FI60019B - FOERFARANDE FOER FRAMSTAELLNING AV ANTIBAKTERIELLA 7 - ((ALPHA- (2-AMINOMETHYL-1-CYCLOHEXENYL-OCH-1,4-CYCLOHEXADIENYL) -ACETAMIDO) -3-HETEROCYCLISK-THIOMETHYL-3-CE-METHYL-3-CEF - Google Patents

Description

U. —I Tul ANNOUNCEMENT, n n „Λ

Wa w ^ utläcgnincsskrift 6001 9 C (45) ~ "11 ^ (51) Kv.ik? /Int.a.3 C 07 D 501/36 SUO MI — FIN LAND (21) PttmttlhakMNM - tamumMcning 2560/7 ** ( 22) H «k« nl * clouds - An * aknlng * d * g 02.09.71 * '** (23) Alkupttvl — GlMghaudag 02.09.7 ^ (41) Tiillut fulklMlul - Blhrtt offwicllg 05.03.75

Patent and Registration Holders .... ........, __,. ...,. . , __, (44) NlhtlvIkiipanM] * moonJulluJew date. -

Patents and Registration ArraMcan utlagd och utl. * Krtft * n puMk * r »d 31.07.8l (32) (33) (31) Requested ecuelkeu * —Begird priori *** OU.09.73 31.10.73 USA 39 ^ 367, I + II559 (71) Bristol-lfyers Company, 3 ^ 5 Park Avenue, New York, NY 10022, USA (72) Takayuki Naito, Tokyo, Hideaki Hoshi, Tokyo, Jun Okumura, Yokohama,

Hajime Kamachi, Yokohama, Japan-Japan (JP) (7I +) Ltd. Kolster Ab (51) Method of antibacterial 7 ~ / "(c <_ (2-aminomethyl-1-cyclohexenyl- and -1,1 + -cyclohexadienyl) -acetamido7- For the preparation of 3 "heterocyclic-thiomethyl-3-cephem-h-carboxylic acids - For the preparation of antibacterial acid 7- [7c <;-( 2-aminomethyl-1-cyclohexenyl- and -1H-cyclohexa-dienyl-acetamido7-3- heterocyclic-tiometyl-3-cefem-U-karbonsyror

The present invention relates to a process for the separation of cyclic thiomethyl-3-cephem-1 * carbonyl from the antibacterial 7 - [(O (- (2-aminomethyl-1-cyclohexenyl) and -1,5-cyclohexadienyl) acetamido] -3 for the preparation of acids and their pharmaceutically acceptable salts ζ ~ γ ** 2 ** 2 ^ CftgCONH —- ^ S \

^ '· -KN-CHg-S-R

o 1

C00H

2,60019 where R is

OH

N = N L_ „- / |

\ V K ~ N I N-N H H n-N

i => w · AJ. ASL, V - (J = »CH3

li— N N N

‘XsJJ XJl JT; Xl Π and the dashed line indicate a possible double bond.

R is preferably h °

rf N — N] H

_Jjb N__J —r y — OH, N, and especially

^ ^ H

jr? f "'CH3

The novel N '(N - (2-aminomethyl-1-cyclohexenyl and -1H-cyclohexadienyl) acetamido] -3-heterocyclic thiomethyl-3-cephem-U-carboxylic acids and their non-toxic, pharmaceutically acceptable acceptable salts are valuable antibacterial agents and are particularly valuable as therapeutic agents in the treatment of infectious diseases caused by gram-positive and gram-regressive bacteria in poultry and other animals and in humans.

Salts include carboxylic acid salts such as non-toxic metal salts with sodium, potassium, calcium and Aluminum as the metal, ammonium salt and substituted ammonium salts, e.g. non-toxic amines such as trialkylamines such as triethylamine, procainamine, procenzyl, dibenzyl salts of phenylethylamine, 1-ephenamine, N, N1-dibenzylethylene-360019 diamine, dehydroabietylamine, N, N-bis-dehydroabiethylethylenediamine, N- (lower) alkylpiperidine, e.g. N-ethylpiperidine salts, and other amines , salts; and in all cases their non-toxic acid addition salts (e.g. amine salts) including mineral acid addition salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, sulfamate and phosphate, and organic acid addition salts such as maleate, acetate, citrate, thialate, oxalate , tartrate, fumarate, malate, mandelic acid salt, ascorbate and the like.

The process according to the invention is characterized in that A) a compound of formula

H -N -SVS

2] II

# - CH ^ -S-R

COOH

wherein R is as defined above, or a salt thereof is reacted with an acid acylation derivative of the formula ch2nhb fjf

\ CH2C00H

wherein B is an amino protecting group used, for example, in peptide syntheses or in the preparation of O 1 -aminobenzylpenicillins from 2-phenylglycine, and the dashed line is the same as above, and said amino protecting group is removed to form the desired compound of formula I or a pharmaceutically acceptable salt thereof, and if desired, either before or after removal of the group B, (a) the product in the form of the free acid or a salt thereof is converted into a pharmaceutically acceptable salt in a manner known per se, or (b) the product in the form of a salt is converted into the corresponding free acid compound or a pharmaceutically acceptable salt thereof in a manner known per se , or B) reacting a thiol of the compound of the formula 11 6001 9 ^ £ H2BH2 ΓΤ = —μη- ^ SN "^ - N \ |

HS - R V

wherein R is as defined above, or a salt thereof, to form a compound of formula I or a pharmaceutically acceptable salt thereof, and if desired (a) converting the product in the form of the free acid or a salt thereof into a pharmaceutically acceptable salt in a manner known per se, or (b) the product in the form of a salt in a known manner to the corresponding free acid compound or a pharmaceutically acceptable salt thereof.

The 3-thiolated-7-aminocephalosporanic acid of formula II can be prepared by replacing the 3-acetoxy group of 7-aminocephalosporanic acid or a salt thereof with an appropriate heterocyclic thiol or a salt thereof. Replacement of the ester group with a thiol group is a known reaction and is preferably carried out in aqueous solution while heating.

Prior to the acylation reaction, the amino group of the acylating agent III is protected by a conventional protecting group B, such as that used in peptide syntheses or in numerous O-amino-benzylpenicillin syntheses using 2-phenylglycine, which can be easily removed at the end of the reaction. Examples of suitable amino protecting groups are t-butoxycarbonyl, carbobenzyloxy, 2-hydroxy-1-naphthycarbonyl, trichloroethoxycarbonyl, 2t-ethoxycarbonyl-1-methylvinyl and 2-methoxycarbonyl-1-methylvinyl. A particularly valuable protecting group is a proton, as in the compound of formula aCH2NH2.HCl

S

CH2C-C1 5 60019 »

Preferred amino protecting groups are t-butoxycarbonyl, proton and β-diketone or β-ketoester as described in GB Patent 1,123,333 or U.S. Patents 3,325 Vf9 and 3,316 Z'k'J, e.g. methyl acetoacetate or / $ -ketoamide described in JP Patent 71/21 + 71 ^. When t-butoxycarbonyl, N-ketoester, N-di-ketone or β-ketoamide is used as a protecting group, it is preferable to convert the amino-protected acylating agent to a mixed anhydride, e.g. using ethyl or isobutyl chloroformate, before reacting with compound II or a salt thereof with. Upon completion of the acylation reaction, the amino protecting group B can be removed in a manner known per se to give the desired compound of formula I. Thus, for example, the t-butoxycarbonyl group can be removed using formic acid, the carbobenzyloxy group using catalytic hydrogenation, the 2-hydroxy-1-naphthcarbonyl group using acid hydrolysis, the trichloroethoxycarbonyl group by treating the amine functional with other zinc dust in glacial acetic acid, etc. and such groups are within the scope of this invention,

Acylation of the 7-amino group of cephalosporin is a well known reaction and any of the functional equivalents of formula III commonly used as acylating agents for primary amino groups can be used. Examples of suitable acylation derivatives of the free acid are the corresponding acid anhydrides, mixed anhydrides, e.g. alkoxy formic anhydrides, acid halides, acid azides, active esters and active thioesters. The free acid may be reacted with Compound II after first reacting said free acid with N, N'-dimethylchloroforminium chloride or using enzymes or N, N'-carbonyldiimidazole or Ν, Ν'-carbonyltriazole or a carbodiimide reagent. , e.g., Ν, Ν'-diisopropylcarbodiimide, Ν, Ν'-di-cyclohexylcarbodiimide or N-cyclohexyl-N '- (2-morpholinoethyl) -carbodiimide, Heh, and Hess, J. Amer . Chem. Soc., 77, 1967 (1955) J, or an alkyllylamine reagent [R. Buijle and H.G. Viehe, Angev. Chem. International Edition 3, 582, (19 & *) Jy or isoxazolium salt reagent /R.B. Woodvard, R.A. Olofson and H. Mayer, J. Amer. CBEM. Soc. 83, 1010 (1961)], or keteneimine reagent, [c.h. Stevens and M.F. Munk, J. Amer. Chem. Soc. 80, U065 (1958) /, or hexachlorocyclotriphosphatiazine or hexabromocyclotriphosphatiazine (U.S. Patent 3,651,050) or diphenylphosphoryl azide, DPPA; J. Amer. Chem. Soc., 9, 6203-6205 (1972). The other equivalent of the free acid is the corresponding azolide, i.e. an amide of the corresponding acid, wherein in the acid the amide nitrogen is a member of a pseudo-aromatic five-membered ring containing at least 2 nitrogen atoms, i. imidazole, pyrazole, benzimidazole, benzotriazol sol and their substituted derivatives. As an example of the preparation of an azolide, N, N'-carbonyldiimidazole is reacted with carboxylic acid in equimolar amounts at room temperature in tetrahydrofuran, chloroform, dimethylformamide or a similar solvent. . Dicarboxylic acids give the diimidazole. The by-product, imidazole, precipitates and can be separated and the imidazolide isolated, but this is not essential. A particularly preferred acylating agent is an acid chloride hydrochloride of the formula vs. ch2nh2.hci CXtvra which both activates the carboxyl and protects the amino. The use of enzymes to couple the free acid with its protected amino groups to Compound II has also been described above. Such methods use a free acid ester, e.g., a methyl ester, with enzymes provided by the various microorganisms described, e.g., in T. Takahashi et al .; J. Amer. Chem. Soc., 9U (11), 1 + 035- 037 (1972) and T. Nara et al .: J. Antibiotics (Japan), 21 + (5), 321-323 (1971) and DE patent 2,216,113. .

Preferred reaction conditions, e.g., temperature, solvent, reaction time, etc. for the selected coupling reaction are determined by the type of acylation method used and are known per se in the art. In general, it is useful to add an organic tertiary amine, e.g. triethylamine, H, N'-dimethylaniline, ethylpiperidine, 2,6-lutidine or quinoline, as a proton accepting or salt-forming agent.

The compounds of the invention may be isolated in the same manner as is conventionally used to separate similar cephalosporins. Thus, the product can be obtained as a neutral molecule, although more likely to be present as an amphoteric ion, or it can be separated as a salt. The formation of the desired pharmaceutically acceptable carboxylic acid or acid addition salt may be carried out in a manner known per se, e.g. by reacting the acid with a suitable base or acid.

Upon completion of the acylation reaction, the product obtained can be converted (either after or before the removal of the amino protecting group) into another desired product of the formula I in a manner known per se. Thus, a compound of formula I in the form of a free acid or a salt thereof can be converted into a pharmaceutically acceptable salt in a manner known per se. Likewise, the product of formula I in the form of a salt can be converted into the free acid or a pharmaceutically acceptable salt thereof.

In Embodiment B, a compound of formula CH2NH2 I-J-O-K-cH2OCOCH3 co2h as a free acid or salt is reacted with a heterocyclic thiol of formula V or a salt thereof, preferably a sodium or potassium salt. The substitution reaction of such an acetoxy group with a thiol is a well known reaction and may be carried out in a solution such as water or aqueous acetone at least at room temperature and preferably at about 50-100 ° C in the presence of a dilute base such as sodium bicarbonate, e.g. preferably near a neutral point such as pH about 6. Excess thiol is preferably used. The reaction product is separated by accurately acidifying the reaction mixture and then extracting with a water-immiscible organic solvent. The product of the substitution reaction may, if desired, be converted into a pharmaceutically acceptable salt by treatment with a suitable acid or base. As in the alternative process described above for the preparation of the compounds of formula I, the product thus obtained in the form of the free acid or a salt thereof may be converted into a pharmaceutically acceptable salt, or alternatively the product obtained in the form of a salt may be converted into the free acid or a pharmaceutically acceptable salt thereof.

The Sehiff bases of the compound of formula I, especially the salicylaldehyde Schiff base, are also useful as active antibacterial agents.

They are prepared by reacting the free amino compound of formula I with an aldehyde and preferably salicylaldehyde. The reaction is usually carried out at ambient or elevated temperature in an inert solvent, e.g. benzene or toluene, the water being removed by azeotropic distillation, or in the presence of a water-binding agent, e.g. potassium carbonate.

The pharmaceutically active compounds of the invention are potent antibacterial agents used to treat a variety of infectious diseases caused by gram-positive and gram-negative bacteria in poultry and other animals, as well as in humans. The active compounds are also valuable as a dietary supplement in animal feed and as a substance in the treatment of bovine 8 60019 mammalian black. The preferred compounds have also surprisingly been found to be effectively absorbed upon oral administration.

The compounds of the invention are formulated into a pharmaceutical dose containing, in addition to the active ingredient, a pharmaceutically acceptable carrier or diluent. The compounds can be administered both orally and parenterally. The pharmaceutical preparations may be solid, such as capsules, tablets or granules, or liquid, such as solutions, suspensions or emulsions. In the treatment of bacterial infections in humans, the compounds of the invention may be administered parenterally in an amount of about 5 to 200 mg / kg / day and preferably about 5 to 20 mg / kg / day in divided doses, e.g. 3 to * + times a day. They are administered in dosage units containing, for example, 125, 250 or 500 mg of active ingredient with a suitable physiologically acceptable carrier or excipient. Dosage units are liquid preparations such as solutions or suspensions.

Certain 3-substituted 7- (6- (2-aminomethylphenyl) -acetamido / cephalosporanic acid derivatives (A; see Dutch Patent 72/06326, Farmdoc 7637 ^ T, corresponding to DE-A-222 95M) form ears for parenteral use. However, their limited water solubility ({2 rag / ml) has caused the formation of crystals in the urine even when antibiotics are administered parenterally as a readily dissociable soluble derivative, and it is therefore an object of the present invention to provide similar active ingredients. compounds with higher water solubility than the amphoteric form.

ex.

CHgCO N ^

-Chg-R-S

co2h (A)

K

i 0 € 0019, »7

The present invention provides certain ~-2-aminomethyl-1-cyclohexenyl- and -1,2-cyclohexadienyl) -acetamido] cephalosporanic acids (B).

B

HgCO-N — y- / »S

-N CH2-S-B

co2h. , (B)

Several of the new compounds have been found to be more soluble in water than the corresponding phenyl derivatives and have a solubility greater than 7 mg / ml as determined in 0.1 M phosphate buffer pH 7 → 0, as shown in the following comparison tables compared to the corresponding phenyl derivatives.

