FI60869B - FRAMEWORK FOR THERAPEUTIC TREATMENT OF THERAPEUTIC EQUIPMENT - Google Patents

Description

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Wfc [B] <”> UTLAOGNINeSSIllUFT 60869 C (45) Patent granted 13 04 1002 latent oeddelat ^ ** ^ (51) Kv.lk?/lnt.CI.3 C 07 D 501/36 ENGLISH | _F IN LAN D ( 21) P »t * nttlh» k «mu« - P »tentM * öknlni 772860 (22) Hakemitpllvi - Ameknlnpdaf 28.09-77 ^ ^ (23) Alkupllvi — Glltlf hetsdag 28.09.77 (41) Tullut | ulklMk« l - Bllvlt offantlig 06.10.78

Patent and Registration Office .... ...._______. . .....

_ '(44) Date of publication and month of publication. -

Patent- och registerstyrelsen 'Anaökan utlagd och utl. «Krlftan publkarad 31.12.8l (32) (33) (31) Pyydetty etuoikeus - Bagird prloritet 05.0l +. 77 USA (US) 781 + 885 (71) Bristol-ltyers Company, 3 ^ * 5 Park Avenue, New York, N.Y. 10022, USA (72) Takayuki Naito, Kawasaki, Jun Okumura, Yokohama, Hajime Kamachi, Chiba, Seiji Iimura, Tokyo, Hideaki Hoshi, Chiba, Masahisa Oka, Yokohama,

Japan-Japan (JP) (7I +) Oy Kolster Ab (5I +) Method for the preparation of new therapeutically useful cephalosporins - Förfarande för framställning av nya therapeutiskt använd-bara cephalosporiner

The invention relates to a process for the preparation of new therapeutically useful compounds of the formula c 1 H, R-NH-CH-CH CHO.

Il I 2 c —N ^> 6 — ch ε I | |

// (CH2) nCOOH I

For the preparation of cephalosporins according to COOH® and pharmaceutically acceptable salts thereof, wherein n is 1 or 2 and is / CH 2 NHR 1 CH NHR 1

/ ------ ^ ^ \ .CH2NHR '_ CH2NHR

R - (_Vch2 "is f j or (i LC" 2C0.

wherein R is hydrogen or hydroxy and R 1 is hydrogen or methyl.

2,60869 These new compounds are particularly useful in the treatment of bacterial infections.

The cephalosporins of formula I and their salts can be prepared by reacting a compound of formula NH 2 CH-CH 2 CH 2? n C - N / C-CH-O-C-CH, / \ C '2 3 0' »

COOH

or a salt thereof to react with a compound of formula

HS-L n. N-ich2) cooh tII

0 wherein n is 1 or 2, and treating the resulting compound with an acylating agent of the formula

R1 - X IV

wherein X is halogen or a functional equivalent thereof and R is as defined above, and if desired, the resulting acid of formula I is converted into a pharmaceutically acceptable salt.

Cephalosporins are well known semisynthetic antibacterial agents which are initially prepared, for example, by acylation of the 7-amino group of 7-aminocephalosporanic acid (7-ACA) and subsequent acylation of its derivative, such as by changing its substituent at the 3-position. Cephalosporins have been extensively described in the literature (e.g., Cephalosporins and Penicillins - Chemistry and Biology, edited by Edwin H. Flynn, Academic Press, New York, 1972, and in particular pages 554-569) and in the patent literature, for example, U.S. Patents 3,687,948; 3,741,965; 3,743,644; 3,759,904; 3,759,905; 3,766,175; 3,766,906; 3,769,281; 3,769,801; 3,799,923; 3,812,116; 3,813,388; 3,814,754 and 3,814,755.

Also known from the literature are 3-thiolated cephalosporins in which the 7-substituent is 3,60869 a) (X-amino- <X-phenylacetamido, US 3,641,021; US 3,734,907; US 3,687,948; US 3,741,965; US 3,757,015 US 3,743,644, JA 71/24400 (Farmdoc 46374S), BE 776 222 (Farmdoc 38983%; GB 1 328 340 for various substituents on the benzene ring), BE 772 592 (Farmdoc 19696T; US 3,687,948; 3,734 907 and 3,757,012), DE 2,202,274 (Farmdoc 50428T) corresponding to US 3,759,904, NL 7205644 (Farmdoc 76309T; US 3,757,014), and b) o-, m- or p-aminoethoxyphenylacetamide, e.g. NL 72 / 13968 (Farmdoc 24740U) corresponding to US 3,759,905 and c) o-aminomethylphenylacetamido e.g. e.g. US 3,766,176 and 3,766,175 (which also refer to the older patent literature relating to substituted 7-phenylacetamidocephalosporinic acids), (phenylacetimidoyl) aminoacetamido, e.g. US 3,692,779; and e) X-amino-X (1,4-cyclohexadienyl) acetamido, e.g. BE 776 222 (Farmdoc 38983%; GB 1 328 340).

Other similar reviews are found in U.S. Patents 3,692,779 (BE 771,189; Farmdoc 12819T), JA 72/05550 (Farmdoc 12921T), Japan 72/05551 (Farmdoc 12922T), U.S. 3,719,673 (BE 759,570;

Farmdoc 39819S), BE 7/93 311 (Farmdoc 39702U) and BE 793 191 (Farmdoc 39684U).

In addition, 3-thiolated cephalosporins in which the 7-substituent is 7-mandelamino (7-X-hydroxyphenylacetamido) are known, US 3,641,021, FR 74,10112, US 3,796,801, GB 1,328,340 (Farmdoc 38983T), US 3,701,775 , JA 48-44293 (Farmdoc 55334U) and Hoover et al., J. Med. Chem. 17 (1), 34-41 (1974) and Wick et al., Antimicrobial Ag.

Chemo., 1 (3), 221-234 (1972).

U.S. Pat. No. 3,819,623 (and also, for example, GB 1,295,841 and DE 1,953,861) deals in particular with the preparation of 2-mercapto-1,3,4-thiadiazole-5-acetic acid and its conversion into 7- (1H-tetrazole-1-acetic acid). -ylacetamido) -3- (5-carboxymethyl-1,3,4-thiadiazol-2-ylthiomethyl) -3-cephem-4-carboxylic acid, which is also discussed in DE-A-2 262 262.

U.S. Patent Nos. 3,766,175 and 3,898,217 disclose compounds of the formula 4,60869 CH_NH_ I Δ Δ M ° / s \ / Λ- CH0-C-NH-CH-CH CH2

\ // 2 'I I

\ - <0 = C- ^ C-CH2-S-R

COOH

where R is T - »-.« s j — s ΓΊ NXN ^ N - tal tj ^

B

and pharmaceutically acceptable salts thereof, and compounds of the formula

R ° S

(CH2) n CC-NH - ^ ^ ^ - ^ 1 or-N \ xJ-Ch2r1 CH9NH9 co2h wherein R is -H or lower alkyl R1 · is -H, (lower) alkanoyloxy, or the group -jr \ p - pii

^, "S- \ cP" CH3 or "S

S I

ch3 and n are an integer from 4 to 7, and pharmaceutically acceptable acid addition salts thereof.

U.S. Patent Nos. 3,883,520 and 3,931,160 and Farmdoc Abstract 22850W refer to 3-heterocycle-methylcephalosporins containing various substituents (including carboxyl) in various heterocycles, but these references are entirely general in nature and do not include physical constants, yields, , and do not even name any compound containing a carboxyl substituent.

U.S. Pat. No. 6,086,339 describes an older cephalosporin study.

U.S. Patent Nos. 3,907,786 and 3,946,000 disclose cephalosporins containing various fused ring bicyclic thiols.

The abbreviation 18830X for Farmdoc comprises a compound of formula R3

S

R1NH -4 - 'N N

i II "/ i-N CH2-S-li j

IS

I I

COOH '

(CH2) n-COOH

i 3 wherein R = acyl or H, R = H or methoxy and n = 1-9.

Compounds with a close chemical structure are further described in FI patent applications 2560/74, 752 434 and 761 035.

Suitable salts of the compounds of formula I include non-toxic carboxylic acid salts, including non-toxic metal salts such as sodium, potassium, calcium and aluminum, ammonium salt and substituted ammonium salts, e.g., non-toxic amines such as -benzyl-betaphenethylamine, 1-ephenamine, N, N1-dibenzylethylenediamine, dehydroabietylamine, N, N'-bis-dehydroabiethylethylenediamine, N- (lower) alkylpiperidine, e.g. N-ethylpiperidine, and other amines used, salts with benzylpenicillin; and their non-toxic acid addition salts (i.e., amine salts) including mineral acid addition salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, sulfamate and phosphate and organic acid addition salts such as melate, acetate, citrate, citrate, oxalate, oxalate , malate, mandelate, ascorbate and the like.

