DE2112058A1 - Chemical compounds and processes for their preparation - Google Patents

Description

"Chemical compounds and processes for their preparation"

The present invention relates to new synthetic compounds, which contribute as antibacterial agents, as feed additives to animal feed and as therapeutic agents Poultry and other animals, as well as humans, are valuable in the treatment of infectious diseases that caused by many gram-positive and gram-negative bacteria. The present invention relates in particular to 7 - [- (aminomethylphenylthio) acetamido] -cephalosporanic acids, 7- (Aminomethylphenylthio) -acetamido-3- (pyridiniummethyl) -ceph-3-em-4-carbo3cylate and their not-

- 2 109844/1937

toxic, pharmaceutically, acceptable salts, as well as that Process for their manufacture.

Sodium cephalothin is a well-known antibacterial agent which has found wide use in medicine by injection. There are many others in the patent literature 7-acyl derivatives of 7-aminocephalosporanic acid (7-ACA) described, for example 7- (p-aminomethylphenylacetaniido) -cephalosporanic acid (U.S. Patent 3,382,241), 7 - (<£ - aminophenylacetamido) -cephalosporanic acid (British patents 985,747 and 1,054,806 and Example 1 of U.S. Patent 3,363,212, this compound is known as Cephaloglyein), 7 - [- (p-aminophenylthio) acetamido] -cephalosporanic acid (U.S. Patent 3,422,100), 7- (halophenylthioacetemido) -cephalosporanic acids (USA Patent 3,335,136) and the almost unlimited number of Variations of such compounds - although often not otherwise described - by the general formulas be covered by patents such as Dutch patent specification 69/02013 (Parmdoc 39172). 7- (p-aminophenylacetamido) -cephalosporanic acid is also described in US Pat. No. 3,422,103 as the corresponding N-irityl derivative, for which, for example, reference is also made to Japanese patent application 2712/67 (Farmdoc 25406).

The cephalosporin compounds of the invention have the common properties of such compounds and are due to their high activity and easy absorption particularly useful in the treatment of bacterial infections after parenteral administration.

The present invention provides a process for the preparation of compounds of the general formula I.

109844/1937

- CH ₀ CNH - CH CH ₀

'- "" ■ O = CN C-CH ₀ A

^ Λ

C COOM

wherein A is acetoxy or pyridinium and M is hydrogen, a non-toxic, pharmaceutically acceptable cation or an anionic charge when A is pyridinium, and of non-toxic, pharmaceutically acceptable Acid addition salts thereof, which is characterized in that a compound of the general formula II

C-CH ₀ A

II

wherein A has the meanings given above, or a salt or a slightly hydrolyzed ester thereof, with an acylating acid of the formula III

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2112958

SCH ₂ COOH

III

wherein the amino group carries a conventional protecting group, or acylated with a reactive derivative thereof in an inert organic solvent at a temperature of about -20 to + 7O ⁰ C, then the amino protecting group is removed and optionally, if A is acetoxy, the acetoxy group by means of per se known working methods converted into a pyridinium group.

Among the pharmaceutically acceptable non-toxic Cations include metal cations such as sodium, potassium, calcium, and aluminum, and organic Amine cations such as trialkylamines such as Triethylamine, procaine, dibenzylamine, N-benzyl-ß-phenethylamine, 1-ephenamine, N, N'-dibenzylethylenediamine, dehydroabietylamine, N, N -Bis-dehydraabietylethylenediamine, N- (lower) -alkylpiperidines, for example N-ethylpiperidine, and other amines that have been used to form salts with benzyl penicillin. The invention Compounds can also form and add non-toxic acid addition salts (i.e. the amine salts) these salts include the addition salts of inorganic acids, such as the hydrochloride, hydrobromia, Hydroiodide, sulfate, sulfamate and phosphate, and the addition salts organic acids such as maleate, acetate, citrate, oxalate, sucoinate, benzoate, tartrate, pumarat, malate, Mandelate, ascorbate and the like. The inventive

109844/1 937

$ 211,205

Convolutions can also exist as "free acid", which of course, present as a zwitterion.

The compounds according to the invention can be prepared by acylation of 7-aminocephalosporanic acid (or a salt or slightly hydrolyzed ester thereof including those in the United States patent. 3,284,451 and including those described in U.S. Patent 3,249,622 for use with 6-aminopenicillanic acid, .written esters) or the pyridinium analogue of 7-aminocephalosporanic acid (produced by reacting 7-AOA with pyridine) with an acylating agent of the selected aminomethylphenylthioacetic acid, with conventional. Acylation methods find application.

The acylation derivatives of the acid of the above! Formula III, which are suitable for the acylation of compounds of the formula II are known to the person skilled in the art and include the corresponding acid halides, acid azides, acid anhydrides, mixed acid anhydrides (and especially the mixed anhydrides obtained from stronger acids such as the lower aliphatic monoesters of carbonic acid, of alkyl and arylsulfonic acids and more hindered acids such as diphenylacetic acid), active esters (for example the p-nitrophenyl ester, the 2,4-dinitrophenyl ester or the Ji-hydroxysuccinimide ester) and active thioesters (for example with thiophenol or thioacetic acid). In addition, the free acid itself can be combined with the compound of formula II, after first the

free acid reacted with N ^ '- Dimethylchlorformiminiumchlorid (see British Patent 1 008 170 and Experientia XXl / 6, 360 (1965)) or by use of enzymes or a !!, N'-carbonyldiimidazole or, a Ν, ίΡ-carbonyltriazole (cf. the South African patent

10984471937

63/2684) or by using a carbodiimide reagent (in particular of !!, N'-dicycloheaylcarbodiimide, N, N * -diisopropyloarbodiimide or ¹ -cyclohexyl-N1- (2-morpliolinoethyl) -carbodiimide (cf. J. Amer. Chem. Soc. 77, 1067 (1955). An alkynyl reagent (cf. R. Buijle and HG Viehe, Angewandte Chemie International Edition 3, 582 (1964)), a ketenimine reagent (cf. CL. Stevens and ME Monk, J . Amer. Chem. Soc. 80, 4065 (1958)) or an isoxazolium salt reagent (cf. Woodward, Olofson and Mayer, J. Amer. Chem. Soc, 83, 1010 (1961)). 4-Dinitrophenyl- and p-Hitrophenylester is a corresponding azolide, ie an amide of the corresponding acid, the amide substance of which is a member of a quasi-aromatic 5-membered ring which contains at least 2 nitrogen atoms, namely imidazole, fyrazole, triazoles, benzimidazole, benzotriazole and their substituted derivatives. As an example of the general method of preparation of an azolide, N, N ^f - Carbonyldiimidazole reacted with a carboxylic acid in equimolar amounts at room temperature in tetrahydrofuran, chloroform, dimethylformamide or a similar inert solvent to form the carboxylic acid imidazolide in practically quantitative yield with the release of carbon dioxide and 1 mole of imidazole. Dicarboxylic acids give diimidazolides. The by-product imidazole precipitates and can be separated off. The <imidazolide can be isolated, but this is not essential.

Particularly preferred acylating agents include Acid halides, most preferably the acid chloride, and activated esters, most preferably the 2,4-dinitrophenyl ester, used in conjunction with a carbodiimide reagent, e.g. lijN ^ dicyclohexylcarbodiimide. The ways of working to carry out these reactions for the production

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21120S8

of a cephalosporin and the procedures for isolating the cephalosporin thus produced are known to the Pachmann.

In order to achieve maximum product yields, the use of an acylating agent is necessary, wherein the free amino group is conventionally protected with an amino protecting group of the type d'er in Peptide synthesis or one of the numerous syntheses of α-aminobenzylpenicillin from 2-phenylglycine is used. Examples of such blocking or amino protecting groups include carbobenzoxy, p-nitrocarbobenzoxy, t-butoxycarbonyl, 2-hydroxy-i-naphthcarbonyl, ß-oxoalkylidene, Furfuryloxycarbonyl, adamantyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl or similar. Alternatively, the amino group can be protonated in "form" during acylation of the salt are protected. So is a particularly preferred acylating agent, the acid halide hydrohalide, for example the acid chloride hydrochloride of the formula.

-S - CH ₂ COCl

HOl'KH ₂ OH ₂ ■

Other particularly preferred amino protecting groups are p-Nitrocarboben ^ oxygruppe and the t-Butoxycarbonylgruppe. Of course, other functionally equivalent protective groups for an amino group can also be used, where including such groups within the scope of the present invention fall.

After completion of the acylation reaction, the amino

109844/1937

protecting group removed in a conventional manner, for example, the p-Hitrocarbobenzoxygruppe by catalytic hydrogenation, the t-butoxycarbonyl group by treatment with cold (ie with about O ⁰ C) trifluoroacetic acid, the 2-hydroxy-1-naphthcarbonyl group by acid hydrolysis and the 2.2 , 2-Trichloräthoxycarbonylgruppe removed by treatment with zinc dust in glacial acetic acid.

The inert solvents which can be used in the acylation reaction are known to the person skilled in the art. This includes solvents such as tetrahydrofuran, dimethylformamide, methylene chloride, Diethyl ether, chloroform, methyl butyl ketone, ethyl acetate and the dimethyl ethers of ethylene glycol and diethylene glycol. The most preferred solvent is methylene chloride.

The preferred temperature range for the AcyIierungsverfahren is about -20 ⁰ C to about +70 ⁰ C. Best results are obtained at temperatures of about ⁰ C O. However, as with most chemical reactions, temperatures higher or lower than those within the preferred limits can be used. Much higher temperatures than 70 ⁰ C lead due to a larger degree of side reactions to reduced yields, while temperatures result in substantially lower than -20 ⁰ C either because of reduced reaction rates in low yields or in excessively long reaction times.

Although some reaction takes place regardless of the molar ratios of the reactants are used, with a view to achieving maximum yields, is to use a molar excess of the acylating agent is preferred.

The compounds of the formula I in which A is pyridinium,

.109 844/1937

can be prepared by first combining 7-ACA with pyridine to form the pyridinium analog of 7-ACA with the formula

₀ N - CH CH CH ₀ ^ ,

² t ι t ² fy / \

O = CI C-CH ₀ -Ii

t
COO

is implemented, whereupon the acylation of the resulting compound with the appropriate acylating agent, as explained above.

The cephalosporin compounds with the 3-pyridiniummethyl substituent can alternatively be prepared by first acylating 7-ACA with the appropriate acylating agent to obtain the desired compound of formula I having the 3-acetoxymethyl substituent, after which the conversion of the acetoxy group into the pyridinium group follows according to procedures known per se.

One known conversion method that can be used involves reacting the aylation product with the 3-acetoxymethyl substituent with an excess of pyridine in a strongly polar medium, most preferred Water.

Another way of working to convert the acetoxy group into the pyridinium group, resulting in higher product yields than the above-mentioned working method, is in the lower

109844/193 7

12058

- ίο -

Patent 6,408,066. This way of working, which is more fully described in the following examples involves first replacement of the acetoxy group in the acylation product by thiopicolinic acid, which in turn by reaction with a mercury perchlorate-pyridine complex is replaced by pyridine. The reaction scheme given below illustrates what takes place Reactions:

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© ■

O H O OJ

OJ

CO

'Ϊ5

O O

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Both of the above conversion methods can either be before or after, the removal of the amino protecting group from the acylation product be performed.

