DE2344095A1 - ANTIBACTERIAL AGENTS AND METHODS FOR THEIR PRODUCTION - Google Patents

Description

PATENT LAWYERS

DR.-ING. WOLFRAM BUNTE DR. KARL GEORG LÖSCH

D-OOOO MUNICH, 13. BAUCRSTRASSE 22, POST BOX 7ΘΟ FERNRUF (08It) 37 β5 83 TELEX S2TB2O8 ISAR d

Munich, August 31, 1973; M / 12 801 j

PATENT APPLICATION j

BRISTOL-MYERS COMPANY. j

345, Park Avenue, New York, N.Y.Y U.S.A. j

ANTIBACTERIAL AGENTS AND METHOD FOR PREPARING THEREOF

The invention relates to certain new 3-thiolated '7-acylamidocephalosporanic acid derivatives, which are used as antibacterial agents, as supplementary feed in animal feed, as agents for the treatment of mastitis in cattle and as therapeutic agents for the treatment of infectious diseases in poultry and others
Animals, as well as humans, which by many gram-positive and
Gram-negative bacteria are valuable. The invention relates in particular to 7-acyl- ^{1 thiolated in the 3-position}
amidocephalosporins in which the 7-substituent is o-aminomethylphenylacetamido , o-aminomethyl-p-hydroxyphenylacetamido or o-amino

409816 / 1U7

methylphenylthioacetamido i.st and the thiol group in the 3-position 3-Hydroxypyridazin-6-ylthiomethyl is, and non-I I toxic, pharmaceutically acceptable salts and simply hydroj lized esters thereof. The invention also relates to this; new intermediate 7-amino-3 (ö-hydroxypyridazin-S-ylthio-I methyl) .- 3-cephem-4-carboxylic acid, as described below-I ben can be acylated and the above-mentioned new active; Cephalosporins yields or alternatively for the production of others ί extremely valuable cephalosporin derivatives can be used.

ι 7-Phenylacetamidocephalosporanic acid, also N-Phenylacetylderii vat of 7-ACA, Cephaloram, PACA and obviously phenasporin

'is well known in the scientific literature. > Below are publications related to the manufacture and / or

to the properties of this compound, with or without substitu- ■ ducks in the benzene ring, and corresponding compounds in which

the 3-acetoxymethyl group by methyl, hydroxymethyl and / or j pyridinium methyl is replaced, indicated:

Chauvette, RR, et al. "Chemistry of Cephalosporin Antibiotics j II. Making a New Class of Antibiotics and ¹ 'the Relationship of Structure to Effectiveness", Journal ! of the American Chemical Society , 84 , 3401-3402 (1962). j Chauvette, RR et al. "Structure-Activity Ratios, Among 7-Acylamidocephalosporanic Acids", Antimicrobial Agents and Chemotherapy - 1962, 687-694. Cocker, JD et al. "Cephalosporansäpren. Part II .. Replacement of the acetoxy group by nucleophiles", Journal of the Chemical Society , 5015-5031 (1965). "Cocker, JD et al." Cephalosporanic Acids. Part IV. 7-Acylamidoceph-2-em-4-carboxylic acids ", Journal of the Chemical Society 1142-1151 (1966).

CuIp, H.W. et al. "Metabolism and absorption of the 7- (phenyl-

14th
acetamido-1-C) -cephalosporanic acid ", Antimicrobial Agents and Chemotherapy - 1963, 243-246.

409816/1 147

Iago, M. '"Antibacterial efficacy of some derivatives of 7-aminocephalosporanic acid against Staphylococcus aureus and synergism between these and other antibiotics", Brit. J. Pharmacol ., 22 , 22-23 (1964). Loder, B. et al. "The Cephalosporin C core (7-aminocephalosporanic acid) and some of its derivatives", Biochemical Journal ' , 79, 408-416 (1961). ·

Nishida, M. et al. "Studies on the Microbial Degradation of Cephalosporin-C Derivatives. II". The Journal of Antibiotics , 21, 375-378 (1968).

Nishida, M. et al. "Studies on the Microbial Degradation of Cephalosporin-C Derivatives I.". The Journal of Antibiotics, 21, -165-169 (1968).
; Spencer, JL, et al. "Chemistry of Cephalosporin Antibiotics - VIII. Synthesis and structure-effectiveness ^{ratios of cephaloridin analogs 1} ", Antimicrobial Agents and J Chemotherapy - 1966, 573-580.

Stedman, R.J. et al. "7-aminodeacetoxycephalosporanic acid and

their derivatives ", J. Med . Chem., 7 (1), 117-119 (1964). j Sullivan, HR et al." Metabolism of oral cephalothin and related cephalosporins in rats ", Biochemical Journa l, 102, 976- 982 (1967).

Vymola, F. et al. "The Classification and Properties of Cephalosporin Antibiotics I. Systematic Study of the Quantitative Sensitivity of Some Pathogenic Microorganisms to Cephaloridin", Journal of Hygiene , Epidemiology, Microbiology and Immunology , 10, 180-189 (1966).

409816/1 U7

There are many other 7-acyl derivatives of 7-aminocephalosporanic acid, including 7-

■ / J- (oC-AminoalkyDphenylacetamido / cephalosporanic acids (US

I Patent 3,382,241), 7- ^ Tp-aminophenylthio) acetamido / -j cephalosporanic acid (U.S. Patent 3,422,100), 7-Halo-I phenylthioacetamido) cephalosporanic acids (US patent; 3,335,13,6) * and the almost unlimited number of variants of those I compounds by the general formulas (and often also ι described differently) of patents, such as j of Dutch patent specification 69/02013 (Farmdoc 39 172),

■ are recorded. 7- (p-aminophenylacetamido) -cephalosporanic acid is disclosed in U.S. Patent 3,422,103, as well

ι the corresponding'N-trityl derivative; see also Japanese Patent Publication 2712/67 ¹ (Farmdoc 25,406).

i U.S. Patent 3,219,662 contains claims to ver; bonds of the structure R - CH ₂ - CO - ACA, in which R is phenyl, nitrophenyl (especially para-nitro), chlorophenyl, alkylphenyl

and is alkoxyphenyl and the corresponding phenoxy and j substituted compounds and for those the corresponding ! Compounds in which the 3-acetoxymethyl group has been replaced by a 3-pyridinium methyl group. A wider group

such compounds, including the series in which R j is phenylthio, and also the compounds in which R

Benzyl / i.e. 7- (ß-phenylpropionamido) cephalosporanic acid /, j is alkoxybenzyl, alkanoyloxybenzyl, aminobenzyl, etc.; are for use as starting materials, at least ! by genus, in British patents 1012

and 1,153,421 (Farmdoc 23,984) and also in British Patent 1,001,478 and U.S. Patent 3,280,118 Further 7-phenylacetamidocephalosporanic acids with Substituents on the benzene ring including hydroxy and

409816/1 147

-δι amino, are used as starting materials in the British patent '

Documents 1,082,943 and 1,082,962 are disclosed.

U.S. Patent 3,341,531 describes, 7- (o-, m- and p-Carboxamidomethylphenylacetamido) cephalosporan acids and

their betaines. A variety of 7 (halo, dihalo, nitro and halonitro-phenylacetamido) cephalosporanic acids are as Starting materials for the reaction with certain Nukleoj philen in US Pat. No. 3,431,259 (Farmdoe 27 715) gej named.-More 7-phenylacetamido) cephalosporan acids with the ! various substituents on the benzene ring are in the yes, Panic patents 2712/67 (Farmdoc 25 406) and 26105/69: (Farmdoc 40 860) ,, of British patent specification 1,178,471 (Farmdoc 27,715) and further in Dutch patent 67/00906 and Japanese patent 25785/69 (Farmdoc 40847) openable "t.

ί The replacement of the 3-acetoxy group of a cephalosporin with the most diverse hetercyclic thiols have been disclosed as follows:. ■

y a> in South African patent specification 70/2290 (see also Dutch patent 70/05519 (Farmdoc 80 188R)), in which j the side chains were for example 7-d-aminophenylacetamido I and typical heterocyclic thiols 2-methyl-1,3,4-thiadiazole-5-thiol and l-methyl-1,2,3,4-tetrazole-5-thiol were,

b) in U.S. Patent 3,516,997 wherein the side chains

3 had structures in the 7-position, such as -R - (alk) m-CO-NH-

3 3 "♦

and R -S- (alk) m-CO-NH -, where R is one of many aromatic Was heterocyclic, and the numerous heterocyclic thiols in the 3-position for example l-methyl-tetrazole-5-thiol and 2-methyl-1,3,4-thiadiazole-5-thiol included, and

C) in U.S. Patent 3,563,983.

409816/1147

Various cephalosporins, occasionally also cephalosporin C,

were reacted with nucleophilic aromatic mercaptans to form I. Preparation of compounds of the formula

Acyl - NH - CH-CH CH ₀

; O = CN A - CH ₂ - S - Ar

COOH

In U.S. Patent 3,278,531, Ar is phenyl or
certain substituted phenyls or certain aromatic
heterocyclic rings, which are listed in column 5, for example. Similar nucleophiles, for example 2-mercaptopyrimidines, are disclosed in US Pat. No. 3,261,832 and US Pat
British Patent 1,101,422 and U.S. Patent Nos. 3,479,350 and 3,502,665. A parallel description can be found in the British patent specification
1 109 525, for example in the definition "h" for R ₃ .
Further nucleophiles of this type are described by Fujisawa in
Belgian patent 714 518 (Farmdoc 35 307, Dutch 68/06129 and South African 2695/68), in Canadian patent 818 501 (Farmdoc 38 845), in British
U.S. Patent 1,187,323 (Farmdoc 31,936, Netherlands 67, 14888) and particularly in U.S. Patent 3,516,997 (Farmdoc 34,328, Netherlands 68/05179) which comprises a compound called cefazoline which has a tetrazolylacetyl side chain on the 7th Amino group and a 5-methylthiadiazolylthiomethyl group in the 3-position, and in detail in the scientific literature, for example in Antimicrobial Agents and Chemotherapy - 1969, American Society for Microbiology, Bethesda,

40981 6/1 U7

Maryland, at pages 236-243 and in J. Antibiotics (Japan) 23 (3), 131-148 (1970).

Recently, the replacement of the 3-acetoxy group of a cephalosporin has been made by various heterocyclic thiols in US Patent 3,563,983 and in Dutch Patent 70/05519 (Farmdoc 80 188R) described, in which the side chains, for example 7-t? (- aminophenylacetamido and typical heterocyclic thiols are 2-methyl-1,3,4-thiadiazole-5-thiol and 1-methyl-ί, 2,3,4-tetrazole-5-thiol; the latter Compound corresponds to U.S. Patent 3,641,021, issued February 8, 1972 for an application filed April 18, 1969. Further Similar disclosures are in U.S. Patent 3,563,983, Belgian Patent 771 189 (Farmdoc 12 817T) ', the Japanese patent specification 72/05550 (Farmdoc 12 921T) and j of the Japanese patent specification 72/0551 (Farmdoc 12 922T) to find! the. ''

Various cephalosporins of the formula ":

■ S \

Acyl - NH - CH - CH CH ₂

0 == c -NC - CH ₂ - S - alkyl

i
COOH

j where acyl has different side chains including o ^ -Ami'no- ! phenylacetyl are in some of the above-mentioned patents and in Belgian patents 734,532 (Farmdoc 41 619) and 734 533 (Farmdoc 41 620) and in the US

409816/1147

U.S. Patent 3,668,203.

; Cephalosporins of the formula

Acyl - NH - CH - CH

O = rC -Ν Λ - CH ₂

COOH

S O

Il Il

wherein X comprises the meanings - S - C - and - S - C are in some of the above-mentioned patents and in the US patents 3,239,515, 3,239,516, 3,243,435, 3,258,461, 3,431,259 and 3,446,803 '.

Corresponding publications in the scientific literature include J. Med. Chem. 8_, 174-181 (19651 and J. Chem. Soc. (London) 1595-1605 (1965), 5015-5031 (1965) and 1959-1963 (1967).

Various disclosures of methanesulfonates from antibiotics have been made in the scientific literature. For example, Belgian patent specification 74 2728 (Farmdoc 41466R) discloses the monomethanesulphonate of ampicillin. U.S. Patents 3,268,508 and 3,295,246 disclose the conversion of one or more of the four free amino groups of kanamycin to the methanesulfonate derivative. Sodium bacitracin methanesulfonate is disclosed in U.S. Patent 3,205,137 and, for neomycin, see J. Antibiotics (Japan) 12 , 114-115 (1959). The injectable antibiotic colistimethat (marketed by Warner-Chilcott under the name "Coly-Mycin") is the sodium

409816 / 1H7

salt of methanesulfonate of colistin; the Merck Index cited
Koyama et al., Japan 4898 (1957). Colistimethat is detailed on pages 315-320 of the US Dispensatory 26th Edition, | JB Lippincott Company, Philadelphia, Penna. described. ι It is called the pentasodium salt of penta (methanesulfonic acid) - .J derivatives of Colistin A on pages 621-622 of USP XVII |

described. The US patent 2 b99 950 discloses in Example VIII the "Polymyxin - formaldehyde sodium bisulfite compound

dung ".. j

The invention includes the amphoteric compounds of the formula '

CH ₂ NH ₂ - I

0 p.!

II / \ I

-I X V- (S) -CHp-C-NH-CH ₂ CH CH _NN;

COOH.

where X is H or OH and m is zero or one
is, with the proviso that when X is OH, m
is zero, which are primarily in the zwitterion form, and non-toxic, pharmaceutically acceptable salts
thereof, including the dimethanesulfonate sodium salts, and
simply hydrolyzed esters. ⁺

'' In addition to the above-mentioned non-toxic, pharmaceutically

Tolerable salts include the non-toxic carboxylic acid salts, including non-toxic metal salts such as sodium,
Potassium, calcium and aluminum salts, the ammonium salt and substituted ammonium salts, for example salts of such

"Simply" hydrolyzed esters are used in the description of the
Invention synonymous with easily hydrolyzing esters.

409816/114 7

non-toxic amines such as trialkylamines / inclusive Triethylamine, procaine, dibenzylamine, N-benzyl-beta-phenethylamine, 1-ephenamine, Ν, Ν'-dibenzylethylenediamine, dehydroabietylr amine, Ν, Ν'-bis-dehydroabietylethylenediamine, N ^ (lower) -alkylpiperidine, for example N-ethylpiperidine, and other amines used in the preparation of salts with benzylpenicillin; and the non-toxic acid addition salts (i.e. the amine salts}, including the mineral acid addition salts, such as the hydrochloride, hydrobromide, hydroiodide, sulfate, sulfamate and phosphate, and the organic acid addition salts, such as maleate, acetate, citrate, oxalate, succinate, benzoate, tartrate, Fumarate, malate, mandelate, ascorbate and the like.

Particularly preferred salts are the dimethanesulfonate sodium salts of the compounds of formula I. Since these salts are water-soluble, they are be'i the preparation of injectables Formulations valuable.

The invention also includes compounds C for use as intermediates or metabolic precursors) in which the amino group is replaced by substituents such as t-butoxycarbonyl, Carbobenzyloxy, formyl, o-nitrophenylsulfenyl, ß, ß, ß-trichloroethoxycarbonyl, 4-oxo-2-pentenyl-2, l-carboethoxy-l-propenyl-2 and the like, is "blocked". Such blocking groups include in particular the ketones (especially acetone) and aldehydes (especially formaldehyde and acetaldehyde), which are described, for example, in U.S. Patents 3,198,804 and 3,347,851 are disclosed and the ß-ketoester and ß-diketones, as disclosed, for example, in U.S. Patent 3,323,479 and the β-ketoamides disclosed in Japanese Patent Publication 71/24714 (Farmdoc 47 321S).

409816 / 1U7

The invention also relates to the process for the preparation of the compounds of the formula

CH ₂ NH ₂ - ·.

- N

where X is H or OH and m is zero or one is,. with the proviso that when X is OH, m is zero, and the non-t.oxis see, pharmaceutically acceptable Salts and singly hydrolyzed esters thereof; the The method is characterized in that a connection of the Formula. -

H ₂ N-CH-

-CH C -H, C

COOH

-N

II

: or a salt or a simply hydrolyzed ester thereof with . an acylation derivative of an acid of the formula.

CH ₂ NHB

(S) - CH ₂ - COOH

III

4Q9816 / 1H7

ί - 12 -

wherein B is an amino protecting group and m and X have the meaning given above, to a compound of the formula

CH ₂ NHB

(S) -OT ₂ -C-NH-CH

wherein B, X and m have the abovementioned meaning, or a salt or a simply hydrolyzed ester thereof is reacted and then the amino protecting group B is removed to give the desired compound of formula I or a non-toxic, pharmaceutically acceptable salt or a simple to produce hydrolyzed esters thereof.

The amphoteric compounds of the present invention are prepared by having a particular one in the 3-position thiolated 7-aminocephalosporanic acid II, that is 7-amino-3 (S-hydroxy-pyridazin-e-ylthiomethyl) -3-cephem-4-carboxylic acid, or a salt or a singly hydrolyzed ester thereof (including but not limited to those of the US patent 3,284,451 and British Patent 1,229,453 and any of the silyl esters described in U.S. Patent 3 24 9 622 for use with 7-aminopenicillanic acid and used in British Patent * 1 073 530 and especially the pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl, p-nitrobenzyl and

409816 / 1U7

β, β, β-trichloroethyl ester) an acid of the formula III or its functional equivalent as an acylating agent for a primary amine group; After coupling becomes the amino blocking group B is removed and the desired product is obtained. The amino protective group B is a blocking group of the Type as used either in peptide syntheses or in any of the numerous syntheses of ampicillin or cephaloglycine or Cephalexin from 2-phenylglycine is used. Particularly valuable Blocking groups are a proton, as in the compound of the formula

CH ₂ NH ₂ * HCl

O (S) _1n -CH ₂ -C-Cl

or a ß-diketone or ß-ketoester, as in the British Patent specification 1 123 333 and US patents 3,325,479 and 3,316,247, for example methyl acetoacetate, or a ß-ketoamide, as in Japanese Patent 71/24714 (Farmdoc 47 321S), in which the blocked arrino group containing Acid is preferably converted to a mixed acid anhydride, such as with ethyl chloroformate, prior to reaction with the compound II or a salt thereof to form the desired product I after cleavage of the blocking group.