The starting materials used for the preparation of the cephalosporins of the formula I can be synthesized by known methods, for example as described in the above-mentioned patents and publications.

The antibacterial properties of the new compounds are shown in Tables 1-4.

6 60869

Nefromyrkyllisyys

A preliminary nephrotoxicity study was performed by administering the test compound to a group of two rabbits at 100 mg / kg intravenously. The results obtained with BB-S469 and BB-S479 indicated that they may have some nephrotoxic effect.

In vitro activity MICs were determined by the serial dilution method using Mueller-Hinton agar against 51 gram-positive and 96 gram-negative bacteria. 147 test organisms were classified into 16 groups based on species and types of anti-biotic resistance, 5 groups for gram-positive bacteria, and 11 groups for gram-negative bacteria. In vitro activity is expressed as the geometric mean MIC. BB-S472 and BB-S479 were overall more active than their non-N-methylated analogs, BB-S483 and BB-S469, respectively. BB-S479 was more preferred than BB-S472 for some gram-negative bacteria. Compared to cefamandole, BB-S479 was more active against most test organisms except Providencia species and Staphylococci.

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Table 2 In vitro antibacterial activity of BB-S493 compared to BB-S479 and cefamandole (determined by the Steer agar dilution method on a Mueller-Hinton Agar plate) MIC (rocg / ml)

Organisms BB-S493 BB-S479 Cefamandole S. aureus Smith A9537 0.4 0.4 0.1 S. aureus A9497 0.2 0.1 0.05 S. aureus BX-1633 A9606 0.4 0.4 0, 2

St. faecalis A9536 100 100 50 E. coli NIHJ 0.1 0.05 0.025 E. coli ATCC 8739 0.2 0.05 0.05 E. coli Juhl A15119 0.2 0.1 0.4 E. coli BX -1373 0.4 0.2 0.4 E. coli A15810 0.2 0.1 0.2 E. coli A9660 0.1 0.05 0.1 E. coli A15147 6.3 3.1 3.1

Kl. pneumoniae A9678 0.4 0.4 1.6

Kl. pneumoniae A9977 0.2 0.1 0.4

Kl. pneumoniae A15130 0.2 0.1 0.4

Kl. pneumoniae A9867 0.2 0.1 0.8

PR. vulgaris A9436 0.4 0.1 0.2

PR. vulgaris A9699 6.3 0.8 25

PR. mirabilis A9554 0.2 0.1 0.8

PR. mirabilis A9900 0.2 0.1 0.8

PR. morganii A9553> 100> 100> 100

PR. morganii A20031 0.4 0.1 0.8

PR. rettgeri A15167 0.1 0.1 0.1

Ps. Aeruginosa A9930> 100> 100> 100

Ps. Aeruginosa A9843> 100> 100> 100

SHIG. dysenteriae 0.05 0.025 0.2

SHIG. flexneri A9684 25 12.5 3.1

SHIG. sonnei A9516 0.05 0.025 0.05

Serr. marcescens A20019 100 25 50

Enterob. cloacae A9656 6.3 1.6 3.1

Sal. enteritidis A9531 0.1 0.05 0.1

Sal. typhosa A9498 0.1 0.05 0.1 B. anthracis A9504 0.0125 0.025 0.2 9 60869 co ro m tr vo oo m co σι 00 ininm ^ vo ^ in ^ On r- ~ * ·· * · » »>«. »» * CO ooooooooo

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u o cd | wi 10 60869 In vitro antibacterial activity of BB-S525 compared to BB-S 479 and MIC (mcg / ml) of cefamandole (determined by the Steer agar dilution method on a Mueller-Hinton agar plate)

Organisms BB-S525 BB-S479 Cefamandole S. aureus Smith 0.4 0.4 0.2 S. aureus 0.2 0.2 0.05 S. aureus BX-1633 0.8 0.8 0.2

St. Faecalis> 100> 100 100 E. coli NIHJ 0.2 0.1 0.1 E. coli ATCC 87-39 6.3 3.1 6.3 E. coli Celebration 0.4 0.2 0.4 E. coli BX-1373 0.4 0.2 0.2 E. coli 0.2 0.1 0.1 E. coli 0.2 0.05 0.05 E. coli 6.3 3.1 1, 6

Kl. pneumoniae 1.6 0.8 3.1

Kl. pneumoniae 0.2 0.1 0.2

Kl. pneumoniae 0.2 0.2 0.8

Kl. pneumoniae 0.2 0.2 0.8

PR. vulgaris 0.2 0.2 0.2

PR. vulgaris 3.1 0.8 50

PR. mirabilis 0.4 0.1 0.4

PR. mirabilis 0.2 0.1 0.2

PR. morganii> 100> 100 3.1

PR. morganii 0.2 0.2 0.8

PR. rettgeri 0.8 0.8 0.1

Ps aeruginosa> 100> 100> 100

Ps aeruginosa> 100> 100> 100

SHIG. dysenteriae 0.1 0.1 0.1

SHIG. flexner 12.5 12.5 3.1

SHIG. sonnei 0.1 0.1 0.2

Serr. marcescens 25 12.5 50

Enterob. cloacae 3.1 3.1 1.6

Sal. enteritidis 0.2 0.1 0.1

Sal. typhosa 0.2 0.1 0.1 B. anthracis 0.2 0.2 0.05 11 60869

The following examples illustrate the invention. All temperatures drink in degrees Celsius. 7-aminocephalosporanic acid is abbreviated to 7-ACA; -ACA- is a substance having the structure -NH-CH CH CH, i T 12 / -Vc ^ C-CH 2 -COOH 5 and thus 7-ACA may be labeled H-ACA-O-C-CH 2.

Methyl isobutyl ketone is designated MIBK. "Skellysolve B" is a Petro-leume ether fraction, b.p. 60-68 ° C, containing mainly n-hexane.

LA-1 resin is a mixture of secondary amines in which each amine has the formula R1 l 2

CH3 (CH2) 10CH2NHC - R2

wherein R, R and R are each a monovalent aliphatic hydrocarbon radical and wherein R, R and R together contain 11 to 14 carbon atoms. This particular mixture of secondary amines, referred to in these examples as "Liquid Amine Mixture No. II", is a clear amber liquid having the following physical properties: viscosity at 25 ° C 70 cpd., Specific gravity at 20 ° C 0.826; refractive index at 25 ° C 1.4554; distillation range at 10 mm, up to 170 ° C - 0.5%, 170-220 ° C. - 3%, 220-230 ° C. - 90% and above 230 ° C. - 6.5%.

lR-12Q is also called Amberlite IR-120 and is a strong ion exchange resin containing sulfonic acid radicals. Amberlite IR-120 is a commercially available cation exchange resin of the polystyrene sulfonic acid type; it is thus a sulfonated polystyrene resin crosslinked with divinylbenzene and obtained by the method described in Kun, Ion Exchange Resing, 2nd. edited (1958), John Wiley and Sons, Inc. See, for example, pages 84 and 87.

Amberlite IRC-50 is a commercially available cation exchange resin of the carboxyl type; it is a copolymer of methacrylic acid and divinylbenzene.

60869

Dicyclohexylcarbodiimide is truncated with DCC, tetrahydrofuran THF, thin layer chromatography TLC, p-toluenesulfonyl Ts and methanol MeOH.

For the following instrument values, infrared nu is written, ultraviolet lambda is written 7 \, molar absorbance epsilon (£), nuclear magnetic resonance (nmr) delta £>, and tau T (6 = 10-T).

Manufacture of starting materials

Example I

6-chloro-2,3-dihydro-s-triazolo [4,3-b] pyridazin-3-one [P. Francavilla and F. lauria, J. Het. Chem., 8, 415 (19717 (1.00 g, 5.9 mmol) in dry DMF (30 mL), sodium hydride (50% in paraffin, 0.3 g, 6.3 mmol) was added with stirring. Ethyl chloroacetate (1.4 mL, 13 mmol) was added to the mixture and the mixture was heated at 90 ° C for 8 hours with stirring.After cooling, the reaction mixture was poured into water (50 mL) and extracted with toluene (5 x 40 mL). The organic extracts were combined, dried over anhydrous sodium sulfate and evaporated under reduced pressure, and the residue was crystallized from benzene-n-hexane to give yellow needles, 6-chloro-2,3-dihydro-2-ethoxycarbonylmethyl-s-triazolo / 4,3- b7pyridazin-3-one (1.16 g, 77%), mp 114-115 ° C (m.p. 110 ° C).

lr: ν '“Js. 1735-1710 cm -1: max. 231 nm (? 26000> nmr. CDCl 3 = 53 J = 10 Hz, pyridazine-H), 6.98 ppm (1H, d, J = 10 Hz) , pyridazine-H), 4.80 (2H, s, -CH 2 CO), 4.27 (2H, q, J = 7.5 Hz, CH 2 CH 3), 1.29 (3H, t, J = 7.5 Hz , CH 2 CH 3).