In the treatment of bacterial infections in humans, the compounds of the invention become parenteral according to conventional methods of administering antibiotics in an amount of about 5 to 200 mg / kg / day and preferably from about 5 to 20 mg / kg / day in divided proportions Dosage, for example 3 to 4 times a day, administered. They are administered in dosage units, the, for example, 125 »250 or 500 mg active ingredient with suitable physiologically acceptable carriers or binders. The dosage units are in the form of liquid preparations such as solutions or Suspensions, before »

Results related to therapeutic activity

It has been found that 7- [- (p-Aminomethylphenylthio) -acetamidoJ-cephalosporanic acid after dissolving in dimethyl sulfoxide (DMSO) after dilution with nutrient broth in a triple experiment the following minimum inhibitory concentrations (M.I.CO in ^ / ml against the specified microorganisms shows, determined by incubation overnight at 37 ° C by serial or tube dilution «,

109844/1937

organism

D. pneumoniae 5% serum St. pyogenes A96o4

S. aureus Smith

S. aureus Smith $ 50 Serum

S. aureus BX-I633-S ^ dilution)

S. aureus BX-1633-2 (10 dilution)

SaI. enteritidis

E. coli Juhl (AI5II9)

E. coli

K. pneumoniae

K. pneumoniae Pr. Mirabilis Pr. Morganii Ps. Aerüginosa Ser. marcescens

MIC in yVml
Attempt no. 2 3 1 <0.02 0.03 0.04 <0.02 0.016 0.04 0.08 0.16 0.08 0.08 0.16 0.16 0.16 0.16 0.08 0.16 0.16 0.3 0.16 0.16 i 0.5 4th 4th 2 125 125 125 0.3 <0.02 <0.5 2 2 1 10 0.04 Ϊ 0.5 250 250 250 250 250 250 > 25O y '250 250

109844/1937

£ 112 058

■ '-H-

7 - [- (p-Aminomethylphenylthio) -ac etamido] -cephalosporanic acid is generally considerably more active than sodium cephalothin or 7- [a- (p-aminomethylphenylthio) -propionamido] -cephalosporanic acid in such tests. This test also shows that 7- [<* ■ - (p-Aminomethylphenylthio) -acetamidο] -cephalosporanic acid is not sensitive to staphylococcal β-lactamase and is not significantly bound to human serum.

7 - [- (p-Aminomethylphenylthio) -ac etamido] -c ephalosporanic acid is well absorbed by mice when administered parenterally but not when administered orally. The blood concentrations in mice after intramuscular administration of 10 mg / kg are about the same as those with cephalothin can be obtained. It is a lower minimum dose (GDcq) of 7 - [- (p-aminomethylphenylthio) -acetamido] -cephalosporanic acid than au cephalothin when given subcutaneously in two doses required to $ 50 by groups of mice to cure those infected with St. pyogenes (A96O4) or E. coli Juhl (A15119).

In duplicate experiments it was found that 7- (p-aminomethyl-phenylthio) -acetamido-3- (pyridiniummethyl) -ceph-3-em-4-carboxylate, which is soluble in water to an extent of at least 2 mg / ml, in Mhr broth, which shows the following minimum inhibitory concentrations (MIC) in y / ml against the specified microorganisms, determined during incubation over power at 37 ^° C. by serial dilution.

109844/1937

organism

D. pneumoniae St. pyogenes

S. aureus Smith

S. aureus Smith 50% serum

S. aureus BX-1633-2 Sal. Enteritidis

E. coli Juhl (A15119) E. coli

K. pneumoniae K. pneumoniae Pr. Mirabilis Pr. Morganii Ps. Aeruginosa Ser. marcescens

K.I.C. in fi ¹ / ml attempt No • 1 2 < 0.02 0 , 004 <0.02 0 , 004 0.04 0 , 04 0.08 0 , 04 0.5 0 , 6 <0.5 0 , 6 1 2 1 1 1 1 2 2 1 1 125 63 250 250 63 63

10 9 8 4 4/1937

7- (p-Aminomethylphenylthio) -acetamido-3- (pyridiniummethyl) -ceph. -3-em-4-carboxylate is generally considerable in such tests, more active than sodium cephalothin and about as active as cephaloridin. Another test also shows that 7 - [^ - (p-Aminomethylphenylthio) acetamido] -3- (pyridiniummethyl) ceph-3-em-4-carboxylate is not significantly bound to human serum. There is preliminary evidence that 7- [- (p-aminomethylphenylthio) acetamido] -3- (pyridiniummethyl) ceph-3-em-4-carboxylate shows lower nephrotoxicity than cephaloridin (allowing higher doses in humans if necessary), while maintaining the same or greater antibacterial activity and breadth of spectrum.

7- (p-Aminomethylphenylthio) -acetamido-3- (pyridiniummethyl) -ceph-3-em-4-carboxylate is well absorbed by mice when administered parenterally, but not when administered orally. The blood concentrations in mice after intramuscular administration of 10 mg / kg are higher than those obtained with cephalothin and about the same as those obtained with cephaloridin. A lower minimum dose (CD ₅₀ ) of 7-C- (p-AMnomethy! Phenylthio) -acetamido] -3- (pyridiniummethyl) -ceph-3-em-4-carboxylate than of cephalothin is required when given subcutaneously in two doses to cure $ 50 from groups of mice infected by St. pyogenes (A96O4) or E. coli Juhl (A15119).

It was found that 7 - [- (m-aminomethylphenylthio) -acetamido] -cephalosporanic acid after dissolving in dimethyl sulfoxide (DMSO) and subsequent dilution with nutrient broth shows the following minimum inhibitory concentrations (MIC) in f / ml against the specified microorganisms, determined at Incubation overnight at 37 ^° C. by serial dilution.

10 9 8 4 4/1 937

Organism 'MI, C. in tf- / ml

Staphylococcus aureus Smith A9537 0.08

Staphylococcus aureus A96O6

(10 "" x ⁵ dilution) 0.16

Staphylococcus aureus AI5097 0.6

Staph aureus + $ 50 serum A9537 0.16

Streptococcus pyogenes A96O4

'+ 5% serum * <0.02

Escherichia coil A9675 8.0

Escherichia coil Juhl A15119 1 * 0

Proteus mirabilis A99OO. 1.0

Proteus morganii A15153 250.0

Pseudomonas aeruginosa A98 * t3A 250.0

Serratia marcescens A2OO19> 250.0

Klebsiella pneumoniae A9977 <0.5

Klebsieila pneumoniae A1513O 2.0

Salmonella enteritidis A9531 0.3

in 45 $ antibiotic test brülie and 50 ^a / <> Mhrbriülie.

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7- [- (m-Aminom ethylphenylthio) acetamido] -cephalo sporanic acid is generally significant in such tests in vitro against most strains of E. coli and Proteus, more active than 7 - [- (p-Aminoniethylph.enylthio) -acetamido] -cephalosporanic acid, that is, it is often four times as active and almost always twice as active. This test also shows that 7 - [- (m-aminomethylphenylthio) -acetamido] -cephalosporanic acid is not sensitive to staphylococcal β-lactamase and is not significantly bound to human serum will.

In duplicate experiments it was found that 7- (m-aminomethylphenylthio) -acetamido-3- (pyridiniummethyl) -ceph-3-em-4-carboxylate, which is soluble in water to an extent of at least 2 mg / ml, in nutrient broth shows the following minimum inhibitory concentrations (MIC) in y / ml against the specified microorganisms, determined during incubation overnight at 37 ^° C. by serial dilution.

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organism

M.I.C. in ^ experiment no.

Staphylococcus aureus Smith A9537

Staphylococcus aureus (BX-1633-2) A96o6 (1O * "- ⁵ dilution)

Staphylococcus aureus A15O97 Staph. aureus + 50 $ serum A9537

Streptococcus pyogenes A96O4 - 5% serum *

Diplococcus pneumoniae A9585 - 5% serum *

Escherichia coli A9675 Escherichia coli Juhl A15H9 Proteus mirabilis A99OO Proteus morganii A15153 Pseudomonas aeruginosa A9843A Serratia marcescens A2OO19 Klebsie11a pneumoniae A9977 Klebsiella pneumoniae A1513O Salmonella emiteritidis A9531 0.02

1.3 0.08

C 0.02

0.016

1.3 1.3 0.03

5 0.002

0.004 <0.002

4th 2 4th 2 2 1 ■ ^ 250 > 125 ^v 250 7 250 125 63 2 1 4th 2 1.3 0.6

in 45% antibiotic test broth and $ 50 nutrient broth

10984A / 1937

7- (m-Aminomethylphenyl thio) -acetamido- 3- (pyridiummethyl) -ceph-3-em-4-carboxylate is generally considerable in such tests, more active than sodium cephalothin and as active as cephaloridin. This test also shows that 7- (m-aminomethylphenylthioJ-acetamido ^ - (pyridiniummethyl) -ceph-3-em-4-carboxylate is not significantly bound to human serum.

In double tests it was found that 7 - [- (o-aminomethylphenylthioj-acetamidoj-cephalosporanic acid, after dissolving in dimethylsulfoxide (DMSO) and subsequent dilution with milk broth) shows the following minimum inhibitory concentrations (MIC) in jf / ml against the specified microorganisms, determined at Incubation overnight at 37 ^° C. by serial dilution.

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organism MIC in
attempt g / ml
No. 1 2 D. Pneumoniae 0.04 0.06 St. pyogenes A96O4 0.02 0.03 S. aureus Smith 0.08 0.13 S. aureus Smith $ 50 Serum 0.08 0.25 S. aureus BX-1633-2 0.16 0.25 SaI. enteritidis <0.5 0.3 E. coli June (A15119) <0.5 0.6 E. coli <0.5 2.5 K. pneumoniae <0.5 0.6 K. pneumoniae <0.5 1.3 Pr. Mirabilis <0.5 0.6 Pr. Morganii > 25O 125 Ps. Aeruginosa > 25O > 250 Ser. marcescens > 250> 250

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7- [- (o-Aminom eth.ylphenylth.io) -ac etamido] -c ephalosporic acid is generally significant in such tests against most strains of E. coli and Proteus in vitro more active than 7 - [- (p-Aminomethyiphenylthio) -acetamido] -cephalosporanic acid, i.e., "It is often four times as active and almost always twice as active. This test also shows that T-C-Co-aminomethylphenylthioJ-acetamido -] - cephalosporanic acid is not sensitive to staphylococcal β-lactamase and is insignificant in human serum is bound.

7 - [- (o-Aminomethylphenylthio) -acetamido] -cephalosporanic acid is well absorbed by mice when administered parenterally, but not when administered orally. The blood concentrations in mice after intramuscular administration γοη 10 mg / kg are about the same as the values obtained with cephalothin . A lower minimum dose (CDc ₀ ) of 7 - [- (o-aminomethy! Phenylthio) -acetamido] -cephalosporanic acid than of cephalothin when administered subcutaneously in two doses is required to cure 505ί of groups of mice infected with St. pyogenes (A9604) or E. coli Juhl (A15119) are infected.

In duplicate tests it was found that 7- (o-aminomethylphenylthio) -acetamido-3- (pyridiniummethyl) -ceph - ^ - em- ^ carboxylate, which in water to an extent of at least 2 mg / ml is soluble in nutrient broth the following minimal Inhibitory Concentrations (M.I.C.) in r / ml versus the stated Shows microorganisms, determined by incubation overnight at 37 ° C. by serial dilution.

1098U / 1937

M.I.C. in ^ - / ml organism experiment no.

in 45 # antibiotic test broth and 50% nutrient broth

Staphylococcus aureus Smith A9537 0.04, 0.04

Staphylococcus aureus A9606

(IO " ⁵ dilution) 0.6 0.3

Staphylococcus aureus A15O97 2.5 0.6

Staph. aureus - 50? Serum A9537 0.08 0.08

Streptococcus pyogenes A96O4

+ 5% serum * <0.02 0.004

Diplococcus pneumoniae A9585 + 5% serum *

Escherichia coil A9675 Escherichia coil Juhl A15119 Proteus mirabilis A99OO Proteus morganii A15153 Pseudomonas aeruginosa A9843A Serratia marcescens A2OO19 Klebsieila pneumoniae A9977 Klebsiella pneumoniae A1513O Salmonella enteritidis A9531

0.016 4th 2 4th 2 2 2 16 63 250 > 25O 32 32 2 2 4th 4th 2.5 2.5

109844/1937

7- (o-Aminome th.ylph.enylth.io) -ac etamido-3- (pyridiniummethyl) -ceph-3-em-4-carboxylate is generally considerable in such tests, more active than sodium cephalothin and about as active as cephaloridin. This test also shows that 7- (o-aminomethylphenylthio) acetamido-3- (pyridiniummethyl) ceph-3-em-4-carboxylate is not significantly bound to human serum.

The following examples are intended to further illustrate the invention, but not to limit it. All temperatures are measured ^{in 0 C.} The protective group "t-butoxycarbonyl" is abbreviated as "BOC", while the protective group "p-l-nitrocarbobenzoxy" is abbreviated as "NOpCbo".

example 1

7 - [-. (P-Aminomethylphenylthio) -acetamidο] -cephalosporanic acid

The synthesis of this cephalosporin is illustrated by the following reaction scheme:

1098U / 1937

PI

Q ι

Ό P-.