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The minimum inhibitory concentrations (MICs) mcg / ml of the compounds prepared according to Examples 3 and 19 are shown in the following table.

aCH2NH2 '- ^ 0 (Γ--

COOH

a a

f S, N N

Organisms M.I.C. (mcg / ml) ~ S τ "* Νϊ ^ _s_ Jj ™ 3 6c (Ex. 3) (Ex. 19) S. aureus Smith + A953T 0.2-0.1 + 0.6 S. aureus Smith + A953T 0.8-1.6 1.6 + 50% Serum S. aureus Russel 0.1 + -0.8 0.8 S. aureus BX1633 A9606 1.6 1.6

Str. Pyogenes * A960I + 0.01 + -0.08 0.16 + 5% Serum D. pneumoniae * A9585 0.01 + -0.08 0.08 + 5% Serum

Mycobacterium 607> 100> .100 E. coli NIHJ 1.6-3.1 3.1 E. coli ATCC 8739 3.1-6.3 3.1 E. coli Jubl + A15119 3.1-6.3 6 , 3 K. pneumoniae + A9977 0.8-3.1 3.1 K. pneumoniae + A15130 3.1-6.3 3.1

PR. mirabilis + A9900 100 25

PR. morganii + A15153> 100> 100

Sal. enteritidis + A9531 1.6-3.1 1.6

Ser. marcescens + A20019> 100> 100

Ps. Aeruginosa + A981 + 3> 100> 100 * 50% broth - 1 + 5% antibiotic broth + -1+ + at 10 dilution i3 60 01 9

After reconstitution with the above samples, the broth was found to have the reported minimum inhibitory concentrations (M.I.C.) mcg / ml against the indicated microorganisms as determined by overnight culture at 37 ° C by tube dilution.

The excellent in vivo activity of the preferred compounds of the invention, e.g. 7c (Example 19, BB-S311) and 6c (Example 3, BB-S336) was found by comparison with MR-S9U by prescribing the lowest curative dose (by subcutaneous administration to an infected mouse). # mice (CD ^ Q) mg / kg compared to lethal doses of the reported microorganisms. The results were as follows: CD50 mg / kg

Organism BB-S311 BB-S336 MR-S9 ^ (per l)

S. aureus BX-1633 1.8 2, U 3, H

St. pyogenes Α96θΗ 1.0 - 0.8

E. coli Juhl 6.25 5, H H, H

E. coli Juhl 5.6 - H, 2

PR. mirabilis A9900 9, H - 8 K. pneumoniae D-ll 8.5 - H, 6 S. aureus Smith 1.8 - 3.1

In rat urine recovery studies, cephalosporins were administered subcutaneously at 200 mg / kg amphoteric ions in aqueous suspension (HO mg / ml; DMSO - 0.5% NaHCO 3 -H 2 O = 1: 1: 8) and salicylaldehyde derivatives of equal potency based on molecular weight. in aqueous solution.

In rat crystallinity studies, BB-S311 gave satisfactorily high serum and urine concentrations without finding crystallization. Studies in dogs showed that BB-S311 provides high crystal-free urine concentrations. Parenteral (IM) absorption studies in mice indicate that peak levels of BB-S311 are higher than cephalothin and that this antibiotic may persist in the blood longer than cephalothin. The amount of JM-administered dose of BB-S311 recovered from rat urine was 31%, a value very close to that obtained with MR-S9U (35.5%). The recovery of cephaloricin was ** 3.1%, while that of cephalothin was 18.1%. BB-S3H was always present in the urine as a bioactive 14,60019 substance, with an RF value identical to that observed in the aqueous solution of the compound itself.

The antibacterial activity of BB-S311 was also compared to the activity of MR-69® using an agar dilution assay system. In Mueller-Hinton experiments involving 18 gram-positive and 58 gram-negative organisms, BB-S311 was found to have the same order of activity as MR-S9 ^ · In additional test series, the relative proportions of MR-S9 ^ and BB-S311 activity was determined using the microorganisms Sarratia marcescens, Enterobacter sp., Providencia stuartii and Proteus sp. for each 32 positions. These compounds had excellent activity against most representatives of the microorganisms Enterobacter sp., Providencia stuartii and Proteus sp. against all representatives except P-morganas.

The compounds of the invention, after being dissolved, e.g. in DMSO or 2 mg / ml in water and then diluted with broth, were found to have the following minimum inhibitory concentrations (MICs) mcg / ml against the indicated microorganisms as determined by overnight culture at 37 ° In C using tube dilution. Results for various known cephalosporins have also been reported. In this and other experiments, salicylaldehyde derivatives are as potent as the corresponding compounds and are also readily soluble in water.

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The excellent in vivo activity of the cowardly preferred compound of the invention (Example 6c, BB-S336) was demonstrated by comparing it to results obtained by essentially the same method from MR-59® (subcutaneous administration to the most infectious mice) for a minimum curative dose of £ 50. mice (CD 2) mg / kg compared to lethal doses of the reported microorganisms. The results were as follows: CD50 mg./kg

Organismi_BB-S336_MR-S9 ^ _ S. aureus BX-1Ö33 2.1+ 3.1+ li coli Juhl 5.1+ 1 +, 1 +

Determination of solubilities

The solubilities Ke, ΙΜρΗ 7.0 in phosphate buffer are determined by bioanalysis and also using a spectroscopic method (where the samples have UV absorption), each using its own standard which had been completely dissolved. The test solution and the standard solution are prepared as follows.

Preparation of the test solution

Place about 10 mg (1) of the sample in a flask and mix with 1 ml of 0,1 m phosphate buffer (pH 7 | 0). Close the flask, seal with a metal capsule, place on a rotary shaker and allow to rotate at 160 rpm for U hours at 25 ° C. The solution is filtered through a microporous filter (IIAWP 01300, average pore size 0.1 + 5 μm). The filtrate is diluted for bioanalysis and UV analysis as follows: 22 6001 9

Soda (min. 1 ml) 0.5 «1. ^ I dilution to a volume of 100 ml • J. with buffer ^ 100 ml.

* ': · Used for UV analysis j. dilution to volume

V 20 ml W

20 ml.

Dilution of the volume to 20 ml with a volume of 20 ml.

Used for bioanalysis

Preparation of standard solution

Dissolve about 2 mg of the same sample, accurately weighed, in 1 ml of 1 M K 2 CO 2 solution (3) (usually within 1 minute). The solution is diluted to exactly the volume in a 50 ml flask with 0.1 M phosphate buffer pH 7.0 and used for the preparation of standard solutions for bioanalysis and UV analysis as follows: _5Q ml._.

^ TJV analysis for bioanalysis __1 lal. w v ·] // h \ | (40 incg./ml.) 10 ml. ' 1 I dilution mode-. ^ \. to a dilution volume of 20 ml · 20 ml · 20 ml · 20 ml. $ - i / m (20 mcg./ml.) (2 mcg./ml.) 5 ml.v '| dilution status * 'i I,. .

--S ieoml.i1 *) used for UV analysis 20 ml.

used as standard solution (0.5 mcg./ml.) for bioanalysis a standard solution of 23 6001 9 (1) 20 πΐβ: η or larger is used when the sample is expected to be more soluble.

(2) 0.1M pH 7 »0 phosphate buffer is used as a diluent in the present experiment and also as a reference solution in UV analysis.

(3) It has previously been found that almost the same result is obtained when the sample is dissolved in DMSO (dimethyl sulfoxide) and then diluted in the same way with buffer.

(H) The volume of the solution is accurately measured using a pipette or flask.

Λ.

A. -50019;

Solubilities (rag / nl) in 0.1M phosphate buffer (pH 7.0) 7-side chain "~~ t“ “SL_coqc :: -CH -S-H'j R '= V ^ | Ϊ-f m-S & 13B- SJ-36 (6c) BB-S311 - (^ c) ~ \ n ^ N M. UV £ A Iö £ BA uv CH 2.0 1 · 9 25.6 24.6 9.7 9.7 _ 25 · 0 26.0 9.4.9.6 7.8 7.8 7.8 N — N m-S56 BB-S341 (6d) BB-S312 (7d) J-Lh 1.2 ° · 9 '· 4 · 3 ή * β 8 * 8 7.6 2 3.4 --------- -: --- N — H BB-S150 BB-S340 (§ 6) BB-SI80 (7a) 4.4 4.4 16.8 16.0 3.1 _u_ N — N BB-S226 BB-S338 (6b) BB-S269 (7b) and I 2 · 5 5.0 3.0 12.5 9.0.,) ^ N 2 · 1 1.7 ^ == / 3.8 3.5 2.2 25 · 6001 9 <*

OH

J EB-S207 B3-S339 (6η) BB-SJ13 (le) M UV ΒΛ irv M uv // 1 \ 1 * .2 3.9 8.0 8.1 5.9 7.7 'O 3.6 * ·> 1.5 3.1 3.7 4.4 BB-S314 (ΖΓ ) Λ J 8.7 8.9 "

n_n

- ({| BB-S324 (Ze)

O

BA: Bioan & lysis, UV: UV analysis

The parallel results reported for the same compound were obtained using samples taken from different batches.

• * •.

λ! ♦ 26 60019

The following examples illustrate the present invention.

All temperatures are reported in degrees Celsius, "Skellysolve B" is a petroleum ether fraction, b.p. 60-68 ° C containing essentially n-hexane. IR-120 is also called Anberlite IR-120 and is a powerful cation exchange resin containing 3 sulfonic acid radicals. Amberlite IR-120 is a commercially available cation exchange resin of the polystyrene sulfonic acid species; thus, it is a ring-sulfonated polystyrene resin crosslinked with diphenylbenzene and obtained according to the method described by Kun, Ion Exchange Resins, 2nd Edition (195), Jonn Wiley and Bons, Inc.

2,4-Dinitrophenol is designated 2,4-DNP, II, ii'-dicyclohexylcarbodiimide is designated DCC, trifluoroacetic acid is designated TFA, tetrahydrofuran is designated THF, t-butoxycarbonyl azide is designated t-BuOCOH 4 and t-butoxycarbonyl is designated t-BOC.

Preparation of starting materials oL (2-Aminomethyl-1,4-cyclohexadienyl) acetic acid (1)

A solution of 16.5 g (0.1 mol) of o-aminomethylphenylacetic acid 1 in> liter of liquid ammonia (treated with 50 mg of lithium to remove traces of moisture) is slowly diluted with 500 ml of dry t-BuOH. 3.4 E (0.5 atoms) of lithium are added in small portions over 4 hours and the mixture is stirred for 16 hours at room temperature to remove liquid ammonia and finally evaporated to dryness below 40 ° C. The residue is dissolved in 500 ml of water and the solution is chromatographed on an IR-120 column (H +, 700 ml) and eluted with 1'*> JH2O2 solution. The ninhydrin-positive fractions of the eluate are combined and evaporated to dryness. The residue is washed four times with 50 ml portions of hot acetone and recrystallized from 500 ml of ethanol / water (1: 1) to give 11.2 g (67%) of colorless needles, 1, m.p. 183 ° C.

IRC / NUJ. i63Q ^ 1520 ^ 138o ^ 1356 cm’1. m & Ks · NMR: - / D20 + K2CO3 2.72 (UH, s,), 3.01 (2H, s, CH CO), 3.20 (2H, a, CH - &), 5.78 (211, s, C =).

Anal, calculated for C 6.65, H, 7 · h, 8.38.

Found: C, 64.77; H, 8.06; H, 8.44.

27 6001 9

An alternative method for the preparation of (2-aminomethyl-1,1-cyclohexadienyl) -acetic acid OUNHo / - /.

^ y — CH 2 CO 2 H LI, tert. BuOH

NH-j - TEA-HCl

O 1 - CH 2 CO 2 H - Li Cl ♦ (C 2 H 5) 2 N

^ CHgNHg

The method used by Welch, Dolfini and Giarrusso in U.S. Patent 3,720,665 (Example 1) to prepare D * 2-amino-2- (1,1 + -cyclohexadienyl) -acetic acid is applied. A solution of 830 ml of distilled liquid ammonia is dried using 10 mg of lithium under an argon atmosphere. To this stirred solution are added 11.0 g (0.07 mol) of 2-aminomethylphenylacetic acid and 3 x 0 ml of tert-butyl alcohol. A total of 1.6 g (0.225 moles) of lithium is added to the vigorously stirred solution over 2 hours. The gray mixture is then treated with 35 g (0.215 moles) of triethylamine (TEA) hydrochloride and stirred overnight at room temperature for 18 hours. The tert-butyl alcohol is removed at 100 ° C (15 mm) to give a white residue which is dried in vacuo overnight using?.> 0. The solid is dissolved in 30 ml of methanol / water 1: 1 while adding 3.5 liters of chloroform / acetone 1: 1 at 5 ° with stirring. The mixture is stirred for 20 minutes and the amino acid 1,1 '(4- (2-aminomethyl-1,1'-cyclohexadienyl) -acetic acid is collected and dried under vacuum for 16 hours using P 2 O 3 to give 6.3 g (56'). ») White crystals, m.p. 190 ° with the same decomposition. The IR and ifMR spectra correspond to the structure.

«3- [2- (t-butoxycarbonylaminomethyl) -3,4-cyclohexadienyl] acetic acid (3) To a stirred solution of 8.0 g (0.08 mol) of q4t (2-aminomethyl-1H-cyclohexane). dienyl) acetic acid and 3.8 g (0.096 mol) of JaH in 150 ml of water are added a solution of 10.3 g (0.072 mol) of t-butoxycarbonyl azide 2β 60019 in 10 ml of TlIF and the mixture is stirred Id hours at room temperature. The THF is removed under reduced pressure and the remaining solution is washed with ether (2 x 100 mL), reconstituted with 6ii HCl and extracted with ether (3 x 100 mL). The combined extracts are washed with water (2 x 100 ml) and saturated JaCl solution (100 ml), dried and evaporated to dryness, the oily residue is taken up in n-n * to give 10.5 g ( 82%) colorless powder 3, m.p. 113UC.

IR: / nU ^ · 3370, 1715, lbUO, 1530, 1280, 1160 cm-1.

ZH & K.S

JMR: <iCDCl3 1.1 * 5 (9H, s, t-Bu-H), 2.73 (1 * H, s, ppm c = c U2Cl3 3.16 (2H'S * CH2CO) '3, 76 (2H * d '6HZ> CH2a)

Utyo (lii, n, JK), 5.66 (2H, s, H 2 -C-), 10.6 (1K, br-s, COOK).

Anal Calcd for C 18 H 18 N 2 O 2: C »62.90; II, 7.92; II, 5.2 days.

Found: C, 63.13; K, 8.21; J, 5.26.

[2- (N-butocycarbonylamino) ethyl] -1-cyclohexen-1-yl] acetic acid (2) Solution of (2- (N-butoxycarbonylamino) methyl-1,1'-cyclohexadien-1-yl) acetic acid (3) (1.33 g, 5 mmol) in 3 l of ammonium hydroxide (10 ml), nitrated at 2.8 kg / cm 3 using palladium on carbon (10 0.2 g), the theoretical amount of hydrogen being absorbed in 3 hours. the filtrate is acidified to pH 2 with dilute HCl and extracted with ethyl acetate (2 x 50 ml) The combined extracts are washed with water (20 ml), dried over Na 2 SO 4 and evaporated under reduced pressure to give an oil (1.3 g). ), which solidifies on standing for several days, is recrystallized from n-hexane / ethyl acetate to give 1.2 g (90 l) of compound 2 as colorless prisms, mp 118-119 ° C.

IR: / nuio11 3 ** 50, 1730, 1660, 1510 cm-1.

m & KS

JMR: C 1 O 3 1.58 (9H, s, t-butyl-H), 1.50 to 1.90 ppm (Un, m, -CK-), 1.90 to 2.20 (UH, m, allyl methylene) -H), 3.10 (2K, s, CH 2 -CO), 3.78 (2H, d, 6 Hz, CH 2 -A), 5.00 (1H, br-s, JK), 8.90 ( 1H, br-s, C00H).