Anal Calcd for C 9 H 9 N 2 O 3 Cl: C, 42.12; H, 3.53; N, 21.83; Cl, 13.81. Found: C, 41.54, 41.46; H, 3.22, 3.49; N, 21.51, 21.53; Cl, 13.88, 13.99.

Example II

To a solution of 6-chloro-2,3-dihydro-2-ethoxycarbonylmethyl-s-triazolo [4,3-b] pyridazin-3-one (2.30 g, 0.12 mol) in ethanol (900 mL) was added. NaSH.2H2O (70% pure, 45.9 g, 0.36 mol) and the mixture was refluxed for 0.5 h. Reaction mixture 13 60869 was evaporated under reduced pressure. The residue was dissolved in water (200 mL) and the pH of the solution was adjusted to 2 by the addition of concentrated HCl. The precipitate, 2-carboxymethyl-2,3-dihydro-6-mercapto-s-triazolo [4,3-b] pyridazin-3-one, was collected by filtration and washed with water. Yield 18.3 g. (69%).

ir: \ / KBr 2900, 2450, 1750, 1660 cm -1 max.

1% NaHCO 3, λ max: 260 nm 1950 °) δ 313 nm (ε / 7000) DMSO-d 6 nmr: δ 7.88 (1H, d, J = 10 Hz, pyridazine-H), 7.45 (1H, d, J = 10 Hz, pyridazine-H), 4.72 (2H, s, CH 2 CO).

Anal Calcd for C 18 H 18 N 2 O 2 S: C, 37.17; H, 2.67; N, 24.77; S, 14.17. Found: C, 37.35, 37.23; H, 2.26, 2.28; N, 23.58, 23.69; S, 14.32.

Example III

To a suspension of 7-aminocephalosporanic acid (8.79 g, 32.2 mmol) in 0.1 M phosphate buffer (pH 7, 149 mL) was added NaHCO 3 (8.14 g, 97.0 mmol) and the thiol from Example II (7 .30 g, 32.2 moles) with stirring. The mixture was heated at 80 ° C for 0.5 h at reflux. The mixture was treated with activated carbon and the pH was adjusted to 3 with concentrated HCl. The resulting precipitate was collected by filtration and washed with water to give 7.59 g (54%) of 7-amino-3- (2-carboxymethyl-2,3-dihydro-s-triazolo [4,3-b] pyridazin-3-one-6 -yylitiometyyli) -3-cephem-4-carboxylic acid.

ir:? KBr 1800, 1720, 1600, 1540, 1470 cm-1 max buffer uv:?) max (pH 7) 252 nm (ε, 19500), 298 nm (ε, 8400) - D 2 O + K 2 CO 3 nmr: £ 7.56 (1H, d, J = 9 Hz, pyridazine-H), 7.05 (1H, d, 2-H Hz, pyridazine-H), 5.45 (1H, d, J = 5 Hz), 6 -H), 5.05 (1H, d, 5 Hz, 7-H), 4.43 (1H, d, J = 14 Hz, 3-CH 2), 4.04 (1H, d, J = 14 Hz, 3 -CH 2), 3.88 (1H, d, J = 18 Hz, 2-H), 3.45 (1H, d, J = 18 Hz, 2-H).

Example IV

To a solution of 6-chloro-2,3-dihydro-s-triazolo [4,3-b] pyridazin-3-one [P. Francabilla and F. Lauria, J. Het. Chem. 8,415 (1971 £ 7 (17 g, 0.1 mol) in dry DMF (300 ml) was added 14,60869 potassium tert-butoxide (0.5 g, 4.5 mmol) with stirring. g, 0.12 mol) in dry DMF (10 ml) was added to the mixture, the mixture was stirred at 100-110 ° C for 24 hours, then poured into water (700 ml) and extracted with ethyl acetate (5 x 400 ml). dried over Na 2 SO 4 and evaporated, the residue was crystallized from ethyl acetate to give 6-chloro-2- (2-cyanoethyl) -2,3-dihydro-s-triazolo [4,3-b] pyridazin-3-one as pale yellow needles. (2.5 g, 11%).

Sp. 166-168 ° C.

ir: KBr 2230, 1720, 1550, 1500 cm -1 max.

uv:? dioxane 373 nm (ε f 2000) max.

nmr: 5DMSO_d6 3.03 (2H, δ, J = 6.0 Hz, CH 2), 4.21 (2H, t, ppm J = 6.0 Hz, CH 2), 7.23 (1H, d, J = 10.0 Hz, pyridazine-H), 7.93 (1H, d, J = 10.0 Hz, pyridazine-H)

Anal Calcd for C 9 H 11 N 2 OCl: C, 42.97; h, 2.70; N, 31.32; Cl, 15.86. Found: C, 42.73, 2.56; H, 2.57, 2.50; N, 31.36, 31.68; Cl, 15.96, 15.81.

Example V

A solution of 6-chloro-2- (2-cyanoethyl) -2,3-dihydro-s-triazolo [4,3-b] pyridazin-3-one (724 mg) in 6N-HCl (15 mL) was refluxed for 6 hours. The reaction mixture was extracted with ethyl acetate (10 x 20 mL). The combined extracts were washed with saturated aqueous sodium chloride (50 ml), dried over Na 2 SO 4 and evaporated to give a pale yellow solid 2- (2-carboxyethyl) -6-chloro-2,3-dihydro-s-triazolo / 4,3- fe7pyridazin-3-one (567 mg, 72%). Sp. > 170 ° C (Sublimation).

and: V "KBr 3400-2400, 1730, 1710, 1540 cm-1 max.

uv: λmax 377 nm (λmax) max.

D 2 O + NaHCO 3 nmr: δ 2.70 (2H, t, J = 7.0 Hz, CH 2), 4.24 ppm (2H, t, J = 7.0 Hz, CH 2), 7.17 (1H, d , J = 10.0 Hz, pyridazine-H), 7.70 (1H, d, J = 10.0 Hz, pyridazine-H).

15 60869

Anal Calcd for C 9 H 11 N 2 O 2 Cl: C, 39.60; H, 2.91; N, 23.09; Cl, 14.61. Found: C, 39.62, 39.48; H, 2.97, 2.67; N, 23.05, 22.70; Cl, 93, 14.12.

Example VI

A mixture of 2- (2-carboxyethyl) -6-chloro-2,3-dihydro-s-triazolo [4,3-h] pyridazin-3-one (567 mg, 2.34 mmol) and 70% sodium hydrosulfide dihydrate (924 mg, 7.02 mmol) in water (10 mL) was stirred at room temperature for two hours. The reaction mixture was adjusted to pH 1 with concentrated HCl, to pH 10 with NaOH, and then to pH 1 with concentrated HCl, respectively. The resulting precipitate of 2- (2-carboxyethyl) -2,3-dihydro-6-mercapto-s-triazolo [4,3-h] pyridazin-3-one was collected by filtration and washed with water. Yield: 418 mg (74%). Sp. 174-176 ° C.

/ KBr 1 is: V max 3600 "2600, 2440, 1730, 1720 (sh) cm -1 uv: 7) pH 7 Buffer 262 nm (£ 17,000), 318 nm (£ 600) max.

nmr: DMSO_d6 2.73 (2H, t, J = 7.0 Hz, CH2), 4.07 (2H, t, J = 7.0 Hz, CH2? m7.30 (1H, d, J = 10.0 Hz, pyridazine-H), 7.74 (1H, d, J = 10.0 Hz, pyridazine-H).

Anal Calcd for C 8 H 8 N 4 O 3 S: C, 40.00; H, 3.36; N, 23.32; S, 13.35. Found: C, 39.08, 39.06; H, 3.12, 3.20; N, 22.65, 22.70; S, 14.23, 14.29.

Example VII

A mixture of 7-ACA (405 mg, 1.49 mmol), thiol-2- (2-carboxyethyl) -2,3-dihydro-6-mercapto-s-triazolo [4,4,3-b] pyridazine -3-one (357 mg, 1.49 mmol) and NaHCO 3 (375 mg, 4.47 mol) in 0.1 M phosphate buffer (pH 7, 8 mL) were stirred at 80 ° C for 30 minutes. The reaction mixture was cooled and filtered to remove insolubles. The filtrate was adjusted to pH 1-2 with concentrated HCl. The obtained 7-amino-3 - [[2- (2-carboxyethyl) -2,3-dihydro-s-triazolo [4,3-b] pyridazin-3-one-6-yl] lithomethyl-117-3-cephem-4 The carboxylic acid precipitate was collected by filtration and washed with water. Yield: 519 mg (77%).