OJ

O O

OJ O

hC C 3 Sh

OJ

τ:

CM

-P

cd ο

cd χ

OJ I

OJ

109844/1937

Original

COPY

- do -

(p-Chloromethylphenylthio) -acetic acid ethyl ester

A rapid stream of dry hydrogen chloride is passed at room temperature through a stirred mixture of 55.4 g (0.28 mol) ethyl (phenylthio) acetic acid ester, 12.0 g (0.133 mol) paraformaldehyde, 5 g anhydrous zinc chloride and 150 ml chloroform, is saturated bis'die mixture (after about 1 hour), the mixture is stirred for another hour at room temperature and then 1.5 Stxraden at 40 ⁰ C and then poured in'400 ml of ice water. The layers are separated and the aqueous layer is extracted with an additional 100 ml of chloroform. The combined chloroform solutions are dried (MgSO,), the solvent is removed, and the residue is fractionally distilled in vacuo to yield 21.1 g ("31 $ >) of the desired product with an Xp = 140 to 150 ° (0.2 mm). NMR spectra (CCl.): ^ 2.72 (s, 4H), 5.53 (3.2H), 5.91 U, 2H), 6.49 (s, 2H) and 8.80 (t, 3K). The infrared spectrum contains a strong band at 1730 cm "which is assigned to the ester carbonyl.

(p-Chloromethylphenylthio) acetic acid

A mixture of 24.5 g (0.10 mol) of ethyl (p-chloromethylphenylthio) acetic acid ester, 320 ml of acetic acid and 125 ml of 6N hydrochloric acid is left at room temperature for 16 hours and then concentrated to a small volume under reduced pressure. The white contaminant is collected by filtration, washed with ice water, dried and recrystallized from a 1: 1 mixture of benzene / n-hexane, 10.5 g (49 #) of solid (p-chloromethylphenylthio) acetic acid with a P = 95 to 103 ° result. Λ ϊ? £ ^θ1 1700 cm "¹; NMR (CDCl,) ι Singlet3 at

ΙΠϋΧ j

7 -0.98 (IH), 2.65 (4H), 5.48 (2H) and 6.35 (2H).

109844/1937.

(p ~ Aminoineth.ylph.enyltJa.io) acetic acid

11.5 g (0.053 Hol) (p-chloromethylphenylthio) -eosetic acid are added proportionally with stirring to 150 ml of concentrated ammonium hydroxide at room temperature. The solution is left at room temperature for 17 hours and then concentrated to dryness. The residue is treated with 100 ml of methanol. The white plague is filtered through, collected, and dried in vacuo over Po ^ s. 5.3 g of (p ~ aminometh.ylplienylth.io) acetic acid '(55? «) With an I? = 203 to 205 ° (decomp.) Obtained. NI-IR (GP ₃ CO ₂ H): T2, 51 (s, 4H), 5.59 (q., 211) and 6.16 (s, 2H).

[p- (p'-liitroca _% rbobenzoxyaminomethyl) phenylthio] acetic acid

To a stirred suspension of 4.53 g (0.023 mol) of (p-aminomethylphenylthio) acetic acid in 120 ml of water is added ' In aqueous sodium hydroxide added "until the solution is pH 10 has reached. The clear solution is diluted with 80 ml of tetrahydrofuran. Then a solution of 5.60 g (0.026 mol) p-Nitrobenzyl chloroformate (NOgCboCl) drop by drop Stirring at room temperature added in about 15 minutes. Simultaneous addition of 1N aqueous sodium hydroxide becomes the pH is regulated at pH 7 to 9. When the addition is complete, the solution is mixed with two 100 ml portions of ethyl acetate extracted. The aqueous solution is cooled and acidified with 20 ml of 3N aqueous sulfuric acid. The failed oil is extracted twice with 100 ml of ethyl acetate, then dried (MgSO.) The ethyl acetate solution is concentrated to a volume of 50 ml and cooled, whereby 5.4 g ($ 63) white solid [p- (p'-ifitrocarbobenzoxyaminomethyl) ~ phenylthioJ-acetic acid with a P = 143 to 147 °. The structure of the compound is determined by the infrared spectrum confirmed.

1098U / 1837

Potassium 7-t - [p- (p f -nitrocarbobenzoxyaminomethylj-phenylthio ""] - acetamido} -cephalosporanate

1 »03 g (0.0050 mol) of N, N'-dicyclohexylcarbodiimide (DCC) are added to a cold solution of 1.88 g (0.0050 mol) [p- (p '~ nitrocarbobenzoxyaminomethyl) -phenylth.io j- acetic acid and 0.92 g (0.005 mol) of 2,4-dinitrophenol (2,4-DNP) in 10 ml of anhydrous tetrahydrofuran. The mixture is left at room temperature for 1 hour, then the JS, N'-dicyclohexyurea is filtered off and the piltrate containing the crude activated ester is concentrated to dryness under reduced pressure. A solution of ¹ »36 g (0.0050 mol) of 7-aminocephalosporanic acid (7-ACA) and 1> 01 g (0.010 mol) of triethylamine in 10 ml of methylene chloride is added to the residue, cooled to 0 °, from the crude activated Ester is given. The reaction mixture is left at room temperature for 4.5 hours, then the solution is diluted with ether. The oily precipitate is redissolved twice in methylene chloride and reprecipitated with ether, then it is dissolved in 15 ml of methanol. The methanol solution is treated with 2.5 ml of a 2.3 molar solution of potassium 2-ethylhexanoate, after which ether is added. The solid precipitate is filtered off and suspended in 30 ml of methanol (only part of it dissolves). After the addition of ether, the product is collected by filtration and dried, giving 3 »0 g (9Ο5έ) yellow-colored solid potassium 7-i- [p- (p'-nitrocarbobenzoxyaminomethyl) -phenylthio! -Acetamido} -cephalosporanate. The infrared spectrum (Nujol muli) contains the expected bands at 3250, 1760, 1725, 1690, 1655 and 1600 cnT ¹ .

109844/1937

7- [- (p-Aminomethylphenylthio) -ac etamido] -cephalosporanic acid

A mixture of 3.0 g (0.0045 mol) of the protected cephalosporin potassium 7- £ - [p- (p '-nitrocarbobenzoxyaminomethyl) -phenylthio] -acetamidol-cephalosporanate, 1.5 g 10 $ palladium-on- Charcoal and 50 ml of water are hydrogenated at atmospheric pressure for 7 hours, after which the reaction mixture is filtered through diatomaceous earth ("Gelite"). The piltrate is cooled in ice and brought to pH 2.0 with dilute hydrochloric acid and then filtered again through "Celite" in order to remove some precipitate. The pale yellow clear filtrate is adjusted to pH 5.9 with dilute aqueous sodium hydroxide and concentrated to dryness under reduced pressure. (The product begins to crystallize from a concentrated solution). The solid residue is washed successively with a 4 ml portion and two 2 ml portions of ice water and then dried in vacuo over PoOc, whereby 0.60 g ($ 30) of pale brown solid 7 - [- (p-aminomethylphenylthlo) - acetamidoj-cephalosporanic acid. A ~ T ^ ⁰¹ . 325O, 1760, 1715, 1650 and 1575 cm "" ¹ . On the basis of the infrared and MMR spectra as well as the thin-layer chromatogram, the purity is estimated at $ 80 or more.

Example 2

Oephalosporin from p-aminomethyl- (thio) -phenoxyacetic acid

p-Aminomethylthiophenoxyacetic acid can be obtained by amidomethylation (H.E. Zaugg and W.B. Martin, Organic Reactions, Vol.14, John Wiley & Sons, Inc., New York, Chapter 2 (1965)) by (Phenylthio) acetic acid and subsequent hydrolysis for example as follows:

109844/1937

211205C

in α.

CM

ΪΠ O

CM

K O il

Q)

about

-p

OJ

DI XQ

CD

O r-H

a ο

<l

109844/1937

ORIGINAL INSPECTED

- 31 -

The use of the tert-butoxycarbonyl group (BOC) as Protecting group is for the production of 7 - [- (p-aminomethylphenylthio) -acetamido] -cephalosporanic acid investigated and found to be very successful. Their use is against the p-nitrocarbobenzoxy group recommended.

109844/1937

> = O OJ

ro

O = O O

O ^N O

ro

Oi ₊
O ^ν - '
Q

• P

OJ

ο
ι

OJ

• p

Rj O Ö

cd X

ω ο

OJ I

OJ O

P * O

109844/1937

(p-Aminomethy! phenyl thio) acetic acid

A mixture of 232.6 g (1.38 mole) of (phenylthio) acetic acid and 172.9 g (1.40 mol) of N-Methylolchloracetaraid is partly ml of concentrated in the course of 1 hour with stirring at ⁰ to 20 C to 375 Given sulfuric acid. The reaction mixture is left at room temperature for 5 days and then poured into 3 liters of a mixture of ice and water with vigorous stirring. The product is extracted with ethyl acetate (one portion of 600 ml and two portions of 200 ml). The combined ethyl acetate solutions are extracted with a total of 700 ml of 2N aqueous sodium carbonate. The carbonate extract is cooled and acidified with 250 ml of 6N hydrochloric acid, followed by extraction with ethyl acetate. The ethyl acetate solution is over MgSO. dried and concentrated to a volume of about 500 ml, followed by cooling at 0 ° overnight. The white solid (241.4 g, P = 85-108 °) is collected by filtration, washed with ether and refluxed with 1 liter of concentrated hydrochloric acid for 2.5 hours. The white hydrochloride which precipitates out on cooling is obtained by filtration and dissolved in 1 liter of warm water. The pH of this solution is adjusted to 5.0 with concentrated ammonium hydroxide. The amino acid, which readily crystallizes out of solution, is collected by filtration after cooling. The white solid (p-aminomethylphenylthio) acetic acid is washed with ice water and methanol and dried in vacuo over P0O5. 45.6 g (17 #) of product with a melting point = 249 to 251 ° (decomp.) Are obtained. The material is identical to the material obtained by treating (p-chloromethylphenylthio) acetic acid with ammonium hydroxide.

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[p- (tert-Butoxycarbonylaminomethyl) phenylthio] acetic acid

A solution of 6.2 g (0.043 mol) of tert-butoxycarbonylazide in 60 ml of tetrahydrofuran is added to an ice-cold solution of 6.9 g (0.035 mol) of (p-aminomethylphenylthio) acetic acid in 80 ml of 1N aqueous sodium hydroxide. The reaction mixture is stirred for 18 hours at room temperature. Some of the starting amino acid has precipitated and is removed by filtration. Most of the tetrahydrofuran is removed under reduced pressure. The aqueous solution is washed once with ether, then covered with a layer of 100 ml of ether and adjusted to pH 3.5 with dilute hydrochloric acid while cooling with ice and stirring. More starting amino acid precipitates at this stage and is removed by filtration (a total of 0.9 g of amino acid is recovered). The layers are separated and the aqueous layer is extracted with an additional 75 ml of ether. The combined ether layers are dried and then concentrated to dryness. The solid residue is washed with 75 ml of cold η-hexane, 7 »0 g (689 ε) of white solid [p- (tert-butoxycarbonylaminomethyl) phenylthio / acetic acid having an F 3 114 ° to 115 ° resulting. The infrared spectrum (Nujol muli) contains the expected maxima at 3350, 1700 and 1680 cm " ¹ .

Potassium 7-i- [p- (tert-butoxycarbonylaminomethylj-phenylthio] -acetamido] -cephalosporanate

2 »47 g (0.012 mol) of N ^ -dioyclohexyloarbodiimide become a solution of 3.56 g (0.012 mol) BOG amino acid [p- (tert, Butoxycarbonylaminomethyl) phenylthio] acetic acid and 2.21g (0.012 mol) 2,4-dinitrophenol in 30 ml tetrahydrofuran, After 1 hour at room temperature, the mixture becomes

109844/1937

filtered and the solvent is removed from the filtrate. The yellow oil that remains is dissolved in 10 ml of methylene chloride and a solution of 3.26 g (0.012 mol) of 7-aminocephalosporanic acid and 3.0 g (0.03 mol) of triethylamine in 25 ml of methylene chloride is added while cooling with ice The reaction mixture is treated with ether for 4 hours at room temperature. The precipitated oil is redissolved twice in methylene chloride and reprecipitated with ether, then taken up in methanol and treated with 5 ml of a 2.4 molar solution of potassium 2-ethylhexanoate in n-butyl alcohol. The potassium cephalosporanate precipitates after the addition of ether. It is redissolved in methanol and then reprecipitated with ether, resulting in 5.0 g ($ 82) of yellow solid potassium 7-i- [p- (tert-butoxycarbonylaminomethyl) -phenylthio] -acetamido] -cephalosporanate, whose infrared spectrum (Nujol mull) shows maxima at 3250, 1755, 1690, 1650 and 1600 cnf ¹ .