Subsequent to the coupling implementation, the "Blockierurfgsgruppe removed in order to prepare the products according to the invention, for example the t-butoxycarbonyl group by treatment with formic acid or trifluoroacetic acid, the carbobenzyloxy group can be removed by catalytic hydrogenation, the 2-hydroxy-l-naphthcarbonyl group by acid hydrolysis, the

409816/1147

Trichlorethoxycarbonyl group by treatment with zinc dust in glacial acetic acid and the l-carboethoxy-l-propenyl-2 group is preferred can be removed with formic acid. Of course, other functionally equivalent blocking groups can also be used can be used for an amino group and "fall within the scope of this of the present invention.

With regard to the acylating agent of the formula III, the functional equivalents include the corresponding acid halides, acid anhydrides, including mixed anhydrides and in particular the mixed anhydrides, which are derived from stronger acids, such as the lower aliphatic monoesters of carbonic acid, or alkyl and arylsulfonic acids and from sterically hindered acids, such as Diphenylacetic acid. In addition, an acid azide or an active ester or thioester (for example with p-nitrophenyl, 2,4-dinitrophenol, thiophenol, thioacetic acid) can be used or the free acid itself can be coupled with compound II after this free acid has first been mixed with N , N ^f -dimethylchloroformiminium chloride (British patent 1 008 170 and Novak and Weichet, Experientia XXI , 6, 360 (1965)) or by using enzymes or a ⁺ Ν, Ν'-carbonylditriazole (South African patent 63/2684) or a carbodiimide reagent (in particular Ν, Ν'-dicyclohexylcarbodiimide, N, N * -diisopropylcarbodiimide or N-cyclohexyl-N ¹ - (2-morpholinoethyl) carbodiimide, Sheehan and Hess, J. Amer . Chem. Soc , 77 , 1067 (1955 )) or an alkylylamine reagent (R. Buijle and HG Viehe, Angew. Chem. International Edition 3, 582, (1964)) or an isoxazolium salt reagent (R..B. Woodward, RA Olofson and H. Mayer, J.Amer.Chem Soc ., 83 , 1010 (1961)) or a ketenimine reagent ens (CL. Stevens and ME Mond, J.Amer.Chem.Soc., 80, (4065)).

Ν, Ν'-carbonyldiimidazole or one

409816/1147

Another equivalent of the acid is a corresponding azolide, namely an amide "of the corresponding acid, the amide nitrogen of which is a member of a quasi-aromatic five-membered ring which contains at least two nitrogen atoms, namely imidazole, pyrazole, the triazoles, benzimidazole, benzotriazole and the substituted derivatives thereof As an example of the general method for producing an azolide, N, N ¹ -carbonyldiimidazole is reacted with a carboxylic acid in equimolar amounts at room temperature in tetrahydrofuran, chloroform, dimethylformamide or a similar inert solvent, the carboxylic acid imidazolide being practically quantitative Yield with release of carbon dioxide and one mole of imidazole gives ·. Dicarboxylic acids give diimidazolide. That

i by-product, imidazole, precipitates and can be separated off} and the imidazolide can be isolated, but this is not essential. The procedures "for carrying out these reactions j

for the production of a cephalosporin and the processes which were used to isolate the cephalosporin so produced, are known to the person skilled in the art.

As mentioned above, enzymes can be used for the coupling of the free acid with its blocked amino group with compound II. In the scope of such methods, the use includes an ester, for example the Methylesters, that free acid with enzymes provided by various microorganisms, for example those described by T. Takahashi et al., J.Amer.Chem.Soc, 94 (11} , 4035-4O37 (1972) and T. Nara et al., J. Antibiotics (Japan) 24 (5), 321τ-323 (1971).

409816/1147

After completion of the coupling and deblocking reactions the compound of the formula I can optionally be converted into non-toxic, pharmaceutically acceptable methods by processes known per se Salts or simply hydrolyzed esters can be converted. The zwitterion products of the formula

ν CH ₂ NHo

0 Il

<S) _m -CH ₂ -C -NH

- CH ₂

COO

in which X and m have the meaning given above, in

their dimethanesulfonate sodium salts of the formula

CH ₂ N (CH ₂ SO ₃ Na) ₂

0 I!

(S) -CH ₂ -C -NH

COONa

being transformed.

The conversion of the zwitterionic cephalosporanic acids into the dimethanesulfonate derivatives can be carried out by methods known to the person skilled in the art. According to a preferred method, the zwitterion is reacted with sodium formaldehyde bisulfite (or a source thereof such as sodium bisulfite and formalin) and water in the presence of a strong sodium base such as sodium 2-ethylhexanoate. The above mixture is cooled to about room temperature, for example at about 40 ⁰ C and preferably in the range of 4O-45 ° C, during a short

0 9 8 1 6/1 U 7

Heated period of time until a solution of the desired product is formed. The product is obtained as a solid by precipitation, for example by adding a water-soluble, essentially anhydrous solvent, preferably j an alcohol such as ethanol or isopropanol.

The invention also relates to a process for the preparation of the compound of the formula

H ₂ N-CH ^ -CH

or their salts or simply hydrolyzed esters, which is characterized in that 7-aminocephalosporanic acid or a salt thereof with a thiol of the formula

or a salt thereof, preferably a sodium or potassium salt, and optionally the 7-amino-3- (6-hydroxypyridazin-3-ylthiomethyl) -S-cephem-'i-carboxylic acid converted into a salt or a simply hydrolyzed ester thereof by methods known per se.

—-4 -irl

The replacement of an ester group, for example the 3-acetoxy group of 7-aminocephalosporanic acid, with a thiol group
is a well-known reaction and can be found in aqueous solution in
Presence of a mild base such as sodium bicarbonate.
This replacement is preferably carried out at a temperature in the range of about 50 ° C to 100 ° C.

The new 7-amino-3- (6-hydroxypyridazin-3-ylthio- | methyl) -3-cephem-4-carboxylic acid intermediate can be used; those extremely valuable in the production of the active cephalosporin compounds j of the formula I or in the production of others! new cephalosporin derivatives. j

It will be clear to the person skilled in the art that the compounds of the formula:

I can alternatively also be prepared by first acylating 7-amino-cephalosporanic acid or ₍ a salt "thereof) with the desired 7-side chain acylating agent,;

followed by nucleophilic replacement of the 3-acetoxy group i

by the S-hydroxy-ß-mercaptopyridazine, whereby the ge j

wanted product. This alternative method also falls within the scope of the invention.

In the treatment of bacterial infections in humans
the compounds according to the invention are parenterally according to the
conventional method of administering antibiotics in
an amount of about 5 to 200 mg / kg / day, and preferably about
5 to 20 mg / kg / day administered in divided doses, for example three to four times daily. They are in dosage units containing, for example, 125, 250 or 500 mg "active ingredient" with suitable physiologically compatible carriers
or containing binders. The dosage units are in the form of liquid preparations such as solutions
or suspensions, before.

0 9 8 1 6/1 U 7

Production of the starting materials

CE ₂ COOH

Pd / C

CE ₃ O- </ y-CH ₂ C00H

NaNO ₂ , KCu (CN) ₂

CH ₂ COOH

PtO ₂ ,

CH ₃ O

CH ₀ NH ₀ -HCl
7 ^

2-cyano-4-methoxyphenylacetic acid

A solution of 12.7 g (0.06 mol) of 4-methoxy-2-nitrophenylacetic acid (D.G. Harvey et al. J. Chem. Soc, 1938, 97) and 2.4 g (0.06 mol) of sodium hydroxide in 100 ml of water is added to

Hydrogenated at room temperature at a hydrogen pressure of 3.52 kg / cm with 1 g of 10% strength palladium on activated carbon as a catalyst. The theoretical amount of hydrogen is absorbed in one hour. The catalyst is removed by filtration using a filter precoated with diatomaceous earth ("Celite"). A solution of 4.2 g (0.06 mol) of sodium nitrite in 30 ml of water at ⁰ ° C. is added dropwise to the filtrate and then 20 ml of conc. Hydrochloric acid (about 0.25 mol) added dropwise. It will be another ten minutes

409816 / 1U7

stirred for a long time. A solution of 4.1 g (0.03 mol) of potassium carbonate in 10 ml of water is added. The reaction mixture becomes one; Solution was added 10 g of potassium cyanide and 7 g of copper cyanide in 3 ml of water 3 \ ser under vigorous stirring at O ⁰ C and the mixture 'is ten minutes at 0 ⁰ C, one hour at 10-15 ⁰ C and finally ¹ Stirred for two hours at 50 ° C, diluted with 300 ml of water and filtered. The filtrate is treated with conc. Chlorine-

: acidified hydrochloric acid, with ethyl acetate (5 χ inn ml) ex- ■ extracted and returned with 5% sodium bicarbonate solution (5 χ 100 ml)

extracted. The combined aqueous extracts are decolorized with a small amount of activated charcoal, cooled, treated with conc. Chlorinated water; acidified and extracted with ethyl acetate (3 × 100 ml). The extracts are washed with water (3 30 ml) and with saturated sodium chloride solution (3 χ 30 ml) and washed with water j ·; dried free sodium sulfate. Removal of the solvent gives 8.7 g (76 % of 2-cyano-4-methoxyphenylacetic acid, which is recrystallized from benzene-ligroin. Melting point j 130-132 ° C.

; .NMR: ο J * Jj [ ^{w U} 6 3.69 (2 H, s, CH ₂ -CO), 3.77 (3 H, 6.9-7.4 (3 H, m, phenyl-H).

Analysis for C _1n H NO

calculated: C, 62.82; H, 4.75; N, 7.33

found: _Cf 62.92; H, 4.74; N, 7-20.

2-aminomethyl-4-methoxyphenylacetic acid hydrochloride

1
1
A solution of 8.2 g (0.043 mol) of 2-cyano-4-methoxyphenylacetic acid in 100 ml of ethanol and 50 ml of 6-n-hydrochloric acid is treated with platinum oxide for two days at room temperature

409816/1 U7

hydrogenated at a hydrogen pressure of 3.52 kg / cm. The catalyst

j is removed by filtration. The filtrate becomes dry

j evaporated. The residue is washed with acetone (3 × 20 ml) j

¹ and gives 5.5 g of 2-aminomethyl-4-methoxyphenylacetic acid hydro-!

; Chloride. "I.

; _f DMSO-d _{6 3e6l} (a H, s, CH ₂ -CO), 3.70 (3 H, s, OCH ₅ ), ¹ 0 ppm ^c ,

! 3.9 (2H, q ^ 6.0 Hz, CH ₂ -N), 6.5 - 7.3 (5 H, m, phenyl-H), 8.5; '(3H, br,

• ^ -aminomethyl ^ -hydroxyphenyl acetic acid

A mixture of 3 g (0.013 mol) of 2-aminomethyl-4-methoxyphenylacetic acid hydrochloride and 40 ml of 48% hydrobromic acid; is held under reflux for five hours. The reaction mixture is treated with activated carbon and with 6-n-sodium \ i hydroxide neutralized (pH of 6-7). You get one; Precipitation of 2-aminomethyl-4-hydroxyphenylessi.gsäure, the; ι collected by filtration, washed with water and acetone \ I and dried in vacuo. Yield 1.9g (81.5%). Melting ΐ

point ^ 300 ⁰ C. \

' 2-t-Butoxycarbonylaminomethyl-4-hydroxyphenylacetic acid

I To a solution of 2.1 g (0.012 mol) 2-aminomethyl 1-4-hydroxy-: ■ phenylacetic acid and 1.4 g (0.035 mol) sodium hydroxide in 50 ml ^:

1 1

! Water becomes a solution of 2.0 g (0.014 mol). t-butoxycarbonyl- i

t azid in 25 ml of THF is added and the mixture is * 22 hours

I stirred for a long time at room temperature, washed with ether, with ver j

; dilute hydrochloric acid and acidified with ether (2x150 ml)!

! extracted. The combined extracts are mixed with water (2 × 50 ml)

4098 16/1147

and a saturated sodium chloride solution (3 5Ο ml) ge j wash and dry. The solvent is removed and I the oily residue with chloroform-carbon tetrachloride (1: 1.50 ml) triturated and gives 1.7 g (52%) 2-t-butoxycarbonylaminomethyl- ■ 4-hydroxyphenylacetic acid as a white solid, which at Melts between 145-147 ° C. i

IR: ^ ^r 5340, 1700, l68O, 12βΟ cm ^"1 -.

^m DMS0-d _{6 1 <57 (9} η, _Sa t-Bu-H), 5-42 (2 H, s, CH ο pm
5.97 (2 H, d, 6 Hz, CH ₂ -N), 6.5-7.1 (3 K, m, phenyl-H), 9.00 (1 H, br, NH), 12.0 (1 H, br, NE).

Analysis for C, .H _1o N0 _c :

14 19 5

calculated: C, 59-77; H, 6.8I; N, 4.98. found: C, 58.84; H, "6.89; N, 4.79-

Potassium o / I-carbomethoxypropen-2-ylaminomethyl7-p-hydroxy phenyl acetate j

J 0.28 mol of o-aminomethyl-p-hydroxyphenylacetic acid, 15.28 g (0.28 mol) of KOH, 64.96 g (0.56 mol) of methyl acetoacetate and 1500 ml of I absolute methanol were introduced. The mixture is then refluxed for three hours. The solution is concentrated to a small volume (100 to 150 ml), filtered and diluted with 400-700 ml of anhydrous ether. Scratching crystallizes the product. The pesticide is filtered and dried in a vacuum desiccator over PoO _1. The yield of the product, which melts at 140-148 ° C., is about

0 9 8 16/1147

Sodium o- (l-ethoxycarbonyl-l-propen-2-ylaminomethyl) -p hydroxyphenyl acetate

To an ethanolic solution of sodium ethylate, prepared from 0/6 g (0.026 atom) of metallic sodium and 50 ml of absolute ethanol / 0.026 mol of o-aminomethyl-p-hydroxyphenylacetic acid and 3.38 g (0.026 mol) of ethyl acetoacetate are added and the mixture is added refluxed for six hours. The mixture is then evaporated to dryness and the residue is recrystallized from ethanol, whereupon about. '. 6 g of sodium o- £ l-ethoxycarbonyl-l-propen-2-ylaminomethyl) -p- j hydroxyphenyl acetate are obtained as colorless needles. ;

t-butoxycarbonylazide . ,, '\

'I: To a cooled solution of 100 g (0.76 mol) of t-butyl carbazate in 87 g glacial acetic acid and i 12Ο ml of water is added dropwise a solu- ι solution of 60 g (0.85 mol) of sodium nitrite in 50 ml of water while! added over a period of 40 minutes, the temperature being kept at 10-15 ° C. When the addition is complete, the mixture is stirred for a further 30 minutes at the same temperature. 100 ml of water are added to the mixture and a separated oil is extracted with five 100 ml portions of methylene chloride. The combined organic extracts are washed with 100 ml of a 10% sodium bicarbonate solution and then with 100 ml of water and dried over anhydrous sodium sulfate. The methylene chloride is removed under reduced pressure on a water bath kept at 40-45 ° C. The remaining azide is distilled and collected at 45 ° C / 20 mm Hg. It weighs 92.7 g (84%).

409816 / 1U 7

3-chloro-6-hydroxypyridaz in

A mixture of 22.47 g (0.15 mol) 3,6-dichloropyridazine and 50 ml acetic acid is refluxed for two hours. The reaction mixture is cooled and diluted with 50 ml of water and then evaporated to dryness under reduced pressure. The residue is crystallized from water and gives 15.8 g (80%) of 3-chloro-6-hydroxypyridazine as colorless prisms which melt at 133-7 ° C (lit. 138-14O ° C). ^! See N. Takabayashi, Yakugaku Zasshi, 7 £, 778 (1955).!
i
3-Hydroxy-6-mercaptopyridazine

^; A mixture of 2.6 g (0.02 mol) of 3-chloro-6-hydroxypyridazine ■ and 5.0 g (0.07 mol) of freshly prepared potassium hydrogen sulfide in 30 ml of ethanol is at 130-140 ° C for six hours in a sealed tube heated. The reaction mixture is cooled and diluted with 200 ml of water. Almost all of the organic solvent is removed by distillation under reduced pressure. The remaining aqueous solution is acidified to pH 3 with dilute hydrochloric acid and extracted with ethyl acetate (6 × 50 ml). The combined extracts are evaporated to dryness and the residue is reprecipitated from 30 ml of ethanol-ligroin (1: 1) and gives 2.2 g (87%) of amorphous 3-hydroxy-6-mercaptopyridazine. Melting point 158-159 ° C (lit. 157-158 ° C). See J. Druey et al. Helv.Chem.Acta, 37. ' ¹²¹ (1954)

S-Hydroxy-S-mercaptopyridazine ■.

To a solution of 5.6 g (0.05 mol) 3,6-dihydroxypyridazine 2.70 g (0.012 mol) of phosphorus pentasulphide are added in portions to 150 ml of pyridine while stirring vigorously and under reflux added. The reaction mixture continues for one hour

409816 / 1U7

kept under reflux and then diluted with 200 ml of water and concentrated to remove the pyridine. The resulting oily residue is suspended in 'water and washed with ethyl acetate extracted. The organic extracts are combined and re-concentrated to give an oily material that works with a small amount of water is triturated to give 3-hydroxy-6-mercaptopyridazine as a yellow solid, Recrystallization from water gives 0.62 g (12%) of the product, which is identical to the product prepared above.

3,6-dihydroxypyridazine

To a boiling solution of 315 g (3 mol) of hydrazine dihydrochloride 295 g (3 mol) of finely ground maleic anhydride are added in portions in 2 liters of water with stirring. After the addition .wi, stirred for a further four hours and then the reaction mixture overnight in a Left the refrigerator. 285 g (85%) of 3,6-dihydroxypyridazine are obtained as massive pillars. ' Melting point ^ 290 ° C.

