gen carbonate (100 ml) and once with brine (100 ml). The washed solution was cried over anhydrous sodium

sulf te. The dried solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give 28.4 g of the objective compound. IR (KBr, cm" ¹ ) :

1493, 1379, 1251, 1241, 1216, 856 NMR (CDC1 ₃ , pom) :

6.9 (1H, s), 6.6 (1H, s), 2.3 (3H, s), 1.7 (6H, s)

Step 2

Preparation of 2,2,6-trimethylbenzodioxol-5- carboxylic acid.

To a suspension of magnesium powder (9.64 g) in dry ether (17 ml) was slowly added a solution of the product of Step 1 (28 g) and ethyl bromide (24.8 g) in dry ether (114 ml). Addition of about 4 ml of the solution was sufficient to cause spontaneous refluxing, and the remaining portion was added drop- wise over a 1.5-hour period to maintain the spon¬ taneous reflux. After completing the dropwise addition, the reaction mixture was refluxed for addi¬ tional 30 minutes, then cooled to room temperature and

poured over crushed pieces of dry ice. Stirring was continued until all pieces of dry ice were lost. The resultant solution was acidified with aqueous hydroch¬ loric acid (20%, 92 ml) under ice cooling and extracted twice with ether (100 ml each). The ether solution was washed 4 times with water (50 ml each), then extracted 5 times with ice-cooled aqueous sodium hydroxide solution (cone. 10%, 50 ml each). The exctract was acidified with 20% hydrochloric acid under ice cooling and stirring to pH 2, and the preci¬ pitated crystals were separated by filtration. The crystals thus obtained were washed, dried and recrystallized with ether-hexane (1:4) to give 10.8 g of the objective compound. IR (KBr, cm" ¹ ) :

3000, 1682, 1498, 1259, 1210, 982 NMR (CDC1 ₃ ; ppm) :

7.5 (1H, s) , 6.6 (1H, s), 2.6 (3H, s), 1.7 (6H, s)

Step 3

Preparation of t-butyl 2,2,6-trimethylbenzo- dioxol-5-carboxylate.

To a suspension of the product obtained in Step 2 (10.6 g) in benzene (50 ml) were added thionyl chloride (15.5 ml) and N,N-dimethylformamide (2 drops) , and the mixture was heated at 60°C for 30 minutes. The solvent was removed under reduced pressure, and the residue was dissolved in methylene chloride (50 ml). The solution was then added drop- wise to an ice-cooled mixture of t-butanol (37.6 g) and pyridine (24.6 ml), and the mixture was stirred at room temperature for 7 hours. After removing the solvent under reduced pressure, ether (200 ml) was added to the residue. The ether solution was washed twice with water (80 ml each), twice with aqueous hydrochloric acid (IN, 80 ml each), once with saturated aqueous solution of sodium hydrogen car¬ bonate (80 ml) and once with brine, and the washed solution was dried over anhydrous sodium sulfate. The dried solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give 11.2 g of the objective compound. IR (KBr, cm" ¹ ) :

2979, 1713, 1498, 1375, 1253, 1165

NMR (CDCI3 / ^* PP ^{m) :}

7.3 (1H, s), 6.6 (1H, s), 2.5 (3H, s), 1.7 (6H, s) , 1.6 (9H, s)

Step 4

Preparation of t-butyl 6-bromomethyl-2,2-di- methylbenzodioxol-5-carboxylate.

To a solution of the product obtained in Step 3 (11.2 g) in carbon tetrachloride (60 ml) were added N- bromosuccinimide (7.6 g) and benzoyl peroxide (30 mg) and the mixture was refluxed for 40 minutes. The reaction mixture was then cooled to room temperature and insoluble matter was removed by filtration. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chroma¬ tography to give 11.5 g of the objective compound. IR (KBr, cm" ¹ ) :

2980, 1689, 1510, 1287, 1157 NMR (CDCI3 * ppm) :

7.3 (1H, S), 6.8 (1H, s), 4.9 (2H, s), 1.7 (6H, s) , 1.6 (9H, s)