Anal. Calcd for. H, 8.6 L; N, 5.20.

Found: C, 62.12; H, 8.77; N, 5.37-4 29001 9

Sodium (2- [N- (1-carbethoxypropen-2-yl) aminomethyl] -1,4-cyclohexadienyl) acetate (4)

To a stirred solution of 460 rag (0.02 moles) of metallic sodium in 100 mL of absolute EtOH is added 3.34 g (0.02 moles) of compound 1 and 3.1 g (0.024 moles) of ethyl acetoacetate and the mixture is heated. under reflux for 4 hours with stirring. The bulk reaction mixture is filtered and the filtrate is allowed to cool overnight to give 2.0 g of colorless needles of compound 4, mp, 264 ° C. The additional product (3.3 g) is obtained by concentrating the mother liquor. The total yield is 5.3 g (88%).

ν max 11,300 »1635, 1600> 157 °> 1300 '1275' 1170 '1090 cm-1 * m: s, O = O-O 2, cis & trans), 2.70 (4H, s, H 2 Cl 2), 3.04 (2H, s, CH 2 CO), 3.66 & 3.95 (2H, s, CH 2 -N, cis & trans), 4.07 (2H, q, 7Hz, CH 2 CH 2), 4.45 & 4.56 (1H, s, =, cis & trans), 5.76 (2H, s, δ jj).

B

Anal Calcd for C 18 H 19 N 2 O 2 Na: C, 59.79; H, 6.69; N, 4.64.

Found: C, 59.69; H, 6.76; N, 4.75 ·

Known starting materials 7-Amino-3- (3-hydroxypyrazin-6-ylthiomethyl) -3-cephem-4-carboxylic acid (4a), 7-amino-3- (tetrat Solo / 1,5-b) pyridazine-6- ylthiomethyl-3-cephem-4-carboxylic acid (4b), 7-amino-3- (1-methyltetrazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid (4c), 7- Amino-3- (5-methyl-1,3,4-adiaziazol-2-ylthiomethyl) -3-cephem-4-carboxylic acid (4d), 7-amino-3- [s- (1,2,3 -triazol-5-yl) thiomethyl) -3-cephem-4-carboxylic acid (4h), 7,7-amino-3- [2- (1,3,4-thiadiazolyl) thiomethyl] -3-cephem-4-carboxylate: Acid (4i) and 7-amino-3- [2- (5-hydroxymethyl-1,3,4-thiadiazolyl) -thiomethyl] -3-cephem-4-carboxylic acid (4j) were prepared according to U.S. Patent 3,516,999r. 'method.

'w »' w I y 30 7-Amino-3- [3,4-c] Libydro-s-triazolo [1,3-b] pyridazin-3-one-6-ylthiomethyl] -3-cephem-U-carboxylic acid (Ue) 7 AC ACA (1.36 g, 5 mmol) is added at 50 ° C to a solution of 6-mercapto-2,3-dihydro-s-triazolo [1,3-b] pyridazin-3-one ( 0.81 * g, 5 mmol) and sodium bicarbonate (0.81 * g, 10 mmol) in 20 ml of 0.1 M phosphate drop buffer solution (pH 6.1 *), and the mixture is heated at 70 ° C for 2 hours. A small amount of insoluble matter is removed by filtration and the filtrate is acidified to pH 5 with dilute HCl to give the product 1 * e which is collected by filtration, washed with water (30 ml) and dried in vacuo using CH 2 Cl 2. 1.25 g of 7-amino-3- [2,3-dihydro-3-triazolo [1,3-b] pyridazin-3-one-6-yl-thiomethyl-5-cephem-1-carboxylic acid are obtained (66%), m.p. > 300 ° C.

e.g., V 10 O 5, 1720, 1620, 1550 cm -1.

257 nm (£ 17700).

NMR: δ D 2 O-K 2 O 3 3jkO (1H, d, 20 Hz, 2-H), 3.78 (1H, d, 20 Hz, 2-H), PPm 1 +, 00 (1H, d , 13 Hz, 3-0¾). U, 35 (1H, d, 13 Hz, 3-0¾). 5.02 (1H, d, k Hz, 6-H), 5.1 * 0 (1H, d, 1+ Hz, 7 ~ H), 6.70 (1H, d, 9 Hz, pyridazine -H), 7.1 * 0 (1H, d, 9 Hz, pyridazine-H).

Anal Calcd for C 13 H 12 N 6 O 1 + S 2, H 2 O: C, 39.19; H, 3.5 **; N 21.09; s, 16.09.

Found: C, 39.1 * 0; H, 3.39; N, 20.36; S, 15.89.

7-Amino-3- (pyrido [2,1-c] s-triazol-3-ylthiomethyl) -3-cephem-1 * -carboxylic acid (1 * f) -N-O-CH2-S - ^

O

To a hot (60 ° C) solution of 1.51 g (10 mmol) of 3-mercapto-s-triazolo-7U, 3-α7-pyridine ^ D.S. Tarhell et al., J. Am. Chem. Soc. 70, 1381 (191 * 8) and 1.68 g (20 mmol) of NaHCO 3 in 0.1 M phosphate buffer (pH 7.1 *, 50 ml) are added portionwise to J-ACA (2.72 g, 10 mmol) and the mixture is heated at 280-85 ° C for 30 minutes. The reaction mixture is treated with carbon, the filtrate is acidified to pH 5 with dilute HCl to give 7-amino-3- (s-triazolo-β-cis-pyridin-4-ylthiomethyl) -3-cephem-1 * carboxylic acid , which is collected by filtration, washed with water (10 ml) and dried in vacuo using P 2 O 3. Yield 1.1 + 0 g (39%). Sp. 215-220 ° (dec.).

®: /iSks.l°°5, 1620, 1530, 11 * 10, 15 ** 5 cm-1.

280 nm (£ 13,200).

31 6001 9 NMR: 1 D 2 O + K 2 CO 3 3.25 (1H, d, 18 Hz, 2-H), 3.63 (1H, d? P? 3 Hz, 3-H), 3.68 (1H , d, 18 Hz, 2-H), U.10 (1H, d, 13 Hz, 3-H), 14-, 7-5 → 3 (2H, m, 6-H & 7'H) .

Anal, calculated for O ^ Sg! C, U6.27; H, 3.61; li, 19.27; 3, 17.65.

Found: C, 1 * 5.81; U5.7U; H, 3.58; 3.69; N, 18.21, 18.13; 3, 17.08.

7-Amino-3-pyridazino [2,1-b] -s-triazol-3-ylthiomethyl) -3'-cephem-U-carboxylic acid (Ug) H2 '- | - β-.1

cvs-f I

0 ΙΓ— CO 2 H \ __ / 3-mercapto-s-triazolo [f], 3-pyridate

A mixture of 1.20 g (8 mmol) of 3-chloro-s-triazolo [1,3-b] pyridazine. Francavilla and F. Lauvia, J. Het. Chem., 8, 1 + 15 (197 μl and 1.20 g (16 mmol) KSH in 20 ml ethanol, is heated for 8 hours at 130 [deg.] C. in a sealed tube. After cooling, the mixture is evaporated to dryness and the residue is dissolved in 100 ml. water, treated with activated carbon, acidified to pH 1 with dilute HCl to precipitate 3-mercapto-β-triazolo [3 '] pyridazine, which is collected, washed with 10 ml of water and dried in vacuo using silica, to give 0.75 S of product (62%), mp 250-270 ° C (decomposes).

3080, 29 ^ 0, 2760, 2760, 1620, 1500, 1280, 1055 cm-1.

max · NMR: / DMSO'd 6 6.99 (1H, dd, k & 10 Hz, 7-H), λ ppm γ, 67 (1H, dd, 2 & 10 Hz, 8-H), δ, 15 (1H, dd, 2 & li Hz, 6-H), 12.3 (1H, br-s, disappears on addition of D ^ Orta).

Anal Calcd for C 18 H 18 N 2 O 2 S-1/2 O 2: C, 37.26; H, 3.13; H, 3, 76.

Found: C, 37.35; H, 2.32; N, 3 * 4.81.

32 € 0019 7-Amino-3- (s-triazolo A, 3-b7-pyridazin-3-ylthiomethyl) -3-cephem-U-carboxylic acid (Ur) 7-ACA (1.36 g, 5 mmol) is added portionwise to a solution of 0.68 g (U, 5 mmol) of 3-mercapto-s-triazolo [1,3-b] pyridazine and 0.8) of 4 g (10 mmol) of NaHCO3 in 20 ml of 0, 1M phosphate buffer (pH 7, * 0) at 40-50 ° C. The mixture is heated at 80-85 ° C) for +0 minutes, treated with a small amount of activated carbon and acidified with dilute HCl to pH k to give 7-amino-3- (3-triazolo [3,3-h] pyridazine). 3-ylthiomethyl) -3-cephemic 4-carboxylic acid precipitates and is collected, washed with 20 ml of water and dried in vacuo using PgO 4 to give 0.81 g (1 * 9%) of product, m.p. ^ 300 ° C.

make 1T ° 5 '1620' 15l * ° 'll + 15' 1350 Cm_1 * UV: λmaxHCO3 275 nm (£ 13100) λma 2-H).

ppm 3.86 (1H, d, 17 Hz, 2-H), U, 80 (1H, dd, U & 2 Hz, 6-H), 5.2 U (1H, d, h Hz, 7 -H), 7.1-8.6 (3H, m, pyridazine-H).

Anal Calcd for C 18 H 18 N 2 O 2 Sg.H 2 O: U 0.83; H, 3.69; N, 21.96; S, 16.78.

Found: C, Ui, 15; H, 3.2U; N, 20.37; S, 17.90.

Example 1

C ^ O00 ”11 I ~ -f N - N

-’H Y ^ ~~ CHg — S - <^ y ~ OH

co2h 6a r-m 2-t-Butoxycarbonylaminomethyl-1-cyclohexenyl) -acetamido-7- (6-hydroxypyridazin-3-ylthiomethyl) -3-cephem-U-carboxylic acid (5a)

A mixture of compound 2 (1.08 g, U millimoles), 2, U-dinitrophenol (0.7 U g, U millimoles) and DCC (0.82 g, U millimoles) in THF (20 mL) stirred for 1 hour at room temperature and filtered to remove precipitated dicyclohexylurea, which is washed with THF (10 mL). The combined filtrate and washings are cooled to 5 ° C and poured in one portion into a cold solution of 7-amino-3- (6-hydroxypyridazin-3-ylthiomethyl) -3-cephem-L-carboxylic acid (1.02 g, 3 mmol) and triethylamine (0.8 g, 8 mmol) in 50% aqueous THF (20 mL). The mixture is stirred overnight at room temperature and washed with ether (2 x 50 mL). The aqueous layer is acidified to pH 2 with 33 mL of dilute HCl to precipitate crude 5a, which is dissolved in THF (100 mL) and filtered to remove insoluble material. The filtrate is treated with a small amount of carbon and dried over anhydrous sodium sulfate. Evaporation of the solvent under reduced pressure gives compound 5a as an oily residue which solidifies on stirring in ether (100 ml). Yield 0.81 g (w / v).

Sp. 18-15 ° C (dec.).

TO: \ (3250, 1780, 1660, 1580, 1530, 1370, 1250, 110 cm -1).

δ ppm δ 6.10 (9H, s, 5-Bu-H), 5.13 (1H, d, 1Ez, 6-H), 5.70 (1H, dd, 1 and 8 Hz, 7 -H), 6.88 (1H, d, 10 Hz, pyridazine-H), 7.15 (1H, d, 10 Hz, pyridazine-H), 9.0 (1H, d, 8 Hz, CONH), 13.2 (1H, br-s, -OH).

Anal Calcd for C 9 H 18 N 2 O Sg.1 / 2 H 2 O: C, 51.98; H, 5.70; N, 11.65; S, 10.67.

Found: C, 51.53; 51.56; H, 5.63, 5.80; N, 11.28, 11.31; S, 11.18, 11.31. 7H-N- (2-Aminomethyl-1-cyclohexenyl) acetamido-3- (6-hydroxypyridazin-3-ylthiomethyl) -3-cephem-k-carbonic acid (6a)

To trifluoroacetic acid (1.5 mL) is added compound 5a (0.80 g, 1.1 mmol) and the mixture is stirred for 30 minutes at room temperature and diluted with ether (50 mL) to precipitate trifluoroacetate of compound 6a, which is collected by filtration and slurried in water (2 mL). mL). The mixture is adjusted to pH 6 with ammonium hydroxide and diluted with acetonitrile (200 mL) to precipitate compound 6a. Yield 0.61 g (93%). Sp. 200-208 ° C (decomposes).

TO: Ϋ nuj ° 1: L 1780, l680 - 1610, 1580 cm-1.

'max.

UV: δ 1.2 ° C 3 255 nm (E, 1100).

lDAiCS #

Anal, calculated for C 21 H 25 N 5 O 5 S? 3 1/2 HgO; C, 15.18; H, 5.82; N, 12.63; S, 11.56.

Found: C, 1 * 5.73, ^ 5.83; H, 1.50, 1.17; N, 12.15, 12.59; S 11.83, 12.06.

Example 2 (2-t-Butoxycarbonylaminomethyl-1-3-cyclohexenyl) -acetanediol-3 - (pyridazino) g. 3- (7-Tetrazol-6-ylthionethyl) -3-cephera-k-carboxylic acid (3b)

A mixture of compound 2 (1.08 g, U mmol), 2, U-dinitrophenol (0.7 ** e »** mmol) and DCC (0.82 g, 1+ mmol) in THF (20 mL) , stirred for 1 hour at room temperature and filtered to remove dicyclohexylurea, which is washed with THF (10 mL). The combined filtrate and washings are cooled to 5 ° C and poured into a solution of 7-amino-3 '(pyridazino-2,3,3-tetrazol-6-ylthionethyl) -3-cephem-1-carboxylic acid (** b) (1.08 g, 3 mmol) and triethylamine (0.81 g, 8 mmol) in 50 L of aqueous THF. (20 ml) 5 ° C: sr. The mixture was stirred overnight at room temperature and washed with ether. (2 x 50 mL). The aqueous layer is acidified to pH 2 with dilute HCl and extracted with ethyl acetate (3 x 50 mL). The combined extracts are washed with water (50 ml), treated with charcoal and dried over anhydrous Na 2 SO 4. Removal of the solvent under reduced pressure gives compound 5t> as an oil which solidifies on stirring with ether (50 nl). The product is collected by filtration, washed with ether and dried. Yield 0.9 ** g (51%) · M.p. 125 "13 ** ° C (decomposes).

IR: / lCBJ 1780, 1680, 1520, 1370, 1250, 1160 cm -1.

IjIcuvS

ιίί ©: Χ0Μδ0 "ά6 1.1 * 0 (9H, s, t-Bu-H), 1 *, 23 (1H, d, li * Hz, 3-CH.S), U, 70 ^ ppm 2 ( lii, d, 15 Hz, 3-CH 2 S), 5.15 (1H, d, 1 * Hz, 6-H), 5.78 (1H, dd, 1 * and 8 Hz, 7 “H), 7, 38 (1H, d, 10 Hz, pyridazine-H), 8.75 (1H, d, 10 Hz, pyridazine-H), 9.0 (1H, d, 8 Hz, 7-COfTK).