3,6-dichloropyridazine

A mixture of 150 g (1.33 moles) of 3,6-dihydroxypyridazine and 250 g of phosphorus xychloride is protected for three hours from moisture, refluxed. The excess of phosphorus oxychloride is removed under reduced pressure and the dark residue is poured into one kg of ice pieces. The resulting precipitate is collected by filtration. the The second crop of the product is obtained from the mother liquor by extraction with five 300 ml parts of chloroform, followed by extraction Treatment with 1 g of activated charcoal and evaporation of the solvent. The first and second yields are com-

4098 16/1147

combined, dissolved in 500 ml of chloroform and again add 1 g of activated charcoal treated and concentrated, whereupon 165 g (83%) 3,6-dichloropyridazine as fine needles with a melting point of 60-61 ° C (in a sealed tube).

6-Chlof-3-hydroxypyridaz in

A suspension of 60 g (0.4 mol) of 3,6-dichloropyridazine in 200 ml of 10% hydrochloric acid is refluxed for two hours until a clear solution is obtained. The clear solution is treated with about 1.5 g of activated charcoal and _t filtered. The filtrate is concentrated under reduced pressure to give 6-chloro-3-hydroxypyridazine as colorless needles. Yield 49.5g (98%). Melting point 137-139 ° C.

3-Hydroxy-6-mercaptopyrj.dazin '·

A mixture of 50 g - {0.38 mol) 6-chloro-3-hydroxypyridazine, 60 g (0.83 mol) potassium hydrogen sulfide in 250 ml ethanol is in a 500 ml autoclave at 140 ^° C. for 14 hours with magnetic stirring heated. The pressure reaches a value of 15-20 kg / cm. The mixture is evaporated to dryness and the residue is dissolved in 300 ml of water. The aqueous solution is acidified to pH 3 with dilute hydrochloric acid and extracted with ten 2OO ml portions of ethyl acetate. The combined extracts are concentrated to give 34.3 g (70%) of amorphous 3-hydroxy-6-mercaptopyridazine. Melting point 151-152 ° C.

Potassium o- / I-carbomethoxypropen-2-ylaminomethyl7-phenylthioacetate

In a 2000 ml round bottom flask fitted with a reflux condenser and is equipped with a stirrer at the top

409816/1 U7

0.28 mole of o-aminomethylphenylthioacetic acid, 15.28 g (0.28 mole) of KOH, 64.96 g (0.56 mole) of methyl acetoacetate and 1500 ml of absolute methanol were introduced. The mixture is then refluxed for three hours. The solution is then concentrated to a small volume (100-150 ml), filtered and diluted with 400-700 ml of anhydrous ether. The product crystallizes out on scratching. ^{The solid is collected by filtration and kneaded} in a vacuum desiccator over P ^^ s 9 etroc.

Sodium o- (lethoxycarbonyl-1-propen-2-ylaminomethyl) phenylthioacetate

Manufactured to an ethanolic solution of sodium ethylate from 0.6 g (0.026 atom) of metallic sodium and 50 ml of absolute ethanol, Ö, Ö2 "6 mol of o-aminomethylphenylthioacetic acid become and 3.38 g (0.026 mol) of ethyl acetoacetate are added and the mixture is refluxed for six hours. That The mixture is evaporated to dryness and the residue is recrystallized from ethanol. Sodium o- ^ l-ethoxycafbonyl-l-propen-2-ylaminomethyl) phenylthioacetate is obtained as colorless needles.

-4

NBS

CH ₂ CO ₂ CH ₅

1. NaN,

2. hydro 1.

CH ₂ Bi

H _o / Pd-C / =

CH ₂ CO ₂ H

BOC-N,

87 *

2,4-DNP

₂ C0 ₂ H

CH NH-BOC 2

DCC

II

-CH ₂ -CO CH ₂ NH-BOC

Methyl o-bromomethylphenyl acetate

A mixture of methyl o-methylphenyl acetate (82.0 g, 0.50 mol), N-bromosuccinimide (89.0 g, 0.50 mol), benzoyl peroxide (1.0 g) and carbon tetrachloride (800 ml) is added under Heated to reflux for two hours while irradiating with a 750 watt light source. The succinimide is removed by filtration, the solvent is removed from the filtrate and the residue is distilled in vacuo. 90.1 g (74%) of a product, boiling point 95-105 ° (0.4 mm, NMR (CCl ₄ ): singlets at · / * 2.85 (4H), 5.50 (2H), 6 , 31 (2H) and 6.38 (3H).

A U3 8TB / 1147

o-Azidoinethylphenylacetic acid

A mixture of methyl o-brominomethylphenyl acetate (90/1 g,. 0.371 moles), sodium azide (26.0 g, 0.40 moles) and 10% aqueous acetone (750 ml) is stirred at room temperature for three hours. The solvent is reduced at Removed pressure and treated the residue with ether (300 ml) and water (100 ml). The crude methyl o-azidomethylphenyl acetate (74.8 g), which one after drying and concentrating the ether solution obtained is dissolved in 150 ml of methanol. This solution is cooled in ice and with 15O ml of 3 n methanolic Sodium Hydroxide Treated. The mixture is allowed to stand at room temperature for one hour, then to dryness concentrated and the residue dissolved in water. The aqueous solution is acidified, the product is collected by filtration, dried and recrystallized from ethyl acetate-n-hexane. 49.5 g (70%) of the acid are obtained, melting point 116-118 °, NMR (CDCl3) -: sharp singlets at -f 2.75 (4H),

5.63 (2H) and 6.28 (2H); \ f "" ^ ^ϋΧ 2100 and -1700 cm " ¹ ,

Analysis for C ₉ H ₉ N ₃ O ₂ : ·. j

calcd t C, 56,535; H, 4.75; N, 21.98. found: ■ C, 56.37; H, 4.65; N, 21.7 *

0-aminomethylphenylacetic acid

A mixture of o-azidomethylphenylacetic acid (9.6 g, 0.050 Mol), 10% Pd on charcoal (2.5 g), methanol (150 ml) and 1 N hydrochloric acid (50 ml) is added at a pressure of 2.11 kg / cm hydrogenated for 3.5 hours. The mixture will

409816 / 1U7

filtered, concentrated under reduced pressure to a volume of about 30 ml and extracted with ether. 1-2 g of impure starting material are obtained from the ether extract. The aqueous solution is adjusted to pH 5.0 with dilute ammonium hydroxide and cooled in ice. The white solid precipitate is collected by filtration, washed successively with ice water, methanol and ether and _{dried in vacuo over P 2} ° 5: Yield 5.4 g (65%), melting point 179-181 ° (dec.); NMR (CF ₃ CO ₂ H): -f 2.54 (s, 4H) 5.48 (q, 2H) and 6.00 (s, 2H).

o-tert-butoxycarbonylaminomethylphenylacetic acid

Triethylamine (14.4 g, 0.143 mol) is an ice-cold suspension of o-aminomethylphenylacetic acid (10.3 g, 0.0624 mol) in 100 ml_ water added, whereupon a solution of tert-butoxycarbonylazide (11.4 g, 0.080 mol) in 75 mL of THF is admitted. The reaction mixture is stirred at room temperature for 16 hours and then most of the THF is added _Removed at reduced pressure.

The aqueous solution is washed with ether, with 125 ml of ethyl acetate layered and with ice-cooling with dilute hydrogen chloride acid brought to a pH of 3.5. The ethyl acetate solution is dried, concentrated and the solid residue from ethyl acetate-n-hexane (1: 1) recrystallized. One counts 14.4 g (87%) of white needles, melting point 114-116 °.

Analysis for ^c i4 ^H i9 ^NO 4 ^:

calculated: C, 63.39; H, 7.22; N, 5.28.

found: c, 63.44; H, 7.21; N, 5.42,

409816/1147

2,4-Dinitrophenyl - o-tert-butoxycarbonylaininophenyl acetate

NjN'-dicyclohexylcarbodiimide (1.0 g, 0.0050 mole) becomes one ice-cold solution of o-tert-butoxycarbonylaminomethylphenylacetic acid (1.33 g, 0.0050 mol) and 2,4-dinitride phenol (0.92 g, 0.0050'Mol) in 12 ml of anhydrous tetrahydrofuran was added. The reaction mixture is left at room temperature for one hour

left, then the precipitated N, N'-dicyclohexylurea removed by filtration. The solvent is removed from the filtrate and the activated ester is obtained as a viscous yellow oil.

o-tert-Butoxycarbonylaminomethylphenylacetic acid can be used in quantitative Yield from tert-butoxycarbonylazide and the amino acid 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) and gives the BOC amino acyl chloride, which is directly linked to the compound of the formula 'j

H ₂ -SR

} Θ

Et, HH

wherein R has the meaning given above, can be coupled in methylene chloride solution in the presence of triethylamine. The protecting group can then be removed by treatment with cold trifluoroacetic acid.

In the description of the present invention, ^{+ B0C. Means o-tert-butoxycarbonyl}

A09816 / 1147

Exactly 200 g of 7-aminocephalosporanic acid (7-ACA) are suspended in 500 ml of acetone and a solution of 240 g of p-toluenesulfonic acid in 500 ml of acetone is added in one batch. The mixture is stirred for five minutes at room temperature, filtered through kieselguhr ("Super CeI") and the bed is washed with 150 ml of acetone (the insoluble substance weighs about 30 g). 80 ml of water are then added to the filtrate and stirred the p-toluenesulfonate salt crystallizes out after the inside of the flask has been scratched with a glass rod. The suspension is stirred in an ice-salt bath for thirty minutes and then filtered cold. It is mixed with 2 × 200 ml of cold acetone ( ⁰ ° C.) Washed and air-dried. Yield 250 g of the salt. This p-toluenesulfonate salt of 7-aminocephalosporanic acid is stirred in 2 liters of methanol and the insoluble substance is filtered through "Super CeI." The filtrate is placed in a five-liter three-necked flask and two liters of water are added. Then adjusted the pH by addition of concentrated ammonium hydroxide while cooling on -a value of 4 and the suspension for one hour at 0 ⁰ C stirred. The product is collected by filtration and _{washed with 2 × 100 ml of H 2} O (O ⁰ C) and 3 × 1 liter of acetone (room temperature). After drying in air, the yield of 7-aminocephalosporanic acid is 145 g.

o-Aminomethylphenylacetic acid is also manufactured by Beckmann rearrangement of 2-indanone oxime with subsequent hydrolysis of the lactam formed according to the following Equation: '

409816/1 147

23Λ4095

PCl _r

NOH

CHCl,

under OC.

J.Org.Ghem. £, 380-391

(1944) and 28, 2797-2804 (1963).

materials

2-indanone oxime

Phosphorus pentachloride chloroform

10% sodium hydroxide solution

"Darko KB" activated carbon '

1. HCl

warmth

2. NH

COOH

Weight g VoI. ml mol

1000

1482

6.78

7.13

56600-680

1000

Procedure

1. 1000 g of 2-indanone oxime are to be dissolved in 31,600 ml of chloroform at 20-25 ° C.

2. The solution must be cooled to -30 ° C. (When cooling the 2-indanone oxime solution · to -30 ⁰ C crystallized part of the oxime).

3. 1482 g of phosphorus pentachloride are to be added in parts to the vigorously stirred suspension. The reaction temperature can be maintained at -28 ° to -32 ° C by the rate of addition of the solid phosphorous pentachloride. (The best results are achieved if the reaction is carried out at -30 ° C. It can also be carried out successfully at -10 to -5 ° C or perhaps even at a higher temperature , but then more tar and by-products appear which subsequently make the isolation of the lactam more difficult.)

4098 16/1147

4. The reaction mixture is at about -3O ° C for 10 minutes to stir after the addition is complete and then to warm to 25 ° C. for 3/4 hours. During this period of time, the Solids and then a new solid precipitates.

5. The reaction mixture is at 25 ° C for an additional three hours to stir and then, with careful mixing, 31,600 ml

Add water at * 0-5 ° C. Can (Following the reaction! I

; thin layer chromatography can be carried out. In the system From 8 parts of benzene and 2 parts of acetic acid, the lactam has an · Rf = 0/36 and the oxime as Rf = 0.64. The stains are through 0.05% potassium permanganate spray. The oxime stain can

does not go away completely, but it should become very weak). Subsequent washing processes are carried out at 20-25 ° C.

6. Separate the layers and wash the chloroform phase with 15800 ml of water.

7. Combine the water fractions and extract with 15800 ml of chloroform.

8. Combine the chloroform fractions, coat with 15800 ml of water and mix well with the mixture to rub to a pH of about 7 with 10% sodium hydroxide solution. This can be about 680 ml of sodium hydroxide solution require and the trituration is low. (This basic washing is important when removing the tar donating by-products. The rubbing can take one or two. Hours).

9. The layers are to be separated and filled with 15800 ml of water to wash.

409816/1147

10. The water fractions are to be combined, it is with Wash 9200 ml of chloroform and combine the chloroform fractions.

11. Carbon treatment of chloroform solution "with 1000 g Activated charcoal ("Darko KB") at about 25 ° for 15-30 minutes.

12. Filter the slurry through diatomaceous earth ("Dicalite"), washing the cake with chloroform and concentrating the filtrate under reduced pressure, whereby o-aminomethylphenylacetic acid lactam remains as a dry solid.

13. The yield of crude lactam is almost 100%. It is a yellow crystalline solid (when the tar gives off Substances had not been removed by the washing process, this product would be dark. It could then be made from hot water, having previously- brought the water slurry to a pH of 7.0, or be recrystallized from toluene-heptane).

Materials Weight g

Crude o-aminomethylphenylacetic acid lactam from 1000 g
2-indanone oxime about 1000

Conc. Hydrochloric acid

"Darko KB" activated carbon 100

Methylene chloride methyl isobutyl ketone (MIBK) 6-n-ammonium hydroxide

Vol. Ml

Mole

about 6.78

8000

6000

as required as required

4098 16/114 7

procedure

1.8000 ml conc. Hydrochloric acid is about 1000 g add the crude lactam obtained by oxime storage.

2. The mixture should be stirred and gently for three hours to reflux. (When this reaction mixture is heated, an excessive amount of foam forms because the excess HCl escapes. This foam can fill the entire device. It can be reduced by using a silicone antifoam. After the initial foaming stage is over, the reaction mixture can be refluxed without difficulty).

3. Cool the dark slurry to about 40-5O ° C and 100 g activated charcoal ("Darko KB") are to be added and stirring is to be continued.

4. Carbon treatment for 15-20 minutes. The slurry is filtered through a "Dicalite" cake and the Wash cakes with about 4000 ml of hot water.

5. The clear yellow filtrate is to be extracted with 6000 ml of methylene chloride (1/2 volume) and the methylene chloride is separated off. The CH ₂ C1 ₂ layer must be retained in order to check whether unchanged lactam can be obtained.

6. Concentrate the water phase at reduced pressure and gives solid o-aminomethylphenylessi.gsäurehydrochlorid.

7. MIBK is to be added to the wet solids for distillation continue at reduced pressure and continue adding MIBK until all of the water is removed from the solids.

409816/1147

8. Continue reduced pressure distillation until all of the methyl isobutyl ketone is removed from the solids is. (In the case of methyl isobutyl ketone distillation, not only ■ the water is removed as an azeotrope, but also excess HCl is also removed).

9. The solids are to be redissolved in 3900 ml of water and 650 ml of methyl isobutyl ketone are to be added.

10. While stirring at 20-25 ° C, the pH value of the solution is with Adjust 6-n-aramonium hydroxide to a value of 5.0. The o-aminomethylphenylacetic acid zwitterion starts at around pH 3.5 to crystallize.

11. The zwitterion slurry should be stirred and brought to 0-5 ° C to refrigerate for an hour.

12. Filter the slurry and carefully wash the cake with about 1000 ml of ice cold water, then 2000 ml of methyl isobutyl ketone and then wash with 5000 ml of ice-cold acetone. The combined filtrate and wash liquids are check for lactam content. ν

13. The cake should be sucked dry and then dried in a circulating air oven at 45 ° C. The yield is 670-730 g; 60-65% based on the oxime.

14. Subsequent to the reactions and processes, thin layer chromatography can be performed using a solvent system of 5 acetone, 1.5 benzene, 1.0 acetic acid, 1.5 water; developed by KMnO ₄ ; R _f lactam = 0.88, R _f amino acid = 0, -69.

098 1 6/1 U 7

o-Aminomethylphenylacetic acid is also produced by Schmidt rearrangement of 2-indanone with subsequent hydrolysis of the lactam formed made according to the following equation:

1.

2.

C.

HC

Warmth ■

CH ₂ COOH

■ Organic

Syntheses' 41, 53 (1961)

2-Indanone is made from indene according to the method described in Organic Syntheses (loc. Cit.) Process made. If a technical grade reagent of indene is used, the yield will decrease of 2-indanone to about 45% (see Note 2 in Organic Syntheses). 2-indanone produced in this way is -as quickly as possible used because it is stated in Note 7 in Organic Syntheses that 2-indanone compared to air at room temperature is inconsistent.

The hydrolysis of the (/ ^ - lactam is successfully used using carried out by hydrochloric acid. The amino acid is obtained in a yield of 79%. Barium hydroxide is also used for the hydrolysis, but the yield is then 32%.

o-aminomethylphenylacetic acid-o-lactam

To a cooled suspension of 12.3 g (0.093 'mol) of freshly prepared 2-indanone and 13.0 g (0.2 mol) of sodium azide in 500 ml of chloroform are carefully 50 ml of conc. Sulfur

409R 1R / 1U7

acid was added in such a way that the temperature is kept below 40 ° C. with stirring. After the addition is complete and the exotherm has ceased, the mixture is stirred at room temperature for a further two hours and then poured into 400 g pieces of ice. The chloroform layer is separated and the aqueous layer is extracted with three 300 ml portions of chloroform. The chloroform layer is with. combined with chloroform extracts and treated with anhydrous sodium sulfate! dries and concentrated under reduced pressure. A crystalline precipitate is obtained which is collected by filtration and recrystallized from 250 ml of n-hexane-benzene (1: 1) and gives 12.0 g (88%) of the desired product as colorless prisms. Melting point 152-153 ° C.