7 - [- (p-Aminomethyl! Phenylthio) -acetamido] -cephalosporanic acid

4.7 g (0.008 moles) of BOC-protected potassium cephalosporanate Potassium 7-t- [p- (tert • -butoxycarbonylaminomethylj-phenylthio J-acetamido] cephalosporanate are dissolved in 150 ml of water. The solution is cooled in ice with 150 ml of ethyl acetate covered and acidified to pH 2.4 with stirring. The layers are separated and the aqueous layer is with extracted two 75 ml portions of ethyl acetate. The United Solutions are dried (MgSO.) And concentrated to dryness. The BOC-protected oephalosporanic acid thus obtained is treated with 25 ml of trifluoroacetic acid at 0 ° for 2 hours treated, after which ether is added. The solid precipitate is collected by filtration, washed with ether and suspended in 125 ml of water. The pH will be below

1098U / 1937

Ice cooling brought to 1.0 with 3N hydrochloric acid. A small amount of insoluble material is removed by filtration through "Gelite". The piltrate is adjusted to pH 5-6 with dilute ammonium hydroxide, after which it is treated with decolorizing charcoal and filtered through "Celite". The almost colorless filtrate is concentrated to a volume of about 15 ml and cooled to 0 °. The white solid precipitate is collected and washed successively with twice 10 ml biswater, twice 15 ml methanol and ether. The product 7 - [^ (p-aminomethylphenylthio) -acetamidoj-cephalosporanic acid is finally dried _{in vacuo over'P 3} O _5. The yield is 2.2 g ($ 61). The infrared spectrum (Hujol muli) contains sharp maxima at 3250 (NH), 1760 (ß-lactam carbonyl), 1740 (acetoxycarbonyl) and 1660 cm (amide carbonyl). The NMR spectrum (in trifluoroacetic acid) is also in full agreement with the assigned structure, the purity of the compound mentioned is estimated at $ 95.

Example 3

In the present example is a somewhat simplified procedure for the preparation of (p-aminomethylphenylthio) acetic acid described a hydrolysis without isolation of the condensation product from N-methylolchloroacetamide and (Phenylthio) acetic acid includes

(p-tert-ButoxycarbonylaminomethylphenylthioJ-acetic acid can be obtained in quantitative yield from tert-butoxycarbony1-azide and the amino acid using triethylamine as the base.

The BOC amino acid reacts with thionyl chloride in the presence

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of Iriäthylamin (methylene chloride as solvent) or
Pyridine (benzene as solvent), resulting in the BOC-aminoacyl chloride, which can be reacted directly with 7-ACA in methylene chloride solution in the presence of triethylamine. The protecting group can then be removed by treatment with cold trifluoroacetic acid.

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CVJ ■ Η O O

ca ο

CM

WHERE

CVJ O O

CVJ

1098U / 1937

(p-aminomethylphenylthio) acetic acid

A mixture of 140.8 g (0.84 moles) of (phenylthio) acetic acid and 103.7 g (0.84 moles) of N-methylol chloroacetamide becomes added proportionately in the course of 1 hour with stirring at 10 to 20 ° to 200 ml of concentrated sulfuric acid. The reaction mixture is left at room temperature for 2 days and then divided into two equal parts. A part will be worked up immediately and the other part will leave at room temperature for a further two days before working up. The mixtures are as follows Worked up: 180 ml of water are added with stirring and the solution is refluxed for 1.5 hours, after which it is cooled in an ice-salt mixture for 50 minutes. The white solid precipitate is filtered through collected and washed with a small volume (60 ml) ice-water. It is dissolved in 400 ml of water by heating and the pH of this solution is adjusted with concentrated ammonium hydroxide set to 5.0. The solution is cooled at 0 ° overnight and the precipitate is collected by filtration collected. The white plague is washed with ice water and methanol and weighs 17.6 g. The second part of the The reaction mixture is worked up in the same way and gives the same amount of product. The total yield is 34.9 g (21%), P = 250 to 252 ° (decomp.).

(p-tert-Butoxyoarbonylaminofflethylphenylthio) -esBig acid

To a stirred and ice-cold solution of 7.9 g (0.140 mol) (p-aminomethylphenylthio) acetic acid and 10.0 g (0.10 mol) of triethylamine in 100 ml of water is a solution of 7.2 g (0.050 mol) of tert-butoxycarbonylazid all at once given in 75 ml of tetrahydrofuran. The mixture will be at

109844/1937

Stirred at room temperature for 16 hours. The main set of Tetrahydrofuran is removed when the pressure is reduced. the aqueous solution is washed with ether, then covered with 125 ml of ether and the solution is stirred and Cooling with dilute hydrochloric acid to pH 2.5 acidified. The layers are separated and the aqueous layer is extracted with an additional 150 ml of ether. The United Ether solutions are over MgSO. dried and concentrated to dryness, giving 11.5 g ($ 97) of whiter solid .Residue with a P = 112 to 114 °, which for the next stage is sufficiently pure.

7-E- (p-aminomethylphenylthio) -ac etamid ο] -c ephalosporanic acid

A solution of 3.00 g (0.025 mol) of thionyl chloride in 25 ml of methylene chloride is added dropwise over 15 minutes to a stirred and ice-cooled solution of 7.43 g (0.025 mol) of (p-tert-butoxycarbonylaminomethylphenylthio) acetic acid and 2.73 g (0.027 mol) of triethylamine in 50 ml of methylene chloride. When the addition is finished, will leave the mixture at 0 ° for an additional 30 minutes and then dropwise over 10 minutes to a stirred Solution of 6.80 g (0.025 mol) 7-aminocephalosporanic acid and 5.05 g (0.050 mol) triethylamine in 50 ml given to -20 to -30 ° cooled methylene chloride «The reaction mixture is gradually over the course of 1 hour allowed to warm and is then treated with 75 ml of water. 25 ml of 1N hydrochloric acid are added while stirring and cooling with ice admitted. The layers are separated and the aqueous layer is added with an additional 25 ml of methylene chloride extracted. The combined methylene chloride solutions are dried (MgSO.) And concentrated to dryness. The yellow-colored foam residue (13.0 g) is partially under Stirring added to 60 ml of trifluoroacetic acid cooled in ice.

1 098AA / 1937

211205a

When the addition is complete (after 10 minutes), the reaction mixture is cooled to 0 ° for a further 30 minutes and then treated with 200 ml of ether. The precipitated salt (10.5 g) is collected by filtration, washed with ether and treated with 150 ml of water. The mixture is filtered through diatomaceous earth ("Celite") and the filtrate is adjusted to pH 5.6 with dilute ammonium hydroxide, after which it is filtered again through "Gelite". The yellow-colored filtrate is concentrated to dryness under reduced pressure and the solid residue is treated with 20 ml of ice water. The whitish solid is collected and washed successively with twice 10 ml ice water, twice 20 ml methanol and ether. The yield is 4.3 g (38%). The assigned structure is confirmed by infrared and NMR spectra. The purity is estimated at $ 90 or more.

Example 4

7 - [- (p-Aminomethylphenylthio) -acetamid ο] -c ephalosporanic acid hydrochloride

A suspension of 0.361 g (0.8 mmol) of the zwitterionic form of 7 - [- (p-aminomethylphenylthio) -acetamido] -cephalosporanic acid in 3 ml of methanol is cooled in ice and treated with a few drops of concentrated hydrochloric acid until a clear solution is obtained will. The hydrochloride precipitates as a pale brown solid after the addition of ether and is collected by filtration and dried in vacuo over Ρρ ^ 5. The yield is 0.354 g ($ 90). The infrared spectrum (Nujol muli) contains maxima at 1770, 1745, 1720 and 1665 cm " ^1. The NMR spectrum is also in agreement with the assigned structure. A solution of the material in D ₉ O is at spatial

109844/1937

temperature stable for at least 1 day as determined by NMR spectroscopy is shown. Me purity is valued at -90 $.

Example 5

. Sodium 7 - [- (p-aminomethylphenylthio) -ac etamido] -cephalosporanate

To a stirred suspension of 0.361 g (0.8 mmol) of the zwitterionic form of 7 - [- (p-aminomethylphenylthio) -acetamidoj-cephalosporanic acid 1N aqueous sodium hydroxide is added at room temperature until a clear solution (pH 10.8) is obtained. This solution is immediately freeze-dried, 0.355 g of slightly colored solid sodium 7 - [- (p-aminomethylphenylthio) acetamido] cephalosporanate result. The infrared and NMR spectra and the M.1.0. Results show that the purity of the material does not exceed $ 50.

example

A. Preparation of 7- [p- (tert-butoxycarbonylaminomethyl) -phenylthioJ-acetamido-3- (picolinoylthiomethyl) -ceph-3- em-4-oarboxylic acid

5.9 g (0.010 mol) potassium 7-t- [p- (tert-butoxycarbonylaminomethyl) -phenylthio] -acetamido} -cephalosporanat are partially with stirring to a solution kept at 75 ° of 2.8 g (0.020 mol) of thiopicolinic acid in 60 ml of water. The reaction mixture is kept at 75 ° for 1 hour, then it is cooled and the brown pesticide is filtered off. A solution of this material in a mixture

109844/1937

100 ml of acetone and 30 ml of water are adjusted to pH 2.0 with concentrated hydrochloric acid and diluted with 250 ml of water. The resulting sticky solid precipitate is dissolved in methylene chloride after washing with water and ether. This solution is dried (MgSO.) And the solvent is removed to give 3.4 g ($ 54) of the crude product as a pale brown solid. The infrared spectrum (Nujol muli) contains bands at 1775, 1700 and 1660 cm "" ¹ .

B. Preparation of 7- [p- (tert-butoxycarbonylaminomethyl) -phenylthioJ-acetamido-3- (pyridiniummethyl) -ceph-3-em-4-carboxylate

A solution of 4.3 g (0.0068 mol) of the picolinoylthiomethylcephalosporin prepared above in 30 ml of pyridine is diluted with 30 ml of water. A solution of 9 »5 g (0.0171 mol) of mercury (II) perchlorate pyridine complex in a mixture of 30 ml of pyridine and 30 ml of water is then added over the course of about 5 minutes at room temperature. The reaction mixture is stirred for 1 hour at room temperature, then hydrogen sulfide is bubbled through for 5 minutes. The mixture is filtered through "Celite" and the filtrate is washed successively with 100 ml of benzene, a 1: 2 mixture of "Amberlite LA-1" resin and benzene (150 ml and 75 ml portions) and 75 ml of benzene . The aqueous solution is concentrated to dryness under reduced pressure to give 1.9 g ($ 49) of a brown residue. The infrared spectrum (Nujol muli) shows strong absorption at 1770 and 1660 to 170 Ω cm " ¹ .

109844/1937

G. Preparation of 7- (p-aminomethylphenylthio) acetamido-3 ^ (pyridiniummethyl) ceph-3-em-4-carboxylate

2 »2 g (0.0039 mol) of BOC-protected cephalosporin are proportionally with stirring at 0 ° to 15 ml of trifluoroacetic acid. given. The reaction mixture is left for 2 hours at 0 ° leave, after which ether is added. The pale yellow pestilent (1.7 g) is collected by filtration, washed with ether and dissolved in 30 ml of water. After removing a By filtering a small amount of insoluble material, the pH of the solution is adjusted to 7 »2 with dilute ammonium hydroxide set. The mixture is treated with decolorizing charcoal and filtered through "Gelite" and then at reduced Pressure concentrated to dryness. The solid residue is mixed with two 20 ml portions and one 10 ml portion of methanol extracted. A small amount of the insoluble material is removed by filtration. The combined methanol solutions are treated with 75 ml of ether. The pale brown solid precipitate is washed with methylene chloride (twice 20 ml) and ether and dried in vacuo over PpOc. His Quantity is 0.73 g ($ 41). The infrared spectrum (Nujol muli) contains the expected absorption bands at 1775, 1675 and

-1
1610 cm, which are ascribed to the ß-lactam, the amide carbonyl and the carboxylate. The NMR spectrum is also in agreement with the proposed structure. A thin-layer chromatogram shows only one component; on the basis of this result and the spectrum results, the material is assigned a minimum purity of 85%.