Step 5

Preparation of t-butyl 2,2-dimethyl-6-(N- phthaloyloxymeth l)benzodioxol-5-carboxylate. To a solution of the product obtained in Step 4 (11.5 g) in acetonitrile (108 ml) was added dropwise a solution of N-hydroxyphthalimide (5.5 g) and triethy- lamine (4.7 ml) in acetonitrile (32 ml) at room tem¬ perature, and the mixture was stirred for 4 hours. The reaction mixture was poured into ice water and extracted twice with ethyl acetate (400 ml each). The organic layer was washed twice with aqueous solution of citric acid (IN, 200 ml each), 4 -times with saturated aqueous solution of sodium hydrogen car¬ bonate (200 ml each) and twice with brine (200 ml). The washed solution was dried over anhydrous sodium sulfate. The dried solution -was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give 11.2 g of the objective compound. IR (KBr, cm" ¹ ) :

2990, 1735, 1499, 1264, 1162, 981, 701 NMR (CDC1 ₃ , ppm) :

7.8 (4H, m) , 7.3 (1H, s) , 7.2 (1H, s), 5.6 (2H, s) , 1.7 (6H, s) , 1.5 (9H, s)

Step 6

Preparation of t-butyl 6-aminooxymethyl-2,2-di- methylbenzodioxo1-5-carboxylate.

To a solution of the product obtained in Step 5 (11.1 g) in methylene chloride (180 ml) and the solu¬ tion was cooled to -30°C. To the cold solution was added a solution of methylhydrazine (1.4 ml) in methy¬ lene chloride (20 ml), and the mixture was stirred for 1 hour with ice cooling. Methylhydrazine (0.3 ml) was added again to the mixture and stirring was continued for 30 minutes. Insoluble matter was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give 7.6 g of the objective compound. IR (KBr, cm" ¹ ) :

3530, 2990, 1703, 1498, 1252, 1160 NMR (CDC1 ₃ , ppm) :

7.3 (1H, s), 6.9 (1H, s), 5.7 - 4.8 (2H, bs) , 5.3

(2H, s), 1.7 (6H, s), 1.6 (9H, s)

Step 7

Preparation of 2-(2-amino-4-thiazolyl)-2-[Z-[(5- t-butoxycarbonyl-2,2-dimethylbenzodioxol-6-yl)- methylloxyimino] acetic acid.

To a solution of the product obtained in Step 6 (7.6 g) in N,N-dimethylformamide (35 ml) was added (2-aminothiazol-4-yl)glyoxylic acid (4.4 g) , and the mixture was stirred at room temperature for 30 minu¬ tes. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate (200 ml). The solution was washed 4 times with water (80 ml each) and once with brine (80 ml) , and dried over anhydrous sodium sulfate. The dried solution was concentrated under reduced pressure, and the residue was recrystallized with ether-hexane (1:1) to give 10.2 g of the objective compound as crystals. IR (KBr, cm" ¹ ) :

2290, 1702, 1617, 1498, 1261 NMR (DMSO-d ₆ , ppm) :

7.24 (1H, s), 7.2 (2H, bs) , 6.94 (1H, s), 6.86

(1H, s), 5.4 (2H, s), 1.7 (6H, s), 1.5 (9H- s)

Step 8

Preparation of (6R,7R)-7-[2-(2-amino-4-thiazol- yl)-2-[Z-[ ( 5-t-butoxycarbonyl-2,2-dimethylbenzo- dioxol-6-yl)meth l]oxyimino] acetamido]-3-[( 2- diphenylmethyloxycarbonyl-5-methyl-s-triazolo- [1,5-a] yrimidin-7-yl)thiomethyl] -8-oxo-5-thia- 1-azabicyclo[4.2.0]oct-2-ene-2-carbox lic acid diphenylmethyl ester.

To a solution of the product obtained in Step 7 (3.0 g) and ( 6R,7R)-7-amino-3-[ ( 2-diphenylmethyloxy- carbonyl-5-meth l-s-triazolo[1,5-a]pyrimidin-7-yl)- thiomethyl] -8-oxo-5-thia-l-azabicyclo[4.2.0] oct-2- ene-2-carboxylic acid diphenylmethyl ester (5.0 g) in methylene chloride (120 ml) was added N,N-dicyclo- hexylcarbodiimide (1.7 g), and the mixture was stirred for 12 hours at room temperature. The reaction mix¬ ture was then concentrated under reduced pressure, and the residue was dissolved in acetone (50 ml) and insoluble matter was filtered off. The filtrate was purified by silica gel column chromatography to give 5.4 g of the objective compound. IR (KBr, cm" ¹ ) :

1791, 1700, 1498, 1377, 1223 NMR (DMSO-d6 ; ppm) :