IR (KBr) i / _Tnav (cm " ¹ ) 3200, 3050, ΐββο, 1500,

NMR (CDCl3,): / (ppm 'from ¹ TMS) 3.50 (2H, t, 1.5 Hz), k Λθ

(2H, br-s, converted to a triplet (J = 1.5 Hz) by an addition of D ₂ O), 7.06 (4H, s), 7.45 (1H, br-s, disappears by a Addition of D ₃ O).

Analysis for C ₉ H ₉ NO:

calculated: c, 73. ^ 5; H, β.ΐβ; Ν, 9.52. found: C. 73.735 H, 6-8; N, 9.23.

6-aminomethylphenylacetic acid - {/ - lactam

Ten milliliters of sulfuric acid are added to a stirred suspension of 1.32 g (0.01 mol) of freshly prepared indanone in 50 ml of chloroform, and 1.30 g (0.02 mol) of sodium azide are added in portions to the mixture at room temperature with stirring. The reaction mixture lasts for half an hour

'409816/1147

stirred, then poured into 200 ml of ice-water and extracted with two 100 ml portions of chloroform. A large amount of insoluble material that appears during the extraction process is filtered off. The chloroform extracts are combined, dried over anhydrous sodium sulfate, treated with activated charcoal and filtered. Evaporation of the filtrate and subsequent recrystallization of the residue from 100 ml of alcohol-n-hexane (1: 1) gives the (/ lactam, the yield melts at 150-152 ⁰ C 0.48 g (32%)..

o-aminomethylphenyl acetic acid

A mixture of 7.3-7 g (0.05 mol) of o-aminomethylphenyl acetic acid lactam and 50 ml of conc. Hydrochloric acid will refluxed for three hours and the reaction mixture treated with 1.0 g of activated charcoal and filtered. The filtrate is concentrated to dryness under reduced pressure to give 8.5 g as a crystalline residue. Recrystallization from 400 ml of aqueous acetone (acetone: water = 10: 1) gives 8.0 g (79%) of the hydrochloride of the desired amino acid, as colorless platelets. Melting point 164-166 C.

IR (KBr): y (cm " ¹ ) 1695, 16IO, 1585, '1230, II80.

UlCL Λ

NMR (DMSO-dg): / (ppm from. TMS) 3.69 (2H, s), 3.9 ¹ * (2H, s), 4.10-4.7 (? H, broad), 7.0-7.5 ( ¹ ^ H, m) , 7.6 - 8.7 (IH, broad). .

Analysis for C ₉ H ₁₁₂

calculated: C, 53.60; H, 6.00; ^ 5 ^ ₉₅ . _C l, 17:58.

found: _c , 53.74. H, 5.98JN, 6.79; Cl, 17.9 ¹ *

409816/1147

-41- 2344U95

o-aminomethylphenyl acetic acid

A mixture of 340 mg - (2.3 mmol) o-aminomethylphenylacetic acid-lactam and 730 mg (2.3 mmol) of barium hydroxide octahydrate in 20 ml of water is placed in a sealed tube at 150 ° C heated for an hour. The reaction mixture is cooled to room temperature and 300 mg (2.5 mmol) of ammonium carbonate become added to precipitate barium carbonate which filters and washed well with 50 ml of water. The aqueous filtrate is combined with the washing liquid and evaporated to dryness. The residue is crystallized from 5 ml of 50% aqueous alcohol and gives 163 mg (30%) colorless Platelets that are identical to the free amino acid obtained from the hydrochloride by neutralizing with ammonium hydroxide is. Melting point 180-182 ° C.

IR (KBr): V "^ m" ¹ ) 3100-2200, ΐββθ-1500, 1400, 1380,

770, 755, 720.

NMR (CF ₃ COOH): ^ (ppm from. TMS) 3.96 (2H, br-s), 4.45 (2H,

br-s), 6.5-8.0 DH, br), 7-38 (4h, s). Analysis for C ₉ H ₁

calculated: C, 65.44; H, 6.7UN, 8.48. found: c, 65.24j, H, 6.47; N, 8.31. o-aminomethylphenylacetic acid lactam

! In a 2 liter three-necked flask with a round bottom

■ is provided and with a reflux condenser, drying tube, above attached stirrer, thermometer and a 100 ml dropping funnel 24.6 g (0.186 mol) of 2-indanone and 26 g (0.4 mol) of sodium azide are placed in 1 liter of chloroform.

409816/1147

The suspension is stirred and 100 ml of conc. Sulfuric acid added in such a way that the temperature is maintained between 33-37 ° C (see note 1). When the addition is complete, the reaction mixture is stirred at room temperature for a further two hours and then poured into 800 g of pieces of ice. The chloroform layer is separated and the aqueous phase is extracted three times with 300 ml portions of chloroform. The aqueous phase is treated with sodium nitrite (Note 2). The chloroform extracts are combined, dried over magnesium sulfate (anhydrous) and evaporated to dryness. The solid is redissolved in 150 ml of hot water and treated with 2.0 g of activated charcoal ("Darko KB") while it is still hot. The solution is filtered and the activated charcoal washed three times with 25 ml portions of hot water. The solution is cooled and the solid is collected by filtration. It is washed with 10-20 ml of ice-cold water and _{dried in a vacuum desiccator over P 2} ° 5. The product Melts at 144-145 ° C, 18.3 g. (yield 69%).

Remarks

1., Caution: During the addition of sulfuric acid. Hydrazoic acid is evolved. Hydrazoic acid boils at 38-39 ° C. Therefore the temperature must be kept below 40 ^Ω c. The entire experiment should be carried out in a well-ventilated fume cupboard.

2. Remaining hydrazoic acid in the aqueous phase is removed by slowly adding sodium nitrite, up with iodine paper and / or ferric chloride solution a positive test is obtained, decomposed.

409816/1 U7

o-aminomethylphenylessic acid hydrochloride

10.1 g (0.075 mol) of o-aminomethylphenyl acetic acid lactam'und 100 ml of conc. Hydrochloric acid introduced. The mixture is then refluxed for three hours. While it is still hot, the reaction mixture is treated with 2.0 g of activated charcoal ("Darko KB") for 5 minutes and filtered. The Piltrat is / 15ΊΜη concentrated to dryness at 50-60 ° and finally in a high vacuum over ^ ₂ ⁰ S ^kon ~ centered (Note 1). The solid is recrystallized from a previously prepared mixture of acetone: water (15: 1) (Note 2). The hydrochloride is dried _{over P 3} O ₅ in a vacuum desiccator. The yield of the pure product, which melts at 188-10 ° C., is 11.4 g (78%).

Analysis for C ₉ H ₁₂₂

calculated: C, 53.73; H, 5.97; N, 6.96; Cl, 17.66. found: C, 53.56; H, 6.02; N, 6.89; Cl, 17.76. Remarks

1. For a successful recrystallization is absolute dry material required. .,.

2. The ratio of acetone to water can change depending on the dryness of the crude hydrochloride.

409816/1147

Production of 'ö-aminomethylphenylacetic acid from indene

HCOOH-H ₂ O ₂

NaN ₃ -H ₂ SO ₄

HCl

2-indanone (2) ...

To a stirred mixture of 370 ml of 80% formic acid and 70 ml of 30% hydrogen peroxide are added dropwise 58 g (0.5 mol) of indene at 35-4O ° C. over a period of one hour. Another 40 ml of formic acid is used to flush the rest of the indene from the dropping funnel into the reaction flask. The reaction mixture is stirred for 15 hours at room temperature. The mixture is concentrated to remove the formic acid under reduced pressure (20-30 mm) below 40 ⁰ C to. The residue is dissolved in 1000 ml of 10% sulfuric acid and this solution is refluxed for 20 minutes and then steam distilled. The distillation is carried out at a rate of approximately two liters per hour until 7 liters of distillate are collected. The distillate is extracted with six 400 ml portions of chloroform. The combined extracts are dried over anhydrous sodium sulfate and filtered. The filtrate is evaporated to dryness and yields 50.2 g (76%)

409816/1147

23U095

2-indanone '. (2) as pale yellow needles with a melting point of 56-57 ° C.

o-aminomethylphenylacetic acid-0-1actam (3>)

To a strongly cooled suspension of 46 g (0.35 mol) of freshly prepared 2-indanone (2) and 27 (0.42 mol) of sodium azide in 75 ml of chloroform, 75 ml of conc. Sulfuric acid is added at such a rate that the temperature j is kept at 30-40 ° C. with stirring. After the addition is complete and the exotherm has ceased, stirring is continued for three hours. The reaction mixture is poured into about one kg of ice pieces. The chloroform layer is separated and the aqueous layer is extracted with three 200 ml portions of chloroform. The chloroform layer is combined with the chloroform extracts _f , dried over anhydrous sodium sulfate and evaporated to dryness. 44.5 g (87 t) of o-aminomethylphenylacetic acid - (/ - lactam are obtained as pale yellow prisms with a melting point of 146-149 ° C.

.Q-aminomethylphenylessyl acid hydrochloride (£)

A mixture of 43.4 g (0.30 mol) of o-aminomethylphenylacetic acid-ci-lactam (3) in 140 ml of 6N hydrochloric acid refluxed for four hours. The reaction mixture is treated with 1 g of activated charcoal and evaporated to dryness. That The remaining oil is triturated with 500 ml of acetone and gives 45 g of (75 1) o-aminomethylphenylacetic acid hydrochloride (£) as colorless Platelets with a melting point of 163-1 £ 4 ° C.

409816/114 7

Manufacture of ^: o-Amlttc-ttieT: nylpheWle'ssl'gic acid from o-nitrotoluene

NO ₂ 1) (COOEt)

2) H ₂ O ₂

CH ₂ COOH

H ₂ (Pd / C)

3) KCu (CN) _-

H ₂ (P <3 / C or PtO ₂ )

CH ₂ COOH

CH ₂ NH ₂ -HCl CH ₂ COOH

o-nitrophenylacetic acid ¹ ξ6)

A mixture of 135 ml (about 1 mole) of ethyl oxalate and 118 ml (about 1 mol) o-nitrotoluene (j5) is added dropwise while cooling to a stirred solution of 23 g (1 g atom) of sodium in 300 ml of absolute ethanol is added and the mixture is left for 1.5 hours stirred at room temperature and then refluxed for 1.5 hours. The mixture is cooled to 60 ° C and approx 500 ml of water are added. The mixture is steam distilled, to remove the unreacted nitrotoluene. The residue is treated with 5 g of activated charcoal and filtered. The clear filtrate is adjusted to a pH of 8-9 with dilute sodium hydroxide. A solution from. , 6% hydrogen peroxide is added at 30-40 ° C until a small sample of the reaction mixture, if alkaline with sodium hydroxide no longer shows the dark color characteristic of alkaline pyruvic acids. The reaction mixture

409816/1 U?

is treated with a small amount of activated charcoal and filtered. The Piltrat is made with conc. Hydrochloric acid acidified and extracted with four 250 mL portions of ethyl acetate. The combined extracts are washed with water (3 × 100 ml) and a saturated sodium chloride solution and dried over anhydrous sodium sulfate. The solvent is removed and the residue is crystallized from ethanol-water (200 ml-600 ml) and gives 81.3 g (45%) of o-nitrophenylacetic acid (.6) with a melting point of 140-141 ° C \ f _CQ ni0 cm. y ^ _N0 1525, 1360 cm " ¹ .

o-cyanophenylacetic acid (7)

A solution of 43.4 g (0.24 mol of o-nitrophenylacetic acid (6) and 9.6 g (0.24 mol) of sodium hydroxide in 200 ml of water

- <· ■ 2- i

at room temperature under a hydrogen pressure of 3.52 kg / cm j

hydrogenated with 1 g of 10% palladium-activated carbon. , The theoretical amount of hydrogen is absorbed for four hours. The catalyst is removed by filtration. A solution of 16.6 g (0.24 mol) of sodium nitrite in 50 ml of water is added to the filtrate while cooling at 0 ° C. The mixture is added dropwise to 80 ml of conc. Hydrochloric acid added at 0-3 ° C with stirring. When the addition is complete, a strongly cooled solution of 16.6 g (0.12 mol) of potassium carbonate in 100 ml of water is added to the reaction mixture with stirring. The diazonium salt solution is / are added with vigorous stirring at 0-5 ⁰ C to a solution of Kaliumcuprocyanid, which is made of 40 g (0.62 mol) of potassium cyanide and 28 g (O, "31 moles) Kupferzyaiiid in 130 ml of water. The mixture is ^{stirred for 30 minutes at 0} ° C., for one hour at 15-20 ° C. and one hour at 50 ° C. and finally left to stand overnight at room temperature The reaction mixture is diluted with 1 liter of water and filtered Hydrochloric acid acidified and extracted with three 300 ml portions of ethyl acetate

«09816/1147

Extracts in turn are extracted with four 200 ml portions of sodium bicarbonate solution. The combined alkaline extracts are treated with a small amount of activated charcoal, acidified with conc. Hydrochloric acid and extracted with three 300 ml portions of ethyl acetate. The combined extracts are washed with 200 ml of water and 200 ml of a saturated sodium chloride solution, dried with anhydrous sodium sulfate and concentrated and give 36 g of the crude product o-cyanophenylacetic acid (J). Recrystallization from benzene gives 29.6 g (77%) dark yellow Needles with a melting point of 121-122 ° C. N ^r _{c == N} 2220 cm " ^1. V _{c = 0} 1690 cm" ¹

o-aminomethylphenylacetic acid hydrochloride {A)

(i) A mixture of 1.6 g (0.01 mol) of o-cyanophenylacetic acid (7), 2.5 ml of ethanol and 15 ml of .6-n-hydrochloric acid is mixed with 1 g of 10% palladium on activated carbon for an ordinary one Pressure and hydrogenated at room temperature. The theoretical volume of hydrogen is absorbed in 18 hours. The catalyst will removed and the filtrate is concentrated and gives 1.6 g (80%) of colorless platelets of o-aminomethylphenylacetic acid hydrochloride (4) with a melting point of 161-163 ° C. The product is with authentic sample obtained by Schmidt rearrangement is made of 2-indanone with subsequent hydrolysis, identical.

(ii) A mixture of 3.2 g (0.02 mol) of o-cyanophenylacetic acid (7), 0.5 g of 10% palladium-on-charcoal, 50 ml of ethanol and 25 ml ö-n-hydrochloric acid is at room temperature at a

Hydrogenated hydrogen pressure of 3.52 kg / cm. The theoretical Amount of hydrogen is absorbed in three hours. The mixture is filtered to remove the catalyst / the filtrate is concentrated and the residue triturated with 100 ml of acetone. 3.0 g (75%) of o-aminomethylphenylacetic acid hydrochloride (4) are obtained with a melting point of 162-163 ° C.

4098 16/1147

(iii) A solution of 3.22-g (0.02 mol) of o-cyanophenylacetic acid

(7) in 50 ml of ethanol and "25 ml of 6-n-hydrochloric acid will '

2 at room temperature with a hydrogen pressure of 3.52 kg / cm hydrogenated using 0.15 g of platinum oxide as a catalyst, the theoretical amount of hydrogen is absorbed in four "hours." Catalyst is removed by filtration and the filtrate is evaporated to give a viscous oil, which with 150 ml of acetone is triturated and 2.1 g (53%) of colorless platelets with a melting point of 161-163 ° C. are obtained.

(iv) Platinum oxide (0.1 g) is a solution of 16. g (0.01 mol) o-Cyanophenylacetic acid (7) in 20 ml of ethanol and 20 ml of 6-N hydrochloric acid are added and the solution becomes ordinary Pressure hydrogenated. The theoretical amount of hydrogen gas is absorbed in 18 hours. The catalyst is filtered off and the filtrate is evaporated to dryness *. The residue is triturated with 50 ml of acetone and gives 1.0 g (50%) of o-aminomethylphenylacetic acid hydrochloride (4) with a melting point of 161-163 ° C. ■ '*

q- Aminomethyl enyl acetic acid

A solution of 76.0 g (0.378 mol) of o-aminomethylphenylacetic acid hydrochloride in 450 ml of water is treated with 6-n-NH.OH until a pH of 5 is reached. The solution will be Chilled overnight at 5-7 ° C. The crystalline solid is collected by filtration to give 56.3 g (92%) pure Product, melting point at 188-19O ° C.

Analysis for CqH.

Calculated: C, 64.45; H, 6.66; N, 8.48.

found: C, 64.98; H, 6.86; N, 8.53.

'4098 16 / 1IU

Potassium-o- / l -carbomethoχypropen-2-ylamlnomethyl7 ~ phenyl acetate

In a 2000 ml round bottom flask equipped with a reflux condenser and a stirrer at the top, 45.92 g (0.28 mol) of o-aminomethylphenylacetic acid, 15.28 g (0.28 mol) of KOH, 64 , 96 g (0.56 mol) of methyl acetoacetate and 1500 ml of absolute methanol were introduced. The mixture is then refluxed for three hours. The solution is then concentrated to a small volume (100-150 ml), filtered and diluted with 400-700 ml of anhydrous ether. The product crystallizes out on scratching. The solid is filtered and dried _{over P 3} O _{5 in a vacuum desiccator.} (The solid material is extremely hygroscopic. It should be filtered as quickly as possible and not air-dried). The yield of the product, which melts at 140-148 ° C., is 84.1 g (99%). <

ι Analysis for C ₁₄ H, / -NO ₄ K:. ·

calculated: C, 54.20; H, 5.49; N, 4.52; Theatrical Version, 2.90.

found: C, 53.72; H, 5.44; .N, 4.56; KF, 2.92. NatriuIn-o- (l-ethoxycarbonyl- ^ l -propen-2-ylaininomethyl) phenyl acetate

To an ethanolic solution of sodium ethylate, which consists of 0.6 g (0.026 atom) of metallic sodium and 50 ml of absolute ethanol is prepared, 4.27 g (0.026 mol) of o-aminomethylphenylacetic acid are obtained and 3.38 g (0.026 mol) of ethyl acetoacetate are added and the mixture is refluxed for six hours. The mixture is evaporated to dryness and the residue is recrystallized from ethanol to give 6.36 g (82%) sodium o- / I-ethoxycarbonyl-1-propen-2-ylaminomethyl) phenyl acetate as colorless needles with a melting point of

23O-232 ° C.