16 60869 KBr is: λmax 3600-2200, 1800, 1725, 1620, 1550, 1480 cm UV nmr: δ 2.20 (2H, t, J = 7.0 Hz, CH 2), 3.40 (1H, d, J = 17.5 Hz, 3.85 (1H, d, J = 17.5 Hz) , 2-H), 4.00-4.50 (4H, m, 3-CH 2 and N-CH 2 -), 5.01 (1H, d, J = 4.0 Hz, 6-H), 40 (1H, d, J = 4.0 Hz, 7-H), 6.94 (1H, d, J = 10.0 Hz, pyridazine-H), 7.44 {1H, d, J = 10, 0 Hz, pyridazine-H).

Anal Calcd for C 18 H 19 N 2 O 9 S 2 • 3H 2 O: C, 40.09, H, 3.99; N, 17.52; S, 13.37. Found: C, 40.06, 40.12; H, 3.33, 3.34; N, 16.96, 16.98; S, 13.87, 13.98.

7-ACA refers to 7-aminocephalosporanic acid and DMF to dimethylformamide.

Example 1 1- [O- (Aminomethyl) phenylacetamide] -3- (2-carboxymethyl-2,3-dihydro-s-triazolo [4,3-b] pyridazin-3-one-6-ylthiomethyl) -3-cephem- 4-carboxylic acid (BB-S469) CH2NH2, -CH.CCK.-,

\ ^ - CH 2 'S ^ / N \ Jj-CH 2 COOH

To a mixture of CO 2 H oa) o- (N-butoxycarbonylaminomethyl) -phenylacetic acid (13 g, 40 mmol) and 2,4-dinitrophenol (9.02 g, 49 mmol) in dry ethyl acetate (123 mL) was added dicyclohexylcarbodiimide (DCC). (10.1 g, 49 mmol) under cooling with water (5-15 ° C), and the mixture was stirred for 30 minutes at the same temperature and then for 40 minutes at room temperature. The resulting precipitate was filtered and the filtrate was evaporated to give 23.9 g of active 2,4-dinitrophenyl-o- (N-butoxycarbonylaminomethyl) phenylacetate, which was used in the next acylation reaction without further purification.

ir: VKBf 1775, 1700, 1600, 1530 cm-1 'max.' '60869 b) To a cold mixture (0 ° C) of the product obtained in Example III (4.38 g, 10 mmol), Et 2 O (4 , 5 mL, 30 mmol), CH 2 CN (20 mL) and water (20 mL), a solution of 2,4-dinitrophenyl-o- (N-butoxycarbonylaminomethyl) phenylacetate (4.79 g) in THF (tetrahydrofuran) was added. ) (20 ml). After the mixture was stirred at room temperature overnight, THF and CH 2 CN were removed from the reaction mixture under reduced pressure, and the pH of the remaining aqueous solution was adjusted to 2 with dilute HCl and extracted with ethyl acetate (10 x 30 mL). The organic extracts were dried over sodium sulfate and evaporated. The residue was chromatographed on a silica gel column (60 g) and eluted with CHCl 3 and 3% MeOH-CHCl 3, respectively, to give 2.40 g. (37%) 7- [3- (N-Butoxycarbonylaminomethyl) phenylacetamido] -3- (2-carboxymethyl-2,3-dihydro-s-triazolo [4,3-b] pyridazin-3-one-6-ylthiomethyl) - 3-cephem-4-carboxylic acid, m.p. > 161 ° C. (Dec.).

ir: Ϋ KBr 1780, 1720 cm -1 max.

uv: Λ PH 7 buffer 252 nm (£ 1, 19800) 298 nm (, 8900) max.

dmso + d20 nmr: / S 7.67 (1H, d, J = 9.0 Hz, pyridazine-H), ppm 7.10 (4H, s, phenyl-H), 7.05 (1H, d, J = 9.0 Hz, pyridazine-H), 5.66 (1H, d, J = 4.5 Hz, 7-H), 5-07 (1H, d, J = 4.5 Hz, 6-H) , 4.71 (2H, s, N-CH 2 -CO), 4.4-4.0 (4H, m, 3-CH 2 & CH 2 -N), 3.8-3.5 (4H, m, 2 -H & CH 2 CO), 1.42 (9H, s, t-butyl-H).

Anal Calcd for C 18 H 18 N 2 O 2 S 2 .2H 2 O: C, 49.62, H, 5.20; N, 14.46; S, 9.46. Found: C, 49.97, 49.95; H, 4.79, 4.62; N, 14.00, 13.84; S, 9.37, 9.32.

c) Trifluoroacetic acid (3.4 ml) was added to the compound obtained in b) (2.33 g) at 0 ° C and the mixture was stirred for 15 minutes at room temperature. Dry ether (100 mL) was added to the mixture and the precipitate was collected by filtration and washed with dry ether (2 x 50 mL). The solid was dissolved in a mixture of CH 2 Cl 2 (100 mL) and water (14 mL) and the pH of the solution was adjusted to 4-5 with concentrated NH 4 OH to give a precipitate which was collected by filtration and washed with CH 2 Cl 2 (2 x 50 mL). to give 1.75 g (82%) of 7- [7β- (aminomethyl) phenylacetamido] -3- (2-carboxymethyl-2,3-dihydro-s-triazolo [4,3-b] pyridazin-3-one-6-ylmethyl) ) -3-cephem-4-carboxylic acid (BB-S469) as the ammonium salt. Sp. > 160 ° C. (Dec.).

18 60869 and: Y KBr 1765, 1710, 1640, 1590cm-1 max.

uv: pH 7 Buffer 252 nm (£ 1, 23900) 298 nm (£, 10900) max.

Anal Calcd for C24H22N7O7S2.NH2O, H2 O: C, 46.51; H, 3.96; N, 18.08; S, 10.35. Found: C, 46.52, 46.26; H, 4.15, 4.19; N, 17.60, 17.48; S, 10.89, 1041.

d) To a suspension of the compound obtained in c) (1.58 g) in 50% acetone water (30 ml) was added a 10% solution to adjust the pH to 7.7. Acetone was added and the precipitate was collected by filtration and washed with acetone to give 1.38 g (87%) of the monosodium salt of BB-S469, m.p. Z170 ° C (dec.).

.KBr - ir: λ max 1765, 1710, 1640, 1600, 1540, 1485 cm -1 pH 7 buffer uv: λ max, 252 nm (ε, 20600), 298 nm (ε, 9200) max. s · d 20 + k_co, nmr: ε> 7.52 (1H, d, J = 9 Hz, pyridazine-H), ppm 7.27 (4H, s, phenyl-H), 7.03 (1H, d, J = 9 Hz, pyridazine-H), 5.62 (1H, d, J = 4.5 Hz, 7-H), 5.07 (1H, d, J = 4.5 Hz, 6-H)

Anal Calcd for C24H22N7O7S2Na.2H2O: C, 44.79; H, 4.07; N, 15.23; S, 9.96. Found: C, 44.44, 44.95; H, 3.68, 3.90; N, 16.50, 16.67; S, 10.38, 1045.