"Amberlite LA-1" resin is a mixture of secondary Amines, wherein each secondary amine has the following formula

R ¹

CH ₅ C (CH ₂ ) ₂ CH ₂ C (CH ₂ ) ₂ CH ₂ CH = CH-CH ₂ NHC-R ²

R ³

109844/1937

12 3

has, where R, R and R ^ each have an aliphatic

12 3

Mean hydrocarbon radical and R, R and R in the mixture contain 11 to 14 carbon atoms. This special mixture of secondary amines, sometimes referred to as "Liquid Amine Mixture No. I", is a clear, amber colored liquid with the following physical properties:
Viscosity at 25 ⁰ C = 70 cP,
specific weight at 2O ⁰ C = 0.845, refractive index at 25 ⁰ C = 1.467,

Distillation range at 10 mm: up to 160 ⁰ C = 74 $,

above 220 ⁰ C =

Example 7

Preparation of 7 - [- (o-aminomethylphenylthio) -aoetamido] - cephalosporanic acid

This cephalosporin is made according to the sequence of reactions below:

10 98 44/1937

LiAlH

-SH

CO ₂ H

-SH

CH ₂ OH

1. After ₃ ZCH ₃ OH

2. ClCH ₂ CO ₂ CH ₃

-SCH ₂ CO ₂ CH ₃
■ CH ₂ OH

SOCl,

-SCH ₂ CO ₂ CH ₃ Hydrol.

CH ₂ Cl

W:

SCH ₂ CO ₂ H

conc.

-CH ₂ Cl

-CH ₃ NH ₂

BOC-N,

-SCH ₂ CO ₂ H

CH ₃ CH ₀ NH-CO-C-CH

2 I! I.

0 CH ₃

1. DCC, 2, M-DNP

3. K-2 ethyl hexanoate

! I

/> - SCH ₂ CNH

CH ₂ NH-BOC

CH ₂ OAc

CO ₂ K

1. CF ₃ CO ₂ H

2. pH 5.6

CH ₂ OAc

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- 47 -

o-mercaptobenzyl alcohol

A solution of 154 _f O g (1.0 mol) of TMosalicylic acid in a mixture of 1 liter of anhydrous ether and 250 ml of tetrahydrofuran is added dropwise over 2 hours to a stirred suspension of 42.0 g (1.1 mol) of lithium aluminum hydride given in 1 liter of anhydrous ether. When the addition is complete, the reaction mixture is refluxed for a further 4 hours and then left to stand overnight. The excess lithium aluminum hydride is destroyed by adding a little ethyl acetate, then the mixture is poured into 1500 ml of ice-cold 3N hydrochloric acid and stirred for 30 minutes . The layers are separated and the aqueous layer is extracted with two 200 ml portions of ethyl acetate. The combined organic layers are dried, concentrated and distilled to give 105.0 g ($ 75) of product with a bp = 94 to 96 ° (0.2 mm), mp = 30 to 32 °. The literature describes Kp = 85 ° (10 mm), i ¹ = 30 to 31 ° [R. Grice and LN Owen, J. Chem. Soc, 1947 (1963)]. The distillation residue consists of 28.0 g of thiosalicylic acid starting material.

(o-Hydroxymethylphenylthio) -acetic acid methyl ester

91f7 g (0.655 mol) of o-mercaptobenzyl alcohol become one Solution of sodium methylate, prepared from 15.1 g (0.655 g Atom) of sodium in 500 ml of methanol, after which a solution of 71.1 g (0.655 mol) of methyl chloroacetate was added dropwise in 100 ml of methanol is added. The reaction mixture is refluxed for 1 hour, cooled and filtered. The filtrate is concentrated and the residue is taken up in ether. The ethereal solution becomes with Washed water, dried and concentrated, whereby

109844/1 937

128.0 g ($ 92) of crude product yielded right for the next Level can be used. In a smaller scale approach, this product is distilled, with a Yield of $ 84 of a colorless liquid with a bp = 136 ° to 138 ° (0.2 mm). The infrared spectrum contains the characteristic hydroxyl absorption at 36ΟΟ to

- 1 - 1

3200 cm and a carbonyl band at 1725 cm (o-chloromethy! Phenylthio) -acetic acid methyl ester

128.0 g (0.604 moles) of crude (o-hydroxymethylphenylthio) acetic acid methyl ester are treated at 0 ° with 250 ml of thionyl chloride. The mixture is at for 30 minutes 0 ° and then leave for a further 30 minutes at room temperature. The excess of thionyl chloride is removed and the residue is distilled in vacuo, with 95.5 g ($ 69) of yellow-colored liquid with a boiling point of 127 to 130 ° (0.1 mm).

(o-Chloromethylphenylthio) acetic acid

113.9 g (0.493 mol) of methyl (o-chloromethylphenylthio) acetic acid are dissolved in a mixture of 1 liter of acetic acid and 550 ml of 6N hydrochloric acid and left at room temperature for 18 hours. The mixture is concentrated to about half of its original volume, diluted with 200 ml of water, cooled and filtered. The solid is washed with ice water, dried and recrystallized from ethyl acetate / n-hexane (1: 2), 84.8 g of ( 80 $) result in white needles with an F = 116 to 118 °. The NMR spectrum (in CDCI,) contains a broad singlet at 7 ~ -1i3 (CO ₂ H), a multiplet at / ^ 2.3 to 2.7 (phenyl protons) and singlets at / 5.16 (-CH ₂ Cl) and 6.32

10984 4/1937

211205a

₂ -) with an integrated area ratio of 1: 4: 2: 2.

(o-aminomethylphenylthio) acetic acid

84.8 g (0.392 mol) (o-chloromethylphenylthio) acetic acid are rapidly added to 1 liter with stirring. given ice-cold ammonium hydroxide. The mixture is left at room temperature for 16 hours and then concentrated to dryness. The solid residue is treated with 200 ml of methanol, cooled and filtered. The solid is washed with methanol and dried, 39.1 g of white solid with a melting point of 182 ° to 186 ° (decomp.) Are obtained. If the filtrate is again concentrated to dryness and the residue is treated with 100 ml of methanol and the procedure as above, 16.9 g of a second fraction of the product with an mp = 182 ° to 190 ° (decomp.) Are obtained. The overall yield is 56.0 g ($ 73). The infrared spectrum (Nujol) shows the characteristic amino acid absorption at about 1550 cm "". The NMR spectrum (in trifluoroacetic acid) contains a multiplet at 7 2.2 to 2.7 (phenyl protons and -ITH ₃ ), a quartet at 7 ^ 5.27 (-CH ₂ -IiH ₃ , coupling constant J = 5.5 Hz) and a singlet at 7 ^ 5.97 (-S-CH ₂ -) with an integrated area ratio of 7: 2: 2.

(o-tert-Butoxycarbonylaminomethylphenylthio) acetic acid

To a stirred and ice-cold solution of 19 »7 g (0.10 mol) (o-aminomethylphenylthio) acetic acid and 24.2 g (0.24 mol) of triethylamine in 200 ml of Waeser is a Solution of 17.2 g (0.12 mol) of tert-butoxycarbonylazide in Given 100 ml of tetrahydrofuran. The reaction mixture becomes stirred at room temperature for 16 hours and then at

109844/1937

. ₅ o- 2112059

reduced! Pressure concentrated to remove the tetrahydrofuran. The aqueous solution is washed twice with ether, layered with 150 ml of ether, cooled in ice and washed with 3N Hydrochloric acid adjusted to pH 2.5. The mixture is filtered and the insoluble white solid becomes with Washed ethyl acetate. The layers of the combined mitrat and wash liquids are separated and the organic layer is dried. After removing the solvent, 17.4 g ($ 59) of oily residue are obtained, which slowly crystallizes on cooling,]? = 64 to 69 ° (after washing with cold ether). The oil will go without further Purification used for the next stage.

Potassium-7- [~ (o-tert, -l) utoxycarbonylaminomethylphenylthio) -acetamido J-cephaloaporanate

12.2 g (0.059 mol) of N, ΪΡ-dicyclohexylcarbodiimide are added all at once to an ice-cold solution of 17.4 g (0.059 mol) of (o-tert.-butoxycarbonylaminomethylphenylthio) acetic acid and 10.9 g (0.059 mol) of 2, 4-Dinitrophenol given in 120 ml of ethyl acetate. The mixture is left at room temperature for 1 hour, then the N, F'-dicyclohexyurea is filtered off and the solvent is removed from the piltrate, leaving the activated ester as a viscous yellow oil. A solution of 16.0 g (0.059 mol) of 7-aminocephalosporanic acid and 12.1 g (0 ₉ 12 mol) of triethylamine in 120 ml of methylene chloride is added to the crude activated ester with ice cooling and the reaction mixture is left at room temperature for 16 hours. A small amount of insoluble material is removed by filtration, after which ether is added to the filtrate. The precipitated viscous oil is redissolved twice in methylene chloride (75 ml) and reprecipitated with ether (250 ml) and is then dissolved in 75 ml of methanol. A 2.4 molar solution of potassium 2-ethylhexanoate in n-butyl

109844/1937.

alcohol (25 ml) is added. To. Adding 200 ml of ether, the yellow solid is filtered off, washed thoroughly with ether and _{dried in vacuo over P 2} ^ 5. The yield is 28.2 g ($ 81). -The infrared and NMR spectra are consistent with the assigned structure. The purity of the material is estimated at $ 75.

7 - [- (o-Aminomethylphenylthio) -acetamido] -cephalosporan acid _________ «_

A solution of 23.6 g (0.040 mol) of the potassium salt of the BOC-protected cephalosporin in 200 ml of water is covered with a layer of 300 ml of ether and brought to pH 3 »0 by adding dilute hydrochloric acid while cooling with ice and stirring. A large amount of sticky, ether-insoluble pesticide precipitates and is extracted with 300 ml of ethyl acetate. After drying (MgSO.) And removing the solvents, 18.5 g of solid foam are obtained from the ether and ethyl acetate solutions. This is treated at 0 ° with 85 ml of trifluoroacetic acid. The solution is left at 0 ° for 1 hour, then ether is added. The solid trifluoroacetate salt is collected by filtration, washed with ether and dried. The white solid (13 »O g) is treated with 75 ml of water and the insoluble material is removed by filtering through diatomaceous earth (" Gelite "). The filtrate is adjusted to pH 5.6 with dilute ammonium hydroxide and again filtered through "Celite" in order to remove a small amount of precipitate. The pale yellow filtrate is cooled to 0 ° for 1 hour, which results in the formation of a voluminous white precipitate which is collected by filtration and washed with ice water, methanol and ether. The material is dried in vacuo over P ₃ O ₅ and makes 3.7 g of 7 - [- (o-Aminome thylphenylthio) -ac etamido] -c ephalosporanic acid. Another 0.3 g are obtained when the filtrate

10 9 8 4 4/1 937

is further concentrated. The overall yield is 4.0 g ($ 22). The infrared spectrum (EFujol muli) contains sharp bands at 3250, 1770, 1730 and 1650 cm " ¹ , which are assigned to amide-NH, ß-lactamcarbonyl, acetoxycarbonyl and amide carbonyl, respectively. The NMR spectrum (in trifluoroacetic acid) is in full agreement with the assigned structure The purity is estimated to be $ 95 or more.