409816/114

, KBr
Max

3320, 1645, I695, 1470, 1395, 1275, 118O cm " ^1. Analysis for C, _C H, _o N0.Na:

calculated: C, 6θ.2Ο; Η, 6.Ο6; N, 4.68.

j found: • _C , 59-95; H, 5,865 N, 4.67.

' t-Butoxycarbonylaz id

, To a cooled solution of 100 g (0.76 mol) of t-butyl carbazate

in 87 g of glacial acetic acid and. 120 ml of water is poured into a solution drop by drop ; solution of 60 g (0.85 'mol) of sodium nitrite in 50 ml of water during

over a period of 40 minutes, the temperature being kept at 10-15 ° C. When the addition is complete, stirring is continued for a further 30 minutes at the same temperature. 100 ml of water are added to the mixture and a separated oil is extracted with five 100 ml parts of methylene chloride. The ; combined organic extracts are washed with 100 ml of 10% sodium bicarbonate solution and then with 100 ml of water and dried over anhydrous sodium sulfate. That ^! Methylene chloride is removed under reduced pressure on a water bath kept at 40-45 ° C. The remaining azide is distilled and collected at 45 ° C / 20 mm Hg. It weighs 92.7 g (84%).

ο- (t-butoxycarbonylaminomethyl) phenylacetic acid

To a solution of 70 g (0.35 mol) of o-aminomethylphenylacetic acid hydrochloride and 116 g (1.15 mol) of triethylamine in 400 ml of water, a solution of 64 g (0.45 mol) of t-butoxycarbonylazide in 300 ml of tetrahydrofuran were ^{added at 0} ° C. with stirring. After the addition has ended, the temperature is allowed to rise

409816 / 1U7

Rise room temperature and stir for a further 20 hours. The tetrahydrofuran is distilled off below 40 ° C and the aqueous solution is washed with 200 ml of ether, coated with 200 ml of ethyl acetate and acidified with diluted hydrochloric acid under cooling at 0 ⁰ C to a pH-value of the third The organic layer is separated and the aqueous layer is extracted with four 200 ml portions of ethyl acetate. The combined ethyl acetate solution is washed with 200 ml of water, over water-

j dried free sodium sulfate and concentrated in vacuo. That

The concentrate is treated with 500 ml of η-hexane "and gives 87.9 g

j (95%) ο- (t-butoxycarbonylaminomethyl) phenylacetic acid as color

loose needles with a melting point of 114-116 ° C.

j 2,4-dinitrophenyl-ot-butoxycarbonylaminomethylphenyl acetate

Dicyclohexylcarbodiimide, (.17.72 g, 0.086 mole) (DCC) is found in j a batch of a mixture of o- (t-butoxycarbonylaminomethyl) phenylacetic acid (22.73 g, 0.086 mol) and 2,4-dinitrophenol (15.82 g, 0.086 mol) (2,4-DNP) in 250 ml of tetrahydrofuran were added. The reaction mixture is for two hours at room

j temperature stirred. The precipitated dicyclohexy! Urea becomes filtered off and washed with 100 ml of tetrahydrofuran. The FiI kicked and the washing liquids are combined and concentrated under reduced pressure below 50 ° C. A viscous one is obtained yellow oil triturated with n-hexane (150 ml) and 2,4-dinitrophenyl-o-t-butoxycarbonylaminomethylphenylacetic acid results as yellow needles. Yield 34.9g (94%). Melting point 76-77 ° C.

IR: γ ™ 33 * 0, 1785, 1685, 1610, 15 * 0, 1530,

Analysis for ^C 2o ^H 21 ^N 3 ° 8 ^:

calculated: C, 55.68; H, 4.91i H, 9.7 *.

found t ^c >55.70; H, 5.05ϊ Ν, 9 93.

409816 / 1U7

description of preferred; Embodiments

The following examples * illustrate the invention without restricting it in any way. All temperatures are given in degrees Celsius . 7-Aminocephalosporanic acid is abbreviated as 7-ACA; 7-Aminocephalosporanic acid is the group with the following formula

MH-CH CH CH ₂

¹ S /

COOH

Il

and consequently 7-aminocephalosporanic acid can be used as H-ACA-OC-CH.,

being represented. j

Example 1 'j

7-Amino-3- (β-hydroxypyridazin-S-ylthiomethyl) -3-cephem-4-carboxylic acid

A mixture of 0.60 g (0.0047 mol) of S-hydroxy-δ-mercaptopyridazine, 1.27 g (0.0047 mol) of 7-aminocephalosporanic acid, 0.78 g (0.0094 mol) of sodium bicarbonate in 25 ml of 0 , 1 M phosphate buffer (pH 6.4) is heated at 60 ° C for five hours. The reaction mixture is filtered to remove all traces of insoluble material and the filtrate is adjusted to pH 5 with acetic acid, whereupon a brown precipitate is obtained which is collected by filtration, washed with water and then

409816/1147

send is washed with acetone and dried in vacuo. 7-Amino-3- (δ-hydroxypyridazin-S-ylthiomethyl) -3-cephem-4-carboxylic acid is obtained; Yield -1.03 g (71%). Melting point 29O-3OO ° C (Decomposition).

>, I65O, I58O, 1415 cm " ¹ .

UV: λ ^ / ^δ0Η 249 im (e 19400). ''

NMR: ^ -D ₂ ^0-1 ^ ⁰⁰ S 3.22 (IH, d, 19Hz), 3.37 (IH, d, 14 Hz), 3.65 (IH, d, Sf ¹ Hz), 3.72 (IH, d, 19 Hz ), 4.9Ο

(IH, d, 4 Hz), 5.30 (IH, d, 4 Hz), 6.75 (IH, d, 10 Hz), 7-30 (IH, d, 10 Hz). ·

! Analysis for C ₁₃ H ₁₃ N ₄ O ₄ S ₃ .1 / 2 H ₃ O: j

calculated: C, 41.25; H, 3.75; N, 04/16; S, 18.36

found: c, 41.45; H / 3.70; N, 15.83; s / 18.03 ^.

1 example 2 j

7- (2-t-Butoxycarbonylaminomethyl-4-hydroxyphenylacetamido) -3 (6-hydroxypyridazin-3-ylthiomethyl) -3-cephem-4-carboxylic acid

To a solution of 1.7 g (6 mmol) of 2-t-butoxycarbonylaminomethyl-4-hydroxyphenylacetic acid and 1.2 g (6.6 mmol) of 2,4-dinitrophenol in 40 ml of tetrahydrofuran are in one batch 1.35 g (6.6 mmol) of dicyclohexylcarbodiimide added and that The mixture is stirred for 1.5 hours at room temperature. The separated urea is removed by filtration. The filtrate a solution of 2.0 g (5.9 mmol) 7-amino-

409816/1147

3- (6-hydroxypyridazin-3-ylthiomethyl) -3-cephem-4-carboxylic acid and 1.7 ml (about 12 mmol) * triethylamine in 30 ml of water was added. The mixture is stirred for 20 hours at room temperature / washed with ethyl acetate (3 150 ml) and with 6N hydrochloric acid acidified to give a precipitate, which is collected by filtration, washed with water (50 ml) and is dried. The precipitate is dissolved in .1 liter of methanol at 50 ° C and filtered to make a small amount insoluble Remove substance. The filtrate is treated with activated charcoal and evaporated to dryness. The residue is washed with ether

• (50 ml) triturated to give 920 mg (25%) 7- (2-t-butoxycarbonylamiriomethyl-4-hydroxyphenylacetamido) -3- (6-hydroxypyridazin-3-ylthiomethyl) -3-cephem-4-carboxylic acid with a melting point of 190-2OO ° C (decomp.).

IRi \ KBr _{1770 1700 #} 1670, x 1580, 1250 cm "" ¹ . Max

Analysis for C ₂₆ H ₂₉ N ₅ O-QS ^ H ₂ O:

calculated: C, 50.23; H, 5.03; N, 11.27 found: C, 50.63; N, 4.69; N, 10.94.

j 7-U-Andnomethyl ^ -hydroxyphenylacetamido) -3- (6-hydroxypyridazin 3-ylthiomethyl) -3-cephem-4-carboxylic acid .

Trifluoroacetic acid (2 ml) and 7- (2-t-butoxycarbopnylaminomethyl-4-hydroxyphenylacetamido) -3- (6-hydroxypyridazin-3-ylthiomethyl) -3-cephem-4-carboxylic acid (850 mg, 1.4 mmol) are mixed with cooling at 0-5 ° C and stirred for one hour. The mixture is diluted with 150 ml of ether and gives the trifluoroacetate

409816 / 1H7

of the desired cephalosporin, 7 ~ (2-aminomethyl-4-hydroxyphenylacetamido) -3- (G-hydroxypyridazin-S-ylthiomethyl) -3-cephem-4-carboxylic acid, which is collected by filtration and suspended in 30 ml of water. The suspension is adjusted to a pH of € with dilute ammonium hydroxide and diluted with 1 liter of acetonitrile while stirring. The solid is collected, washed with acetonitrile (3 × 100 ml) and dried in vacuo. 546 mg (77% ') of the desired 7 - ^ - aminomethyl ^ -hydroxyphenylacetamido) -3- (6-hydroxypyridazin-3-ylthiomethyl) -3-cephem-4-carboxylic acid with a melting point of 210 ° C (dec. ). IR: ^ 5 * 177Q, 1660, 1580, 1390, cm " ¹ .

j analysis for

j calculated: C, 43.82; H, 4.73; N, 12.56

I ■ ·

j found: C, 43.85; H, 4.28; N, 12/13. . *

Example 3

i 7- (o-aminomethyl-p-hydroxyphenylacetamido) -3- (3-hydroxy pyridazin-6-ylthlomethyl-3-cephem-4-carboxylic acid -

To a stirred suspension of 2.1 mmol sodium-o- (l-ethoxycarbonyl-l-propen-2-ylaminomethyl) -p-hydroxyphenyl acetate in 5 ml of dry acetonitrile containing a drop of N ₁ N-dimethylbenzylamine ¹ are 0, 25 g (2.3 mmol) Äthylchlorofonniat added with stirring at -15 ° C and stirred for further 20 minutes at -10 to -15 ⁰ C. A solution of 0.71 g (2.1 mmol) 7-amino-3- (3-hydroxypyridazln-6-ylthiomethyl) -3-cephem-4-carboxylic acid and 0.21 g (2, 1 mmol) of triethylamine in 3 ml of acetonitrile and 3 ml of water

409816/1147

added and the mixture is stirred at OC for one hour. The reaction mixture is mixed with a small amount of activated carbon treated and filtered. 0.5 ml of formic acid are added to the filtrate with shaking and the mixture is filtered, a small amount of unreacted 7-amino-3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid to remove. 100 ml of acetonitrile are added to the filtrate and the mixture is allowed to stand at room temperature for one hour. The precipitate is collected by filtration, washed with 10 ml of water and dried in vacuo. You get about 0.5 g of 7- (o-aminomethyl-p-hydroxyphenyiacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid.

Example 4

7-o-Andnomethyl-o-hydroxyphenylacetamido-3- (3-hydroxy-pyridazin- 6-ylthiomethyl) -3-cephem-4-carboxylic acid _

In a 1 liter ore-necked flask, which is equipped with a round bottom, a drying tube, at the top with a stirrer and thermometer, 0.08 mol of well-powdered potassium-o- ^ I-carbomethoxypropen-2-ylaminomethyl7-p-hydroxyphenyl acetate in 350 ml of dry tetrahydrofuran (THF) and 4 drops of N, N-dimethylbenzylamine were added. The suspension is stirred vigorously and cooled to -40 ⁰ C with an acetone-dry ice bath. 13.6 g (0.06 mol of isobutyl chloroformate are added to the suspension all at once, while the temperature is kept below -35 C. After 2.5 minutes, the acetone-dry ice bath is removed and a solution of 0.036 mol of 7-amino-3- (3-hydroxypyrida3in-6-ylthiomethyl) -3-cephem-4-carboxylic acid and 5.04 g (0.050 mol) of N * -methylmorpholine in 250 ml of water are added. The reaction mixture is stirred until it reaches room temperature.

409816/1147

enough (1-2 hours) .. The tetrahydrofuran is removed under reduced pressure. The aqueous solution is coated with 100 ml of ethyl acetate and treated with conc. HCl acidified to pH 1.7-2.0. The mixture is treated with 3.5 g of activated charcoal ("Darco KB") and filtered. The layers are separated off and the aqueous phase is extracted with 2 × 100 ml parts of ethyl acetate. The aqueous phase is then filtered through filter paper and adjusted to a pH of 4.0. After your scratching, the solution is left to crystallize overnight at 5-7 ° C. The product is collected by filtration, washed with a small amount of water and _{dried in a vacuum desiccator over P 2} Oe. The yield of 7- (o-aminomethyl-p-hydroxyphenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid is approximately 30%.

Example 5 . . . '

A suspension of the zwitterionic form of 7- (o-aminomethyl-phydroxyphenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid (0.361 g) in 3 ml of methanol is cooled in ice and with a few drops of conc. Treated hydrochloric acid until a clear solution is obtained. 7- (o-Aminomethylp-hydroxyphenylacetaimido) -3- (3-hydroxy-pyridazin-6-ylthipmethyl) 3-cephem-4-carboxylic acid hydrochloride precipitates as a pale brown solid on addition of ether j and is collected by filtration and in vacuo dried over P ₂ Or.

Example 6

! To a stirred suspension of the zwitterionic form of 7- (οι Aminomethyl-p-hydroxyphenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid In aqueous sodium hydroxide (0.361 g) is added at room temperature until a clear. Solution (pH 10.8) is obtained. This solution will be immediate

409816 / 1U7

Freeze-dried and gives impure, solid sodium 7- (oj aminomethyl-p-hydroxyphenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylate.

Example 7

CH ₂ NH ₂

Il

-CH ₂ -C-NH-CH-

fs>

i ·

COOH

HIGH ₂ SO ₅ Na

Sodium 2-ethylhexanoate in acetone)

1. Heat in water

2. Treat with activated charcoal and filter

3. Add the Piltrat to anhydrous alcohol

CH ₂ N (CH ₂ SO ₃ Na),

11

CH ₂ -C -NH-CH

EHA.

0 9 8 16/1147

procedure

2.0 moles (approximately 1010 g on an anhydrous basis) 7- (o-aminomethyl-p-hydroxyphenylacetamido-3- {3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid _/ 540 g sodium formaldehyde bisulfite (4 .03 mol), 3000 ml of water and 2700 ml (4.87 mol) of 30% sodium 2-ethylhexanoate (SEH) in acetone are placed in a suitable vessel and the mixture is heated to 40-45 ° C. while stirring . The mixture dissolves to a yellow solution in about 10 minutes.

After 15 minutes of heating, 50 g of activated carbon ("Darco KB") added to the solution and stirring is continued for 15 minutes at 40-45 ° C.

After heating the reaction mixture at 40-45 ° C for a total of 30 minutes is filtered through diatomaceous earth ("Dicalite").

The activated charcoal cake is washed with 2000 ml of 50% ethanol-water. The filtrates are combined, adjusted to 25 ° C and the solution is rapidly stirred at 25 ° C to 112 liters ^; 100% ethyl alcohol added. A fine white amorphous precipitate of the di (sodium methanesulphonate) of sodium 7- (o-aminomethyl-p-hydroxyphenylacetamido- 3- (3-hydroxypyridazin-6-ylthiomethyl) -S-cephem ^ -carboxylate forms.

The suspension is stirred for about 10 minutes and then filtered and the cake with 15 liters of 100% ethyl alcohol washed.

The cake is dried in a circulating air oven at 50-55 ° C for approximately two hours and then dried in vacuo at 4 × 6 mm for 24 hours.

409816/1147

The yield is approximately 1200-1400 g of amorphous, white, solid di (sodium methanesulfonate) of sodium 7- (o-aminomethylp-hydroxyphenylacetamido-α-iS-hydroxypyridazin-e-ylthiomethyl) -3-cephem-4-carboxylate. The product usually contains a few percent water and possibly a trace of ethanol.

This product is also sodium-7- ^ ö-N, N-ibis (natriuinsulfoj methyl) aminoπιethyl-p-hydroxyphenylacetamido7-3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylate.

Example 8

The following slurry is made:

2.19 g sodium formaldehyde bisulfite (2 equivalents), 3.5 g 7- (o-aminomethyl-p-hydroxyphenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid zwitterion (100-200 stitches),

25 ml of water (the amount can be changed), 14 ml of 30% sodium 2-ethylhexanoate isopropanol (SEH isopropanol).

Nearly a solution is obtained in about 0.5 hours of rapid stirring at 24 ° C. The temperature of the mixture is raised rapidly to 40-43 ° C, held for about two minutes and then quickly cooled to 20-23 ° C.

The solution is filtered to remove a small amount of insoluble matter (the total time in solution should be two hours not exceed).

The solution with a pH of 7.3 is used for a period of time of 5 minutes to 600 ml of very rapidly stirred absolute ethanol (other alcohols such as anhydrous isopropanol, can be used). It forms

409816/1 U?

an amorphous precipitate of the di (sodium methanesulfonate) of Sodium 7- (o-aminomethyl-p-hydroxyphenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylate. The mixture is stirred for 5 minutes. The precipitate is collected by filtration, with 60 ml of ethanol (or isopropanol) washed and dried in vacuo at 50 ° C. for 24 hours. The yield is approximately 4.3 g.

j The product is soluble in water at a pH of approximately 7 to at least 200 mg / ml. Such a solution is min-

stable for at least two hours at room temperature; still ! more dilute solutions are stable even longer. The product exhibits the same antibacterial spectrum as that Mother zwitterion and is completely biologically effective, whether it is hydrolyzed back into the zwitterion or not.