Example 2 7- (3-Methylaminomethyl-2-thienyl-acetamido) -3- (2-carboxymethyl-2,3-dihydro-s-triazolo [4,3-b] pyridazin-3-one-6-ylthiomethyl) - 3-cephem-4-carboxylic acid (BB-S472) --------- CH-NHCH,

! I Q

'y — CH „C0HH \ ^ 2 Ί ----- f

Qsy-2 “S ^ N / Nv ^ Jl-CH2COOH

CO 2 H oa) A mixture of (3-Nt-butoxycarbonyl-N-methylaminomethyl-2-thienyl) acetic acid (513 mg, 1.8 mmol), 2,4-dinitrophenol (400 mg, 2.16 mmol) and DCC (445 mg, 2.16 mmol) in THF (5 mL) was stirred at room temperature for 12 hours. The precipitated urea was removed and the filtrate was added to a mixture of 7-amino-3- (2-carboxymethyl-2,3-dihydro-s-triazolo [4,3-b] pyridazin-3-one-6-ylthiomethyl) -3- cefem-4-carboxylic acid (800 mg, 1.8 mmol) and triethylamine (0.76 mL, 5.4 mmol) in water (5 mL) at 0 ° C with stirring. Stirring was continued until the active ester had disappeared in tlc (silica gel plate; Rf 0.95; solvent system, CHCl 3: MeOH = 3: 1). The reaction mixture was diluted with water (20 mL), layered with AcOEt (50 mL), and the pH was adjusted to 2 with concentrated HCl at 5 ° C. The organic layer was separated and the aqueous layer was extracted with AcOEt (3 x 50 mL). The AcOEt extracts were combined, washed with saturated aqueous NaCl, dried over MgSO 4 and concentrated under reduced pressure. The residual oil (1.9 g) was chromatographed on silica gel (40 g). The column was eluted with CHCl 3 (400 mL), 3% MeOH-CHCl 3 (100 mL) and 10% MeOH-CHCl 3, respectively, while detecting by tlc (silica gel plate, solvent system MeOH: CHCl 3 = 1: 2, found J 2: with). A mixture of 2,4-DNP and the starting amino acid and 3% MeOH-CHCl eluate was recovered from the CHCl 3 eluate. Desired product, 7- (3-Nt-butoxycarbonyl-N-methylaminomethyl-2-thienylacetamido) -3- (2-carboxymethyl-2,3-dihydro-s-triazolo [7,3-b] pyridazin-3-one-6-ylthiomethyl) -3-cephem-4-carboxylic. (Rs 0.4, solvent system CHCl 3: MeOH = 3: 1) was obtained by evaporating the eluate with 10% MeOH-CHCl 3. Yield 490 mg (39%). Sp. 215-220 ° C.

KBr _1 is: λmax 3400 '1780' 1720 '1680' 1550 crtl pH 7 buffer uv: λmax 245 nm (c, 23000) 260 nm (ε, 18000) DMSO, 300 λmax 790 °) nmr: δ 6.42 (9H, s, BOC-H), 2.75 (3H, s, N-CH 3), ppm H pj 3.80 (4H, br-s, 2-H & S II CO ), 4.35 (H, br-s,

SJ

& 3-CH 2), 4.72 (2H, s, BOC-N-CH 2), 5.10 (1H, d, J = 4.5 Hz, 6-H), 5.70 (1H, dd, J = 4.5 & 10.5 Hz, converts to ublet J = 4.5 by addition of D 2 O, 7-H), 6.85 (1H, d, J = 4.5 Hz, thiophene H) 7.19 (1H, d, J = 9 Hz, pyridazine H), 7.34 (1H, d, J = 4.5 thiophene H), 7.72 (1H, d, J = 9 Hz, pyridazine-H), 9 , 11 (1H, d, J = 10.5 Hz, disappears with addition of D 2 O, NH).

20 60869

Anal. Calcd for C 28 H 18 N 2 O 2 S 2. H2O: C, 46.46; H, 4.60; N, 13.55; S, 13.29. Found: C, 46.67; H, 4.71; N, 12.79; S, 12.81.

b) Trifluoroacetic acid (0.4 ml) was added to the compound obtained in a) (400 mg, 0.57 mmol) at 0 ° C and the mixture was stirred at room temperature for 15 minutes. Anhydrous ether (10 mL) was added to the reaction mixture to separate the precipitate, which was collected by filtration, washed with anhydrous ether (2 x 10 mL) and suspended in acetonitrile (10 mL). The pH of the suspension was adjusted to 4 with concentrated NH 4 OH and stirred for 10 minutes. The solid was collected by filtration, washed with acetonitrile (2 x 5 mL) and dried at 60 ° C / 1 mm Hg for 7 hours to give 310 mg (90%) of 7- (3-methylaminomethyl-2-thienylacetamido) -3- ( 2-Carboxymethyl-2,3-dihydro-s-triazolo [4,3-b] pyridazin-3-one-6-ylthiomethyl) -3-cephem-4-carboxylic acid (BB-S472), at 188-191 ° C ( dec.).

/ KBr _1 is: V, 3400, 1770, 1720, 1680, 1550 cm-1. S · pH 7 buffer uv: / \. 245 nm (ε1, 22400), 26 nm (ε ', 18700), λmax · 300 nm (ε, 8600).

Anal Calcd for C 23 H 23 O 7 O 7 • 3.3H 2 O: C, 41.87; H, 4.43; N, 14.86; S, 14.58. Found: C, 42.03; H, 3.59; N, 14.79; S, 14.35.

c) To a suspension of the compound from b) (230 mg, 0.38 mmol) in 0.5 mL of deionized water was added N NaOH to adjust the pH to 8.9. Acetone (15 mL) was added to the solution. The precipitate was collected by filtration, washed with acetone (2 x 5 mL) and dried at 60 ° C / 1 mm Hg for 7 hours to give 170 mg (71%) of the monosodium salt of BB-S472, m.p. > 210 ° C (dec.).

KBir is:? , 7800).

nmr: δ 2.72 (3H, s, N-CH 2), 3.45 (1H, d, J = 18 Hz, 2-H), ppm 3 h 3.75 (1H, d, J = 18 Hz, 2-H), 3.95 (2H, s, S 2 -CH 2 CO 2), 4.18 (4H, m, N-CH 2 N & 3-CH 2), 4.57 (2H, s, N-CH 2) , 5.00 Γ

S

21 60 8 6 9 (1H, d, J = 4.5 Hz, 6-H), 5.53 (1H, d, J = 4.5 Hz, 7-H), 6.97 (1H, d, J = 9 Hz, pyridazine-H), 7.03 (1H, d, J = 4.5 Hz, thiophene-H p), 7.34 (1H, d, J = 4.5 Hz, thiophene-H oi) .), 7.48 (1H, d, j = 9 Hz, pyridazine-H).

Anal Calcd for C23H22N7O7S3Na * ^ / 2H2O: C, 43.40; H, 3.64; N, 15.40; S, 15.11. Found: C, 43.26; H, 4.08; N, 14.18; S, 13.91.

Example 3 7- (2-N-Methylaminomethyl-4-hydroxyphenylacetamido) -3- (2-carboxymethyl-2,3-dihydro-s-triazolo [4,3-b] pyridazin-3-one-6-ylthiomethyl) - 3-Cephem-4-carboxylic acid (BB-S478) HO _ 2NH-CH3 ^ J_CH2CC «h γ '* \

N 2 - CH 2 -S 2 N-CH 2 CO ° H

OT Π CO 2 H 0 a) Mixture of 2-Nt-butoxycarbonyl-N-methylaminomethyl-4-hydroxyphenylacetic acid, (708 mg, 2.4 mmol), 2,4-dinitrophenol (478 mg, 2.6 mmol) and DCC (536 mg, 2.6 mmol) in dry THF (20 mL) was stirred at room temperature for 2 h. The precipitated urea was removed by filtration. The filtrate was added to a solution of 7-amino-3- (2-carboxymethyl) -2,3-dihydro-s-triazolo [4,5-b] pyridazin-3-one-6-ylthiomethyl) -3-cephem-4 carboxylic acid (876 mg, 2 mmol) in 20 mL of water containing triethylamine (0.84 mL, 6 mmol) and the mixture was stirred at room temperature for 18 hours. After concentrating the aqueous solution to 20 mL, it was washed with ether, acidified with 6N HCl and extracted with 200 mL of ethyl acetate. The extract was filtered to remove insolubles, washed with water and saturated aqueous NaCl solution, and dried. The solution was evaporated to dryness and the oily residue was chromatographed on silica gel (Wago gel C-200, 25 g) eluting with chloroform and 3% chloroform-methanol. Fractions containing the desired product (detected by tlc;

Rf 0.3; solvent system CHCl 3: MeOH = 2: 1) was collected and evaporated to dryness, the oily residue was triturated with ether-n-hexane to give 630 mg (44%) of 7- / 2- (Nt-butoxycarbonyl-N-methylaminomethyl). ) -4-Hydroxyphenylacetamido [-3- (2-carboxymethyl-2,3-dihydro-s-triazolo [4,5-b] pyridazin-3-one-6-ylthiomethyl) -3-cephem-4-carboxylic acid, which melt at 200-2.10oc. at 22,608 6 9 (slowly dispersed).

KBr is: λmax 1780, 1720, 1660, 1400, 1240, 1150 cm-1 pH 7 buffer UV λmax 252 nm (ε, 13000), 300 nm (f, 5400) cDMSO-dfi nmr δ 1.39 (9H, s, C-CH 2,), 2.73 (3H, s, N-CH-) ppm 3.3-3.9 (4H, m, CH 2 CO & 2-H), 4 , 35 (4H, m, CH 2 N & 3-H), 4.48 (2H, s, NCH 2 CO), 5.03 (1H, d, 4.5 Hz, 6-H), 5.61 (1H, dd, Δ & 4.5 Hz, 7-H), 6.4-7.2 (3H, m, phenyl-H), 6.98 (1H, d, 10 Hz, pyridazine-H), 7.61 (1H, d, 10 Hz, pyridazine-H), 8.87 (1H, d, 8 Hz, NH).