Example 8

7 - [- (o-Aminom ethy! Phenylthio) -ac etamido] -c ephalo sporanic acid

The synthesis of this cephalosporin takes place according to the following reaction scheme:

109844/1937

-SCH ₂ CO ₂ H

CH ₂ NH ₂

NOpCboCl

O
-CH ₂ OCNHCH ₂

2,4-DNP

SCH ₂ CO ₂ H DCC

NO ₂ CbO-NHCO ₂

Il

1. 7ACi

SCH ₂ CO-2,4-DNP

2. K-2 ethyl hexanoate

UI

0 NO ₂ CbO-NHCH ₂ "SCH ₂ CNH

0
CH ₂ OCCH ₃

H ₂ / Pd-C

V \ // - ^SCH 2 ^CNH

CH ₂ NH ₃

[o- (p ^t -nitrocarbobenzo3yaminoinethyl) phenyltMo] acetic acid

To a stirred suspension of 4.53 g (0.023 mol) ' (o-Aminomethy! phenylthio) acetic acid in 120 ml of water Add 1N aqueous sodium hydroxide until the solution reaches pH. The clear solution is made with 80 ml of tetrahydrofuran diluted. Then a solution of 5.60 g (0.026 mol) of p-nitrobenzyl chloroformate (NOgCboCl) is added dropwise Stirring at room temperature added over about 15 minutes. By simultaneous addition of 1N aqueous Sodium hydroxide, the pH is regulated at 7 to 9. When the addition is complete, the solution is made in two 100 ml portions Ethyl acetate extracted. The aqueous solution is cooled and acidified with 20 ml of 3N aqueous sulfuric acid. The precipitated oil is extracted twice with 100 ml of ethyl acetate. After drying (MgSO.) The Ethyl acetate solution concentrated to a volume of 50 ml and cooled to form white solid [o- (p'-nitrocarbobenzoxyaminomethyl) phenylthio] acetic acid results.

Potassium ^ -C-Co-ip ^ nitrocarbobenzoxyaminomethylJ-phenyl thioj-acetamidol-cephalosporanate

1 »03 g (0.0050 mol) of NjN'-dicyclohexylcarbodiimide (DCO) are added to a cold solution of 1.88 g (0.0050 mol) of [o- (p ^t -nitrocarbobenzoxyaminomethyl) -phenylthioj-acetic acid and 0.92 Added g (0.005 mol) of 2,4-dinitrophenol (2,4-DNP) to 10 ml of anhydrous tetrahydrofuran. The mixture is left at room temperature for 1 hour, then the Ν, Ν'-dicyclohexylurea is filtered off and the piltrate containing the crude activated ester is concentrated to dryness under reduced pressure. A solution of 1 »30 g (0.0050 mol) of 7-aminocephalosporanic acid (7-ACA) and 1.01 g (0.010 mol) of triethylamine in 10 ml of methylene chloride is added to the residue, which has been cooled to 0 °

109844/1937

the crude activated ester is given. The reaction mixture is left at room temperature for 4.5 hours, then the solution is diluted with ether. The oily precipitate is redissolved twice in methanol (15 ml). The methanol solution is mixed with 2.5 ml of a 2.3 molar solution treated by potassium 2-ethylhexanoate in n-butanol, after which Ether is added. The solid precipitate is filtered off and suspended in 30 ml of methanol. After adding the product is collected from ether by filtration and dried, solid potassium 7-i- [o- (p'-nitrocarbbenzoxyaminomethyl) -phenylthioj-acetamidoj-cephalosporanate being formed results.

7 _ [_ (o-Aminomethylphenylthio) -ac etamido] -c ephalosporanic acid

A mixture of 3 »0 g (0.0045 mol) of the protected cephalosporin potassium-7- £ - [o- (p'-nitrocarbobenzoxyaminomethyl) -phenylthio] -acetamido ^ -cephalosporanate, 1.5 g of 10? 6-palladium on charcoal and 50 ml of water is hydrogenated at atmospheric pressure for 7 hours, then the reaction mixture is filtered through diatomaceous earth ("Celite"). The filtrate is cooled in ice and brought to pH 2.0 with dilute hydrochloric acid and then filtered again through "Celite". The pale yellow clear filtrate is adjusted to pH 5.9 with dilute aqueous sodium hydroxide and concentrated to dryness under reduced pressure. The solid residue is washed successively with a 4 ml portion and two 2 ml portions of ice water and then in vacuo over P ₂ ⁰ S dried, resulting in solid 7 - [- (o-aminomethylphenylthio) -acetamido] -cephalosporanic acid.

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example

(o-tert-Butoxycarbonylaminomethylphenylthio) acetic acid can in good yield from tert-butoxycarbonylazide and the amino acid can be prepared using triethylamine as the base.

The BOC amino acid reacts with thionyl chloride in the presence of triethylamine (methylene chloride as solvent) or pyridine (benzene as solvent), whereby the BOC-Aminoaeylchlorid results in the presence of triethylamine directly with 7-ACA in methylene chloride solution can be implemented. The protecting group can then be removed by treatment with cold trifluoroacetic acid will.

1098U / 1937

-S-CH ₂ CO ₂ H

CH ₂ NH-BOC

SOCl _2} Ät ₃ N Π =

0 -S-CH ₂ CCl

CH ₂ NH-BOC

1.

2.

0 -S-CH ₂ CNH

CH ₂ NH-BOC

CH ₂ OAc

1. CP ₃ CO ₂ H,

2. pH 5.6

-S-CH ₂ CNH

CH ₃ NH ₃

"" CH ₂ OAc

1098U / 1937

(o ~ tert-Butoxyoarbonylaminomethylphenylthio) acetic acid

To a stirred and ice-cold solution of 7.9 g (0.040 mol) (o-aminomethylphenylthio) acetic acid and 10.0 g (0.10 mol) triethylamine in 100 ml of water is added at once a solution of 7.2 g (0.050 mol) of tert-butoxycarbony1-azide given in 75 ml of tetrahydrofuran. The mixture is stirred at room temperature for 16 hours · the bulk the tetrahydrofuran is removed under reduced pressure. The aqueous solution is washed with ether, then with 125 ml of ether are covered and acidified with stirring and cooling with dilute hydrochloric acid to pH 2.5. The layers are separated and the aqueous layer is extracted with an additional 150 ml of ether. The United Ether solutions are dried (MgSO.) And concentrated to dryness, whereby (o-tert-butoxycarbonylaminomethylphenylthio) -acetic acid as a syrupy residue that is sufficiently pure for the next stage.

7 - [- (o-Aminomethylphenylthio) -ac etamido Jc ephalo sporic acid

A solution of 3.00 g (0.025 mol) of thionyl chloride in 25 ml of methylene chloride is added dropwise over 15 minutes to a stirred and ice-cooled solution of 7.43 g (0.025 mol) of (o-tert-butoxycarbonylaminomethylphenylthio) acetic acid and 2.73 g (0.027 mol) of triethylamine in 50 ml of methylene chloride. When the addition is finished, will leave the mixture at 0 ° for an additional 30 minutes and then add dropwise to one over the course of 10 minutes -20 to -30 ° cooled stirred solution of 6.80 g (0.025 mol) 7-aminocephalosporanic acid and 5.05 g (0.050 mol) Triethylamine given in 50 ml of methylene chloride. The reaction mixture will gradually warm over the course of 1 hour

1098U / 1937

left and then treated with 75 ml of water. While stirring and ice cooling, 25 ml of 1N hydrochloric acid are added. The layers are separated and the aqueous layer is extracted with an additional 25 ml of methylene chloride. the combined methylene chloride solutions are dried (MgSO,) and concentrated to dryness. The one left behind Foam (13.0 g) is added partially with stirring to 60 ml,. Chilled in ice: · Trifluoresaetic acid added. If so, the encore is complete (after 10 minutes), the reaction mixture is cooled to 0 ° for a further 30 minutes and then with 200 ml of ether treated. The precipitated salt is collected by filtration, washed with ether and washed with Treated 150 ml of water. The mixture is filtered through diatomaceous earth ("Celite") and the filtrate is diluted with Ammonium hydroxide adjusted to pH 5.6, after which again is filtered through "Gelite". The Piltrat is at concentrated to dryness under reduced pressure and the solid residue is treated with 20 ml of ice-water. The fixed one 7 - [_. (o-aminomethylphenylthio) -ac etamido] -c ephalo sporanic acid is collected and washed successively with twice 10 ml of ice water, twice 20 ml of methanol and ether.

Example 10

7 - [- (o-Aminomethylphenylthio) -acetamido] -cephalosporan acid hydrochloride

A suspension of 0.361 g (0.8 mmol) of the zwitterionic form of 7 - [- (o-aminomethylphenylthio) -acetamido] -oephalosporanic acid in 3 ml of methanol is cooled in ice and treated with a few drops of concentrated hydrochloric acid until a clear solution is obtained. The hydrochloride precipitates as a pale brown colored plague

109844/1937

Add ether and is collected by filtration and dried ^{in vacuo over Pp 0 E}

Example 11

Sodium 7-C- (o-aminomethylphenylthio) -ac etamido] cephalosporanate

To a stirred suspension of 0.361 g (0.8 mmol) of the zwitterionic form of 7 - [- (o-aminomethylphenylthio) -acetamido] -cephalosporanic acid 1N aqueous sodium hydroxide is added at room temperature until a clear solution (pH 10.8) is obtained. This solution is immediately freeze-dried, resulting in impure solid sodium 7 - [- (o-aminomethylphenylthioj-acetamidoj-cephalosporanate results.

Example 12

7- (o-aminomethylphenylthio) acetamido-3- (pyridiniummethyl). ceph-3-em-4-carboxylate

The acetoxy group in potassium 7-i- [o- (tert-butoxycarbonylaminomethyl) -phenylthio] -acetamido3-cephalosporanate is replaced by the pyridinium group by a procedure described in the Dutch patent 6 408 066 from GLAXO LABORATORIES ITD. is described. The preparation is illustrated by the following sequence of reactions:

109844/1937

-SCH ₂ CNH

CH ₂ NH-BOC

CH ₂ OAc

-3CH ₂ CNH

CH ₂ NH-BOC

CH ^ SC-

CO ₂ H

2ClO

It

-SCH ₂ CNH

-N ^ _c

CH ₂ NH-BOC _approx

CF ₃ CO ₂ H, O ^c

-SCH ₂ CNH

CH ₂ NH ₃

-2CF ₃ CO ₂

pH 7 - 7.5

O -SCH ₂ -CNH

CH ₂ NH ₂

CO,

^0Η2 Λ \ //

1098AA / 1937

Example 13

(m-Aminomethylphenylthio) acetic acid is converted from m-aminobenzoic acid prepared according to the reaction sequence below.

The first stage in the synthesis, which involves the implementation of the diazonium salt of m-aminobenzoic acid with potassium ethylxanthate is already described in the literature (R, Grice and 1.N. Owen, J. Ghem. Soc, 1947 (1963)) <>

109844/1337

OJ H

HO U

ΚΛ

O O

co

CM O O

OJ

OJ

ro

1098U / 1937

Me implementation of the amino acid with tert-butoxycarbonylazide, results in the BQC-protected amino acid, which via an activated ester in potassium-T-C-Cm-tert.-butoxycarbonylaminometh.ylph.enylth.io) -acetamido] -cephalosporanate can be converted as illustrated in Scheme B. Removal of the BOC protecting group with cold trifluoroacetic acid and then Adjustment of the pH of the aqueous solution of the trifluoroacetate salt to 5.0 to 5.5 gives 7 - [- (m-Aminomethylph.enylth.io ) -acetamido] -cephalosporanic acid (Scheme B), which by. Treatment of a methanolic Suspension with the minimum amount of concentrated hydrochloric acid and subsequent addition of ether is converted into the hydrochloride salt.

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H ₂ NCH ₂

-SCH ₂ CO ₂ H + tert ..- C ₂₁ HgOCN, H ₂ O-THP

BOC-NHCH,

BOC-NHCH,

W //

-SCH ₂ CO ₂ H

2,4-DNP, DCC / =

-NO,

NO

BOC-NHCH,

Θ Θ

₅ ) NH 7-ACA

2. K-2 ethyl hexanoate

-SCH ₂ CNH-

• Ν

CH ₂ OAc

CO ₂ K

1.