9.8 (1H, d, J » 8 Hz), 7.6 - 7.1 (25H- m) , 7.0 (1H, s), 6.96 (1H, s), 6.8 (1H, s), 5.9 (1H, dd, J = 8, 5 Hz), 5.4 (2H, s), 5.3 (1H, d, J = 5 Hz) 4.3 (2H, s), 3.7 (2H, ABq) , 2.6 (3H, s), 1.6 (6H, s) , 1.5 (9H, s)

Step 9

Preparation of (6R,7R)-7-[2-(2-amino-4-thiazol- yl)-2-[Z-[(2-carboxy-4,5-dihydroxyphenyl)meth- yl] oxyimino] acetamido]-3-[(2-carboxy-5-methylrs- triazolo[1,5-a]pyrimidin-7-yl)thiomethyl]-8-oxo- 5-thia- ^' l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid.

To a suspension of the product of Step 8 (5.4 g) in dichloroethane (3.5 ml) was added anisole (3.5 g) . To the mixture was added trifluoroacetic acid (14 ml) under ice cooling, and the resultant mixture was stirred at room temperature for 6 hours. The reaction mixture was then poured into ether (300 ml). The pre¬ cipitated crystals were suspended in water (45 ml) and the pH of the suspension was adjusted to 8.2 with sodium hydrogen carbonate. The resultant solution was applied to a Diaion HP-20 column and eluted with

water. Fractions containing the objective compound were collected and concentrated under reduced pressure. The residue was recrystallized by pouri'ng into ethanol (70 ml) to give 1.6 g of the purified crystals.

IR (KBr, cm" ¹ ) :

1762, 1598, 1515, 1376, 1314° NMR (D ₂ O; ppm) :

7.2 (1H, s), 7.1 (1H, s), 7.0 (2H, s) , 5.7 (1H, d, J = 5 Hz), 5.4 (2H, s), 5.1 (1H, d, J = 5 Hz)

4.3 (2H, ABq) , 3.5 (2H, ABq), 2.6 (3H, s)

Example 2

Preparation of (6R,7R)-7-[2-(2-amino-4-thiazol- yl)-2-[Z-[(2-carboxy-4,5-dihydroxyphenyl)meth¬ yl]oxyimino] acetamido]-3-[(2-carboxy-5-methyl-s- triazolo[l,5-a]pyrimidin-7-yl)thiomethyl]-8-oxo- 5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid.

Step 1

Preparation of ethyl 2-(2-amino-4-thiazolyl)-2-

[Z-[( 5-t-butoxycarbonyl-2,2-dimethylbenzodioxol-

β-yl)methyl]oxyimino] acetate.

To a solution of ethyl 2-(2-amino-4-thiazolyl)- 2-( Z-hydroxyimino)acetate (1 g) in dry dimethyIform- amide (10 ml) was added 60% aqueous solution of sodium hydride (200 mg) under ice cooling, and the mixture was stirred for 15 minutes. To the mixture was added dropwise a solution ³ of the product obtained in Example 1, Step 4 (1.9 g) in dimethylformamide (10 ml). The mixture was stirred under ice cooling for 1.5 hours. The reaction mixture was then poured into an ice- cooled mixture of cone, hydrochloric acid and ethyl acetate (200 ml each) and stirred thoroughly. The organic layer was separated and washed once with saturated aqueous solution of sodium hydrogen car¬ bonate (100 ml) and thrice with brine (100 ml). The washed solution was dried over anhydrous sodium sulfate. The dried solution was concentrated under reduced pressure, and the residue was recrystallized with hexane (50 ml) to give 1.85 g of the objective compound. NMR (CDC1 ₃ , pp ) :

7.3 (1H, s), 6.9 (1H, s), 6.7 (1H, s), 5.7 (2H, bs), 5.6 (2H, s) , 4.4 (2H, q, J = 7 Hz), 1.7

(6H, s), 1.6 (9H, s), 1.4 (3H, t, J = 7 Hz)

Step 2

Preparation 2-(2-amino-4-thiazolyl)-2-[Z-[(5- t-butoxycarbon l-2,2-dimethylbenzodioxol-6-yl)- methyl]oxyimino] acetic acid.