Example 9

Sodium 7- ^ ö-N, N-bis (sodium sulfomethyl) aminömethyl-p-hydroxy- ] phenylacetamido7-3- (S-hydroxy-pyridazin-ö-ylthiomethyl) -3-! cephem-4-carboxylate. Manufactured using hydroxy; methanesulfonate

A mixture of 2.05 mmoles of 7- (o-aminomethyl-p-hydroxyphenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid zwitterion, 1.56 g (10 mmoles) of sodium hydroxymethanesulfonate monohydrate, 6 ml (6 mmol) of 1 M sodium 2-ethylhexanoate solution in ethyl acetate, 10 ml of isopropanol and 10 ml of water is stirred at room temperature for 3.5 hours. The. The resulting solution is treated with 1 g of activated charcoal and poured into 300 ml of absolute ethanol with stirring and the mixture is stirred at room temperature for 30 minutes and gives the crystalline product, which is collected by filtration, washed with three 50 ml parts of absolute ethanol and poured over P ₂ ⁰ S is ^{dried at} 45-52 / 1 mm for 20 hours. 4 0 9 8 16/1147 are obtained

23U095

about 1.5 g of sodium 7- ^ ö-N, N-bis (sodium sulfomethyl) aminomethyl-p-hydroxyphenylacetamido? ^ - (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylate, which is easily soluble in water (> 1 g / ml).

j Example 10

Sodium 7- ^ o-N, N-bis (sodium sulfomethyl) aminomethyl-p-hydroxy- ! phenylacetamido7-3- (S-hydroxy-pyridazin-e-ylthiomethyl-S-cephem-4-carboxylate. Manufactured using formalin and

j sodium bisulfite

J (a) To a solution of 1 ml (10 mmol) of 30% formalin and 1 g of sodium bisulfite in 10 ml of water, add 2 mmol of 7- (o-aminomethyl-p-hydroxyphenylacetamido) -3- (3- hydroxypyridazin (6-ylthiomethyl) -3-cephem-4-carbobiotisic acid zwitterion _/ 6 ml of 1 M sodium 2-ethylhexanoate solution and 10 ml of isopropanol were added. The mixture is stirred for 2.5 hours at room temperature and poured into 300 ml of ethanol. The resulting sodium 7- ^ o- N , N-bis (sodium sulfomethyl) aminomethyl-p-hydroxyphenylacetamido? - 3- (3-hydroxypyridazin-6-ylthiomethyl-3-cephem-4-carboxylate is collected by filtration, with three 50 ml portions of ethanol and dried in vacuum dried. The yield is about 1.5 g.

(b) To a mixture of 2 mmol of 7- (o-aminomethyl-p-hydroxyphenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid zwitterion, 6 ml of 1M sodium 2-ethylhexanoate in 1 ml (10 mmol) of 30% formalin is added to ethyl acetate, 10 ml of isopropanol and 10 ml of water. The mixture is stirred for two hours at room temperature and gives a clear solution with a small amount of oily precipitate. After 1 g of sodium bisulfite has been added, the solution is stirred for an additional two hours, during which time the oily precipitate dissolves in the solution. The reaction mixture is dissolved in 300 ml of ethanol

409816/1147

Poured with vigorous stirring and yields about 1.8 g of sodium 7- ^ ö-N, N-bis (sodium sulfomethyl) aminomethyl-p-hydroxyphenylacetamidcy-S-O-hydroxypyridazin-e-ylthiomethyl) -3- j cephem-4-carboxylate, which is collected by filtration, with three j 50 ml parts of ethanol are washed and dried in vacuo.

• Example 11

^A ·
: 7- (o-tert-Butoxycarbonylaminomethylphenylthioacetamido) -3- (3-

hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid

j To a solution of 2.19 g (4.7 mmol) of 2,4-dinitrophenyl ester of the BOC-protected amino acid in 50 ml of tetrahydrofuran, a solution of 1.02 g (3 mmol) of 7-amino ^{is added at 0 ° C.} -3- (3-hydroxypyri-

; dazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid and 0.85 ml of Trij ethylamine in 50 ml of tetrahydrofuran and 20 ml of water were added. ! The reaction mixture is left at room temperature for 17 hours

j stirred, treated with activated charcoal and filtered. The filtrate! is evaporated in vacuo to remove tetrahydrofuran and the concentrate is washed with five 100 ml portions of ether. The aqueous solution is coated with 100 ml of ethyl acetate and acidified to a pH value of 2 at 0 ° C. with dilute hydrochloric acid while stirring vigorously. The aqueous layer is extracted with three 100 ml portions of ethyl acetate. The organic layer is combined with the ethyl acetate extracts, dried over anhydrous sodium sulfate and treated with activated charcoal. The filtrate is evaporated at 30 ° C. under reduced pressure and gives an oily product which is triturated with 100 ml of ether. 7- (o-tert-Butoxycarbonylaminomethylphenylthioacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) ^L 3-cephem-4-carboxylic acid is obtained as a pale yellow solid.

409816/1 U7

By collecting the precipitate by filtering and then washing with 100 ml of ether, 0.71 g (38%) of the
desired 7- (o-tert-butoxycarbonylaminomethylphenylthioacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid.

I ^B · ■ ■

• 7- (o-aminomethylphenylthioacetamido) -3- (3-hydroxypyridazine-6-
! ylthiomethyl) -3-cephem-4 ~ carboxylic acid

j 7- (o-tert-butoxycarbonylaminomethylphenylthioacetamido) -3- {3- ^!

! hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid (0.50 g,;

; 0.8 mmol) is added in one batch to 3 ml of trifluoroacetic acid under j

'. Stirring at ⁰ ° C. added. It will continue for an hour j

; stirred at 0-5 ° C. When adding 100 ml of ether, a j appears

ι!

white precipitate. He ₍ is collected by filtration and placed in;

ι. ι

\ 4 ml of water suspended at room temperature. The suspension i

is diluted with ammonium hydroxide while stirring to an I.

\ pH value of 5 is set. By collecting the precipitation |

filtration gives 7- (o-aminomethylphenylthioacet- j

amido) -3- (S-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carbon- I

acid as a brown powder mixed with water and then with;

Acetonitrile washed and in vacuo over phosphorus pentoxide i

is drying. It weighs 220 mg (52%). Melting point 200-210 ° C '

(Decomposition). I.

IR; γ KBr 1770, ΐβγο, 1580, 1210 cm " ^1. \

Max .. -. j

^UVi λ ™? L ^NaHC S 250 nm ( ε 2θ8θθ), 270 nm (, ε 14900). ·
Analysis for C ₂₁ H ₃₁ N ₅ O ₅ S ₃ ^ 1/2 H ₃ O:

calculated: C, 44.67; H, 4.64; N, 12.40; S, April 17
found: C, 44.43; H, 3.85; N, 10.84; S, 17.89.

4098 16/1147

23UQ95

Example 12

7- (o-tert-Butoxycarbonylaminomethylphenylthioacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid

(Herge.stellt as described <in US Patent 3,657,232) to a solution of 1.85 g (4 mmol) of 2,4-dinitrophenyl (o-tert-j butoxycarbonylaminomethylphenylthio) acetate in 50 ml of tetrahydrofuran is added a solution of 1.02 g (3 mmol) of 7-amino-3- j (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-'4-carboxylic acid,
0.85 ml (about 6 mmol) of triethylamine in a mixture of 30 ml of tetrahydrofuran (THF), 30 ml of acetonitrile and 50 ml of water were added while cooling at ⁰ ° C. The mixture is allowed to slowly reach room temperature and is stirred for 12 hours. The reaction mixture is treated with a small amount of activated charcoal and filtered. The piltrate is evaporated to remove the organic solvent in vacuo below 35 ° C. The ent-; standing aqueous solution is mixed well with ether (3O ml χ 4)! wash, covered with 50 ml of ethyl acetate and acidified with dilute hydrochloric acid with stirring at 0-5 ° C to a pH of 3 j. The precipitate that appears between the two layers is collected by filtration. It weighs 0.6 g. i That is the desired 1- (o-tert-butoxycarbonylaminomethyl-: phenylthioacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) 3-cephem-4-carboxylic acid. Another amount of the product is obtained from the filtrate, which comprises two layers. the
The aqueous layer is extracted three times with 100 ml of ethyl acetate. The organic layer is combined with the organic extracts, dried on anhydrous sodium sulphate and used
Evaporated to dryness. The residue becomes good with 100 ml of ether
washed and dried in vacuo on phosphorus pentoxide. He
weighs 0.42 g. The total yield of 7- (o-tert-butoxycarbonylaminomethylphenylthioacetamido) -3- O-hydroxypyridazin-e-ylthiomethyl) -3-cephem-4-carboxylic acid is 1.02 g (55%). Melting point 15 0-1 6 O ⁰ C (decomp.).

4098 16/1147

1770, 1690, '1590, 1535, 1175 cm " ¹ .

250 nm (ε 21700), 270 nm (sh) (€ 15500).

Analysis for C ₃₆ H ₃₉ N ₅ O ₇ S ₃ .1 / 2 H ₃ O:

Calculated. · C, 49.67; H, 4.97; N, 11.13; S, 15.30

found: -C, 49.95; H, 4.60; N, 10.70; S, 15:52. '

B. .. j

7- (o-Aminomethylphenylthioacetamido) -3- (3-hydroxypyridazine-e- I

ylthiomethyl-3-cephem-4-carboxylic acid 1

A mixture of 0.98 g (1.58 mmol) of the BOC-protected Cepha- · losporins 7- (o-tert-Butoxycarbonylaminomethylphenylthioacetamido) \ - 3- (S-hydroxypyridazine-o-ylthiome'thyl) -3-cephem- 4-carboxylic acid and 4 ml of trifluoroacetic acid are stirred at 0 ° C. for one hour. \ By adding 100 ml of ether to obtain a white low- j blow, which was collected by filtration and washed with 100 ml of ether overall! wash -will. A suspension of the precipitate in 10 ml of water is adjusted to a pH value of 5 with dilute ammonia and gives the desired cephalosporin 7- (o-aminomethyl- i phenylthipacetamido) -3-O-hydroxypyridazin-e-ylthiomethyl) -3- j cephem-4-carboxylic acid, collected by filtration, with 50 ml
Acetonitrile is washed and dried in vacuo. The yield is 0.46 g (56%). The product is with the product
identical to Example 11 above (IR).

'409816/1 U7

- 68 - 23UQ95

Example 13

7- (o-Aminoraethylphenylthioacetamido) -3- (3-hydroxypyridazin-6- ylthiomethyl-S-cephem- ^ - carboxylic acid

0.25 g (2.3 mmol ) Ethylchlorfomiat added with stirring at -15 ° C and it is further stirred for 20 minutes at -15 ° C. A solution of 0.71 g (2.1 mmol) 7-amino-3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid and 0.21 g (2, 1 mmol) of triethylamine in 3 ml of acetonitrile and 3 ml of water are added and the mixture is ^{stirred at 0} ° C. for one hour. The reaction mixture is treated with a small amount of activated charcoal and filtered. 0.5 ml of formic acid are added to the filtrate with shaking and the mixture is filtered to remove a small amount of unreacted 7-amino-3- (3-hydroxypyridazin-6-ylt-hiomethyl) -3-cephem-4-carboxylic acid . 100 ml of acetonitrile are added to the filtrate and the mixture is allowed to stand at room temperature for one hour. The precipitate is collected by filtration, washed with 10 ml of water and dried in vacuo. 0.4 g of 7- (o-aminomethylphenylthioacetamido) -3-O-hydroxypyridazin-o-ylthiomethyl) -3-cephem-4-carboxylic acid is obtained.

Example 14

7-o-Aminomethylphenylthioacetamido-3 (3-hydroxy-pyridazin-6- ylthiomethyl) -3-cephem-4-carboxylic acid

In a 1 liter three-necked flask with a round bottom, a drying tube, equipped with a stirrer and thermometer at the top, is 0.08 mol of well-powdered potassium -ο / ϊ-carbomethoxypropen-2-ylaminomethyl7phenylthioacetate in 350 ml

409 8 16/1 14 7

dry tetrahydrofuran (THF) and 4 drops of N, N-dimethylbenzylamine brought in. The suspension is stirred vigorously and cooled to -40 ° C. with an acetone-dry ice bath. Of the ! 13.6 g (0.06 mol) of isobutyl chloroformate are added to the suspension all at once, while the temperature is kept below -35 C. " will. After 2.5 minutes, the acetone-dry ice bath j is removed and a solution of 0.036 mol of 7-amino-3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid is removed and 5.04 g; (0.050 mol) of N-methylmorpholine in 250 ml of water is added. The reaction mixture is stirred until it has reached room temperature (1-2 hours). Tetrahydrofuran is made under reduced pressure j away. The aqueous solution is coated with 100 ml of ethyl acetate and with conc. HCl to a pH of 1.7-2.0

'leavened. The mixture is mixed with 3.5g activated carbon ("Darco KB")

j treated and filtered. The layers are separated and the aqueous phase is ex- ■ with 2 100 ml parts of ethyl acetate . . .

; traced. The aqueous phase is then passed through filter paper filtered and the pH adjusted to 4.0. After scratching, the solution will stand overnight at 5-7 ° C to crystallize.

ί serene. The product is collected by filtration, washed with a small amount of water and _{dried in a vacuum desiccator over P 2} ° 5. The yield of 7-o-aminomethylphenylthioacetamido-3-O-hydroxypyridazin-e-ylthiomethyl) -3-cephem-4-carboxylic acid is approximately 30%.

Example 15

A suspension of the zwitterion form of 7- (o-aminomethylphenylthioacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid (0.361 g) in 3 ml of methanol is cooled in ice and concentrated with a few drops. Treated hydrochloric acid, until a clear solution is obtained. 7- (o-Aminomethylphenylthioacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid hydrochloride precipitates as a pale brown solid on addition of ether and is filtered off

409816/1147

collects and dried in a vacuum over 'P ₂ ^ ς.

Example 16

To a stirred suspension of the zwitterion form of 7- (o-aminomethylphenylthioacetamido) -3- (3-hydroxypyridazin-6-yl-

thiomethyl) -3-cephem-4-carboxylic acid (0.361 g) is 1 η aqueous j

Sodium hydroxide added at room temperature until a clear j

Solution (pH 10.8) is obtained. This solution is immediately applied

freeze-dried and results in unclean, solid. Sodium 7- (o-amino- ' methylphenylthioace tamido) -3- (3-hydroxy-pyridazin-6-ylthiomethyl) -

3-cephem-4-carboxylate. j

409816 / 1U7

Manufacturing process "of pimethanesulfonate

Example 17

O φ
• CH ^y \ C. Il I. C. COOH -C-NH-CH 1 C ₂ H ₅ O

+ HIGH ₂ SO ₅ Na -f- CH ₅ (CH ₂ ) ₅ OH-COONa

Sodium 2-ethylhexanoate in * acetone)

1. Heat in water

2. Treat with activated charcoal and filter

3. the filtrate to anhydrous \ i / add alcohol

fixed

CH ₂ N (CH ₂ SO ₃₁ Na) ₂ 0

S-CH-c -NH-CH-CH N

CH,

N-N

C-CH ₂ -S- ^

COONa

H ₂ O

EHA.

A0981 6 / 1H7

procedure

2.0 moles (about 1020 g 'on an anhydrous basis) 7- (o-aminomethylphenylthioacetamido-3-O-hydroxy-pyridazin-e-ylthiomethyl) -3-cephem-4-carboxylic acid, 540 g sodium formaldehyde bisulfite (4.03 mol), 3000 ml water and 2700 ml (4.87 mol) 30% Sodium 2-ethylhexanoate (SEH) in acetone are placed in a suitable vessel and the mixture is increased while stirring Heated 40-45 ° C. The mixture dissolves in about 10 minutes to a yellow solution.

After 15 minutes of heating, 50 g of activated carbon ("Darco KB") are added to the solution:
touched.

added to the solution and another 15 minutes at 40-45 ° C

After heating the reaction mixture at 40-45 ° C for a total of 30 minutes is' filtered through diatomaceous earth ("Dicalite").

The activated charcoal cake is washed with 2000 ml of 50% ethanol-water. The filtrates are combined, adjusted to 25 ° C and the solution is rapidly stirred at 25 ° C to 112 liters 100% ethyl alcohol added. A fine white amorphous precipitate of the di (sodium methanesulfonate) forms of sodium 7- (o-aminomethylphenylthioacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylate.

j The suspension is stirred for about 10 minutes and then filtered and the cake is mixed with 15 liters of 100% ethyl j alcohol washed.

j The cake is cooked in an air-circulating oven at 50-55 ° C dried for about two hours and then dried in vacuo at 4-6 mm for 24 hours.

AO 98 1 6/1 H?

The yield is approximately 1200-1400 g of amorphous, white, solid di (sodium methanesulfonate) of sodium 7- (o-aminomethylphenylthioacetamido) -3- (S-hydroxypyridazin-ö-ylthiomethyl) -3-cephem-4-carboxylate. The product usually contains a few percent water and possibly a trace of ethanol.

This product is also used as sodium T- ^ ö-lS ^ N-bis (sodium sulfomethyl) aminomethylphenylthioacetamido7-3- (3-hydrqxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylate called.

Example 18

The following slurry is made: 2.19 g sodium formaldehyde bisulfite (2 equivalents), 3.5 g 7- (o-aminomethylphenylthioacetamido) -3- (3-hydroxypyridaziri-6-ylthiomethyl) -3-cephem-4-carboxylic acid zwitterion (100-200 stitches),

25 ml water (volume can be changed), ' \

14 ml of 30% sodium 2-ethylhexanoate isopropanol. j

Almost a solution is obtained in about 0.5 hours of rapid stirring at 24 ° C. The temperature of the mixture is quickly raised to 40-43 ° c. This will take about two minutes maintained for a long time and then quickly reduced to 20-23 ° C.

The solution is filtered to remove a small amount of insoluble matter (the total time in solution should be two hours not exceed).