Anal Calcd for C 26 H 32 I 18 ° 6 S 2: C * 5 °, 6 **; H, 5.23; 2, 10.1 * 0.

Found: C, 50.36, 50.1 * 2; H, 5.11, 5.21; S, 9.60.

7- [6- (2-Aminomethyl-1-cyclohexenyl) -acetamido] -3- (pyridazino [2,3-b] tetrazol-6-ylthiomethyl) -3-cephem-1 * -carboxylic acid (6h)

To trifluoroacetic acid (1.5 mL) is added compound 5b (0.90 g, 1.5 moles) at 0 ° C and the mixture is stirred for 30 minutes at room temperature. The mixture is diluted with ether (50 mL) to precipitate trifluoroacetate, which is collected by filtration and suspended in water (2 mL). The suspension is adjusted to pH 6 with ammonium hydroxide and diluted with acetonitrile (200 ml) to precipitate compound 6b, which is collected by filtration and dried. Yield 0.65 g (δ 3 l).

Sp. 18U-188 ° C (decomposes).

IR: / nujoli 1T8o, 1620, 1570 cm -1, max.

UV: 21 * 5 nm * λ * 18500), 255 nm (sh) max (ε, 160000), 275 nm (sh) (ε, 10700), 315 nm (sh) (ε, 1 + 700).

35 6001 9

Anal Calcd for C 18 H 18 N 2 O 2 Sg.1 / 211 ^ 0: C, U7.99; K, U, 79; N, 21.32; S, 12.20. Found: C, U 8.21, 1 + 7.90; H, U, 6l, 1+, 67; N, 20.68; 20.63; S, 11.1 + 7.

Example 3 c 0 -1 CO 2 CO 2 H-β- (2- / Th-t-butoxycarbonylamino) methyl-1-cyclohexenyl-1-yl] methylamino) -3- (1-methyltetrazole-3 -ylthiomethyl) -3-cephem-1 + carbonylamino (5c)

A mixture of compound 2 (1.30 g, U, 8 mmol), 2,1 + -dinitrophenol (0.88 g, 1, 8 mmol) and DCC (0.99 g> U, 8 mmol) in THF: (20 mL), stirred for 1 h at room temperature and filtered to remove dicyclohexylurea, which is washed with THF (5 mL). The combined filtrate and washings are poured into a cold solution of 7 "amino-3-1-methyltetrazol-5'-thiomethyl) -3-cephem-1 + carboxylic acid (Uc) (1.31 g, 1+ mmol) and triethylamine (1, 01 g, 10 mmol) in $ 50 aqueous THF at 5 ° C. The mixture is stirred overnight at room temperature, evaporated under reduced pressure below U0 ° C and washed with ether (3 x 30 ml) .The aqueous layer is acidified to pH 2. dilute HCl and extracted with ethyl acetate (1+ x 500 mL) The combined extracts were treated with charcoal and dried to remove the solvent under reduced pressure below 100 ° C to give the title compound as an oil which solidified on stirring with ether (100 mL) Yield 1.33 g · Mp 16-159 ° C (dec.).

IpVnujoii 178o> 1700 (sh)> 1668 ° 1520> 12k0t 1155 cm -1 NMR: 1 DMSO_d6 1, U 2 (9H, s, t-butyl-H), 1.5-1.7 (UH , m, aliphatic ppm methylene-H), 1.8-3.3 (UH, m, allyl methylmethylene-H), U, 12 (3H, s, N-CH 2), 5.15 (1H , d, 5 Hz, 6-H), 5.80 (1H, dd 5 and 8 H:, 7 "H), 6.85 (1H, br-s, NHBOC), 9.00 (1H, d, 8 Hz, CONH).

Anal Calcd for C 28 H 30 N 2 O 3: C, U 9.73; H, 5.7U; N, 16.91 »S, 11.06.

Found: C, U9.U6, U9, U6; H, 5.53, 5.62; N, 16.00, 15.97; S, 11.01.

• 36,600,9,7 - [(2-aminomethyl-1-cyclohexenyl) acetamide] -3- (tetrazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid (6c)

To trifluoroacetic acid (2 mL) is added compound 5c (1.30 g, 2, h mmol) and the mixture is stirred for 30 minutes at room temperature. The mixture is diluted with ether (50 or) to precipitate trifluoroacetate of compound 6c, which is suspended in water (2 ml) and the suspension is adjusted to pH 6 with ammonium hydroxide and diluted with acetonitrile (200 ml) to precipitate compound 6c which is washed with acetonitrile and dried. Yield 0.80 g (69), m.p. 19 DEG -207 DEG C. (decomposes).

IR:? Max ^ iTTO, 1630, 1590, 1370 cm -1. · UV: 711 ° ° 3 270 nm (f, 10000).

TTISLiCS a ^

Anal Calcd for C 18 H 18 N 2 O 2 Sg · H 2 O: C, * + 5.86; H, 5.7 ° .i, 19.70; 3, 12.89.

Found: C, 1 * 5.09, ^ 6.18; H, 5.26, 5.28; N, 19.71 *, 19.82; S, 12.1 * 9.

Example h

| -I

COgH

M

N- [1- (2-t-butoxycarbonylaminomethyl-1-cyclohexenyl) -acetamido ") - 3- (5-methyl-1,3,1 * -thiadiazol-2-ylthiomethyl) -3-cephem-U -carboxylic acid _ (5d)

A mixture of compound 2 (1.08 g, 1 mmol), 2,1 * -dinitrophenol (0.71 * g, 1 * mmol) and DCC (0.82 g, 1 * mmol) in THF (20 mL) , stirred for 1 hour at room temperature and filtered to remove dicyclohexylurea, which is washed with ThF (10 mL). The combined filtrate and washings are cooled to 5 ° C and poured into a solution of 7-amino-3- (5-methyl-1,3,3-thiadiazol-2-ylthio-acetyl) -3-cephem-1 * carboxylic acid. (kd) (1.9 g, 3 mmol) and triethylaxinine (0.81 g, 8 mmol) in 50 S aqueous THF (20 mL). The mixture was stirred overnight at room temperature and washed with ether (2 x 50 mL). The aqueous layer is acidified to pH 2 with dilute HCl and extracted with ethyl acetate (3 x 50 mL). The combined extracts are washed with water (50 ml), treated with charcoal and dried over H 2 SO 4 and evaporated under reduced pressure to give an oil which solidifies on stirring with ether / n-hexane (1: 1, 100 ml). The product is collected by filtration, washed with n-hexane (50 ml) and dried. Yield 1.29 ° C (72%). Sp. 95 "102 ° C (decomposes).

IR: '/ ^ 011 1780, 1680, 1520, 12 ^ 5, 1160 cm -1.

1 naics.

: «·«: JDx430'd6 1.30 (9H, s, t-Bu-H), 2.58 (3H, s, CH_), U, 03 (1H, d, lh y? ..

ppm 3 3-CH23), 1.38 (1H, d, lit Hz, 3 "CH2S), 1 +, 88 (1H, d, 1» HZ, 6-H), 5.U6 (1H, dd, u and 8 Hz, 7-H), 8.5 U (1H, d, 8 Hz, COHH).

Anal, calculated for compound c »50.1 + 0; H, 5.58; H, 11.76.

Found: C, 50.76, 50.98; H, 5.67, 5.67; N, 11.27, 11.28.

7 - [(2-Aminomethyl-1-cyclohexenyl)-] -ethamido] -3- (5-methyl-1,3,11-thiadiazol-2-ylthiomethyl) -3-cephem-tert-carboxylic acid (6d) In trifluoroacetic acid (2 ml) ) is added compound 5d (1.20 g, 2 mmol) and the mixture is stirred for 30 minutes at room temperature. The mixture is diluted with ether (50 ml) to precipitate trifluoroacetate 6d, which is suspended in water (2 ml). The suspension is adjusted to pH 6 with ammonium hydroxide and diluted with acetonitrile (200 ml) to precipitate compound 6d. Yield 0.75 E (76%).

Sp. 215-220 ° C (decomposes).

: Rf 1τ6 ° · 161 °> 1580 UV-276 nm (1.13U00).

'max.

Anal Calcd for C 12 H 9 N 2 O 2 S 2 · 1 / 2H 2 O: C, 1 + 7.60; H, 5.19; H, 13.87; S, 19.06.

Found: C, 1 + 7.H +, ** 7.23; H, 1 +, 96, 5.07; N, 13.61, 13.70; 3, 18.66.

38 6001 9

Example 5

OH

OCln-L, r

W2H

& L

7- [N- (2-t-Butoxycarbonylaminomethyl-1-cyclohexenyl) -acetamide] -3- (3-hydroxypyrrolidino) -3- (2-7-3-triazol-6-ylthiomethyl) -3-cephem-U-ceronic acid (5e ).

A mixture of compound 2 (1.08 g, U millimoles), 2, U-dinitrophenol (0.7¾ g, k millimoles) and DCC (0.82 6, 1 millimoles) in THF (20 ml), stirred for 1 hour at room temperature and filtered to remove dicyclohexylurea. washed with THF (10 mL). The combined filtrate and washings are cooled to 5 ° C and poured into a cold (5 ° C) solution of 7-amino-3- (3 "hydroxy-pyridazino-5,2'-s-triazol-6-ylthiomethyl) -3- cephem-U-carboxylic acid (1.1¾ g, 3 mmol) and triethylamine (0.8 g, 8 mmol) in 50 2 aqueous THF (20 mL) The mixture was stirred overnight at room temperature and washed with ether (2 x 50 The aqueous layer is acidified to pH 2 with dilute HCl, the precipitate is dissolved in THF (100 ml) at 50 ° C, treated with charcoal and dried over anhydrous H 2 SO 4. which solidifies on stirring with ether (50 ml) to give the product 5e (yield 0.85 g (5%)) ♦ mp 190 ·· 198 ° C (decomposes).

IR: max 178 ° * 17 ° ° '152 ° * 135 ° * 125 ° * 1160 Cra1': 1MR: jCDM3 ° 'd6 1.0 (9H, s, t-Bu-H), 5.20 , d, ppm U Hz, β-Η), 5.80 (1H, m, 7 “H), 7.10 (1H, d, 10 Hz, pyridazine-H), 7.80 (1H, d, 10 Hz, pyridazine-H).

Anal Calcd for C 18 H 18 N 2 O 2 S 2 O 2: 2, C, 50.6 L; H, 5.35; S, 10.01.

Found: C, 50.59, 50.68; H, 0.5 ^ 5.59; S, 9.51, 9.55.

N- (2-aminomethyl-1-cyclohexenyl) -ethamido] -3- (3-hydroxypyridazino- {3,2-c3-3-triazol-6-ylthiomethyl) -3-cephem-U-carboxylic acid (6e)

To trifluoroacetic acid (2 ml) is added compound 5e (0.8 l g, 1.3 mmol) and the mixture is stirred for 30 minutes at room temperature. The mixture is diluted with ether (50 mL) to precipitate trifluoroacetate, which is suspended in water (2 mL).

6001 9 39

The suspension is adjusted to pH 6 with ammonium hydroxide and diluted with acetonitrile (200 ml) to precipitate compound 6e. Yield 0.57 S (82%). Sp. 225-23U ° q (decomposes).

IR: * / nUi011 1770, 1710, 1630, 15 ^ 5 cm -1.

.

Anal, calculated for α22Η25Ιί7 ° 5δ2 * 3H2 °: C '** 5'12' H, 5.3 ^; ίί, lö, 7U; 3, 10.9¾.

Found: C, 1 * 5.28, U5, UU; H, U, 35 »Mol N, 16.56, 16.66; 3, 11.75.

Example 6 CHpNHr »ex ^ cO-N-p-f ^" I N-N ·.

& ^ 11 \ J

7 - [(N-N-butoxycarbonylaminomethyl-1-cyclohexenyl) -acetamide] -3- (pyrido [2,1-b] thiazol-3-ylthiomethyl) -3-phenyl-N-carboxylic acid (5f) DCC (7I + 0 mg, 3.6 mmol) is added to a solution of 950 mg (3.3 mmol) of (t-butoxycarbonylaminomethyl) -1-cyclohexenyl} acetic acid (2) and 660 mg (3.6 mmol). 2.1 + dinitrofen in 30 ml of THF, and the mixture is stirred for 1 hour at room temperature and the urea is filtered off. To the filtrate is added a solution of 1.1 g (3 mmol) of 7-amino-3- (pyrrolo [2,1-c7-s-triazol-3-ylthioethyl) -3-ylthiomethyl) -3 "cephem-U-carboxylic acid (bf ) and 1.25 ml (9 mmol) of triethylamine in 30 ml of water, and the mixture is stirred for 18 hours at room temperature, the THF is removed under reduced pressure below U0 ° C and the residue is washed with ether (2 x 10 ml), acidified with 6N HOl and extracted with ethyl acetate. (5 x 10 ml) The combined extracts are washed with water (2 x 10 ml) and saturated liaCl solution (10 ml) and evaporated to dryness below 0 DEG C. Mixing the residue with ether gives about 1.2 g of solid compound 5f.

uo 6 001 9 7- [2-Aminomethyl-1-cyclohexenyl-acetamide] -3- (pyrido {2,1-cis-triazol-3-yl-thiomethyl) -3-cephem-U-keroic acid (6f)

Trifluoroacetic acid (2.5 ml) is added to the BOC-protected cephalosporin 5f (1.1 e, 1.0 mmol) with stirring over 1.5 hours and cooling, and the mixture is diluted with 100 ml of ether to separate the trifluoroacetate of compound 6f, which is dissolved in 5 ml. to 1 ml of water, adjust to pH 6 with concentrated NK 2 OH and dilute with 100 ml of acetonitrile. The resulting yellow precipitate was collected by filtration and washed with acetonitrile to give about 700 mg (76%) of solid compound 6f.

Example 7 CH2NH2

O

** o 7 - [(2-N-t-butoxycarbonylaminomethyl-1-cyclohexenyl) -acetemido] -3-pyridazino-2,1-g-s-triazol-3-ylthiomethyl) -3-cephem-U-carboxylic acid (5ε)

To a solution of o (-? - (t-butoxycarbonylaminomethyl) -1-cyclohexenyl} acetic acid (2) (1.07 g »U millimoles) and 2, U-dinitrophenol α (0.7 g, 1+ millimoles) in dry THF (10 mL), DCC (0.82 g, 1+ millimoles) is added and the mixture is stirred for 1 hour at room temperature to precipitate urea, which is removed by filtration, and a cold solution of 7-amino-3 -pyridazino (1,1'-cis-triazol-3-ylthiomethyl) -3 "cephem-U-carboxylic acid (Ug) (1.09 g> 3 mmol) and triethylamine (0.8 g, 8 mmol) 50% in aqueous THF (20 mL) and sexed for 20 h at rt The reaction mixture was washed with ether (2 x 50 mL), acidified to pH 2 with dilute hydrochloric acid and extracted with ethyl acetate (6 x 50 mL). the extracts are washed with water (2 x 30 ml), treated with charcoal and dried * 60019 over anhydrous sodium sulphate., and the solvent is evaporated off under reduced pressure to give an oily residue which solidifies on stirring with ether (50 ml) to give about 0.6 g of solid 5g.

7- (2-Aminomethyl-1-cyclohexenyl-acetoido) -3- (pyridazino-1,1-cis-triazol-3-yl-thiomethyl) -3-cephem-β-carboxylic acid (6g)

A mixture of 5g (0.53 g, 0.86 mmol) in trifluoroacetic acid (1 mL) was stirred for 1 h at 0-5 ° C and diluted with ether (30 mL) to precipitate 6g of trifluoroacetate, which was collected. by filtration and dissolved in water (H ml). The solution is adjusted to pH 6 with ammonium hydroxide and diluted with acetonitrile (100 ml) to precipitate compound 6g, which is collected by filtration, washed with acetonitrile (30 ml) and dried in vacuo over P 2 O 2 to give 0.3 g of solid compound 6g.