To a suspension of the product of Step 1 (1 g) in ethanol (6 ml) was added an aqueous solution of sodium hydroxide (cone. 2N, 2.1 ml), and the mixture was stirred for 2 hours at room temperature then for 1 hour at 63°C. The reaction mixture was concentrated under reduced pressure, and water (20 ml) was. added to the residue. The mixture was acidified with aqueous hydrochloric acid (1 N) to pH 3, then extracted with ethyl acetate (50 ml). The extract was washed twice with brine (30 ml each) and dried over anhydrous sodium sulfate. The dried solution was concentrated under reduced pressure, and the residue was recrystallized with ether-hexane (1:1, 30 ml) to give 870 mg of the objective compound as crystals. IR (KBr, cm" ¹ ) :

2290, 1702, 1617, 1498, 1261 NMR (DMSO-d _s , pom) :

7.24 (1H, s), 7.2 (2H, bs), 6.94 (1H, s), 6.86 5.4 (2H, s), 1.7 (6H, s), 1.5 (9H, s) These data are in complete agreement -with those of Step 7 of Example 1.

Step 3

Preparation of (6R,7R)-7-[2-(2-amino-4-thiazol- yl)-2-[Z-[(2-carboxy-4,5-dihydroxyphenyl) th¬ yl] oxyimino] acetamido]-3-[(2-carboxy-5-meth 1-s- triazolo[1,5-a]pyrimidin-7-yl)thiomethyl]-8-oxo- 5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid. ^" .

The compound of the title given above was pre¬ pared from the product of Step 2, according to the methods of Steps 8 and 9 of Example 1. IR (KBr, cm" ¹ ) :

1762, 1598, 1515, 1376, 1314 NMR (D ₂ 0; ppm) :

7.2 (1H, s), 7.1 (1H, s), 7.0 (2H, s), 5.7 (1H, d, J = 5 Hz), 5.4 (2H, s), 5.1 (1H, d, J = 5 Hz)

4.3 (2H, ABq), 3.5 (2H, ABq), 2.6 (3H, s)

These data are in complete agreement with those of Step 9 of Example 1.

According to the method described in Example 1, compounds of Examples 3 and 4, described below, were prepared. According to the method described in Example 2, compounds of Examples 5 and 6 were pre¬ pared.

Example 3

(6R,7R)-7-[2-(2-amino-4-thiazolyl)-2-.[Z-[(2- carboxy-4,5-dihydroxyphenyl)methyl]oxyimino]- acetamido] -3-[ (5-methyl-s-triazolo[1,5-a]pyri- midin-7-yl)thiomethyl]-8-oxo-5-thia-l-azabi- cyclo[4.2.0]oct-2-ene-2-carboxylic acid

IR (KBr, cm" ¹ ) :

1762, 1595, 1514, 1380 NMR (D O; ppm) :

8.5 (1H, s), 7.2 (1H, s), 7.1 (1H, s), 7.0 (2H, s), 5.7 (1H, d, J = 5 Hz), 5.4 (2H, s), 5.1 (1H, d, J = 5 Hz) , 4.3 (2H, ABq), 3.5 (2H, ABq), 2.6 (3H, s)

Example 4

( 6R,7R)-7-[2-(2-amino-4-thiazolyl)-2-[Z-[(2-

carboxy-4,5-dihydroxyphenyl)methylloxyimino]- acetamido]-3-[(5-carboxy-s-triazolo[1,5-a]pyri- midin-7-yl)thiomethyl]-8-oxo-5-thia-l-azabi- cyclo[4.2.0] oct-2-ene-2-carboxylic acid

IR (KBr, cm" ¹ ) :

1762, 1598, 1534, 1396

NMR (D ₂ 0; pp ) :

8.5 (IH, s), 7.3 (IH, s), 7.1 (IH, s) , 7.0 (2H, S), 5.7 (IH, d, J = 5 Hz), 5.4 (2H, s), 5.1 (IH, d, J = 5 Hz), 4.3 (2H, ABq), 3.5 (2H, ABq)

Example 5

(6R,7R)-7-[2-(2-amino-4-thiazolyl)-2-[Z-[(2- carboxy-4,5-dihydroxyphenyl)methyl]oxyimino]- acetamido]-3-[(5-carboxymethyl-s-triazolo- [1,5-a]pyrimidin-7-yl)thiomethyl]-8-oxo-5-thia- l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

IR (KBr, cm" ¹ ) :

1762, 1595, 1515, 1390

NMR (D ₂ 0; ppm) :

8.5 (IH, s), 7.2 (IH, s), 7.1 (IH, s), 7.0 (2H, s), 5.7 (IH, d, J = 5 Hz), 5.4 (2H, s), 5.1 (IH, d, J = 5 Hz), 4.3 (2H, ABq), 3.5 (2H, ABq), 2.6

(2H, s)