Anal Calcd for H30H33N7®102.2: C, 0.34; H 4.65; N, 13.70; S, 8.96. Found C, 50.98; H, 5.36; N, 11.88; S, 7.60.

b) A mixture of the compound obtained in a) (570 mg, 0.7 mmol) and trifluoroacetic acid (1.5 ml) was stirred at 10 ° C for 30 minutes and the mixture was diluted with ether (50 ml) to separate trifluoroacetate of BB-S478. , which was collected by filtration, was then dissolved in a mixture of 10 ml of acetonitrile and 5 ml of water and then filtered. The filtrate was adjusted to pH 6 with concentrated ammonium hydroxide and then the solution was diluted with acetonitrile (100 mL). The resulting precipitate was collected by filtration, washed with cetonitrile and dried in vacuo over P 2 O 4 to give 370 mg (75%) of 7- (2-N-methylaminomethyl-4-hydroxyphenylacetamido) -3- (2-N-carboxymethyl-2,3- dihydro-s-triazolo [4,5-b] pyridazin-3-one-6-ylthiomethyl) -3-cephem-4-carbonic acid (BB-S478), melting at 215-220 ° C (dec.).

KBx is; Vmax 1770, 1710, 1600, 1380, 1350 cm -1 pH 7 buffer uv: 7), 252 nm (ε, 19000) 300 nm (ε, 9100) nidK S · d20 + k2co,

nmr: δ 2.75 (3H, s, N-CH 2), 2.9-3.3 (4H, m, CH 2 CO

ppm j * & 2-H), 4.0-4.3 (4H, m, CH 2 N & 3-H), 4.57 (2H, s, NCH 2 CO), 4.81 (1H, d, 4.5 Hz, 6-H), 5.53 (1H, d, 4.5 Hz, 7-H), 6.6-7.5 (5H, m, phenyl-H & pyridazine-H).

23 60869

Anal Calcd for C 25 H 25 N 7 O 8 S 2.5 / 2H 2 O: C, 45.45; H, 4.58; N, 14.84; S, 9.71. Found: C, 45.69; H, 4.21; N, 15.03; S, 9.46.

c) To a suspension of the compound obtained in b) (308 mg, 0.5 mmol) in water (2 ml) was added 0.3-0.4 ml of N NaOH and the mixture was stirred at room temperature; the pH of the resulting solution was 9.2. Acetone (20 mL) was added slowly to the solution. The resulting precipitate was collected by filtration, washed with acetone (10 mL) and dried in vacuo over P2 <0g to give 290 mg (91%) of the monosodium salt of BB-S478 melting at 230-235 ° C (dec.).

rKBr is: λ max 1770, 1700 '1600,1390, 1350 cm pH 7 buffer uv: λ max 250 nm (λ f 18000) 300 nm (ε, 8400)

Anal. H, 4.28; N, 14.36; S, 9.39. Found: C, 43.96; H, 4.14; N, 13.51; S, 9.34.

Example 4 7- [O- (Methylaminomethyl) phenylacetamide] -3- (2-carboxymethyl-2,3-dihydro-s-triazolo [4,3-b] pyridazin-3-one-6-ylthiomethyl) -3- cephem-4-carboxylic acid (BB-S479) CH2NH-CH3 L :. ^ J— n

CO2-N 2 N 2 N 2 A-CH 2 COOH

co2h a) To a cold (0 ° C) mixture of the compound from Example III (5.4 g, 12 mmol), Et 2 N (5.5 mL), CH 2 CN (25 mL) and water (25 mL). a solution of 2,4-dinitrophenyl-o- (n-butoxycarbonyl-N-methylaminomethyl) phenylacetate in THF / prepared from o- (N-butoxycarbonyl-N-methylaminomethyl) -phenylacetic acid (3.48 g, 13 .5 mmol) from 2,4-dinitrophenol (2.49 g, 13.5 mmol) and DCC (2.79 g, 13.5 mmol) in dry THF (37 mL). The mixture was stirred at room temperature overnight. The THF and CH 2 CN were removed from the reaction mixture by evaporation under reduced pressure and the resulting aqueous solution was washed with ether (3 x 30 mL), the pH was adjusted to 2-3 with dilute HCl and extracted with ethyl acetate 24 60869 (4 x 30 mL). The organic extracts were combined, dried over sodium sulfate and evaporated. The residue was chromatographed on a SiO 2 (100 g) column. After washing with CHCl 3, the column was eluted with 3%

MeOH in CHCl 3 to give the desired fraction containing 1- [o- (N-butoxycarbonyl-N-methylaminomethyl) phenylacetamido] -3- (2-carboxymethyl-2,3-dihydro-s-triazolo [4]). 3-b7pyridatsiini-3-one-6-ylthiomethyl) -3-cephem-4-carbonic acid. Yield 3.8 g (45%) m.p. > 200 ° C (dec.).

KBr.

ir: λ max 1780, 1720, 1680 cm pH 7 buffer uv: λ 250 nm (k., 18800) 297 nm (ε, 8400) dmso + d20 nmr: <3 m 7.62 (1H, d, J = 10 , 5 Hz, pyridazine-H), 7.14 (4H, s, phenyl-H), 6.98 (1H, d, J = 10.5 Hz, pyridazine-H), 5.61 (1H, d, J = 4.5 Hz, 7-H), 5.03 (1H, d, J = 4.5 Hz, 6-H), 4.67 (2H, s, N-CH 2), 4.42 (2H , s, CH 2 -N), 4.4-4.0 (2H, m, 3-CH 2), 3.8-3.4 (4H, m, 2-H & CH 2 -CO), 2.72 ( 3H, s, NaCH 2), 1.38 (9H, s, BOC-H).

Anal Calcd for C 20 H 23 N 2 O 5 S 2.5 / 2H 2 O: C, 48.38; H, 5.14; N, 13.16; S, 8.61. Found: C, 48.25, 48.23; H, 4.52, 4.46; N, 12.93, 12.86; S, 8.68.

b) Trifluoroacetic acid (7 ml) was added to the compound obtained in a) (3.8 g, 5.5 mmol) at 0 ° C and the mixture was stirred at room temperature for 20 minutes. Dry ether (100 mL) was added to the mixture. The resulting precipitate was collected by filtration and washed with dry ether (3 x 100 mL). The precipitate was dissolved in a mixture of CH 2 CN (120 mL) and water (18 mL) and the solution was adjusted to pH 5-6 with concentrated NH 4 OH to give an oily precipitate which was triturated with CH 2 Cl 2 to give a solid. Product, 7-7.0- (methylaminomethyl) phenylacetamide-7- 3- (2-carboxymethyl-2,3-dihydro-s-triazolo [7,3-b] pyridazin-3-one-6-ylthiomethyl) -3-cephem-4-carboxylic acid (BB-S479), collected by filtration, washed with CH 2 Cl 2 and dried. Yield 2.55 g (77%).

ir: V max 1770, 1710, 1600, 1550 cm -1 60869 c) To a solution of BB-S479 (2.54 g, 4.3 mmol) in water (25 mL) was added n NaOH (ca. 3 mL) while cooling (solution pH was 10). A large amount of acetone was added to the solution and the precipitate was collected by filtration and washed with acetone to give 1.94 g (84%) of the monosodium salt of BB-S479 m.p. > 200 ° C (dec.).

K3r is: λmax 1770 '1710' 1600, 1550 cm -1 pH 7 buffer UV: λmax 250 nm (/ 1940 °) 297 nm (?

Anal Calcd for C 25 H 24 N 7 O 7 S 2 Na * 1/2 → 0: C, 47.61; H, 4.00; N, 15.55; S, 10.17. Found C, 47.43, 47.43; H, 4.68; N, 15.97, 15.70; S, 9.25, 9.84.