Example 8 OU1H0 CT »_ Ναι2σοΜ ^ γ ^ Jf | j

6i ^ NNy ^ L "CH2" S '* CV. / CH

COOH S

7-ZTP-tert-butoxycarbonylaminomethyl-1-cyclohexenyl) -acetamido-3- (1,3'-thiadiazole-2-mercaptomethyl) -3-cephem-h-carboxylic acid (5i) To a stirred solution of 1.10 g (0.001 mol) of compound 2 and 0.80 g (0.00 mol) of 2, U-dinitrophenol in 10 ml of ethyl acetate are added in one portion. 0.90 g (0.00UU mol) of N, N'-dicyclohexylcarbodiimide and the mixture. . o.

stirred for 3 hours at 25 ° C. The dicyclohexylurea is recovered and the filtrate is evaporated at U5 ° C (15 ° C) to give the activated ester as an oil. The oil is dissolved in 30 ml of THF. To the solution of the activated ester is added a solution of 1.22 g (0.0037 moles) of 7-amino-3 '' (1,3'-thiadiazole-2-ylcaptoptoethyl) -3 "cephem-h-carboxylic acid and 1.03 nl (0.007 .mu.mol) of triethylamine in 20 ml of 50 .mu.l of THF water, and stirred for 18 hours at 25 DEG C. The TIIF is removed at 0 DEG C. (15 mm) and the H2 O 600 concentrate (10 ml) is washed with ether (5 .mu.l). x 100 ml) and acidified to pH 2 with 1> 0 # phosphoric acid The mixture is extracted with ethyl acetate (6 x 100 ml) and the combined extracts are washed with water and finally with saturated sodium chloride solution The ethyl acetate solution is evaporated at U0 (C) (15 mm) to a volume of 20 ml and diluted with 30 ml of Skellysolve 3 to precipitate a solid product 5i, which is collected and dried under vacuum for 16 hours at Pirilä 25 ° C to give about 1.0 g of product.

Τίβΐ - Aminomethyl-1-cyclohexenyl) ace dQ? * - 3 ~ (1,3 »V ti adi at s ol-2-mercaptomethyl) -3-cephem-U-carboxylic acid (6i)

A solution of 1.30 g (0.00226 mol) of compound 5i and 3.0 ml of trifluoroacetic acid is stirred for 1 hour at 0 ° C. The solution is diluted with 200 ml of ether and the filtrate is collected by filtration. The trifluoroacetate salt is suspended in U0 ml of water and adjusted to pH 6.0 with dilute ammonium hydroxide. The resinous residue is mixed with 25 ml of acetonitrile to give about 350 mg of compound 6i as a white powder. The product is dried for 16 hours under vacuum in Pj, 0.11 at 25 ° C.

Example 9

OC

CHgCOiTir-j — t "'' * 'S N-— jj

q / ^ C-CH2OH

C00H S

7 - [(12-aminomethyl-1-cyclohexenyl) acetamido] -3- (5-hydroxymethyl-1,3,1 * -thiadiazole-2-mercaptomethyl) -3-cephem-h-carboxylic acid (6j)

The procedure of Example 8 is followed except that the product 6j is recovered from water to give about 3 mg of brown powder. The second fraction is obtained from the filtrate to give about 200 mg of compound 6j as a yellow crystalline substance.

ö 60019

Example 10 OG-q- ^ JT "0 // U ^ - ^ 2-s \ n.n

. / H

COgH

6h 7- (2-t-Butoxycarbonylaminomethyl-1-cyclohexenyl) -acetamido} -3-1H-1,2,3-triazol-U-ylthiomethyl) -3-cephem-U-carboxylic acid (5h )

A mixture of compound 2 (1.08 g, U millimoles), 2, U-dinitrophenol (0.7 U e. Millimoles) and DCC (0.82 g, U millimoles) in THP (20 ml) is stirred for 1 hour. at room temperature and filtered to remove dicyclohexylurea, which is washed with THF (10 mL). The combined filtrate and washings are cooled to 5 ° C and poured into a solution of 7-amino-3- (1H-1,2,3-triazol-U-yl-thiomethyl) -3-cephem-U-carboxylic acid (Uh). (1.2 U, U mmol) and triethylamine (0.8 g, 8 mmol) in 50% aqueous THF (20 mL). The mixture is stirred overnight at room temperature and washed with ether (2 x 50 mL). The aqueous layer is acidified to pH 2 with dilute HCl and extracted with ethyl acetate (3 x 50 mL). The combined extracts are washed with water (50 ml), treated with charcoal and dried over anhydrous Na 2 SO 4. Removal of the solvent under reduced pressure gave an oil which solidified on stirring with ether / n-hexene (1: 1, 100 mL) to give the product 5h. Yield 0.60 g (26%). Sp. 120-128 ° C (decomposes).

1780, 1720, 1680, 1520, 1250, 1160 cm -1.

NMR: λmax · 1.38 (9H, s, t-Bu-H), 5.08 (1H, d, U Hz, 6-H), 5.65 (1H, dd, U and 8 Hz .7 * H), 8.00 (1H, s, triazole-H), 8.92 (1H, d, 8 Hz CO11H).

Anal Calcd for C 21 H 32 N 6 O 6 S 2 C »5 °» 25i H, 5.80; N, lU, 65; S, 11.18.

Found: C, 50.51, 50.69; H, 5.73, 5.65; N, IU.57, 1U, 25; S, 10.05, 10.16.

»60019 ω 2- [¾ (2-Aminomethyl-1-cyclohexenyl) acetamido) -3- (1H-1,2,3-triethol-h-ylthiomethyl) -3-cephem-4-carboxylic acid (6h)

To trifluoroacetic acid (1 mL) is added compound 5h (0.55 g> 0.98 millinol) and the mixture is stirred for 30 minutes at room temperature. The mixture is diluted with ether (50 mL) to precipitate trifluoroacetate, which is collected by filtration and suspended in water (2 mL). The suspension is adjusted to pH 6 with ansionium hydroxide and diluted with acetonitrile (200 ml) to precipitate compound 6h, which is washed with acetonitrile. Yield 0.36 g (80%). Sp. 203-215 ° C (decomposes).

IR: / NUi ° 1: L 1760, 1630, 1570 cm -1.

UV: λ max K 2 O 3 266 nm ((, 8000).

max.

Anal, calculated for C 18 H 19 N 2 O 3 S 2. 1 1/2 11 ^ 0: C, U6.U2; K, 5.51 *; N, 17.10; S, 13.0U.

Found: C, 1 »6.06, H6, 16; H, 5.13, 5.28; N, 18.06, 18.02; S, 12.37.

Example 11 7 - {[2- (8-N-methyl-1-3-cyclohexenyl) -acetamide] -3- (3-hydroxypyridazine- (3,2-9β-triazol-1-yl-thiomethyl) -3-cephem-U-carbonic acid) salicylaldehyde adduct (7c)

HO

V)

~> k ^ jk ^ CO-NH-j-S-jJJ

J-IL-2-H2-S-kN / N

öo2K ch5

To a stirred suspension of 6c (Example 3, 766 mg, 1.6 mmol) and triethylamine (300 mg, 3 mmol) in methanol (8 mL) is added salicylaldehyde (370 mg, 3 mmol) and the mixture is stirred for 30 minutes at room temperature until clear. to obtain a dark yellow solution. The solution is treated with a small amount of carbon and KEH (2 mL, 1M solution of potassium 2-ethylhexanoate in ethyl acetate) is added to the filtrate. The mixture is diluted with a large amount of ether (100 ml) to precipitate solid product 7c, which is collected by filtration, washed with ether (30 ml) and dried. The yield is about 750 mg.

, s 6001 9 Ιί5

Example 12 7- (T2-Aminomethyl-1-cyclohexenyl) -acetamid97-3 * (5-methyl-1,3,3-thiadiazol-2-ylthiomethyl) -3-cephem-U-carboxylic acid salicylaldehyde - dduk Tue

. (7d) .HO

64—>. . H — s y / U! J_Ch2-sj ^ SJ-ch? , TS. C0aK,

To a stirred mixture of 6d (Example U., * 778 mg, 1.6 mmol) and triethylamine (300 mg, 3 mmol) is added salicylaldehyde (370 mg, 3 mmol) and the suspension is stirred for 15 minutes at room temperature until clear. to obtain a dark yellow solution which is treated with a small amount of carbon. KEH (2 mL, 1M solution in ethyl acetate) is added to the filtrate. The mixture is diluted with a large amount of ether (100 ml) to precipitate product 7d, which is collected by filtration, washed with ether (30 ml) and dried. The yield is about 85Ο mg.

Example 13 7- (2-Aminomethyl-1-cyclohexenyl) acetamido-3- (3-hydroxypyridazino-, 2-s-triazol-6-ylthiomethyl) -3-cephem-U-carboxylic acid 3alicylaldehyde adduct (7c )

> = \ 0H

IS C02K

To a stirred suspension of compound 6e (Example 5, 75 U mg, 1.1 U 2 mol / l) and triethylamine (300 mg, 3 mmol) in N, dimet-dimethylformamide (7 μl) was added salicylaldehyde (370 mg, 3 mmol). ) and the suspension is stirred for 1 1/2 hours at room temperature to give a clear dark yellow solution which is treated with a small amount of carbon. KEII (1.5 ml, 1M solution in ethyl acetate) is added to the filtrate. The mixture is diluted with a large amount of ether (100 ral) to precipitate the product Te, which is washed with ether (30 ml) and dried. The yield is about 900 mg.

Example lU

The potassium salt of the compound of Example 3 is prepared by adding a solution of potassium 2-ethylhexanoate (XEI1) in ethyl acetate to a (amphoteric) solution of cephalosporin (6c) in DMSO to precipitate the desired potassium salt.

Example 15

The sodium salt of the compound of Example 3 is prepared by first forming its diethylammonium salt in methanol and then adding a solution of sodium 2-ethylhexanoate (SFH) in ethyl acetate and then diluting with isopropanol to precipitate the desired sodium salt having a solubility in water greater than 250 rag / ml.

Example 16 7 - [(2-t-Butoxycarbonylamino-ethyl-1-cyclohexenyl) -acetamido} -cephalosporanic acid (5v) rr * -N-LcH2 "CC0CH5

6w C ° aH

A mixture of compound 2 (1.08 g, 1 * mmol), 2, U-dinitrophenol (0.7 * g, U mmol) and DCC (0.82 g, U mmol) in THF (20 mL ), stirred for 1 hour at room temperature and filtered to remove dicyclohexylurea, which is washed with THF (10 mL). The combined filtrate and washings are poured into a solution of 7-ACA (Uv) (0.8 L g, 8 mmol) and triethylamine (0.8 L g, 8 mmol) in 50% aqueous THF (20 mL). 5 ° C. The mixture is stirred overnight at room temperature and washed with ether (2 x 50 mL). The aqueous layer is acidified to pH 2 with dilute HCl and extracted with ethyl acetate (3 x 50 mL).

„60019

The combined extracts are washed with water (50 ml) and dried over anhydrous Na 2 SO 4. Removal of the solvent under reduced pressure gives 5W as an oil which solidifies on stirring with ether / n-hexane (1: 1, 100 ml) and is washed with n-hexane (50 ml). Yield 1.2 U g (79%) · M.p. 100-110 ° C.

: R: / aujo11 1780, 1730 - 16U0, 1520, 1355, 1225 en * 1, naks.

.7MR: δ DMSO "d6 1.23 (9H, s, t-Bu-H), 1.96 (3H, s, 3" OAc), U, U8 ppin (1H, d, 13 Hz, 3-CH ), U, 83 (1H, d, 13 Hz, 3- (¾), U, 89 (li, d, h Hz, 6-Ii), 5, U8 (1H, dd, it and 8 Hz, 7 1 H), 8.5 (1H, d, 8Hz, CONH).

Anal. Calcd for C 18 H 19 N 2 O 2 S 2 O 3 S / P 1 O 2: C, 5, 12; H, 6.1 * 3; N, 7.88; S, 6.02.

Found: C, 5U, 16.5U, 07; H, 6.19, 6.22; N, 8.36, 8.31 *; S, 5.61, 5.79.

7- [N- (2-Aminomethyl-1-cyclohexenyl) acetamide] -7-cephalosporanic acid (6v)

To trifluoroacetic acid (2 mL) is added compound 5v (1.20 g, 2.3 mmol) and the mixture is stirred for 30 minutes at room temperature. The mixture is diluted with ether (50 mL) to precipitate trifluoroacetate, which is suspended in water (2 mL). The mixture is adjusted to pH 6 with ammonium hydroxide and diluted with acetonitrile (200 ml) to precipitate compound 6w. Yield 0.68 g (69%). Sp. 250-260 ° C (decomposes).

IR: / nU ^ 011 1800, 1735, 1625, 1570, 1230 cm-1, max.

UV: 255 nm (ε, 7100).

sweet.

Anal. Calcd for C 18 H 18 N 2 O 2 • 3.1 / 2 H, 6.05; N, 9.71; S, 7, Ui.

Found: C, 52.36, 52.15; H, 6.09 »6, Ob; N, 9.7U, 9.71, 3, 7.71, 7.81.

The solubility ratios were 5 »U (BA) and 5.3 (UV).

is 6001 9

Example 17 aCH2NH2

CHgC O-H r ...... S 'SX

JCHg-S- ^ -OH

CO H

7a

2-t-Butoxycarbonylaminomethyl-1,1-cyclohexadienyl) -acetamide N- (3- (3-hydroxypyridazin-6-yl) isomethyl) -3-cephem-h -carboxylic acid ( 6a)

To a stirred solution of 1.1 g (H, 1 millimole) of o - (E - (t-butoxycarbonylaminomethyl) -1,10-cyclohexadienyl] -acetic acid (3) and 0000 mg (t, U millimole) 2, U-dinitrophenol in 1 ml of ethyl acetate, 0.9 g (U, 4 mmol) of Ν, Ν-dicyclohexylcarbodiimide (DCC) are added in one portion. to obtain an oily activated ester which is dissolved in 30 ml of DHF A solution of 1.26 g (3.7 milol-1 mol) of 7-amino-3- (3-hydroxypyridazin-6-ylthiomethyl) -3- k ephememe-U-carbon i- 6001 9

It9 acid (5a) and 1.03 ml (7.1 * mmol) of triethylamine in 20 ml of 50 * THF. The mixture is stirred at room temperature for 18 hours and concentrated in vacuo to a volume of 10 ml. The concentrate is washed five times with 100 ml portions of ether, acidified with 6 l of hydrochloric acid and extracted six times with 100 nl portions of ethyl acetate. The combined extracts are washed with water and saturated IfaCl solution. The dried solution is concentrated to dryness to give 720 mg (33%) of il-BOC-protected cephalosporin 6a, m.p. 170 ° C (decomposes).