Example 6

(6R,7R)-7-[2-(2-amino-4-thiazolyl)-2-[Z-[(2- carboxy-4,5-dihydroxyphenyl)methyl]oxyimino] - acetamido]-3-[(2-hydroxysul onyl-5-meth 1-s-tri- azolo[1,5-a] yrimidin-7-yl)thiomethyl] -8-oxo-5- thia-1-azabicyclo[4.2.0] oct-2-ene-2-carboxylic acid

IR (K3r, cm" ¹ ) :

1763, 1596, 1510, 1395, 1368

NMR (D ₂ 0; ppm) :

7.2 (IH, s), 7.1 (IH, s), 7.0 (2H, s), 5.7 (IH, d, J = 5 Hz), 5.4 (2H, s), 5.1 (IH, d, J = 5 Hz)

4.3 (2H, ABq), 3.5 (2H, ABq), 2.6 (3H, s)

Example 7 ,

Preparation of formic acid solvate of (6R,7R)-7- [2-(2-amino-4-thiazolyl)-2-[Z-[(2-carboxy-4,5- dihydroxyphenyDmethyl] oxyimino] acetamido]-3- [(2-carboxy-5-methyl-s-triazolo[l,5-a]pyrimidin- 7-yl) thiomethyl]-8-oxo-5-thia-l-azabicyclo- [4.2.0]oct-2-ene-2-carboxylic acid.

Trifluoroacetic acid salt of (6R,7R)-7-[2-{2- amino-4-thiazolyl)-2-[Z-[(2-carboxy-4,5-dihydroxy- phenyl)methyl] oxyimino] cetamido]-3-[(2-carboxy-5- methyl-s-triazolo[1,5-a]pyrimidin-7-yl)thiomethyl]-8- oxo-5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (3.0 g) was dissolved in formic acid (15 ml) at room temperature. To the solution was added water (15 ml) and the mixture was allowed to stand for 1 hour at room temperature. Additional water (5 ml) was then added and the mixture was stirred for 30 minutes at room temperature. To the mixture were added seed crystals, and stirring was continued for 1 hour. Precipitated crystals were separated by filtration, washed with water (20 ml) and dried in air to give 1.95 g of the objective compound. IR (Nujol, cm" ¹ ) :

3600 - 2200, 3269, 1770, 1654, 1596, 1517, 1509, 1303, 1188, 1159, 1101, 1061, 1025, 962, 904, 853, 794, 770 NMR (DMSO-d ₆ , ppm):

9.61 (IH, d, J = 8 Hz), 9.44 (IH, bs), 9.18 (IH, bs), 8.12 (s; HCOOH) , 7.40 (IH, s), 7.35 (IH,

s) , 7.17 (2H, S) , 6.89 (IH, s), 6.75 (IH, s) , 5.88 (IH, dd, J = 8, 4 Hz) , 5.39 (2H, s), 5.20 (IH, d, J = 4 Hz) , 4.43 (2H, s) , 3.70(2H, ABq) , 2.62 (3H, s) X-ray diffraction pattern [given as d spacings in Angstrom units and percentage intensities] :

d (A) I (%) d (A) I (%)

18.87 39 3.63 52

10.01 22 3.51 88

9.17 22 3.32 60

8.25 20 3.12 48

7.54 23 3.00 28

6.18 40 2.76 32

5.71 22 2.67 27

5.05 34 2.52 ^" 32

4.77 41 2.49 31

4.53 55 2.47 29

4.24 42 2.31 26

3.97 57 2.23 25

3.77 100 2.03 21

Example 8

Preparation of formic acid solvate of (6R,7R)-7- [2-( 2-amino-4-thiazolyl)-2-[Z-[ (2-carboxy-4,5- dihydroxyphenyl)methyl] oxyimino] acetamido] -3- [ (2-carboxy-5-methyl-s-triazolo[1,5-a] pyrimidin- 7-yl) thiomethyl]-8-oxo-5-thia-l-azabicyclo- [4.2.0] oct-2-ene-2-carboxylic acid, j

Trisodium salt of (6R,7R)-7-[2-(2-amino-4- thiazolyl)-2-[Z-[(2-carboxy-4,5-dihydroxyphenyl) eth¬ yl]oxyimino] acetamido]-3-[(2-carboxy-5-methyl-s-tri- azolo[1,5-a] yrimidin-7-yl)thiomethyl]-8-oxo-5-thia- l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (1.1 g) was dissolved in water (3 ml), and formic acid (1.5 ml) and water (2 ml) were added to the solution. The mixture was stirred for 5 minutes at room temperature. Additional water (5 ml) was then added and the mixture was stirred for 30 minutes at 0°C. Precipitated crystals we ^' re separated by filtration, washed with water (5 ml) and dried ^' .in air to give 0.89 g of the objective compound. IR (Nujol, cm" ¹ ):