2. CP ₃ CO ₂ H,

3. Ph 5.0 to 5.5

H ₃ NCH ₂

O-SCH ₂ CNH

-N

CH OAc

CO ₂ Θ

REACTION SUBJECT B

1Q98U / 1937

m-mercaptobenzoic acid

A solution of 25.1 g (0.35 mol) of sodium nitrite in 100 ml of water is added dropwise at 0 ° to a stirred suspension of 48.0 g (0.35 mol) of m-aminobenzoic acid in 360 ml of 2N hydrochloric acid. When the addition is complete, the mixture is kept at 0 ° for a further 30 minutes, then a solution of 60.0 g (0.375 mol) of potassium ethyl xanthate in 200 ml of water is added over the course of 5 minutes, with copious evolution of gas. The reaction mixture is left to stand at room temperature overnight. The yellow plague is filtered off, washed with water and heated on a steam bath for 1 hour with 250 ml of 4N aqueous sodium hydroxide. The solution is cooled and acidified with concentrated hydrochloric acid. The solid precipitate is taken up in 300 ml of ethyl acetate and this solution is washed with water and dried. Removal of the solvent and washing of the solid residue with cold η-hexane give 51.9 g ($ 96) of grayish solid with Έ = 132 to 136 °, which is used for the next stage without further purification. A Έ = 146 to 147 ° is described in the literature (S, Smiles and J. Stewart, J. former Soc, 119, 1792 (1921)).

m-meroaptobenzyl alcohol

A solution of 60.4 g (0.392 mol) of m-mercaptobenzoic acid in 400 ml of anhydrous ether is added dropwise with a such rate to a stirred suspension of 22.3 g (0.588 moles) of lithium aluminum hydride in 400 ml anhydrous ether given that without the application of external Heat return flow is maintained. When the addition is complete (in about 30 minutes) the reaction mixture becomes The excess was refluxed for a further 4 hours

109844/1937

21-2058

Lithium aluminum hydride is destroyed by carefully adding a little ethyl acetate. The reaction mixture will then poured into 700 ml of ice-cold 2N hydrochloric acid and the resulting mixture becomes 30 minutes touched. The layers are separated and the aqueous layer is extracted with two 150 ml portions of ethyl acetate, The combined organic layers are washed with a saturated aqueous solution of sodium bicarbonate, dried and focused. The residue is distilled in vacuo, with 32.1 g (58%) of product as a colorless liquid with a bp = 94 to 96 ° (0.1 mm) result «, A Kp = 88 ° (10 mm) is described in the literature (R. Grice and L.K. Owen, J. Ohem. Soc, 1947 (1963)).

(m-Hydro3tymethylphenylthio) acetic acid methyl ester

30.8 g (0.22 mol) of m-mercaptobenzyl alcohol become a · Solution of iatrium methylate prepared from 5.1 g (0.22 g atom) of sodium in 200 ml of methanol, followed by a solution of 23.9 g (0.22 mol) of methyl chloroacetate in 25 ml of methanol is added dropwise. The reaction mixture is refluxed for 1 hour, cooled and filtered. The piltrate is concentrated and the residue is taken up in ether. The ethereal solution becomes with Washed water, dried and concentrated to give 42.2 g ($ 90) of the crude product, which was obtained without further Cleaning direlct is used for the next stage.

109844/1937

(m-Chloromethylphenylthio ^ acetic acid methyl ester

42.2 g (0.20 mol) of crude methyl (m-hydroxymethylphenylthio) acetic acid ester are carefully treated with 50 ml of thionyl chloride at 0 °, with an immediate reaction. The excess thionyl chloride is removed and the residue is distilled in vacuo, with 40.7 g ($ 88) of a pale yellow colored liquid with result in a Kp = 132 to 136 ° (0.2 mm).

(m-Ohlormethylphenylthio) acetic acid

4-0.7 g (0.171 mol) (m-chloromethylphenylthio) -acetic acid methyl ester are dissolved in a mixture of 400 ml of acetic acid and 200 ml of 6N hydrochloric acid. The solution is left at room temperature for 18 hours, then concentrated to approximately half its original volume, diluted with 100 ml of water, cooled and filtered. The pesticide is washed with ice water, dried and recrystallized from ethyl acetate / n-hexane (1: 2), giving 27.3 g (74%) of white crystals with ¥ = 79 to 82 °. The infrared spectrum (Nujol muli) contains a garbonyl band at 1695 cm. The NMR spectrum (in CDCI,) consists of singlets at / 1.0 (CO ₂ H), 2.64 and 2.76 (phenyl protons), 5.52 (-CH ₂ Cl) and 6.37 (-S -CH ₂ -) with an integrated area ratio of

(m-Aminomethylphenylthio) acetic acid

13.4 g (0.062 mol) of (m-chloromethylphenylthio) -acetic who achieved the concentrated ammonium hydroxide in 130 ml of ice-cooled. The solution is left at room temperature for 19 hours and then concentrated to dryness. Der.feste return

109844/1937

stand is treated with 50 ml of methanol, cooled and filtered. The solid is washed thoroughly with methanol and dried. The yield is 6.0 g ($ 49) whiter Solid with an mp = 197 to 200 ° (decomp.). The infrared spectrum (Nujol muli) shows the characteristic amino acid absorption at about 1550 cm. The NMR spectrum (in trifluoroacetic acid) contains a multiplet at / 2.1 to 3.3

(Phenyl protons and -MH,), a quartet at / 5 »62 (-CH ₀ -NH ,, coupling constant J = 6 Hz) and a singlet at / 6.21 (-SCHp-) with an integrated area ratio of 7: 2j2 .

(m-tert-Butoxycarbonylaminomethylphenylthioj-acetic acid

To a stirred and ice-cooled solution of 4 »7 g (0.024 mol) of (m-aminomethylphenylthio) acetic acid and 5.5 g (0.054 mol) of triethylamine in 50 ml of water, a solution of 4.3 g (0.030 mol) of tert. -Butoxycarbonylazid in 40 ml! Tetrahydrofuran given all at once. The reaction mixture is stirred for 16 hours at room temperature and then extracted with two 75 ml portions of ether. The aqueous solution is covered with a layer of 75 ml of fresh ether, cooled in bis and acidified to pH 2.5 with 3N hydrochloric acid while stirring. The ether layer is dried and the solvent is removed, 5.3 g of the BOC amino acid being a colorless, viscous one yield oil. The infrared spectrum of the undiluted oil contains an NH band at 3350 cm and a strong, broad carbonyl band at 1700 cm " ¹ .

109844/1937

Potassium 7 - [- (m-tert, -butoxycarbonylaminomethylphenylthio) -acetamidoj -cephalosporanate

3 »7 S (0.0178 mol) NjN'-dicyclotiexylcarbodiimide are added all at once to an ice-cold solution of 5.3 g (0.0178 mol) (m-tert.-butoxyoarbonylaminomethylphenylthio) acetic acid and 3 * 3 g (0, 0178 mol) 2,4-dinitrophenol in 30 ml ethyl acetate. given. The mixture is left at room temperature for 45 minutes, then the N, Έ * -diyclohexy! Urea is filtered off and the solvent is removed from the filtrate, leaving the activated ester as a viscous yellow oil. A solution of 4.8 g (0.0178 mol) of 7-aminocephalosporanic acid and 3.6 g (0.0356 mol) of triethylamine in 35 ml of methylene chloride is added to the crude activated ester while cooling with ice, and the reaction mixture is added for 2 hours Leave room temperature · A small amount of insoluble material is removed by filtration and ether is added to the filtrate. The oily precipitate is redissolved twice in methylene chloride (15 ml) and reprecipitated with ether (150 ml) and then dissolved in 30 ml of methanol. A 2.4 molar solution of potassium 2-ethylhexanoate in n-butyl alcohol (8 ml) is made added, then 200 ml of ether are added. The yellow solid is collected by filtration, washed with ether and _{dried in vacuo over P 2} Of ₅ The yield is 8.6 g ($ 82) The infrared spectrum (Nujöl mull) contains strong bands at 3250 (NH), 1750 ( β-lactam carbonyl), 1680 (tert-butoxycarbonyl), 1650 (amide carbonyl) and 1600 cm (carboxylate). Thin layer chromatography shows that only one component is present. On the basis of this result and the infrared spectrum, the product is assigned a purity of at least 90 #.

109844/1937

7 - [- (m-Aminomethylphenylthio) -ac etamid ο] -c ephalo sporanic acid

An ice-cold solution of 8.6 g (0.0146 mol) of potassium 7 - [- (m-tert-butoxycarbonylaminomethylphenylthioj-acetamido] -cephalosporanate in 75 ml of water is covered with 75 ml of ethyl acetate and 3N hydrochloric acid to pH 2, 5 Acidified The layers are separated and the aqueous layer is extracted with a further 50 ml of ethyl acetate. The combined ethyl acetate solutions are dried and concentrated to dryness. The remaining foam (8.1 g) is cooled in ice and treated with 30 ml of trifluoroacetic acid The solution is left at 0 for 1 hour and then mixed with 150 ml of ether. The pale yellow solid precipitate is collected by filtration, washed with ether and _{dried in vacuo over P 2} ^° C. This trifluoroacetate salt (7.6 g) is mixed with 50 ml Treated water and some insoluble material is removed by filtration o The piltrate is cooled in ice and adjusted to pH 5.4 with dilute ammonium hydroxide The product precipitates easily and becomes after Collected by filtration after cooling to 0 ° for 30 minutes. The pale yellow solid is washed successively with ice water, methanol and ether and finally dried _{over P 2 O in vacuo.} The yield is 3.9 g ($ 60). The infrared spectrum (Nujol muli) contains bands at 1755, 1735 and 1655 cm " ¹ , which are assigned to β-lactam carbonyl, acetoxycarbonyl and amide carbonyl, respectively. The NMR spectrum (in trifluoroacetic acid) is also in full agreement with the assigned structure thin layer chromatography shows that only one component is present. On the basis of this result and the spectral results, the product is assigned a minimum purity of $ 90 «

1098AW1937

- 72 Example 14

7 - [- (m-Aminomethylphenylthio) -ac etamido] -c ephalosporan acid hydrochloride

A stirred suspension of 0.90 g (2.0 mmol) of 7 - [- (m-aminomethylplienylthio) -acetamido] -cephalosporanic acid in 15 ml of methanol is cooled in ice and treated with a slight excess of concentrated hydrochloric acid. Initially the material goes into solution, then a voluminous white precipitate of the hydrochloride is formed. 30 ml of ether are added and the pesticide is collected by filtration. The product (0.87 g, 895ε) is only sparingly soluble in water and 40 ml of water and slight heating are required to dissolve it. A very small amount of insoluble material is removed by filtration, the filtrate is concentrated to dryness and the white solid residue (0.75 g) is dried _{in vacuo over P 3} O _5. The product obtained in this way is still only slightly soluble in water. The infrared spectrum (Nujol muli) contains sharp bands at 3300, 1765, 1700, 1690 and 1640 cm "and also the NMR spectrum (in DMS0-d6) is in full agreement with the assigned structure. A thin layer chromatogram shows the presence of only one Component. The purity of the product is estimated with «,

1 0 9 84 A / 1937

Example 15

Sodium 7 - [- (m-aminomethylphenylthio) -acetamido] - cephalosporanate

To a stirred suspension of 0.361 g (0.8 mmol) of the zwitterionic form of 7- [~ (m-aminomethylphenylthio) -acetamidoj-cephalosporanic acid 1N aqueous sodium hydroxide is added at room temperature until a clear solution (pH 10.8) is obtained. This solution is immediately freeze-dried, whereby impure solid sodium-7- [~ (m-aminomethylphenylthio) acetamido] cephalosporanate gives.

Example 16

A. 7- (m-tert-Butoxycarbonylaminomethylphenylthio) -acetamido-S-ipicolinoyl-thiomethylJ-oeph-J-em- ^ · - carboxylate

11.7 g (0.020 mol) of potassium 7- [~ (m-tert. ~ Butoxyoarbonylaminomethylphenylthio) acetamido] cephalosporanate are proportionately in the course of 15 minutes to a stirred solution kept at 70 to 75 ° of 5.6 g (0.040 mol) Thiopicolinic acid is added to 115 ml of water. The reaction mixture is kept at this temperature for 1 hour. then it is cooled and the precipitate is collected by filtration. The solid is with 200 ml of acetone treated and a small amount of insoluble material is removed by filtration. Become the aoetone solution 60 ml v / water are added, then the mixture is cooled in ice and diluted with concentrated hydrochloric acid pH 2.0 acidified «600 ml of water are added and the product is extracted with 200 ml of methylene chloride · The

109844/1937

Methylene chloride solution is mixed with two 75 ml portions of water washed, dried, and concentrated to dryness to give 7.5 g ($ 59) of pale brown solid. The infrared spectrum (liujol muli) of the product contains a strong ß-lactam carbonyl band at 1770 cm "* ^ and a broader carbonyl band at 1660 to 1720 cm.