IR: max 1 TO 0, 171 ° '169 ° * 167 ° »158 °' 152 ° '187 °' 1255> 1170 1.3¾ (9H, s, CH -C ~ 1.27 (UH, s, H 2 C ^), 3.03 (2H, s, CH 2 CO), 3.1 * - 3.7 (1 + H, m, 2-H <k CH 2 -J), 3.9 - b, 2 (2H, m , 3-CH 2), U, 98 (1H, d, U, 5Hz, 6-H), h, 9 - 5.2 (3H, m, & C-NK), 6.29 (1H, d, 9 .5Hz, pyridazine-H), 7.23 (1H, d, 9.5Hz, pyridazine-H), 8.77 (1H, d, 7.5Hz, CONH).

Anal. Calcd for C26H31: I-7S2 * H2O: C> 51.56; II, 5.16; If, 11.56; S, 10.59.

Found: C, 51.3 H 3, 5.13; N, 11.66; S, 10.79.

7 - [(2-Aminomethyl-1,1'-cyclohexadienyl) -acetamide? -3 "(3-hydroxypyridazin-6-ylthiomethyl) -3-cephem-U-carboxylic acid (7a) (Λ) Solution containing 670 mg (1.13 mmol) of BOC-protected cephalosporin 6a in 1.5 ml of trifluoroacetic acid, stirred for 1 hour at 0 [deg.] C. 100 ml of dry ether are added to the solution and the precipitate obtained is collected by filtration and suspended in 20 ml of water. and adjusted to pH 6 with dilute ammonium hydroxide to give a viscous oil which is mixed with acetonitrile to give 3δθ mg (75.5%) of the desired product 7 ** »9P« 200-205 ° C (decomposes).

IR: 1765, 161 * 0, 1580, 1390, 1350 cm-1, make ·

Anal Calcd for C 21 H 23 I 5 ° 5 S 2 · 1 · 2H 2 O: C, 50.59; H, M5; H, li », 05.

Found: C, 50.95; H, U, 79; N, 13.10.

(B) To a stirred suspension of 1.1 g (3.6 mmol) of sodium (2-? N- (1-carbethoxypropen-2-yl) aminomethyl] -1,1-cyclohexadienyl) acetate (1 ») 20 In 1 ml of dry tetrahydrofuran (TIIF) containing a drop of ΓΙ, Ν-dimethylbenzylamine, 0.38 ml (3.95 mmol) of ethyl chloroformate are added at 50 6001 9 -10 to -5 ° C. To the mixed anhydride solution is added in one portion a solution of 1.02 g (3 mmol) of 7 ', N, N-cyclohexadienyl-acetamido-3- (3-hydroxypyridazin-6-ylthiomethyl) -3- cefera-1 + carboxylic acid (5a) in 10 ml of 50 .mu.l of aqueous THF containing 0.1 + 2 ml (3 mmol) of triethylamine. The mixture is stirred for 30 minutes at room temperature. Treat with activated carbon and filter. 3 ml of formic acid are added to the filtrate, and the mixture is stirred for 30 minutes at room temperature. The resulting precipitate is filtered, washed with water and acetonitrile to give 570 mg of compound 7a.

Example 18 aCHgNHg

H * M

ch „co-Nt — f" i JM * | 0 ^ VLcVs <==> ^

COgH

TSL

7 - (β- (N-t-butoxycarbonylaminomethyl) -1H-cyclohexadienyl) betamido) -3- (tetratol [5-b] pyridazin-6-ylthiomethyl) -3-cephem-1-carboxylic acid (6b)

To a stirred solution of 3.2 g (0.012 mol) of ck (2- (t-butoxycarbonylaminomethyl) -1U-cyclohexadienyl-4-acetic acid (3) and 0.3 g (0.0125 mol) of 2, U-dinitrophenol 50 in THF, 2.6 g (0.0125 mol) of DCC are added and the mixture is stirred for 1.5 hours at room temperature, the precipitated urea is removed by filtration, and a solution of 3.65 g (0.01 mol) of ) 7-Amino-3 "(tetrazolo [1,5-b] pyridatein-6-ylthiomethyl-3-cephem-β-carboxylic acid (5b) and 2.3 ml (0.02 mol) of triethylamine in 100 ml of water The mixture is stirred for 18 hours at room temperature. Most of the THF is removed under reduced pressure below i * 0 ° C and the remaining solution is washed with ether (3 x 50 ml) and acidified with 6.i HCl and extracted with ethyl acetate (8 The combined extracts are washed with water (3 x 100 ml) and saturated NaCl solution (2 x 100 ml) and evaporated to dryness, the oily residue is taken up in ether to give 2.2 g (52; seat 6b, m.p. 165 ° C (decomposes).

51 6001 9 s IR: / nu-011 1770, 1710, 1670, 1510, 121 * 0, 1150 cm ”1, naks«

Anal Calcd for C 26 K 30 N 8 O 622: 50.18; H, U, 92; N, 18.23; S, 10.1 * 3.

Found: C, 51.05; H, 5.97; N, 17.2U; S, 9.09.

7- [2-Aminoacetyl-1,1 * -cyclohexadienyl) acetamide [7- (tetrazolo [1,5-b] pyridazin-6-ylthioethyl) -3-cephem-1 * carboxylic acid (7b)

A mixture of 3.1 g (5.05 millinoles) of compound 6b and 6 nl of trifluoroacetic acid is stirred for 1 hour while cooling and diluted with 200 ml of ether to separate trifluoroacetate 7b which is dissolved in 5 ml of water, the pH is adjusted to 6 with concentrated 1 H 2 O and diluted with 900 mL of acetonitrile. The yellow precipitate was collected by filtration and washed with acetonitrile to give 2.3 g (88.5%) of compound 7b, m.p. 210 ° C (decomposes). Crystallization of 2 g of compound 7b from 350 ml of 50) 5 gives 1.19 g of pale yellow needles, m.p. 220-230 ° C (decomposes).

IR: ν ° U 1785. 161.0, 1610, 1570 cm-1.

^ Al. Calculated for 1/20 2 O: C, 50.17; 1, 76; j, 20.35; s, 11.61 *.

Found: C, 50.17; H, 1 *, 56; N, 19.59; S, 11.30.

Example 19

Ο ^ α ^ ΟΟ-Ν-τ-γ3 ^ K — N

0 COgH (¾ Ζίϊ.

7 * (N 2 - (N-t-butoxycarbonylaminomethyl) -1,1-cyclohexadienyl-7-acetamido) -3- (1-ethyltetrazol-5-ylthiomethyl) -3'-cephem-U-carboxylic acid (6c).

To a stirred solution of 3.2 g (C, 012 moles) of (t-butoxycarbonylaninomethyl) -1,1 * -cyclohexadienyl} 7-acetic acid (3) and 2.3 g (0.012 moles) of 2,1-dinitrophenol In 50 ml of THF, 2.6 g (0.012 mol) of DCC are added and the mixture is stirred for 1.5 hours at room temperature. The precipitated urea is removed by filtration. To the filtrate is added a solution of 3.28 g (0.01 mol) of 7-8-amino-3- (1-methyl-tetrazolo-5-ylthiomethyl) -3-cephem-U-carboxylic acid (5c) and 2.3 ml (0, 02 moles) of triethylamine in 100 ml of water. The mixture is stirred for 18 hours at room temperature. Most of the THF is removed under reduced pressure, below 10 ° C and the remaining solution is washed with ether (2 x 50 mL) and acidified with 6N HCl and extracted with ethyl acetate (h x 50 mL). The combined extracts are washed with water (2 x 50 ml) and saturated NaCl solution (2 x 50 ml) and evaporated to dryness. The residual residue is mixed with ether (100 ml) to give 2.3 g (1 * 3%) of compound 6c, m.p. ll * 5 ° C (decomposes).

IR: / nUj011 1780, 1700, 1510, 12U0, 1150 cm-1.

TSlcLK S ·

Anal Calcd for C 18 H 19 N 2 O 2 S 2 S 2: C,> +9.90; H, 5.1 * 1; J, 16.97; S, 11.10.

Found: C, 50.23; H, 5.23; N, 15.80; S, 12.1 * 3.

7- [2-Aminomethyl-1,1'-cyclohexadadienyl) acetamide] -3- (1-methyltetrazol-5-ylthiomethyl) -3-cephem-1 * carboxylic acid (7c).

A mixture of 2.2 g (3.8 mmol) of compound 6c and 5 ml of trifluoroacetic acid is stirred for 1 hour under the same cooling and diluted with 200 ml of ether to separate trifluoroacetate 7c, which is dissolved in 5 ml of water, the pH is adjusted to 6 with concentrated NH 4 OH and diluted with 200 mL of acetonitrile. The yellow precipitate is collected by filtration and washed with acetonitrile to give 1.5 g (82 g (82.5%) of compound 7c, mp 200 ° C (decomposes).) Crystallization from 50 .mu.l of 1HF gives pale yellow needles, m.p. 210 ° C (decomposes).

IR: V ^ nUi011 1785, I6U0, I615, 1570 cm-1, rustics ·

Anal, calculated for C 11 * 9.11; H, 5.30; d, 19.09; 3, 12.1 * 8.

Found: C, * * 9.85; II, 5.20; N, 18.53; S, 12.26.

! '6001 9! 53! • f i t

Example 20 j

° C02H

Zä.

7 - ([2- (N-t-butoxycarbonylaminomethyl-tert-cyclohexadienyl) -acetamide] -3- (5-methyl-1,3,3-thiadiazol-2-ylthiomethyl) -3-cephem-α-carboxylic acid (6d)

To a stirred solution of 900 mg (3.3 mmol) of (t-butox8 * · carbonylaminomethyl) -1,1 + -cyclohexadienyl-4-acetic acid (3) and 66Ο mg (3.3 mmol) of 2,1 + -dinitrophenol 1 + 0 in 1 ml of THF, 7 → 0 mg (3 * 6 mol) of DCC are added and the mixture is stirred for 2 hours at room temperature. The precipitated urea is removed by filtration. To the filtrate is added a solution of 1.03 g (3 mmol) of 7-amino-3- (5-methyl-1,3,1-thiadiazol-2-ylthiomethyl-3-cephem-U-carboxylic acid (5d) and 1 , 3 ml (9 mmol) of triethylamine in 30 ml of water, the mixture is stirred for 18 hours at room temperature, the THF is removed under reduced pressure below 1 + 0 ° C and the remaining solution is washed with ether (3 x 10 ml) and acidified with 6.1 HCl and extracted with ethyl acetate (5 x 10 ml) The combined extracts are washed with water (2 x 10 ml) and saturated NaCl solution (10 ml), evaporated to dryness and stirred with ether to give 980 mg (55%) of compound 6d.

IR: / nUfo11 1770, 1710, 1660, 1610, 1510, 12fc0, 1150 cm -1.

IQoLKS ·

Anal Calcd for C 25 H 31 N 5 O 6 S 3: c> 50.57; H, 5.26; N, 11.80.

Found: C, 50.51; H, 5.20; N, 11.1 + 6.

7-Aminomethyl-1,3 * -cyclohexadienyl) acetamido-3- (5-methyl-1,3,1 * -thiadiazol-2-ylthiomethyl) -3-cephem-H-carboxylic acid (Jd) (A ) A mixture of 9 x 0 mg (1.58 mmol) of compound 6d and 2 ml of trifluoroacetic acid is stirred for 1 hour under cooling and diluted with 100 ml of ether to separate trifluoroacetate 7d, which is dissolved in 5 ml of water and 5 ml of acetonitrile:. The pH is adjusted to 6 with concentrated NH 4 OH and diluted with 100 ml of acetonitrile. The pale yellow precipitate was collected by filtration and washed with acetonitrile (5 ml) to give 600 mg (77.5%) of compound 7d, m.p. 230-2l * 0 ° C (decomposes).

5, 6001 IR: V nujo11 1790, 1650, I58O ca "1.

max.

Anal Calcd for C 18 H 12 N 2 O 2 S 2 S: C, U 8.66; H, U, 70; N, lU, 19i S, 19.1 * 9.

Found: C, U9.2U; H, U, 59; B, 13.88; S, 20.16.

(B) To a stirred suspension of 1.1 c (3.6 mmol) of sodium (2- [1- (1-carbethoxypropen-2-yl) aminonethyl] -1,1 * -cyclohexadienyl) acetate (1 *) 0.3 ml (3.95 mmol) of ethyl chloroformate are added at -10 to -15 [deg.] C. in 1 ml of dry tetrahydrofuran (THF) containing a drop of N, N-dimethylbenzyl azine. To the mixed anhydride solution is added in one portion a solution of 1.03 G (3 mmol) of compound 5d in 10 ml of 50% aqueous THF containing 0.1 U2 ml (3 mmol) of triethylamine. The mixture is stirred for 30 minutes at room temperature. A few drops of formic acid are added to the mixture and the mixture is filtered. To the filtrate are added 200 ml of ether and 3 ml of formic acid. The precipitate is collected by filtration, washed with water and cetonitrile to give 1.1 g (62%) of compound 7d.

Example 23.

7 - (Z2- (± -butoxycarbonylaminomethyl-1, 2-cyclohexadienyl) -acetemid-3 * (3-hydrocapyridazinoyl-2'-s'-triazol-6-ylthiomethyl) -3-yl .phemene-k-carboxylic acid (6e)

pH

ί jT H Ln

2-S- () = H

O bo2H ^ == 6

You

To a stirred solution of 900 mg (3.3 mmol) of N, N-2- (t-butoxycarbonylaminomethyl) -1,1 * -cyclohexadienyl-7-acetic acid (3) and 660 mg (3.6 mmol) 2, U -dinitrophenol in 30 ml of THF, 7 ** 0 mg (3.6 mmol) of DCC are added and the mixture is stirred for 2 hours at room temperature. The precipitated urea is removed by filtration. To the solution of the activated ester is added a solution of 1.13 g (3 mmol) of 7-amino-3 "(3-hydroxypyridazino] 5,2-s7-s * triazol-6-ylthioine-style-3-cephem-β-carboxylic acid (5e) and 1.25 ml (9 mmol) of triethylamine in water, stirred for 18 hours at room temperature, the THF is removed under reduced pressure below U0 ° C and the noble solution is washed with ether (2 x 10 ml) and acidified with 6ii HCl: The combined extracts are washed with water (2 x 10 ml) and saturated NaCl solution (10 ml) and evaporated to dryness, the residue is taken up in ether to give 505 mg (27%) of compound 6e, mp 175-180 ° C (dec.).

"60019 55 IR: v / nujo11 1770, 1700, 1510, 1200, 1150 cm-1".

Anal Calcd for C H, U, 96; n, 15.57; s, 10.18.

Found: C, 51.61; H, U, 80; N, IU, U7; S, 10.25.

7 * Q-aminomethyl-1,1'-cyclohexadienyl) acetemido-3- (hydroxypyridazino- [3,2-b] s-triazol-6-ylthiomethyl) -3-cephem-U-carboxylic acid (7e)

A mixture of U70 mg (0.7-7 mmol) of compound 6e and 1 ml of trifluoroacetic acid is stirred for 1 hour under cooling and diluted with 100 ml of ether to separate trifluoroacetate 7e, which is dissolved in 5 ml of water, the pH is adjusted to 6 with concentrated NH 4 O and diluted with 100 μl of acetonitrile. The yellow precipitate is collected by filtration and washed with acetonitrile to give 360 mg (91%) of compound 7e, mp 210-220 ° C (dec).

IR: / nujo11 1760, 1710, 16U0, 1580 cm-1.

Anal Calcd for c22H23ii705S2, H2O: C, ** 8.25; H, U, 60; il, 17.91; 3, 11.71.

Found: C, U8.75; K, 1 + .60; II, 16.56; S, 12.17. '

Example 2g.