3600 - 2200, 3255, 1764, 1701, 1653, 1597, 1542, 1517, 1305, 1268, 1220, 1203, 1186, 1172, 1157, 1103, 1065, 1020, 962, 907, 852, 795, 771 NMR (DMSO-d _s , ppm) j

9.61 (IH, d, J = 8 Hz), 9.44 (IH, bs), 9.18(1H, bs), 8.12 (s; HCOOH) , 7.40 (IH, s), 7.35 (IH, s), 7.17 (2H, s), 6.89 (IH, s), 6.75 (IH, s), 5.88 (IH, dd, J = 8, 4 Hz), 5.39 (2H, s) , 5.20 (IH, d, J = 4 Hz), 4.43 (2H, s), 3.70 ^" (2H, ABq),

2 . 62 ( 3H , s ) X-ray diffraction pattern [g iven as d spacings in Angstrom uni ts and percentage intens ities] :

d ( A) I ( % ) d ( A) I ( % )

15.94 29 3.53 74

8.48 32 3.42 99

7.10 37 3.24 80

5.90 45 3.05 62

4.84 42 2.81 41

4.58 44 2.70 45

4.38 61 2.47 46

3.83 75 2.27 41

3.66 100 2.20 39

Example 9

Preparation of hydrate of (6R,7R)-7-[2-(2-amino- 4-thiazolyl)-2-[Z-[( 2-carboxy-4,5-dihydroxy- phenyl)methyl]oxyimino]acetamido]-3-[(2-carboxy- 5-meth l-s-triazolo[1,5-a] yrimidin-7-yl)thio¬ methyl]-8-oxo-5-thi -l-azabicyclo[4.2.0] oct-2- ene-2-carboxylic acid.

Trifluoroacetic acid salt of (6R,7R)-7-[2-(2- amino-4-thiazolyl)-2-[Z-[ ( 2-carboxy-4,5-dihydroxy- phenyl)methyl] oxyimino] acetamido]-3-[(2-carbαxy-5- methyl-s-triazolo[1,5-a]pyrimidin-7-yl)thiomethyl]-8- oxo-5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic

acid (3.0 g) was dissolved in formic acid (15 ml) at room temperature. To the solution was added water (15 ml) and the mixture was stirred for 1 hour at room temperature. Additional water (5 ml) was then added and the mixture was stirred for 30 minutes at room temperature. To the mixture were added seed crystals and water (10 ml), and stirring was continued for 1 hour. ^" Precipitated crystals were separated by filtra¬ tion, washed once with water (20 ml) and once by stirring in 30 ml of water, then dried in air to give 1.85 g of the objective compound. IR (Nujol, cm" ¹ ) :

3600 - 2200, 3261, 1768, 1653, 1595, 1517, 1509 1305, 1269, 1188, 1158, 1103, 1063, 1024, 962, 903, 853, 795, 770 NMR (DMSO-ds , ppm) :

9.54 (IH, d, J = 8 Hz), 7.41 (IH, s), 7.36 (IH, s), 6.89 (IH, s), 7.4 - 6.9 (2H, bs), 6.76 (IH, s), 5.81 (IH, dd, J = 8, 5 Hz), 5.39 (2H, s) , 5.21 (IH, d, J = 5 Hz), 4.43 (2H, bs), 4.1 (2H, bs) , 2.6 (3H, s) X-ray diffraction pattern [given as d spacings in Angstrom units and percentage intensities] :

d (A) I (%) (A) I (%)

18.95 44 4.22 36

9.48 27 3.95 74

8.23 12 3.77 100

7.47 21 3.65 17

7.14 21 3.50 63

6.21 60 3.36 31

5.70 33 3.22 13

5.20 16 3.10 41

4.75 88 2.84 16

4.51 61 2.75 12

Example 10

Preparation of hydrate of (6R,7R)-7-[2-(2-amino- 4-thiazolyl)-2-[Z-[(2-carboxy-4,5-dihydroxy- phenyDmethyl]oxyimino] acetamido]-3-[(2-carboxy- 5-methyl-s-triazolo[1,5-a]pyrimidin-7-yl)thio¬ methyl]-8-oxo-5-thia-l-azabicyclo [4.2.0] oct-2- ene-2-carboxylic acid.