The solution with a pH of 7.3 is used for a period of time from 5 minutes to 600 ml of very quickly stirred absolute ethanol (other alcohols such as anhydrous Isopropanol, can be used). An amorphous precipitate of the di (sodium methanesulfonate) of Sodium 7- (o-aminomethylphenylthioacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylate. The mixture is stirred for 5 minutes. The precipitation is

409816/1147

trier collected / washed with 60 ml of ethanol (or isopropanol) and in vacuo
The yield is 4 g.

Wash and dry in vacuo at "50 ° C for 24 hours.

Ί
I The product is soluble in water at a pH of about 7 to I at least 200 mg / ml. Such a solution is stable for at least two hours at room temperature; even more

j «

j thin solutions are stable for a longer time. The product has ^! exhibits the same antibacterial spectrum as the mother zwitterion

and is completely biologically effective, whether it is in the hybrid • ion is back hydrolyzed or not.

Example 19

: Sodium 7- / oN, N-bis (sodium sulfomethyl) aminomethylphenylthio-

-i acetamido / - 3- (S-hydroxy-pyridazin-ö-ylthioinethyl) -3-cephem-4-

! carboxylate production using hydroxymethane

i sulfonate

j A mixture of 2.05 mmol 7- (o-aminomethylphenylthioacetamido) -

; 3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid

: zwitterion, 1.56 g (10 mmol) sodium hydroxymethanesulfonate mono-

I hydrate, 6 ml (6 mmol) of 1 M sodium 2-ethylhexanoate solution in

j Ethyl acetate, 10 ml isopropanol and 10 ml water are

j stirred temperature for 3.5 hours. The resulting solution is treated with 1 i g activated carbon and with stirring in 300 ml absolute ethanol poured ■ th and the mixture is stirred at room temperature j Hinuten long and yields the crystalline product; collected by filtration, washed with three 50 ml portions of absolute ethanol and _{dried over P 2} ^O S ^{at 45} ~ ⁵² ° ^c / l ro ™ for 20 hours. One receives_Natrium-7- ^ 5-N, N-bis (sodium sulfomethyl) aminomethylphenylthioacetamidoZ-S- (-3-hydroxy-pyridazi ι 6-ylthiomethyl) -3-cephem-4-carboxylate, which is easily soluble in water (J. > 1 g / ml).

409816/1147

M / 128O1 -.75-234

Example 20

Sodium 7- / oN, N-bis (sodium sulfomethyl) aminomethylphenylthioacetamido73- (3-hydroxy-pyridazin-6-ylthiomethyl-3-cephem-4-carboxylate. Manufactured using formalin and! Sodium bisulfite \

i.

j (a) To a solution of 1 ml (10 mmol) of 30% strength formalin and; 1 g of sodium bisulfite in IO ml of water are successively 2 mmol ι _f 7- (o-Aminomethylphenylthioacetamido) -3- (3 ~ hydroxy-pyridazin-5-j, ylthiomethylJ-S-cephem ^ -carbonsäurezwitterion, 6 ml of 1 M sodium; 2-ethylhexanoate solution and 10 ml of isopropanol are added, the mixture is stirred for 2.5 hours at room temperature and poured into 300 ml of ethanol.

j (sodium sulfomethyl) aminomethylphenylthioacetamido7-3- (3-hydroxy-!

pyridazin-e-ylthiomethyl-S-cephem ^ -carboxylate is given by FiI- I

j trieren collected, washed with three 50 ml portions of ethanol and j

^: dried in vacuum .. ^!

■ (b) To a mixture of 2 mmol of 7- (o-aminomethylphenylthio- j '. Acetamido) -3- (3-hydroxvpyridazin-6-ylthiomethyl) -3-cephem-4-

¹ carboxylic acid zwitterion, 6 ml of 1 M sodium 2-ethylhexanoate in

, Ethyl acetate, 10 ml of isopropanol and 10 ml of water becomes 1 ml

', (10 mmol) 30% formalin added. The mixture becomes two

Stirred for j hours at room temperature and gives a clear

I solution with a small amount of an oily precipitate.

After; 1 g of sodium bisulfite has been added, the solution becomes more stirred for two hours, during which the oily precipitate

■ strike dissolves in the solution. The reaction mixture is in

Poured i 300 ml of ethanol with vigorous stirring and gives solid

y mm

i Sodium-7- ^ o-N, N-bis (sodium sulfomethyl) aminomethylphenylthioj acetamido7-3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylate, which is collected by filtration, washed with three 50 ml portions of ethanol and dried in vacuo.

409816 / 1U7

j Example 21

7- (o-tert-Butoxycarbonylaminomethylphenylacetamido) -3- (3-hydroxy-j pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid, j

To a stirred solution of 1.9 g (3/75 mmol) of 2,4-dinitrophenyl-ot-butoxycarbony.laminomethylphenyl acetate in 15 ml of tetrahydrofuran, a cooled solution of 1/14 g (3.3 mmol) of 7- Amino-3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid and 0.62 g (6.6 mmol) of triethylamine in 15 ml of acetonitrile and 5 ml of water were added while cooling at ⁰ ° C. The reaction mixture is allowed to slowly reach room temperature and is stirred overnight. The mixture is evaporated to remove the organic solvent under reduced pressure below 30 ° C. and the remaining aqueous solution is washed with ether (10 ml χ 3). The aqueous layer is covered with 20 ml of ethyl acetate and washed with dilute chlorine water! chemical acid is acidified to pH 2.5 with stirring and the organic layer is separated. The aqueous layer is extracted with ethyl acetate (100 ml χ 3). Insoluble material appearing during the extraction is collected by filtration and washed with a small amount of water. 7- (o-tert-Butoxycarbonylaminomethylphenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid is obtained as a pale yellow powder (0.30 g) (A). The combined organic extracts are treated with a small Mönge activated charcoal and filtered. The filtrate is dried over anhydrous sodium sulfate and the solvent is evaporated. An oily substance is obtained which is washed well with 50 ml of ether and 0.59 g of 7- (o-tert-butoxycarbonylaminomethylphenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid as a gives pale yellow powder (B). Both

Yields (A) and (B) show the same IR spectra.

409816/1 U 7

Overall yield 0.89 g (46%). Melting point 160-170 ° C (dec.).

IR: Ύ JJBr 1780; 1710, 1690, I68O, 1530 cm " ^1. WX ^ _x ^NaHC0 3 250 nm ( _e 18400)

° ¹³⁵ < ^{9 H 3} ^ ³⁵⁵

^NMR & ppm ³ <^ ⁽

3.3-4.7 (5H, not out aufaelöpt ^ .95 (IH, d, 4 Hz), 5.50 * (1 H, d-d, 9, 4 Hz), 6.64 (IH, d, 10 Hz), 7.04 (4 H, s), 7.18 (1 H, d, 10 Hz), 8.9O ** (1 H, d, 9 Hz), 12.'60 ** (1 H, br-s). (* doublet (J = 4 Hz). by adding D ^ O; * · # disappears through

Addition of D ₂ O). .

Analysis for C ₂₆ H ₂₉ N ₅ O ₇ S ₂ -1/2 H ₃ O:

calculated: C, 52.34 ;. H, 5.07, -N, 11.74; S, 10.75

found: C, 52.03; H, 4.98; N, 11.37; S, 09/10.

7- (o-aminomethylphenylacetamido) -3- (3-hydroxypyridazine-6-

ylthiomethyl) -3-cephem-4-carboxylic acid

7- (o-tert-Butoxycarbonylaminomethylphenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid (500 mg, 0.87 mmol) is added to 3 ml of trifluoroacetic acid with stirring at 0 ° C. the mixture is ^{stirred below 0 °} C. for a further hour. Ether (100 ml) is added to the reaction mixture and the resulting precipitate is collected by filtration.

40 9816/1147

The precipitate is suspended in 10 ml of water and the pH of this suspension is adjusted by adding dilute ammonium hydroxide adjusted to a value of 5 with stirring. The desired product 7- (o-aminomethylphenylacetamido) -3- (3-hydroxy-i pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid is obtained by FiI- ι trieri collected, with each 5 ml of water and acetonitrile;

washed each other and dried with vacuum on phosphorus pentoxide. It weighs 286 mg (67%). Melting point 222-230 ° C (dec.).

IR: / *? *! 77O, 1665, 1640, 1580, 1-395, 1005 cm "* ¹ .

ΓΠ3Χ »

.UV: h # ^NaHC0 3 250 nm (£ 18400), 270 nm (sh) (ε ΐ46θθ)

Max

NMR £ D ₂ ⁰ ~ ^K 2 ^C0 3 3.70 (2 H, s), 4.15 (2H, s), 3.0-4.3 ppm

well resolved), 4.90 (l H, d, 4Hz), 5. ^ 5 (1 H, d, 4 Hz), 6.63 (1 H, d, 9Hz), 7: i5 (^ H, s), 7.20 ( IH ₁ d, Hz).

Analysis for C ₂₁ H ₃₁ N ₁ -O ₅ S ₂ . 1 1/2 H ₃ O ι

calculated: C, 49.02; H, 4.71; N, 13.61; S, 12.46.

found: C, 48.66; H, 4.52; N, 13.04; S, 12.43.

Example 22

7- (o-tert-butoxycarbonylaminomethylphenylacetamido-S- (3-hydroxy-,

pyridazin-6-y11hiomethy1) -3-cephem-4-carboxylic acid.

To a solution of 1.0 g (3.7 mmol) of o-tert-butoxycarbonylaminomethylphenylacetic acid and 0.69 g (3.7 mmol) in 8 ml of dry ethyl acetate are in one batch 0.78 g of Ν, Ν'-di-

2,4-dinitrophenol

409816 / 1U7

M / 128O1-79-2344Q95

cyclohexylcarbodiimide is added and the mixture is stirred for one hour at room temperature. Dicyclohexylurea is separated off and filtered off, the filtrate being evaporated under reduced pressure and giving the activated ester as a yellow oil j. The active ester is dissolved in 30 ml of tetrahydrofuran ^! and chilled at 0 ° C. The solution is a mixture in one batch! mixture of 7-amino-3- (3-hydroxypyridazin-6-ylthiomethyl) -3-

i ·

^! cephem-4-carboxylic acid (1.02 g, 3 mmol), 0.85 ml of triethylamine, 30 ml of tetrahydrofuran and 10 ml of water were added. The reaction mixture is stirred at room temperature for 16 hours and treated with activated charcoal. The filtrate is diluted with 50 ml of water and washed twice with 100 ml of ether. The aqueous solution is covered with 100 ml of ethyl acetate, with dilute chlorine! ; hydrochloric acid while stirring vigorously at 5-10 ° C

acidified pH 2. The aqueous layer is combined twice with 100 ml of ethyl acetate on anhydrous sodium sulfate I dried, treated with activated charcoal and filtered. The Ver- ; Evaporation of the solvent gives 7- (o-tert-butoxycarbonyl- : aminomethylphenylacetamido) -3- (3-hydroxypyridazin-6-ylthio- : methyl) -3-cephem-4-carboxylic acid as a pale yellow oily solid which is triturated with 100 ml of ether. The BOC-protected Product is collected by filtration, washed with 30 ml of ether and dried in vacuo. It weighs 0.95 g (54%).

7- (o-aminomethylphenylacetamido) -3- (3-hydroxypyridazin-6-ylthio-

methyl) -S-cephem ^ -carboxylic acid

A cold mixture of 0.95 g (1.6 mmol) of BOC-protected cephalosporin , 7- (o-tert-butoxycarbonylaminomethylphenylacetamido) ■ ! 3- (S-hydroxypyridazin-ö-ylthiomethyl) -S-cephem ^ -carboxylic acid,

4098 1 67 1 1-4 7

m / 12801 - 80 - 2344U95

and 6 ml of trifluoroacetic acid for one hour at 0-5 ° C touched. Ether (100 ml) is added to the reaction mixture and the resulting white precipitate is filtered off collected and washed with 100 ml of ether. The suspension of the precipitate in 10 ml of water is diluted with ammonium hydroxide adjusted to a pH of 5. The desired product, 7- (o-aminomethylphenylacetamido) τ3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid, is filtered, washed with 50 ml of acetonitrile and in vacuo over phosphorus pentoxide dried. Yield 0.50 g (61%),

Example 23

7- (o-t-butoxycarbonylaminomethylphenylacetamido) -3- (3-hydroxy-

pyridazin- »6-ylthiomethyl) -3-cephem-4-carbanic acid

To a cooled (-3 to ⁰ ° C) and stirred solution of 93 g of 2,4-dinitrophenyl-ot-butoxycarbonylaminomethylphenylacetat in 600 ml of tetrahydrofuran in a batch, a cooled solution of 62 g (0.18 mol) of 7-amino 3 ~ (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid and 46 g (0.46 mol) of triethylamine in 250 ml of water were added. The reaction mixture is stirred overnight and the temperature is allowed to rise to room temperature. The mixture is treated with 10 g of activated charcoal and filtered. The filtrate is concentrated to about 300 ml under reduced pressure at 40 ° C. The concentrate is washed with five 200 ml parts of ether and treated again with approx. 20 g of activated charcoal. The piltrate is covered with a liter of ether and acidified with dilute hydrochloric acid to a pH of 3.5 while stirring vigorously. Crude 7- (ot-butoxycarbonylaminomethylphenylacetamido) -3- (S-hydroxypyridazin-e-ylthiomethyl) -3-cephem-4-carboxylic acid is obtained, which is collected by filtration with 2 liters of ether and then with 2 liters of water and 0.5 Liters of ether is washed.

409816/1147

The raw product is divided into two parts. The first part
(50 g) is used for the next stage without further "purification".

The second part (40 g) is dissolved in 1 liter of methanol to obtain the

j to remove dark "insoluble material (3.9 g) and the piltrate,

! is concentrated to approximately 50 ml. 300ml of the concentrate:

Acetonitrile is added and 17.4 g of a pale gel are obtained

; ben precipitate, which is collected by filtration and with 100 ml:

Acetonitrile is washed. The concentration of the mother liquor j

gives an additional 12.5 g of product. :

B. - ^l

7- (o-aminomethylphenylacetamido) -3- (3-hydroxypyridazin-6-ylthio- '.

methyl) -3 ~ cephem ~ 4-carboxylic acid !

! (A) 50 g of the crude 7- (o-tert-butoxycarbonylaminomethylphenyl- i

acetamido) -3- (S-hydroxypyridazin-δ-ylthiomethyl) -3-cephem-4- j

: carboxylic acid are dissolved in 100 ml of trifluoroacetic acid with cooling j

] solved. The solution is stirred for 1.5 hours at 0-5 ° C. j

ί By adding a liter of ether to the solution, trifluorine is obtained;

acetate of the desired cephalosporin, which is obtained by filtering ί

ί is collected and washed with 300 ml of ether. The trifluoro i

; acetate is dissolved in 100 ml of water and the solution is with. [

j 10% ammonium hydroxide adjusted to a pH value of 4 j

ί and gives the colored product (26.6 g), which with 300 ml of water, j j 300 ml of acetonitrile washed successively and gej in vacuo is drying. The crude product is added in 50 ml of formamide

Room temperature dissolved and the solution are added dropwise
150 ml of water added. A small amount of colored ones is obtained
Precipitation 'which is filtered off. The filtrate is 1.5
Liters of acetonitrile are added and 7- (o-aminomethylphenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid is obtained as a pale yellow powder. It weighs
9.8 g. -

4 Ö 9 8 1 6/1 U 7

ίκ / ΐίβοί -82- 23UÜ95

ι (b) The same method as described above is used : two fractions (17.4 g and 12.5 g) of the reprecipitated

ι; 7- (o-tert-Butoxycarbony-laminomethylphenylacetamido) -3- (3- j

; hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid at j

j and receives 12.3 g of the desired product 7- (o-aminomethyl- j

phenylacetamido) -3- (S-hydroxypyridazin-e-ylthiomethyl) -3-cephein- ι

; 4-carboxylic acid. · I

The two products of (a) and (b) described above; show almost the same IR and MIC spectra. you will be ; combined and analyzed. Melting point 208 C (decomp.). j

'Analysis for ^C 21 ^H 21 ^N 5 ° 5 ^S 2 * ^{2 1 // 2 H} 2 ° ^: ''

. calculated: C, 47.36; H, 4.92; N, 13.15; S, 04/12!

j found: C, 47.30; .47 * 56; H, '4.37; 4.50; N, 13.67, 13.71: 'S, 12:38.

Example 24,:

7- (p-aminomethylphenylacetamido) -3- (3-hydroxypyridazin-6-ylthio- ■. Methyl -3-cephem-4-carboxylic acid . Ι

; To a stirred suspension of 0.63 g (2.1 mmoles) of sodium-o- ^! (l-ethoxycarbonyl-l-propen-2-ylaminomethyl) phenyl acetate in ^; 5 ml of dried acetonitrile with a drop of Ν, Ν'-dimethyl- 'benzylamine are added to 0.25 g (2.3 mmol) of ethyl chloroformate! Stirring at -15 ° C added and stirring is continued for 20 minutes at -10 to -15 ° C. A solution of 0.71 g (2.1 mmol) of 7-amino-3- (3-hydroxy-1 pyridazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid and 0, 21 g (2.1 mmol) of triethylamine in 3 ml of acetonitrile and 3 ml of water are added and the mixture is ^{stirred at 0 °} C. for one hour.

409816/1. H?

OWQfNAL FNSPECTfD

j M / 12801-83-23 <U035

<The reaction mixture is mixed with a small amount of activated charcoal

! treated and filtered. "0.5 ml of ants are added to the filtrate

'acid added with shaking and the mixture is filtered /

'' A small amount of unreacted 7-amino-3- (3-hydroxy-

■ to remove pyridazin-6-ylthiomethyl) -3 ~ cephem-4-carboxylic acid.