CH2NH2

U-U H / SN

^ C ^ ^ CH2CO-N * p- ^ ^ / N

N, O-CO 2 H- [7 - ([2- (Nt-butoxycarbonylaminoethyl-1,1'-cyclohexadienyl) acetamido] -3- (pyrido [2,1-c] -s-triazole-3 - ylthiomethyl) -3-cephem-U-carboxylic acid (6f)

To a solution of 950 mg (3.3 mmol) of o (, -? 2- (t-butoxycarbonylaminonethyl) -cyclohexadienyl] -acetic acid (3) and 660 mg (3.6 mmol) of 2,1 * -dinitrophenol a 30 In 7 ml of TIIF, 7 ** 0 ng (3.6 moles) of DCC are added and the mixture is stirred for 1 hour at room temperature, the precipitated urea is removed by filtration, and a solution of 1.1 g (3 mmol) of 7-emino-3- (pyrido [1,1-c] -5-triazol-3-ylthiomethyl-3 * cephetene-U-carboxylic acid (5f) and 1.25 ml (9 nilinols) of triethylamine in 30 ml of water. The mixture is stirred for 18 hours at room temperature. The TIIF is removed under reduced pressure below U0 ° C and the 56,6009 solution remaining on step 11c is washed with ether (2 x 10 mL) and acidified with 6 'HCl and extracted with ethyl acetate (5 x 10 mL). x 10 ml) and saturated NaCl solution (10 ml) and evaporated to dryness below 10 DEG C. The residue is taken up in ether to give 1.15 g (63 DEG C.) of compound 6f, m.p.

ip.jnujoii i630, 1510 i2Uo, 1150 cm -1.

f max.

Anal. Calcd for 0.5: 89; H, 5.26; II, 13.72; S, 10, UT.

Found: C, 53.8 U, H, 5.13; N, 13.12; S, 9.26.

N- (% -aminomethyl-1,1 * -cyclohexadienyl) acetamido [3 "(pyrido [5,1-g] -s-triazol-3-ylthiomethyl) -3-cephem-U-carboxylic acid (7f) 2, 5 ml of trifluoroacetic acid are added to BOC-protected cephalosporin 6f (1.1 g, 1.8 nillimoles) and stirred for 1.5 hours under cooling and diluted with 100 ml of ether to separate trifluoroacetate Jf, which is dissolved in 5 ml of water, the pH is adjusted to 6 with concentrated NH 4 OH and diluted with 100 mL of acetonitrile The yellow precipitate is collected by filtration and washed with acetonitrile to give 700 mg (76%) of 7f, mp 200-210 ° C (dec).

^^ mäs11 1790 '1660 »l63 °» 1580 e® ”1 ·

Anal Calcd for C22H24N6O1 * S2'H2O: C * $ 52.9; : ·, U, 33; 11, 16.11; 3, 12.29. Found: C, 5.3.08; H, U, 65; H, 15.16; 3, 12.29.

Example 23

B

- · CHoCO-N-r-f> N— N

· (O-α2-U-t-butoxycarbonylaminomethyl-1,1'-cyclo] lexadienyl) -acetamide (-3-pyridazino) -2,1-g7-8-triazol-3-ylthiomethyl-3-cephem-14-carboxylic acid (6ε).

To a solution of dc Q- (t-butoxycarbonylaminomethyl) -1H-cyclohexadienyl / acetic acid (3) (1.07 g, 1 mmol) and 2,1-dinitrophenol (0.7 U g, 1 mmol) in dry THF (10 mL), DCC (0.82 g, U mmol) is added? 60019 and the mixture is stirred for 1 hour at room temperature to precipitate the urea, which is removed by filtration. To the filtrate is added in one portion a cold solution of 7 "amino'3-pyridazino (1,1'-pyriazol-3-ylthiomethyl) -3" cephem-U-carboxylic acid (5c) (1.09 g → 3 mmol). ) and triethylanine (0.8 L, 8 mmol) in 50 overnight aqueous THF (20 mL), and the mixture is stirred for 20 hours at room temperature. The reaction mixture is washed with ether (2 x 50 mL), acidified to lead 2 with dilute hydrochloric acid and extracted with ethyl acetate (6 x 50 mL). The combined extracts were washed with water (2 x 30 mL), treated with charcoal and dried over anhydrous sodium sulfate. Evaporation of the solvent under reduced pressure gave an oily residue which solidified on stirring with ether (50 ml) to give 0.63 g (3U%) of compound 6g, m.p. 202-213 ° C (decomposes).

IR: ν max 11 1785 »1780» 1520 »1250» 1160 «h" 1 · NMR:? (1H, d, UHz, 6-H), 5.67 (3H, br-s, 7Ή & ^ H =), 7.20 - 8.90 (3H, m, pyridazine-H), 9.05 ( 1H, d, 8Hz, HHCO).

Anal Calcd for C 9 H 8 N 2 O 2 S 2 S 2 • H 2 O: C, U 8.08; ia, U, 95; N, 17.83; S, 11.67- Found: C, U8.32; H, 3.9; 17.19, S, 11.90.

7 * (2-Aminomethyl-1,1'-cyclohex8-dienyl-acetamido) -3- (pyridazino-1,1'-cis-triazol-3-ylthioethyl) -3-cephem-U-caronic acid (7g).

A mixture of 6g (0.53 g, 0.86 mmol) and trifluoroacetic acid (1 mL) is stirred for 1 h at 0-5 ° C and diluted with ether (30 mL) to precipitate trifluoroacetate 7g, which is collected by filtration and dissolved in water (U ml). The solution is adjusted to pH 6 with ammonium hydroxide and diluted with acetonitrile (100 ml) to precipitate compound 7g, which is collected by filtration, washed with acetonitrile. (30 ml) and dried in vacuo over P 2 O 4. Yield 0.38 g (86%), m.p. 208-216C (decomposes).

IR: H011 1780, 1620, 1570, 13 ** 0 cm -1.

ground UV: 71 ^ 2 ^ 3. 276 nm (£ 5600).

Anal Calcd for 7 ° US2'K2O: c * 51.33; H, 5.27; H, 15.52; 3, 10.15.

Found: C, 51.00; H, 5.10; N, 15.00; S, 10.12.

58 6 0 01 9

Reaction scheme for Examples 2k, 25 and 26 f1? j} ^ -CHgCOOH -} _ CHj-C-O-C-Nj _ ^ k · O CH,

11 1 A

/ —Tt-CH “NH-C-O-C-CH ..“ τ I

CK «y i, / + 2 no2 3 4- ho ^ ko2 + <J) -N; c; nh ^)

DCC

CH2-NH-C-O-C (CH) r - / O NO2 0 w "no2 o

A

o

II

A + ^, Α, ®2-νη-ο-ο-ο (οη3) 3 - 0ÄJ-ch2-s-r — 5-

COOH 0 o ^ LÄ> fL “CH2-S-R

COOH

2 6 - 59 60019, -Λ® * 6 - + - CP, CO „H -> Λ ycHo-C-NH-r-f ^ 'l> 2 ^ - / 2 H ^ Lk ^ c ^ -s.u

£ COOH

„K'5 '!! t * W * fY ’

N-N N-H -N

A 1 h.

f *. · 6001 9

Example 2h if.

<Μ2 < »ΝΗτ-j | } [

COOK

4 · · * * “21 7 - [N-tert-butoxycarbonylaninomethyl-1,1 + -cyclohexadienyl) -8-acetane-3- (1,3,3-k-thiadiazole-2-mercaptamethyl) -3-cephem-U carboxylic acid (6i)

To a stirred solution of 1.10 g (0.001 + 1 mol) of compound 3 and 0.30 g (0.0Q + 1 + mol) of 2,1 + - diitrophenol a in 1 + 0 ml of ethyl acetate is added in one portion A mixture of 0.90 g (0.001 + 1 + mol) of N, N'-dicyclohexylcarbodiimide and 8 is stirred for 3 hours at 25 ° C. The dicyclohexylurea is collected and the filtrate is evaporated at + 5 ° C (15 mm) to give the activated ester as an oil, the oil is dissolved in 30 ml of THF. To a solution of the activated ester is added a solution of 1.22 g (0.0037 moles) of 7-amino-3- (1,3,1 + “thiadiazole-2-mercaptomethyl) -3 * cephera-1 + carboxylic acid and 1, 03 ml (0.0071 * mol) of triethylamine in 20 ml of 50 2 THF water, and stirred for 18 hours at 25 ° C. The THF is removed at U0 ° C (15 mm) and the concentrate (10 mL) is washed with ether (5 x 100 mL) and acidified to pH 2 with 1 + 0 2 phosphoric acid. The mixture is extracted with ethyl acetate (6 x 100 ml) and the combined extracts are washed with water and finally with saturated sodium chloride solution. The ethyl acetate solution is evaporated at 1 ° C (15 mm) to a volume of 20 ml and diluted with 30 ml of Skellysolve B. The product 6i is collected and dried for 16 hours in vacuo over P 2 O 4 at 25 ° C to give 1.1+ g (65.1+%) of the product as a white powder. The IR and NMR spectra correspond to the structure. Sp. 135 ° C, decomposes slowly.

7 - [(2-aninomethyl-1,1 + -cyclohexadienyl) -8-etamide-3- (1H-thiazole-2-mercaptomethyl) -3-cephem-1 + carboxylic acid (7i)

A solution of 1.30 g (0.00226 mol) of compound 6i and 3.0 ml of trifluoroacetic acid is stirred for 1 hour at 0 ° C. The solution is diluted with 200 ml of ether and the precipitate is collected by filtration. The trifluoroacetate salt is suspended in a 1 + 0 strip of water and. adjusted to pH 6.0 with dilute ammonium hydroxide.

61 6001 9

The resinous residue is mixed with 25 ml of acetonitrile to give 370 nig (3.5) of the compound as a white powder. The product is dried under vacuum for 16 hours at 25 ° C. The IR and NMR spectra correspond to the structure. Sp. 135 ° C, (decomposes slowly. Anal. Calcd for ν 9 ^ 21 ^ 5 ^ 1 ^ 3 * H 2 O: C »** 5.85; H * 1», 65; H, 1U.07.

Found: C, 1.71i H, 1 », 52; N, Ib, 12.

The equilibrium solubility of the compound in mg / ml was U, 28 in distilled water and 5.85 in pH 7.0 phosphate buffer.

Example 25 ny \ ^ xCH2CONHi {> n n

/ -N 2 O 2 -S-S 2 -C 2 CH 2 OH

° COOH

• · ·· * ’*

II

7 - [(2-Allylmethyl-1,1'-cyclohexadienyl) acetamido] -3- (5-hydroxymethyl-1,3'-thiadiazole-2-mercaptomethyl) -3-cephem-U-carbonic acid (7j)

The procedure of the example is followed, but the product is recovered from water to give 3U0 mg (29.8%) of a brown powder. The second fraction is obtained from the filtrate to give 230 mg (20.0%) of compound 7j as a yellow crystalline hemihydrate. The IR and HMR spectra correspond to the structure, the melting point is 150 ° C while slowly decomposing.

Anal. Calcd for. , 75 1

The second fraction is as pure as the first. The IR and HMR spectra correspond to the structure.

, 60019 62

Example 26 .ry «^ ^ CHgCO-m-r - ^^ j f ||

/ ^ y ^ CH ^ S-C ^ N

COOH y H _._.

ZTL

N- (2-aminomethyl-1,4-cyclohexadienyl) -acetyl-3- (1,2,3-triazole-5-mercaptomethyl) -3-cephem-U-carboxylic acid (7h)

The procedure of Example 1 is followed, but the product is recovered from 15 ml of acetonitrile to give 100 mg (19.1%) of Th as a light brown powder. The IR and fiMR spectra correspond to the structure. The melting point is 160 ° C with slow decomposition.

Anal. Calcd for C 18 H 18 N 2 O 2 • 2 · O: C, U 6.1 T, H, E, 88; S, 16.38.

Found: C, 1 * 5.76; H, 5> 25; N, 16.85.

63 6001 9

Example 27 Preparation of 7- [2-aminomethyl-1,1'-cyclohexadienyl) -acetamido] -3- (1-methyltetrazol-5-ylthiophthyl) -3-cephem-14-carboxylic acid (7c), batch 2 BOC ^ FJ ^ V "NH-BOC ^ xJ ^ COOH ^ COO-Q-NO2

NOp J

'«> 28.

every n — n Z £

Dicyclohexylcarbodiimide (DCC) (9.0 g, 0.0M moles) is added to a mixture of 11.2 g (0.0U2 moles) of (2-Nt-t-butoxycarbonylaminomethyl-1,1 + -cyclohexadienyl) acetic acid (3) and 8.03 g, (0.010 mol) of 2.0 U-dinitrophenol in 250 ml of THF. The mixture is stirred at room temperature for 1.5 hours and the precipitated urea is filtered off. To the filtrate is added a solution of 11.5 g (0.035 moles) of 7-amino-3- (1-methylethetrazol-5-ylmethyl) -3-cephem-U-carbonyl acid and II +, 7 ml (ca. 0.105 moles) of triethylamine in 250 ral of water. The mixture is stirred at room temperature α 6001 9 for 20 hours and the THF is removed under reduced pressure below 1 → 0 ° C. The aqueous solution is washed with ether (200 mL), acidified with 6N HCl and extracted with ethyl acetate (3 x 200 mL). The extracts are washed with water and saturated aqueous NaCl solution, dried over Na 2 O 4 and evaporated to dryness, the oily residue is taken up in ether (500 ml) to give 8.05 g (1.0%) of the N-3-protected compound To (i.e. 6c).

HR: / nUi011 1770, 1710, 1650, 1510 cm-1, max ·

A mixture of 8.0 g (0.0138 mol) of compound 6c and 16 ml of trifluoroacetic acid (TFA) is stirred at 0-5 ° C for 1 hour and then diluted with 300 ml of ether to separate the trifluoroacetate of compound 7c, which is dissolved in 30 ml of 50 # acetonitrile, adjusted to pH 6 with concentrated NH 4 OH and diluted with 500 ml of acetonitrile. The separated yellow precipitate is washed well with acetonitrile (200 ml) to give 5.2 g (79 / ») of the amorphous compound 7c (batch 2). 3p. 195 DEG-205 DEG C. (decomposition).

IR: 1780, 1650, 1620, 1560 cm-1.

UV;

Anal, calculated for C H, 5.08; N, 19.78; S, 12.91 ».

Found: C, 1 »6.61»; H, 1 », 60; N, 18.63; S, 12.59.

Solubility in 0.1 M phosphate buffer (pH 7): 9.1 mg / ml.

i,, 65 6001 9

Example 28 7- [2-Aminomethyl-1-cyclohexadienyl) -ethane] -3- (5-methyl-1,3 - [- thiadiazol-2-ylthiomethyl) -3-cephera-k-carbonic acid batch 2, completed ocSö! p- „oa] * · L no2

bo2H

M

Uk.CO ^ s Ϊ-f / ^ - 0Η2-3 <8Λ®5 i »2d 66 6001 9

To a stirred solution of 8.02 g (0.03 moles) of 2- (Nt-butoxycarbonylaminomethyl) -1,1'-cyclohexadienylacetic acid (3) and 5.6 g (0.0301+ moles) 2, 1 + -dinitrophenol 80 ml: get dry THF, add 6.2 g (0.0301+ moles) of DCC O ...