Trisodium salt of (6R,7R)-7-[2-(2-amino-4- thiazolyl)-2-[Z-[(2-carboxy-4,5-dihydroxyphenyl)meth- yl]oxyimino]acetamido]-3- [(2-carboxy-5-methyl-s-tri- azolo[1,5-a] yrimidin-7-yl)thiomethyl]-8-oxo-5-thia- l-azabicyclo[4.2.0] oct-2-ene-2-carboxylic acid (1.1 g) was dissolved in water (3 ml), and formic acid (1.5 ml) was added to the solution. The mixture was stirred for 5 minutes at room temperature. Additional

water (5 ml) was then added and the mixture was stirred for 30 minutes at 0°C. Precipitated crystals were separated by filtration and washed with water (once each with 2 and 1 ml) . These washed crystals were redissolved in formic acid (5ml). To the solu¬ tion was added water (5 ml) , and the mixture was stirred for 1 hour at room temperature. Precipitated crystals were separated by filtration, washed once with water (20 ml) and once by stirring in 30 ml of water, then dried in air to give 0.89 g of the objec¬ tive compound. IR (Nujόl, cm" ¹ ) :

3600 - 2200, 3275, 1769, 1652, 1596, 1542, 1521, 1519, 1307, 1272, 1190, 1160, 1103, 1064, 1026, 964, 901, 854, 795, 770 NMR (DMSO-d ₆ , ppm):

9.54 (IH, d, J = 8 Hz), 7.41 (IH, s), 7.36 (IH, s), 6.89 (IH, s), 7.4 - 6.9 (2H, bs), 6.76 (IH, s), 5.81 (IH, dd, J = 8, 5 Hz), 5.39 (2H, s) , 5.21 (IH, d, J = 5 Hz), 4.43 (2H, bs), 4.1 (2H, bs) , 2.6 (3H, s) X-ray diffraction pattern [given as d spacings in Angstrom units and percentage intensities] :

d (A) I (%) (A) I (%)

19.11 55 3.79 74

14.34 11 3.66 44

9.48 48 3.56 73

8.73 13 3.36 47

7.19 37 3.22 22

6.27 82 3.15 13

5.73 58 3.11 26

5.34 20 2.85 28

5.21 28 2.75 12

4.78 77 2.62 13

4.53 42 2.49 10

4.26 25 2.12 13

3.97 100 1.98 16

Example 11

Preparation of hydrate of (6R,7R)-7-[2-(2-amino- 4-thiazolyl)-2-[Z-[ (2-carboxy- ,5-dihydroxy- phenyDmethyl]oxyimino] acetamido]-3-[(2-carboxy- 5-methyl-s-triazolo[1,5-a] yrimidin-7-yl)thio¬ methyl]-8-oxo-5-thia-l-azabicyclo[4.2.0]oct-2- ene-2-carboxylic acid.

Formic acid solvate of (6R,7R)-7-[2-(2-amino-4- thiazolyl)-2-[Z-[(2-carboxy-4,5-dihydroxyphenyl)meth- yl] oxyimino] cetamido]-3-[(2-carboxy-5-methyl-s-tri- azolo[l,5-a]pyrimidin-7-yl)thiomethyl] -8-oxo-5-thia- l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (1.85 g), obtained in either Example 7 or 8 , was dissolved in formic acid (10 ml). To the solution was added

water (12 ml) and the mixture was stirred for 30 minu¬ tes at room temperature. Additional water (10 ml) was then added and the mixture was stirred for 1 hour at room temperature. Precipitated crystals were separated by filtration, washed with water (30 ml) and dried in air to give 0.89 g of the objective compound. IR (Nujol, cm" ¹ ) :

3600 - 2200, 3275, 1769, 1652, 1596, 1542, 1521, 1519, 1307, 1272, 1190, 1160, 1103, 1064, 1026, 964, 901, 854, 795, 770 NMR (DMSO-d ₅ , ppm): 9.54 (IH, d, J = 8 Hz), 7.41 (IH, s), 7.36 (IH, s), 6.89 (IH, s) , 7.4 - 6.9 (2H, bs), 6.76 (IH, s), 5.81 (IH, dd, J = 8, 5 Hz), 5.39 (2H, s) , 5.21 (IH, d, J = 5 Hz) , 4.43 (2H, bs), 4.1 (2H, bs) , 2.6 (3H, s) X-ray diffraction pattern [given as d spacings in Angstrom units and percentage intensities] Ϊ

d ( A) I ( % ) ( A) I ( % )