B. 7- (m-tert-Buto2ycarbonylaminomethylphenylthio) -acetamido -3-pyridiniummethyl) -oeph-3-em- $ - carboxylate

A solution of 7.5 g (0.012 mol) of 7- (m-tert-butoxycarbonylaminomethylphenylthio) -ac etamido-3- (pie olinoylthiomethyl) -ceph-3-em-4-carboxylate in 50 ml of pyridine is diluted with 50 ml of water. A solution of 14.6 g (0.026 mol) of the mercury-II-perchlorate-pyridine complex in a mixture of 50 ml of pyridine and 50 ml of water is stirred for about 5 minutes at room temperature added »The reaction mixture is stirred for 1 hour at room temperature, then a rapid flow of hydrogen sulfide bubbled through for 5 minutes. The mixture is filtered through "Gelite" and the filtrate is successively with benzene (140 ml), a 1: 3 mixture of liquid ion exchange resin Amberlite LA-1 and Benzene (120 ml and 60 ml portions) and 125 ml benzene washed The aqueous solution is concentrated to dryness to give 4.0 g (58ji) of a brown solid residue.

C.7- (m-Aminomethylphenylthio) -aoetamido-3- (pyridinium methyl) -ceph-3-a-4-carboxylate

40 g (0.007 moles) of 7- (m-tert, -butoxycarbonylaminomethylphenylthio) -aoetamido-3- (pyridiniummethyl) -oeph-3-em-4-carboxylate are treated at 0 ° with 40 ml of trifluoroacetic acid. The resulting solution will be at for 30 minutes

109844/1937

O ⁰ held, after which. 150 ml of anhydrous ether are added. The solid precipitate is collected by filtration, washed with ether and dried in vacuo. It weighs 3/2 g. The trifluoroacetate salt is dissolved in 20 ml of water and this solution is cooled in ice and adjusted to pH 7-3 with dilute ammonium hydroxide. A small amount of solid contaminant is removed by filtration and the filtrate is concentrated to dryness under reduced pressure to give 3> 2 g of pale brown colored solid foam. This is treated with 50 ml of methanol (30 ml and 20 ml portions) and a small amount of insoluble material is removed by filtration. The filtrate is treated with 100 ml ether and the solid precipitate is collected by filtration, washed with ether and dried in vacuo over ϊρΟκ S ^e "b ^r ocknet to give 1» 8 g (55 $) beige colored solid yield. Contains the Infrarοtspektrum strong bands at 1770, 1665 and 1600 cm "", which are ascribed to ß-lactam carbonyl, amide carbonyl and carboxylate, respectively. The NMR spectrum (in D ₂ O) is also in full agreement with the assigned structure. Thin-layer chromatography shows that that only one component is present. The material is therefore assigned a purity of at least $ 90.

The resin "Amberlite LA-1" is a mixture of secondary Amines, wherein each secondary amine has the general formula

E ^1. ) ₂ 0H ₂ 0 (OHj) ₂ 0H ₂ 0H = 0H-0 H ₂ NHO-E ²

109844/1937

12 3
has, wherein R, R and R each an aliphatic

12 mean 3 hydrocarbon radical and R, R and R ^ in the aggregate contain 11 to 14 carbon atoms. This special mixture of secondary amines, sometimes referred to as "Liquid Amine Mixture Kr.I", is a clear amber colored liquid with the following physical properties:
Viscosity at 25 ⁰ C = 70 cP,
specific weight at 20 ⁰ C = 0.845, refractive index at 25 ⁰ C = 1.467,

Distillation range at 10 mm: up to 160 ⁰ C = 74 $

above 22O ⁰ C =

1098U / 1937

Claims (1)

■ ·. 211205a "* II" PATENT CLAIMS Process for the preparation of compounds of the general Formula I. o / S - GH ₀ GNH - CH GH CPI ₀ ^ t Γ t ^c O = C-N C-CH ₂ A COOM v / orin A Ac et oxy or pyridinium and M Viass er stoff, a
non-toxic, pharmaceutically acceptable cation or
mean an anionic charge when A is pyridinium,
and of non-toxic, pharmaceutically acceptable acid addition salts thereof, characterized in that a compound of the general formula II H w OH CH CH ₀ O = CN C-CH ₉ A COOH II 1098AA / 1937 wherein A has the meanings given above, or a salt or an easily hydrolyzed ester thereof with a acylating acid of the general formula III -SCH ₂ COOH wherein the amino group carries a conventional protecting group, or acylated with a reactive derivative thereof in an inert organic solvent at a temperature of about -20 ⁰ C to +70 ⁰ C, then the amino protecting group is removed and, if A is acetoxy, optionally the acetoxy group by means of converts known working methods into a pyridinium group, 2. The method according to claim 1 for production of compounds of the general formula 2 x ^ ^s \ -S - CH ₀ C - NH - CH - CH CH ₀ ^ * Il I ^ C ± J O = CN C-CH ₂ -N H ₂ NCH ₂ coo © one of the non-toxic, pharmaceutically acceptable acid addition salts of it, characterized in that one takes place one after the other 109844/1937 (Λ) 7-aminocephalosporanic acid or a salt or a easily hydrolyze esters thereof with an acylating agent Acid of the general formula -S-CH ₂ OOOH H ₂ NCH _{2 4} wherein the Amiiiogruppe carries a conventional protecting group, or acylated with a reactive derivative thereof in an inert organic solvent at a! Temperature of about -20 ⁰ C to +70 ⁰ C, (B) the amino-protected cephalosporanic acid thus produced or a salt thereof is reacted with thiopicolinic acid to form an amino-protected intermediate the formula \ O
-S-CH ₂ -C -NH - 'CH-CH
ι t CH ₉
ι ^c O
(I. Λ = ZJ O = C - ^ 0-CH ₂ -sc H ₂ NCH _{2 t} \ * C
t COOH or a salt thereof is formed, (G) this intermediate with a mercury-II-per- ■ reacts chlorate-pyridine complex, with the amino-protected Compound of formula 109844/1937 -S-CH ₂ -C-NH-CH O = C - CH, Ψ ~ Χ H ₂ NCH ₂ COO or a salt thereof .is formed, and (D) removed the amino protecting group, leaving the desired . Product or a non-toxic, pharmaceutically acceptable Acid addition salt is formed thereof. 3. The method according to claim 1 for the preparation of compounds of the general formula - ö - OJtIr) - Il NH
0 CH t CH
ι CIL C. = C - II C - H ₂ NCH ₂ t
COO ¹ or the non-toxic, pharmaceutically acceptable acid addition salts thereof, characterized in that one consecutively (A) 7-aminocephalosporanic acid or a salt or a slightly hydrolyzed ester thereof with an acylating one Acid with the formula H ₂ NCH S - CH ₂ COOH 10 4/1937 wherein the amino group carries a conventional protecting group, or acylated with a reactive derivative thereof in an inert organic solvent at a temperature of about -2O ⁰ O to + 7O ⁰ O, (B) the amino-protected cephalosporanic acid thus produced or a salt thereof is reacted with pyridine in an aqueous medium to form an amino-protected compound the formula -S-CH ₉ -C-IH - CH - CH \ jlL ^c it ι * · HoNCH or a salt thereof is formed, and (C) Removed the amino protecting group to give the desired product or a non-toxic, pharmaceutical grade to form a compatible acid addition salt thereof. 4. The method according to claim 1 to 3, characterized in that that the acylating acid in the form of its acid halide or its activated 2,4-dinitrophenyl ester is present. 5.. Process according to Claim 4, characterized in that the acylation reaction is carried out at a temperature of about ⁰ ° C in methylene chloride as the solvent. 1 0 9 8 AA I 1 9 3 7 6. The method according to claim 1 to 5, characterized in that the amino protective group is the t-butoxycarbonyl group which is then removed by treatment with trifluoroacetic acid. 7. Process according to claims 1 to 5, characterized in that the amino protective group is the p-nitrocarbobenzoxy group which is then removed by catalytic hydrogenation. 8, method according to claim 1, characterized in, that 7-aminocephalosporanic acid or a salt thereof with an acylating acid of IOrmel -S - CH ₂ COOH in which the amino group carries a conventional protective group, or acylated with a reactive derivative thereof » 9. The method according to claim 1, characterized in that that 7-aminocephalosporanic acid or a salt thereof with an acylating acid of iOrmel S - CH ₂ COOH or in which the amino group carries a conventional protective group, ler acylated with a reactive derivative thereof. 109844/1 937 10. The method according to claim 1, characterized in, that 7-aminocephalosporanic acid or a salt thereof with an acylating acid of the formula -S - OH ₂ COOH wherein the amino group carries a conventional protecting group, or acylated with a reactive derivative thereof. 11, compounds of the general formula 0 y Sv -S - CH ₀ C - NH - CH - CH CH ₀ N .C-CH ₂ A COOM wherein A is acetoxy or pyridinium and M is hydrogen non-toxic, pharmaceutically acceptable cation or anionic charge when A is pyridinium, and non-toxic, pharmaceutically acceptable acid addition salts * thereof. 12, compound of formula 109844/1937 .S-CH ₀ -C -NH-CH - CH CH ₉
cc \\ // 2 _f ,, 2 ο CN C-CH ₉ OC-CH, COOH and their non-toxic pharmaceutically acceptable salts. 13. Compound of Formula H ₉ N-CH ₉ - / .Vs-CH ₉ -C-FH-CH - CH CH ₉ C-CH ₀ OC-CH ² COOH 14 · sodium salt of the compound according to claim 15. Potassium salt of the compound according to claim 16. Hydrochloride of the compound according to claim 13 · 17 · Secondary ion form of the compound according to claim 13, 18. Non-toxic, pharmaceutically acceptable acid addition salts of the compound according to claim 13, 10984 W1937 The compound of the formula H ₂ IT-CH ₂ - .S-CH ₀ -O-NH-CH * ι CH COO ¹ and non-toxic, pharmaceutically acceptable acid addition salts of that* Compound of formula H ₂ N-CH ₂ - Il -S-CHo-C-NH-CH The hydrochloride of the compound of claim 20. Compound of formula O -S-CH ₉ -C-NH-CH , σ CH
ι 0 η CH ₂ NH ₂ C-CH ₀ OC-CH COOH and non-tozish, pharmaceutically acceptable salts thereof. 1098U / 1937 Compound of formula O
) -S-CH ₂ -C-NH-CH y
CH
I. ' ^s \ O J ⁵ '
CH ₂ -NH ₂ O N C-OH,
t 0- (5-CH ₃ COOH Compound of formula Ii -S-CH ₉ -C-HH-CH * ι CH ι CH "ι t CH ₂ NH ₂ COO ⁽ and non-toxic, pharmaceutically acceptable acid addition salts of that. Compound of formula -S-CH ₉ -C-NH-CH * · ι CH ₂ NH ₂ CH CH ₉ ι t ^£ H C-CHoN COO ¹ Hydrochloride of the compound according to claim. 20, 109844/1937 27 · Compound of the formula -S-CH ₀ -C-NH-CH-CH CH _{9 n} 2,, 2 O CN C-CH ₉ OC-CH, S ^ o ' ² ' COOH and non-toxic, pharmaceutically acceptable salts thereof « 28. Compound of Formula 0
S-CHp-C-ÜH - CH - CH CHp CN C-CH ₉ OC-CH ογ COOH 29 «Hatriiimsalz of the connection according to claim 30. Potassium salt of the compound according to claim 28. 31. Hydrochloride of the compound according to claim 32. Zwitterionenfora the compound according to claim 28. 33 · Hioht-toiische, phaxiiacetic compatible Acid addition salts of the compound according to claim 28. 109844/1937 Connection of the IOrmel H ₂ N-CH ₂ -S-CHo-S-NH-CH O' CH t CH, C-CH ₂ N COO and non-toxic, pharmaceutically acceptable acid addition salts of that. Compound of formula H ₂ N-CH ₂ -S-CHo-C-NH-CH , 0 CH ₀ C-CH ₂ N COO ' Hydrochloride of the compound according to claim 35 109844/1937