At 5-10 ° C at a time and the mixture is stirred for 2 hours at room temperature. After removal of the precipitated urea, the reaction mixture is stirred at 0-5 ° C with a solution of 9.1 g (0.027 mol) of 7-8mino-3- (5-methyl-1,3,1 + -thiadiazol-2-ylthiomethyl) - 3 "cefene-α-carboxylic acid and 7-7 ml (0.055 mol) of triethylamine in 30 ml of water and 50 ml of THF. The mixture is stirred for 20 hours at room temperature and concentrated under reduced pressure to remove most of the THF. The aqueous solution is treated. With 2 g of activated carbon, the filtrate is covered with 10 ml of ethyl acetate and acidified with 10% HCl, the aqueous layer is extracted twice with 50 ml portions of ethyl acetate, the ethyl acetate layer is combined with ethyl acetate extracts, washed twice with 100 ml portions of water, dried anhydrous sodium urea sulfate and concentrated to dryness below 1 + 0 ° C to give compound 6d as a viscous oil which solidifies on stirring with ether to give 12.5 6 (70, ί) of compound 6d.

IR: max 1 ° C> 1700 ° C 633 ° C, 1C ° C-protected cephalosporin 6d (12.5 g> 0.021 mol) is treated with 25 ml TFA at 0-5 ° C with stirring. 1 hour at the same temperature The mixture is diluted with 300 ml of ether to give a white precipitate which is dissolved in 30 ml of 50 [mu] l of aqueous acetonitrile and treated with 1 g of activated carbon, the filtrate is adjusted to pH 6 with concentrated NH4OH and diluted with 250 ml of acetonitrile to give 8 g (77%) of compound 7d (batch 2) as a pale yellow powder, mp 230-235 ° C (dec).

IE: / ^ 1790, I65O, 1620, 1570 cm-1.

m & K s · UV: λ max pH 3 - pH 7 buffer 225 nm (sh) (ε, 31800), 277 nm (ε, 121 + 00).

Anal Calcd for C 20 H 23 N 5 O 1 + S 3: C, 1 + 8.67; H, i +, 70; ä, 11 + .19; s, 19.1 * 9.

Found: C, 1 + 8.55; H, U, 3U; H, 13.7 **; S, 19.00.

Solubility in 0.1M phosphate buffer (pH 7): 3.1 * ug / ml.

67 6001 9

Example 29 7- [2-Aminomethyl-1,1'-cyclohexadienyl) -acetamide-3- (3-hydroxy-pyridazino-3,2-c7-s-triazol-6-ylthioethyl) -3-cephera] -k8rbonic acid (le), batch 2, preparation a ·

(PV ^ NH-BOC

fj ^ jl ^ COOH ^ C00- {j NO2 L NO2 * »t

OH

Ln ✓'V ^ nh-boc q /

COOH

§ £.

*

^ OH

- / i

COC

For 66 6001 9

To a stirred solution of. 10.1 g (0.038 mol) of N- [4- (t-butoxycarbonylaminomethyl) -1,1 + -cyclohexadienyl] -acetic acid (3) and 8.1 g (0.0¾¾ mol) of 2,6-dinitrophenol in 300 ml . TiIF, 9 * 1 g (0.0i + U moles) of DCC are added in one portion and stirred for 2 hours at room temperature. The separated urea is filtered off. To the filtrate is added a solution of 1¾ g (0.038 mol) of β-amino-3- (3-hydroxypyridazino-β, 2-c7-S-triazol-6-ylthiomethyl) -3-cephem. Acidic acid and 12.2 g (0.12 mol) of triethylamine in 250 ml of water, and the mixture is stirred for 16 hours at room temperature, the reaction mixture is evaporated under reduced pressure below U0 ° C and the aqueous concentrate is washed with ether (5 x 200 ml) and The filtrate is acidified with dilute HCl to pH 2 to give a precipitate which is washed with 200 ml of water and extracted with hot THF (5 x 500 ml) .The THF extracts are evaporated under reduced pressure below 1 * 0 ° C and the residue is stirred with 500 ml with ether to give the N-protected 7e (i.e. 6e).

IR: 1765, 1700, 13½. 12½. 1150 cm’1.

To BOC-protected cephalosporin 6e (7 * 8 g, 0.012¾ mol) is added dropwise 16 ml of TFA at 5 ° C and the mixture is stirred for 1.2 hours at room temperature. The mixture is diluted with 1000 ml of ether and the precipitate obtained is dissolved in 50 ml of 50% acetonitrile. The solution is adjusted to pH U-5 with concentrated NH 4 OH and diluted with 200 ml of acetonitrile to give 5.55 g (8%) of compound Te (batch 2). 3p. 220-230 ° C (decomposes).

ΣΕ: IT60, 1710, 16U0, 15½. 1U60 cm'1.

UV: ^ 2C ° 3 - pH 7 buffer. 253 nm (£ 20,000) nax.

Anal, calculated for Ο ^ Η ,, ^ ΝγΟ ^ ε ,, ^^ Ο: C, U7.06; H, 5.03; II, 17, ½.

Found: C, U6.28; U6.37; H, 3.73; 3.71; N, 17.20; 16.93.

Solubility in 0.1 M phosphate buffer (pH 7): 1.1 mg / ml.

Recrystallization: Trifluoroacetic acid (0.5 ml) is added to compound 7e (batch 2, 100 mg) at 5 ° C and the mixture is stirred for 5 minutes at room temperature.

The solution is diluted with 50 ml of ether. The trifluoroacetate obtained is dissolved in 10 ml of 50 5J aqueous acetonitrile solution. The solution is adjusted to pI 6 using 10/5: JH 2 O to give a light brown precipitate (91 mg), m.p. 22 * »- 23l» ° C (decomposes).

IR: / nu0o11 1760, 1710, I630, 15½. lU60 cm'1.

fflftX s »UV: ^ 2003 + pH 7 buffer 253 nm (£, 19,000).

max.

6001 9

Anal Calcd for C, 47.06; H, 5.03; II, 17.U6.

Found: C, 1 * 7.30; 1 * 7.25; H, 4.23; 4.21; N, 17.89; 17.82.

Solubility in 0.1 M phosphate buffer (pH 7) i 1.1 mg / ral.

Example 3Q

7 - [(2-Allylmethyl-1,4-cyclohexadienyl) acetamide] -3- (1- (1-methyltetrazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid salicylaldehyde adduct (7c? 'Hqd »(X-ivl CO2K ®3

You

To a stirred suspension of 7c (Example 27, lot 2, 766 mg, 1.6 mmol) and triethylamine (300 mg, 3 mmol) in methanol (3 N) was added salicylaldehyde (370 mg, 3 mmol) and the mixture was stirred. 30 minutes at room temperature to give a clear dark yellow solution. The solution is treated with a small amount of carbon and ICEH (2 ml, 1M solution of potassium 2-ethylhexoenoate in ethyl acetate) is added to the mixture. The mixture is diluted with a large amount of ether (100 ml) to precipitate a solid product 8c which is collected by filtration, washed with ether (30 ml) and dried. The yield is 788 mg (78 2).

Sp. I65 · 173 ° C (decomposes).

IR: ^ ni ^ sl1 1760 '1660 »1625»! 6 ° 5, 1280 cm * 1.

λmax Pekuri 256 nm (C 19500), 275 nm (C 14000, sh), 325 nm (ε2600).

3.95 (3H »s» w'ch3) »k * 96 d» k, Iz> 6_H ^ 5.50 (1H, m, 7-H), 5.69 (2H, s, C = CH) , 6.7-7.6 (4H, m, phenyl-H), 8.63 (II, s, -CH = H-), 9.00 (1H, d, 8 Hz, 7-CONH ).

το 6 001 9

Anal, calculated for C,> 48.96; H, U, U3; L !, 15.37; S, 10.06.

Found: C, U 8.96; U8,85; H, U, 01, U, 10; N, 1.03, 114.26; S, 9.31, 9, TO.

Example 31 N - [(2-Aninomethyl-1,1'-cyclohexadienyl) acetamide] -3- (5-methyl-1,3,3-thiadiazole-g-thiomethyl) -3-cephem-U-carboxylic acid salicylaldehyde adduct (7d )

HO

2a-> y I ^

\ X ^ 'CONH- | -] N — N

^> Ni ^ LCH2-S-l! V JLro COgH ·

M

To a stirred mixture of Td (Example 28, lot 2, 778 mg, 1.6 mmol) and triethylismine (300 mg, 3 mmol) in methanol (8 mL) was added salicylaldehyde (370 mg, 3 mmol) and the suspension was stirred. minutes at room temperature to obtain a clear dark yellow solution which is treated with a small amount of carbon. KEH (2 mL, 1M solution in ethyl acetate) is added to the filtrate. The mixture is diluted with a large amount of ether (100 mL) to precipitate product 8d, which is collected by filtration, washed with ether (30 mL) and dried. Yield 355 mg (1%) · M.p. 163 DEG-370 DEG C. (decomposes).

λ max λ max δ δ δ λ λ λ λ λ λ ° ° ° ° ° ° Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ

2.68 (3H, s, -CH 3), U, 9U (lii, d, U Kz, 6-li), 5.50 (1H, m, 7-H), 5.68 (2H, s, C = CH), 6.70 - 7.60 (UH, m, phenyl-K), 3.65 (1H, s, CH = N), 8.92 (1H, d, 8 Hz, -CONH-) .

71 6001 9

Anal. Calcd for C 18 H 18 N 2 O 3 S 3/2 1 H 2 O: C, + 8.92; H, Ji, l »l; N, 10.57; S, li », 51.

Found: C, 1 * 8.91, l »9.2l»; H, 3.71 ». N, 0.1 N, 10.15, 10.3 U; S, 12.75, 13.57.

Example 32 7- (2-Aninonethyl-1,1'-cyclohexadienyl) -? 3-emidium [N- (3-hydroxypyridazino- [3,2-b] -3-triazol-6-ylthiomethyl) -3-cephem-1 * -carboxylic acid salicylaldehyde adduct (7e) J?

co2K

Ss.

To a stirred suspension of 7e (Example 29, batch 2.7, 1 mg, 1.0 U2 mmol) and triethylamine (300 mg, 3 mmol) in H, N-dimethylformamide (7 mL) was added salicylaldehyde (370 mg, 3 mmol) and the suspension is stirred for 1 1/2 hours at room temperature to give a clear dark yellow solution which is treated with a small amount of carbon. To the filtrate is added • jdn (1.5 ml, 1M solution in ethyl acetate). The mixture is diluted with a large amount of ether (100 ml) to precipitate product 8e, which is washed with ether (30 ml) and dried. The yield is 906 mg (95 5). 3p. 215-222 ° C (decomposes).

IR: 1776, 1720, 1660, 1615, 1600. 13U5, 1275 cm-1.

υν: λρΗν7 Pu8kuri 255 nm (£ .23800) 310 rm (£ 8500, sh), males · -, MR: ^ Ppm 6 5.00 <1H · d »k Hz» <> - «), 5.50 ( 1H, m, 7-H), 5.72 (2H, s, C = CH), 6.7-7.6 (61: m, phenyl-H), 8.70 (1H, s, Cl il), 9.00 (1H, d, 811z, -CONH).

T2 6001 9. Anal Calcd for C 26 H 60 N 7 S 2 K, H 2 O: C, 50.1 + 9; H, U, 09; N, li *, 21; S, 9.30.

Found: C, 50.3 ^ +, 50.30; H, U, 15, H, 00; N, li *, 02, il *, 03; S, 9.36, 9.31.

Example 33

The potassium salt of the compound of Example 1T is prepared by adding a solution of potassium 2-ethylhexanoate (KEH) in ethyl acetate to a solution of cephalosporin (amphoteric ion) (7a) in DMSO to precipitate the desired potassium salt.

Example 3l *

The sodium salt of the compound of Example 19 is prepared by first forming its diethylammonium salt in methanol and then adding a solution of sodium 2-ethylhexanoate (SFH) in ethyl acetate and then diluting with isopropanol to precipitate the desired sodium salt having a solubility in water greater than 250 mg / ml.

Example 35 11.2 g (0.075 mol) of 5-mercapto-1,2-N, N-triazolium potassium salt are added with stirring to a suspension of 31.8 g (0.07 mol) of 7H-alpha- (2-aminomethyl-1-cyclohexenyl) acetamido-7-cephalosporanic acid (6w; prepared according to Example 16) and 5.9 g (0.07 mol) of NalEO ^ in 350 ml of 0.1 M phosphate buffer (pH 6, k), the mixture is heated to 55 ° C and stirred stirred at this temperature for 3.5 hours under a nitrogen atmosphere. The resulting solution is cooled to 22 ° C, the pH is adjusted to 5.5 with 0.0% H 2 PO 4, and the solution is diluted with acetonitrile to give 7- [alpha- (2-aminomethyl-1-cyclohexenyl) acetamide] - (1,2,3-triazol-5-yl-thiomethyl) -3-cephem-1 * -carboxylic acid precipitates (6h). This is washed with acetonitrile (50 ml). Melting point 203-215 ° C (decomposes). The IR, UV and analytical results are the same as in Example 10.

Claims (1)

73 6001 Patents: Method for the treatment of antibacterial T-ΓΜ- (2-aminomethyl-T-cyclohexenyl and -1H-cyclohexadienyl) -acetamido-3-heterocyclic-thiomethyl-3 -cephem-1 + carboxylic acids and their pharmaceutically acceptable salts, aCVNH2 CHgCONH - p ^ ^ - ch2-sr COOH wherein R is OH NN ^ * \ i = T · -U- 'ΑΛ - & - 1 · Clt3 NN n - N _ I - <r il fi NN — N l Ϊ / A / Λ XJ '· H and the dashed line indicates a possible double bond, characterized in that A) a compound of formula H2 »—-- p ^ f - CH2 -SR II COOH 71, 6001 9 wherein R is as defined above, or a salt thereof is reacted with an acid acylation derivative of the formula ί Y ^^^ CH2C00H wherein B is an amino protecting group used, for example, in peptide syntheses or in the preparation of 0 ( aminobenzylpenicillins from 2-phenylglycine, and the dashed line is the same as above, and said amino protecting group is removed to form a compound of formula I or a pharmaceutically acceptable salt thereof, and if desired either before or after removal of the group B, (a) converting the product in the form of the free acid or a salt thereof into a pharmaceutically acceptable salt in a manner known per se, or (b) converting in a manner known per se a product in the form of a salt to the corresponding free acid compound or a pharmaceutically acceptable salt thereof, or B) reacting a compound of the formula OC 2 CH 2 C NH - ^ IV f - --CH 2 OCOCH 3 c ° 2h or a salt thereof with a thiol of the formula HS - RV wherein R is as defined above, or a salt thereof, to form a compound of formula I or a pharmaceutically acceptable salt thereof, and if desired (a) converting the product in the form of the free acid or a salt thereof into a pharmaceutically acceptable salt in a manner known per se, or (b) in a manner known per se, a note in the form of a salt corresponding to the corresponding free acid compound or a pharmaceutically acceptable salt thereof. Patent patent: 6001 9 Τ5 For the preparation of antibacterials of 7- (2-aminomethyl-1- (β-cyclohexenyl-och-1,1'-cyclohexadienyl) -acetamido) -3-heterocyclyl-thiomethyl-3-cephem-U- carbonsyror with formula I, aCH2-BH2 X CH2CONH —-- ^ CH2 -sr COOH color R är OH - €> »-Cp- -y! -l> ,. -o-I CH3 <X -g. ' -o. -c / · and streklinjen betyder en eventuell dubbelbindning, och deras pharmaceutiskt godtagbara salter, kännetecknat därav, att man A) omsätter en förening med formuleln V * - | - / SVs # - CI ^ -3-R 11 COOH