■ 100 ml of acetonitrile are added to the filtrate and the mixture

is left to stand for one hour at room temperature. Of the

ι Precipitate is collected by filtration with 10 ml of water

i washed and dried in vacuo. 0.49 g (48%) are obtained

7- (o-aminomethylphenylacetamido) -3- (S-hydroxypyridazin-e-yl- ; thiomethyl) '- 3 »-cephem-4-carboxylic acid. Melting point 200-210 ° C ! (Decomposition)! . ,

! Example 25

I T-o-aminomethylphenylacetamido-S- (3-hydroxy ~ pyridazin-6-ylthio-

j I

j methyl) -S-cephem ^ -carboxylic acid _,

^! In a 1 liter three-necked round bottom flask fitted with a drying tube,

_■: overhead stirrer and thermometer is equipped, 24.0 g ι (0.08 mol) of powdered potassium well o- ~ / I-carbomethoxypropen-: 2-ylaminomethyl7phenylacetat in 350 ml of dry tetrahydrofuran \ (THF), and 4 drops Ν, Ν-Dimethylbenzylamine introduced. The I _: suspension is stirred vigorously and cooled to -35 to -40 ° with an acetone-dry ice bath. The suspension is 13.6 g

(0.06 mol) isobutyl chloroformate added all at once while the temperature is kept below -35 °. After 2.5 minutes, the acetone-dry ice bath is removed and a solution of 0.036 mol 7-Amino-3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4carbon- ' : acid and 5.04 g (0.050 mol) of N-methylmorpholine in 250 ml of water

! · Is added. The reaction mixture is stirred until it reaches room temperature (1-2 hours). Tetrahydrofuran is removed under reduced pressure. The aqueous solution is covered with a layer of 100 ml of ethyl acetate and treated with conc. HCl acidified to pH 1.7-2.0. The mixture is mixed with 3 _Λ 5 g of activated charcoal

4,098 16/1147

>("DarcoKB") treated and filtered. The layers are separated from-I and the aqueous phase is extracted with 2 × 100 ml parts of ethyl acetate. The aqueous phase is then filtered through filter paper and the pH is adjusted to 4.0. After scratching, the solution is left to crystallize overnight at 5-7 °. The product is collected by filtration, washed with a small amount of water and _{dried in a vacuum desiccator over P 3} Oc. The yield of 7-o-amino -: methylphenylacetamido-3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4carboxylic acid is approximately 30%.

! Example 26

■ A suspension of the zwitterionic form of 7- * (ö-aminome thy! Phenyl ¹ acetamido) -3- (3-hydroxy ~ pyridazin-6-ylthiomethyl) -3-cephem-4- 'carboxylic acid (0.361 g) in * 3 « ml of methanol is cooled in ice and a few drops of conc. Treated hydrochloric acid until a clear solution is obtained. 7- (o-Aminomethylphenylacetamido) - j 3-O-hydroxy-pyridazin-ö-ylthiomethyl) -S-cephem ^ -parboxylic acid- | hydrochloride falls as a pale brown colored solid; after the addition of ether and is collected by filtration / and _{dried in vacuo over P 2} O ς.

Example 27

; To a stirred suspension of the zwitterion form of 7- (o-

Aminomethylphenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) '■ 3-cephen \ -4-carboxylic acid (0.361 g) becomes 1 η aqueous sodium hydroxide

1 added at room temperature until a clear solution (pH 10; 8) er-I hold is. This solution is immediately freeze-dried and gives I impure solid sodium 7- (o-aminomethylphenylacetamido) -3- (3-hydroxy-pyridazin-6-ylthiomethyl) -S-cephem ^ -carboxylate,

409816/1 U7

M / 128ol

Manufacture of bimethanesulfonate

Example 28

V /

-CH ₂ -C-NH-CH

CH -CH,

II '

N.. C-C

COOH

HIGH ₂ SO ₃ Na - + "CH ₅ (CH ₂ ) ^ CH-COONa

. (30% sodium 2-ethylhexanoate in acetone)

τ in "heat water-

₂ treat with activated charcoal and

filter
-z the filtrate to anhydrous

Add alcohol

solid

CH ₂ N (CH ₂ SO ₃ Na) ₂

Il

CH ₂ -C -NH-CH

COONa

H ₂ O

EHA.

4 0 9 8 1 6/1 U

ι procedure

2.0 moles (approximately 980 g ^- on an anhydrous basis) 7- (o-amino- | methylphenylacetamido-3- (3-hydroxypyridazin-6-ylthiomethyl) - j

3-cephem-4-carboxylic acid, 540 g sodium formaldehyde bisulfite j

(4.03 mol), 3000 ml of water and 2700 ml (4.87 mol) of 30% ^:

Sodium 2-ethylhexanoate) in acetone are in a suitable i

Into the vessel and the mixture is heated to 40-4 5 ° C. while stirring _:

heated. The mixture dissolves in about 10 minutes to a \ yellow solution. . '

After 15 minutes of heating, 50 g of decolorizing activated charcoal are added
("Darco KB") is added to the solution and the mixture is stirred at 40-45 ° C. for a further 15 minutes.

After heating the reaction mixture at 40-45 ° C during
a total of 30 minutes is provided by diatomaceous earth ("Dicalite")
filtered. ^:

The activated charcoal cake is washed with 2000 ml of 50% ethanol-water. The filtrates are combined, adjusted to 25 ° C \ and the solution is rapidly stirred at 25 ° C to 112 liters
100% ethyl alcohol added. It forms a fine \ white amorphous precipitate of the di (Natriummethansulfonats) j of sodium 7- (o-aminomethylphenylacetamido-3- (3-hydroxy-pyridazin-6-ylthiomethyl i) -S-cephem ^ -carboxylate i.

The suspension is stirred for about 10 minutes and then ' filtered and the cake with 15 liters of 100% ethyl alcohol j washed.

The cake is cooked in an oven with air circulation at 50-55 ° C
dried for about two hours and then im
Vacuum dried at 4-6 mm for 24 hours.

409816/1147

I. ■ ■ ι

I The yield is approximately 1200-1400 g of amorphous, white, j solid di (sodium methanesulfonate) of sodium 7- (o-aminomethy1- ; phenylacetamido-3-O-hydroxypyridazin-g-ylthiomethyl) -3cephemi i

'4-carboxylate, The product usually contains several percent j

Water and possibly a trace of ethanol. '■

'■ This product is also sodium 7- ^ oN, N - bis (natriumsulfomethyl) -; aminomethylphenylacetamido7-3- (S-hydroxy-pyridazin-e-ylthiome-; ^1- thyl) -3-cephem-4-carboxylate. ^;

! Example 29 !

The following slurry is prepared ^N

2.19 g sodium formaldehyde bisulfite (2 equivalents), I.

3.5 g of 7- (o-Aminomethylphenylacetamido) -3- ^{(3-hydroxy-pyridazine-1} 6-ylthiomethyl) _{-3-cephem-4-carbonsäurezwitterion} (100-200 mesh), ^; 25 ml water (the volume can be changed),; 14 ml of 30% sodium 2-ethylhexanoate isopropanol.

; Almost a solution is obtained in about 0.5 hours ι stirring at 24 ° C. The temperature of the mixture quickly increases to j > o

40-43 C raised. This is held for about two minutes and then quickly cooled to 20-23 ° C. ;

^! The solution is filtered to remove a small amount of insoluble I j substances (the total time in solution should be two hours

not to exceed). · |

ι i

ι i

The solution with a pH of 7.3 is for a time j ; span from 5 minutes to 600 ml of very quickly stirred, absolute Ethanol added (other alcohols such as anhydrous isopropanol can also be used). A j is formed amorphous precipitate of the di (sodium methanesulfonate) of sodium 7- (o-raminomethylphenylacetamido) -3- (3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-4-carboxylate. The mixture is five minutes

409816/1 U7

j long stirred. The precipitate is collected by filtration /

i washed with 60 ml of ethanol (or isopropanol) and stored at 50 ° C

; Vacuum dried for 24 hours. The yield is approx

'G 4.3.

[ The product is at least 200 mg / ml soluble in water at a pH of about 7. Such a solution is at least

: stable for at least two hours at room temperature; even more diluted solutions are stable even longer. The product shows the same antibacterial spectrum as the mother zwitterion and is completely biologically active whether or not it is hydrolyzed back to the zwitterion.

Example 30

ι Sodium-7- ^ ö-N, N-bis (natriumsulfomethyl) aminomethylphenyl-

I acetamido7-3- (S-hydroxy-pyridazin-e-ylthiomethyl) -3-cephem-

! 4-carboxylate. Manufactured using hydroxymethane

! sulfonate ; ; * ■

ι * ■

> A mixture of 1 g (2.05 mmol) 7- (o-aminomethylphenylacetamido) -j 3-O-hydroxypyridaziri-e-ylthiomethyl) -3-cephem-4-carboxylic acid- 'zwitterion, 1.56 g (10 mmol ) Sodium hydroxymethanesulfonate mono-! ^: hydrate, 6 nil (6 mmol) 1 M sodium 2-ethylhexanoate solution in ethyl acetate, 10 ml isopropanol and 10 ml water is at room temperature; stirred for 3.5 hours. The resulting solution is with; Treated 1 g of activated charcoal and poured with stirring into 300 ml of absolute ethanol and the mixture is stirred at room temperature for 30 minutes and gives the crystalline product, which is collected by filtration, washed with three 50 ml parts of absolute ethanol and over P ₂ ⁰ B ^ ^e "^ 45-52 ° / l mm is dried for 20 hours. 1.64 g of sodium 7- / ö-N, N-bis (sodium sulfomethyl) aminomethylphenylacetamideg7-3- (3- hydroxy-pyridazin-6-ylthiom.ethyl) -3-cephem-4-carboxylate, which is easily soluble in water (^ 1 g / ml), melting point ^ 270 °.

409816 / 1H7

" ¹

^IR; / Ξ? 5 ¹ ^ oO, Ιβ8θ-ΐβ2θ, 1580, 1400, 1200, 1040 cm " ¹ .

UV: ^ ^H 2 °. 249 nm (ε 14,9OO), 272 ran (ε 10,900, sh),

XiliiX

XiliiX.

I (£ 2,100, sh).

NMR: ^ D ₂ O 3.30-4.0 (βΗ, τη, S-CH ₂ , 0-CH ₂ -CO and 0-CH ₂ -N or

jS), 4.04 (2H, s, N-CH ^ -0 or 3-CH.-S), 4.30 (4H, s, N-CiU- ■ SO ₃ Na), 4.89 (IH, d, 4 Hz, 6 -H), 5.40 (IH, d, 4 Hz, 7 "H), 6.78 (IH, d, 9.5 Hz, pyridazine-H), 7.1-7.4 (4H, phenyl-H), 7.32 (IH, d, 9.5 Hz, pyridazine-H). ...

Example 31

: Sodium 7- ^ ö-N, N-bis (natriumsulfQmethyl) aminomethylphenyljacetamidg73- (S-hydroxy-pyridazin-e-ylthiomethyl-S-cephem ^ - carboxylate. Manufactured using formalin and sodium bisulfite . \

: (a) To a solution of 1 ml (10 mmol) of 30% formalin and ¹ 1 g of sodium bisulfite in 10 ml of water, 0.975 g; (2 mmol) 7- (o-aminomethylphenylacetamido) -3- (3-hydroxypyridazin- ^; 6-ylthiomethyl) -3-cephem-4-carboxylic acid zwitterion, 6 ml 1 M Na-1 trium-2-ethylhexanoate solution and 10 ml isopropanol added. I The mixture is stirred for 2.5 hours at room temperature • and poured into 30 ml of ethanol. The resulting sodium 7- '■■ ^ oN, N-bis (sodium sulfomethyl) aminomethylphenylacetamido7-3-. (3-hydroxypyriaazin-6-ylthiomethyl-3-cephem-4-carboxylate is: collected by filtration, washed with three 50 ml portions of ethanol and dried in vacuo. Yield 1.60 g.

AO 9 8 16/1 14.7

M / I28ol

(b) To a mixture of o; 975 g (2 mmol) 7- (o-aminomethyl-

approx. ^. 'ra - 4: -zaz ~ zG-s'i ~ zaz- »z. ± -zer ± zr. _r 6 -1 1 M} 7acriu ^: - 2-ethylhexanoate in ethyl acetate, 10 ml of isopropanol and 10 ml of water, 1 ml (10 mmol) of 30% formalin is added. The mixture is stirred for two hours at room temperature and gives a clear solution with a small amount of oily precipitate. After 1 g of sodium bisulfite has been added, the solution is stirred for a further two hours, during which time the oily precipitate is dissolved in the solution. The reaction mixture is poured into 300 ml of ethanol with vigorous stirring and gives 1.84 g of sodium 7- / ö-N, N-bis (sodium sulfomethyl) aminomethylphenylacetamido7-3- (^ -hydroxy-pyridazin-g-ylthiomethyl) -3- cepher: -4-carboxylate, which by. Filtration collected, washed with three 50 ml portions of ethanol and dried in vacuo. . ,.

SAD ORIGINAL

409816/1147

Claims (28)

M / 128ol Claims Process for the preparation of compounds of the formula CH ₂ NH ₂ wherein X is H or OH and m is zero or one, with the proviso that when X is OH, m is zero, and the non-toxic, pharmaceutically acceptable salts and slightly hydrolyzed esters thereof, characterized in that a compound of the formula H ₂ N - CH I
C CHr C - CH ₂ - S COOH II 409816ΛT U7 M / 128O1-92-23440 or a salt or slightly hydrolyzed ester thereof with an acylation derivative of an acid of the formula CH ₂ NHB X - / V- (S) _1n - ^CH 2 - ^C00H III wherein B is an amino protecting group and m and X have the meaning given above, to a compound of the formula CH ₂ NHB O / S Il ^x -CH ₂ -C-NH-CH CH CH _{2 N} -N ^ OH IV ^! COOH wherein B / X and m have the abovementioned meaning, or one Salt or a slightly hydrolyzed ester thereof reacted and then the amino protecting group B is removed to give the desired compound of formula I or a nontoxic, pharmaceutically acceptable salt or easily to produce hydrolyzed esters thereof ". 2. The method according to claim 1, characterized in that the acylating agent of the formula III in the form of the acid halide, S-acid anhydride or activated ester is present. 409816/1147 M / 12801 3. The method according to claim 1 or 2, characterized in that the amino protective group B is t-butoxycarbonyl , 1-carboethoxy-1-propenyl-2 or 1-carbomethoxy-1-propenyl-2. 4. The method according to any one of claims 1-3 ', characterized in that the amino protective group is removed by acid hydrolysis. 5. The method according to claim 1, characterized in, that the acylating agent 9 \ CH ₂ NH2 · HCl - CH ₂ - - Cl where X has the meaning OH or H and m zero or one is, with the proviso that when X is OH, m is zero, then » 6. The method according to any one of claims 1-5, characterized in that that the dimethanesulfonate sodium salt of the formula CH ₂ N (CH ₂ SO ₃ Na) ₂ 0 ii (S) ■ - CH ₂ - C - NH 16/114? where X is H or OH and m is zero or one is, with the proviso that when X is OH, m is zero, is prepared by removing the zwitterion the formula (S) _n , - CH ₂ - C - NH wherein X and m are as defined above, with water, sodium formaldehyde bisulfite or a source thereof and with a strong sodium base. 7. The method according to claim 6, characterized in that the strong sodium base is sodium 2-ethylhexanoate. 8. Process for the preparation of the compound of formula H ₂ N-CH CH CH ₂ NN Js N.. C-CH-S- / COOH or a salt or slightly hydrolyzed ester thereof, characterized in that 7-aminocephalosporanic acid or a salt thereof with a thiol of the formula 0 9 8 1 6/1 U 7 M / 128O1 23U095 or a salt thereof and optionally the one thus prepared 7-AmInO-S- (6-hydroxypyridazin ~ 3-ylthiomethyl) -3-cephem-4-carboxylic acid to a salt or a readily hydrolyzed ester thereof by methods known per se. 9. Compounds of the formula Z - COOH wherein Z is H or CH ₂ NH ₂ Il - c Wherein X is H or OH and m is zero or one, with the proviso that when X is OH, m is zero, has, and non-toxic, pharmaceutical 4 09816/1147 M / 12801 - 96 - 23U095 ί compatible salts and readily hydrolyzed esters thereof. 10. Compounds of the formula
CH ₂ NH ₂ wherein X is H or OH and m is zero or one, with the proviso that when X is OH, m is zero, and non-toxic, pharmaceutically acceptable salts and slightly hydrolyzed esters thereof. 11. Compound of Formula CH ₂ -C -NH-CH CH C COOH- and non-toxic, pharmaceutically acceptable salts thereof. 12. The zwitterionic form of the compound according to claim 11. 13. The sodium or potassium salts of the compound according to claim 11. · AQ9816 / 1 147 M / 128O1 14. The hydrochloride of the compound according to claim 11. 15. Compound of formula CH ₂ N (CK ₂ SO ₃ Na) ₂ CH ₂ -C-NH-CH- -CH CH ₂ > - - S. COQNa 16. Compound of Formula ! I CH ₂ -C-NH-CH- -CH CH, Ν — Ν - S. COOH and non-toxic, pharmaceutically acceptable salts thereof. 17. The zwitterionic form of the compound according to claim 16. 18. The sodium or potassium salts of the compound according to claim 16. ■. 409816/1 U? M / 128ol 19. The hydrochloride of the compound according to claim 16. 20. Compound of Formula Il CH-C-NH-CH- ² I. CH- 21. Compound of the formula CH ₂ COONa - S. S-CH ₂ -C-NH-CH- • CH CH ₂ 0_ _N C-CH ₂ - S. COOH and non-toxic, pharmaceutically acceptable. salts thereof. 22. The zwitterionic form of the compound according to claim 21. 23. The sodium or potassium salts of the compound according to claim 21. 40981 6/1147 M / 128ol 24. The hydrochloride of the compound according to claim 21. 25. Compound of Formula H ₂ N-CH CH K-K - CH ^ - S COOH OH 26. Compound of Formula 0 ti S-CHo-C-NH-CH I and non-toxic, pharmaceutically acceptable salts and easily hydrolyzed esters thereof. 27. The sodium or potassium salts of the compound according to claim 26. 28. A pharmaceutical agent which comprises a compound, a salt or an ester according to any one of claims 10-25 in association with at least one pharmaceutical carrier or binding agent. th.- 409816/1147