Example 5 7- (3-Aminomethyl-2-thienylacetamido) -3- (2-carboxymethyl-2,3-dihydro-s-triazolo [4,3-b] pyridazin-3-one-6-ylthiomethyl) -3-cephem- 4-carboxylic acid (BB-S483) ........ J-CH2NH2

i— N J-CH -S -> v N-CH-COOH

N g "

CO 2 H

a) A mixture of (3-Nt-butoxycarbonylaminomethyl-2-thienyl) acetic acid (410 mg, 1.56 mmol), 2,4-dinitrophenol (313 mg, 1.7 mmol) and DCC (353 mg, 1.7 mmol) in THF (5 mL) was stirred at room temperature for 12 hours. The precipitated urea was removed and the filtrate was added to a mixture of 7-amino-3- (2-carboxymethyl-2,3-dihydro-s-triazolo [4,3-b] pyridazin-3-one-6-yl-thiomethyl) - 3-Cephem-4-carboxylic acid (683 mg, 1.56 mmol) and triethylamine (0.62 mL, 4.68 mmol) in water (5 mL) at 0 ° C with stirring. Stirring was continued at room temperature until the active ester disappeared at tlc (silica gel plate; Rf 0.95; solvent system CHCl 3: MeOH = 3: 1). The reaction mixture was diluted with water (20 mL), layered with AcOE (50 mL), and the pH was adjusted to 2 with concentrated HCl at 5 ° C. The organic layer was separated and the aqueous layer was extracted with AcOEt (3 x 50 mL). The AcOEt (ethyl acetate) extracts were combined, washed with saturated aqueous NaCl, dried over MgSO 4 and concentrated under reduced pressure. The remaining oil (1.8 g) was chromatographed on silica gel (40 g). The column was eluted with CHCl 3 (400 mL) and 3% MeOH-CHCl 3 (500 mL), respectively. The eluate was detected by tlc (silica gel plate, solvent system CHCl 3: MeOH = 2: 1, detected by I 2). Desired product, 7- (3-Nt-butoxycarbonylaminomethyl-2-thienylacetamido) -3- (2-carboxymethyl-2,3-dihydro-s-triazolo [3,3-b] pyridazin-3-one-6- ylthiomethyl) -3-cephem-4-carboxylic acid, (Rf 0.2) was obtained by evaporation of the MeOH-CHCl3 eluate. Yield 450 mg (42%), melting at 155-160 ° C.

V, KBr -1 is: λmax 3300 '1775, 1720, 1680 cm pH 7 buffer uv: λmax 245 nItl (' 23900) 260 nm (6, 19200) 300 nm (ε, 8700).

c DMSO-d 6 nmr: δ 1.39 (9H, s, BOC-H), 3.76 (4H, br-s, il ppm 2-H & s' CH 2 CO), 4.05 (2H, d, J = 6 Hz, converted to singlet by addition of D 2 O, BOCNH-CH 2), 4.20 (2H, m, 3-CH), 4.69 (2H, s, N-CH 2 CO 2), 5.06 (1H, d, J = 4.5 Hz, 6-H), 5.62 (1H, dd, J = 4.5 & Hz, converts to doublet J = 4.5 Hz by adding D 2 O, 7-H), 6.83 (1H, d, J-4.5 Hz, thiophene-H fb), 7.00 (1H, m, disappears with addition of D 2 O, NHBOC), 7.04 (1H, d, J = 9 Hz, pyridazine-H), 7.12 (1H, d, J = 4.5 Hz, thiophene-H rk), 7.65 (1H, d, J = 9 Hz, pyridazine-H), 8.97 (1H, d, J = 9 Hz, disappears with addition of D 2 O, 7-NH).

Anal Calcd for C27H29N7O9S3: C, 46.88; H, 4.23; N, 14.17; S, 13.90. Found: C, 46.42; H, 4.37; N, 13.49; S, 13.61.

b) Trifluoroacetic acid (0.4 ml) was added to the compound obtained in a) (410 mg, 0.59 mmol) at 0 ° C and the mixture was stirred at room temperature for 15 minutes. Anhydrous ether (10 mL) was added to the reaction mixture to separate the precipitate, which was collected by filtration, washed with anhydrous ether (2 x 10 mL) and suspended in acetonitrile (10 mL). The pH of the suspension was adjusted to 4 with concentrated NH 4 OH and stirred for 10 minutes. The precipitate was collected by filtration, washed with acetonitrile (2 x 5 mL) and dried at 60 ° C / 1 mm Hg for 7 hours to give 310 mg (88%) of 7- (3-aminomethyl-2-thienylacetamido) -3- (2-Carboxymethyl-2,3-dihydro-s-triazolo [4,3-b] pyridazin-3-one-6-ylthiomethyl) -3-cephem-4-carboxylic acid (BB-S483 melting above 200 ° C (decomposes slowly).

27 60869 1 KBr is: V max 3400, 3150, 1760, 1700, 1680, 1600 cm. pH 7 buffer UV: max 245 nm ('1710 °) 260 nm <ε, 14100) 300 nm (ε, 6500).

Anal Calcd for C22H21N7O7S3C / 40.90; H, 4.21; N, 15.17; S, 14.89. Found: C, 40.39; H, 3.62; N, 15.87; S, 14.35.

c) To a suspension of the compound obtained in b) (280 mg, 0.48 mmol) in 0.5 ml of deionized water was added N NaOH to adjust the pH to 9.5 and the insoluble matter was collected by filtration. Acetone (15 mL) was added to the filtrate to separate the precipitate, which was collected by filtration, washed with acetone (2 x 5 mL) and dried at 70 ° C / 1 mm Hg for 7 hours to give 220 mg (76%) of the monosodium salt of BB-S483. .

Sp. 210 ° C (slow decomposition).

KBr is: V max 3400, 3250, 1760, 1710, 1650, 1600, 1550 cm -1 pH 7 buffer uv: λ v 245 nm (ε, 19900) 260 nm (ε, 16400) 300 nm (ε, 6900) D90 nmr ^ pp ^ 3.60 (2H, m, 2-H), 3.91 (2H, s, CH 2 CO), 4.12 (2H, s, CH 2 -NH 2), 4.20 (2H, m, 3 -CH 2), 4.55 (2H, s, N-CH 2 CO), 4.95 (1H, d, J = 4.5 Hz, 6-H), 5.50 (1H, d, J = 4.5 Hz 7-H), 6.94 (1H, d, J = 9 Hz, pyridazine-H), 6.99 (1H, d, J = 4.5 Hz, thiophene-H p), 7.28 (1H , d, J = 4.5 Hz, thiophene-H, ^.), 7.32 (1H, d, J = 9 Hz, pyridazine-H).

Anal Calcd for C 22 H 20 N 7 O 7 S 3 Na * 1 / 2CH 3 COCH 3: C, 43.92; H, 3.61; N, 15.26; S, 14.97. Found: C, 43.48; H, 4.56; N, 15.28; S, 13.91.

Example 6-N- (2-Aminomethyl-1,4-cyclohexadienyl) acetamide-3 - (2-carboxymethyl-s-triazolo [4,3-b] pyridazin-3-one-6-ylmethyl) -3-cephem- 4-carboxylic acid (BB-S493) po 60869

I ^ NH-BOC

{'.L COOH ---> | j. v '^' Nh-boc

,, CONH-p-f / SN

^ -N ^ J-CH2S-Wn> ^ -CH2CO2H

co2h if Y '^ NH2 ^ Y \ /' C0NH ~ r ~ C I ί ^ Ύ »! O 2 -N-γ-N 2 -N 2 -N-CH 2 CO 2 H CO 2 H 0 a) A mixture of 2-Nt-butoxycarbonylaminomethyl-1,4-cyclohexadienylacetic acid (640 mg, 2.4 mmol), 2.4 -dinitrophenol (422 mg, 2.4 mmol) and DCC (49 mg, 2.4 mol) in 10 mL of dry THF were stirred for 1.5 h at room temperature. The precipitated urea was removed by filtration. The filtrate was added in one portion to a solution of 7-amino-3- (2-N-carboxymethyl-s-triazolo [4,5-b] pyridazin-3-one-6-ylthiomethyl) -3-cephem-4-carboxylic acid in 10 ml of water. contained triethylamine (0.56 mL, 4 mmol) and the mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated to 10 mL under reduced pressure, washed with ether (3 x 10 mL), acidified with 6N hydrochloric acid and extracted with ethyl acetate (5 x 10 mL). The combined extracts were washed with saturated saline and dried over anhydrous Na 2 SO 4. The solvent was evaporated and the residue was chromatographed on silica gel (Wako-gel C-200, 30 g) eluting with chloroform-methanol (0-50%). Fractions containing the desired product were collected. The solvent was removed and the residue was triturated with ether-n-hexane to give 410 mg (30%) of 7 - [(2-Nt-butoxycarbonylaminomethyl-1,4-cyclohexadienyl) acetamido] -3- (2-N- carboxymethyl-p-triazolo 4,5-b ^ / seek-pyridazine-3-one-6-ylthiomethyl) -3-cephem-4-carbonic acid. Sp. 110 ° C (dec.).

KBr, irs Y max 1780, 1730, 1610, 1530, 1250, 1160 cm -1 Buff (pH 7) uv: λ, 252 nm (, 19000) 300 nm (C, 8600, sh).

max.