19.11 55 3.79 74

14.34 11 3.66 44

9.48 48 3.56 73

8.73 13 3.36 47

7.19 37 3.22 22

6.27 82 3.15 13

5.73 58 3.11 26

5.34 20 2.85 28

5.21 28 2.75 12

4.78 77 2.62 13

4.53 42 2.49 10

4.26 25 2.12 13

3.97 100 1.98 16

The following examples detail typical pharma¬ ceutical preparations containing the cephalosporin derivatives of the present invention. These examples are not intended to limit the types of compounds to be used, but the methods are applicable to all the com¬ pounds of the present invention.

Example A (Method of manufacturing parenteral injec¬ tions)

(6R,7R)-7-[2-(2-amino-4-thiazolyl)-2-[Z-[(2-car- boxy-4,5-dihydroxyphenyl)methyl]oxyimino] acetamido] - 3-[(2-carboxy-5-methyl-s-triazolo[1,5-a]pyrimidin-7- yl)thiomethyl] -8-oxo-5-thia-l-azabicyclo[4.2.0] oct-2- ene-2-carboxylic acid (500 g) and 3 equivalent amounts of sodium hydrogen carbonate were uniformly mixed and

distributed into 15-ml hermetic containers each weighing 500 mg by ordinary methods.

Example B (Method of manufacturing fraeze-dried paren¬ teral injections)

(6R,7R)-7-[2-(2-amino-4-thiazolyl)-2-[Z-[(2-car- boxy-4,5-dihydroxyphenyl)methyl]oxyimino] acetamido]- 3-[(2-carboxy-5-methyl-s-triazolo[1,5-a]pyrimidin-7- yl)thiomethyl]-8-oxo-5-thia-l-azabicyclo[4.2.0] oct-2- ene-2-carboxylic acid trisodium salt (550 g) was dissolved in 2.2 1 of sterile water, and 2 ml each of this solution was poured into 10-ml ampoules, freeze- dried and sealed by ordinary methods, to produce a freeze-dried preparation for parenteral injections.

Example C (Method of manufacturing freeze-dried paren¬ teral injections)

(6R,7R)-7-[2-(2-amino-4-thiazolyl)-2-[Z-[(2-car- boxy-4,5-dihydroxyphenyl)methyl]oxyimino]acetamido]- 3-[(5-methyl-s-triazolo[1,5-a] yrimidin-7-yl)thio¬ methyl]-8-oxo-5-thia-l-azabicyclo[4.2.0]oct-2-ene-2- carboxylic acid trisodium salt (500 g) was dissolved in 1.0 1 of sterile water, and 2 ml-each of this solu-

tion was poured into 10-ml ampoules, freeze-dried and sealed by ordinary methods, to produce a freeze-dried preparation for parenteral injections.

Example D (Method of manufacturing tablets for oral administration)

Granules were prepared by ordinary methods using 100 g of (6R,7R)-7-[2-(2-amino-4-thiazolyl)-2-[Z- [(2-carboxy-4,5-dihydroxyphenyl)meth l]oxyimino]- acetamido]-3-[(5-carboxymeth l-s-triazolo[1,5-a]- pyrimidin-7-yl) thiomethyl]-8-oxo-5-thia-l-azabicyclo- [4.2.0]oct-2-ene-2-carboxylic acid, 100 g of lactose, 30 g of starch, and 10 g of polyvinyl pyrrolidone. Starch (30 g) and magnesium stearate (5 g) were further added to the granules, and the resulting mix¬ ture was compressed into tablets, each piece weighing 275 mg.

Example E (Method of manufacturing gelatin capsules for oral administration)

(6R,7R)-7-[2-(2-amino-4-thiazolyl)-2-[Z-[(2-car- boxy-4,5-dihydroxyphenyl)methyl] oxyimino] acetamido]- j

3-[(2-carboxy-5-meth l-s-triazolo[1,5-a] pyrimidin-7-

yl)thiomethyl]-8-oxo-5-thia-l-azabicyclo[4.2.0]oct-2- ene-2-carboxylic acid (100 g) , water-soluble polyvinyl pyrrolidone (15 g) , mannitol (15 g) , talc (15 g) and magnesium stearate (5 g) were uniformly mixed, and filled into gelatin capsules each weighing 150 mg.