Anal Calcd for C29H33N7O9S2-2H2O: C, 48.12'H, 5.15; N, 13.54; S, 8.86. Found: C, 48.07; H, 4.64; N, 12.70; S, 8.39.

b) The N-BOC-protected cephalosporin obtained in a) (350 mg, 0.51 mmol) was treated with trifluoroacetic acid (TFA) (1 mL) for 30 minutes at room temperature. Ether (50 mL) was added to the mixture to give the TFA salt of compound 3, which was collected by filtration and then dissolved in a mixture of acetonitrile (5 mL) and water (2 mL). The solution was treated with a small amount of activated carbon and the pH was adjusted to 6 with concentrated ammonium hydroxide. The precipitate was collected, washed with acetonitrile (5 ml) and dried in vacuo to give 235 mg (78%) of 7 - [(2-aminomethyl-1,4-cyclohexadienyl) acetamide] -3- (2-N-carboxymethyl-s-). tri-azolo [4,5-b] pyridazin-3-one-6-ylthiomethyl) -3-cephem-4-carboxylic acid (BB-S493). Sp. 220-230 ° C (decomposes).

KBr is: λmax 1770, 1740, 1710, 1650, 1600, 1550 cm-1.

Anal Calcd for C24H25N7O7S2.H10O: C, 47.60; H, 4.49; N, 16.19; S, 10.59. Found: C, 47.77; H, 4.06; N, 16.49; S, 10.64.

30 60869

Example 7 7- [3- (N-Methylaminomethyl) phenylacetamido] -3- [2- (2-carboxymethyl) -2,3-dihydro-s-triazolo [4,3-b] pyridazin-3-one 6-ylthiomethyl-3-cephem-4-carboxylic acid (BB-S525) a) To a mixture of 7-amino-3 - [2- (2-carboxyethyl) -2,3-dihydro-s-triazolo] 7,3-pyridazine -3-One-6-ylthiomethyl--3-cephem-4-carboxylic acid (452 mg, 1 mmol) and triethylamine (0.46 mL, 3.3 mmol) in 50% aqueous acetonitrile (4 mL) were added 2.4 a solution of -dinitrophenyl-o- (Nt-butoxycarbonyl-N-methylaminomethyl) phenylacetate in THF (3 mL) prepared from o- (n-butoxycarbonyl-N-methylaminomethyl) phenylacetic acid (283 mg, 1.1 mmol), 2,4-dinitrophenol (202 mg, 1.1 mmol) and DCC (227 mg, 1.1 mol). The mixture was stirred at room temperature overnight and concentrated under reduced pressure to remove organic solvents. The aqueous concentrate was washed with ether (3 x 20 mL), acidified with concentrated HCl to pH 1-2 and extracted with ethyl acetate (5 x 20 mL). The combined extracts were dried over anhydrous Na 2 O 4 and evaporated to dryness. The residue was chromatographed on a silica gel column (Wako-gel C-200, 10 g) eluting with MeOH-CHCl 3 (MeOH: 0-3%). The combined eluates containing the desired product were evaporated to give 359 mg (50%) of 7- [1- (N-butoxycarbonyl-N-methylaminomethyl) phenylacetamido] -3- [2- (2-carboxyethyl) -2,3- dihydro-s-triazolo / 4,3-b7pyri-datsin-3-one-6-yylitiometyyli7-3-cephem-4-carbonic acid. Sp. > 150 ° C (dec.).

r KBr is: N max 3600-2400, 1780, 1720, 1680, 1550, 1490 cm 1 pH 7 buffer UV! λ max 254 nm (ε, 19800) 298 nm (ε, 9400) δ DMSO-d 6 nmr: λ max 1.37 (9H, s, t-Bu-H), 2.70 (3H, s N-CH 2), ppm j 2.70 (2H, t, J = 7.0 Hz, -CH 2 -), 3.2 - 4.5 (10H, m), 5.01 (1H, d, J = 5 Hz, 6-H), 5.60 (1H, dd, J = 5 & 8 Hz, 8 Hz bond disappears with addition of D 2 O, 7-H), 6.93 (1H, d, J = 10 Hz , pyridazine-H), 7.58 (1H, d, J = 10 Hz, pyridazine-H).

Anal, calculated for the compound. 5 / 2H2O: C, 49.08; H, 5.31; N, 12.92; S, 8.45. Found; C, 49.32; 49.36; H, 4.70, 4.63; N, 12.52, 12.53; S, 8.44, 8.43.

31 60869 b) A mixture of trifluoroacetic acid (1 ml) and the BOC-protected cephalosporin prepared in a) (302 mg, 0.42 mmol) was allowed to stand at room temperature for 15 minutes and then diluted with ether (10 ml). The resulting precipitate was collected by filtration and washed with dry ether (2 x 10 mL) to give 263 mg of a solid which was dissolved in a mixture of water (6 mL) and acetonitrile (3 mL). The stirred mixture was adjusted to pH 4 with 1 N NOH (0.36 mL) and diluted with acetonitrile (100 mL) to give a precipitate (187 mg) which was suspended in water (4 mL) and the pH was adjusted to 9 with sodium hydroxide (1 N , 0.3 ml). The solution was treated with a small amount of activated carbon and lyophilized to give 7- [2- (N-methylaminomethyl) phenylacetamide] -3- [2- (2-carboxyethyl) -2,3-dihydro-s-triazolo [4,3] - pyridazin-3-one-6-ylthiomethyl-3-cephem-4-carboxylic acid (BB-S525) monosodium salt. Yield 106 mg (39%). Sp. > 180 ° C (dec.).

rKhr -1 is: Y makg 3600-2400, 1770, 1710, 1600, 1490, 1400 cm pH 7 buffer uv: λmax 253 nm (λ '19800> 298 nm (ε / 880 °) λ max 2 ° nmr: o 2.70 (2H, m, -CHO-), 2.75 (3H, s, NäCH.), Ppm z, 3.4.4-3.4 (10H, m), 4.92 (1H, d, J = 4.0 Hz, 6-H), 5.55 (1H, d, J = 4.0

Hz, 7-H), 6.93 (1H, d, J = 9.5 Hz, pyridazine-H), 7.28 (4H, s, pH-Η), 7.40 (1H, d, J = 9.5 Hz, pyridazine-H).

Anal, calculated for C26H26N7O7S2 · Na. 3H2O: C, 4.28; H, 4.68; N, 14.22; S, 9.30. Found: C, 45.34, 44.84; H, 4.01, 3.85; N, 14.14, 14.08; S, 9.76.

The monosodium salts prepared in the above examples are all more than 10% soluble in water.

Claims (4)

32 6 0 8 6 9 A process for the preparation of novel therapeutically useful cephalosporins of the formula I and their pharmaceutically acceptable salts S R 1 -NH-CH-ciT 2 CHO II] 2 II / -V "CH 2 S- (CH 2> nC00H τ ^ C-OH 0 II o wherein n is 1 or 2 and r 'is CH2NHR' CHjNBR '____ch2nhr' R - <^ _ y-CH2CO- or 1H-CH2co- or 1H-CH2c; o- wherein R is hydrogen or hydroxy and R 'is hydrogen or methyl, characterized in that a compound of the formula (S) NH-CH-CH CH-O Min 11 .CN "^ C-CH-OC-CH-, // CM2 3 O j COOH is obtained. or a salt thereof to react with a compound of the formula ^ N = tN II TTT HS -Sn / «γ ^ - (CH 2) nCOOH O wherein n is 1 or 2, and treating the obtained compound with an acylating agent of the formula R 1 - X IV 60869, wherein X is halogen or a functional equivalent thereof and R is as defined above, and if desired, the resulting acid of formula I is converted into a pharmaceutically acceptable salt. Process according to Claim 1, characterized in that a compound of the formula I is prepared in which R is CH 2 NHCH. CH2CO- 34 60869 A process for the preparation of a therapeutically active cephalosporin according to formula I and a pharmaceutical composition containing r1— «h-ch-ciT" fcH2 __M / C_N \ -....... ^% N, CH2 »nC00H j C-OH O h color n är 1 eller 2 och är ^^^ ch2nhr '^ \ ch2nhr' ____ ch2nhr- R _ \ ^ - CH2CO-eller | l ^ jL_CH2C0- eller l! ^ s Jl CH2CO- color R är väter eller hydroxyl and R 'are methyl or methyl, which may be treated with NH2-CH-CjH 0 e —c <^ c-ch2o-c-ch3 0 | I ii COOH or sait därav med in the case of formulas hs_L n ii - (CH?) COOH \ / Δ nnmo color n is 1 or 2, and in the case of a mixture with an acylation ring with formulas R1 - X IV