FI62306B - FOERFARANDE FOER FRAMSTAELLNING AV SUBSTITUERADE N-METYLEN-DERIVAT AV TIENAMYCIN ANVAENDBARA SAOSOM ANTIBIOTISKA AEMNEN - Google Patents

Description

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Patant · och registerstyrelsan Anseksn utlagd och uti.skriftsn pubiictrtd 31.08.82 (32) (33) (31) Pjrydutty stuoikuus — Bugtrd priorkut 21.11.75 i2.0U.76, 18.10.76 USA (US) 63U296,

67626I, 73365U

(71) Merck & Co., Inc., 126 E. Lincoln Avenue, Rahway, New Jersey, USA (72) Burton Grant Christensen, Metuchen, New Jersey, William Joseph Leanza, Berkeley Heights, New Jersey, Kenneth James Wildonger,

Somerville, New Jersey, USA (US) (7¾) 0y Kolster Ah (5U) Method for the preparation of substituted N-methylene derivatives of thienamycin useful as antibiotics The present invention relates to a process for the preparation of substituted N-methylene derivatives of thienamycin useful as antibiotics, having the formula

OH

^ X, -1-SCH0CH_N = C-R

1 I 2 2 f 1 2

I I NR R II

^ -N - C00H

O

wherein R is a hydrogen atom, a lower alkyl group, a phenyl, pyridyl, thiazolyl, trifluoromethyl, amino, azidoalkyl or aminoalkyl group or group, R 1 is a hydrogen atom, a lower alkyl group, a lower alkenyl group, a lower alkylthioalkyl group, a benzyl, dialkylarino, trifluoroethyl, carboxy, methyl, alkoxy or nitro group and R is a hydrogen atom or a lower alkyl group, or R and R together with a nitrogen atom form a piperidyl ring, and to prepare pharmacologically acceptable acid addition salts of these compounds.

Thienamycin is described in U.S. Patent 3,950,357. Thienamycin has the following structure:

OH

—Sch2ch2nh2 I

N - COOH

O ^

Substituted N-methylenethienamycin derivatives prepared according to the invention can also be presented as an internal salt of the formula

° H Q

^> -f N- SCHOCH_NiJ-i- C - ^ NR R

I o R 11 '

^ -N-j —- COO W

For simplicity, the compounds of the invention are represented in the examples by the symbol:

QH

Th - N = C-X II ·

Y

* -COOH

wherein "Th" represents the bicyclic backbone of thienamycin and wherein the hydroxyl, amino and carboxyl groups of the compound are indicated.

New antibiotics are constantly needed. Unfortunately, there is no static efficacy of a particular antibiotic because the continued large-scale use of any such antibiotic selectively results in the emergence of resistant strains of pathogens. In addition, the disadvantage of the known antibiotics is that they are effective only against certain species of microorganisms. Therefore, research to find new antibiotics continues.

62306

Surprisingly, it has been found that the compounds according to the invention are antibiotics with a broad spectrum of action which can be used in animal and human therapy and in non-living systems. The new antibiotics are very broad-spectrum and are effective against both gram-positive bacteria (such as S. aureus, Strep, pyogenes, and B. subtilis) and gram-negative bacteria (such as E. coli, Proteus morganii, Klebsiella, Serratia, and Pseudomonas).

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

X

111

# N - COOH

o or a suitable O- and / or carboxyl derivative thereof is reacted with the formula. 0

NR

'11 © IV RCX with an imidoester according to R ", wherein X ° R" is a leaving group, wherein X ° is O or S and R "is lower alkyl or corresponds to 1 2 va, and R, R and R have the same meaning as above, or the formula r1r2n ® = cx 'r \

! x · ^ V

R

With an imidohalide of formula 2, wherein R, R and R are as defined above, or X 2 R "of formula 2"

1 2 I VI

R R N-ON-R

wherein X 0, R ", R, R 1 and R 2 have the same meaning as above, after which the compound obtained is converted into an acid addition salt.

Suitable solvents include water, dioxane, tetrahydrofuran (THF), dimethylformamide (DMF), chloroform, acetonitrile, acetone or mixtures thereof. The reaction is carried out at 62306 ° C to 25 ° C for a reaction time of 1 to 6 hours. Examples of suitable reagents are: a) Imidoesters: R1 R1R2

N N

Il Θ II o R-C-XR "R-C-X ° R"

X ° = O or S

methylformimidate, ethylformimidate, methylacetimidate, ethylacetimidate, methylbenzimidate, ethyl 4-pyridylcarboxyimidate, methylphenylacetimidate, methyl-3-thienylcarboxyimidate, methyl azidoacetimidate, methylchloroacetate, methylchloroethylate p-nitrobenzimidate, methyl 2,4-dimethoxybenzimidate, ethyl N-methylformimidate, methyl N-methylformimidate, methyl N-isopropylimidate and the like.

Imidoesters are prepared in a manner known per se, such as: 1) RCN reaction of nitrile with a lower alkanol in the presence of HCl (Pinner synthesis).

2) RCN reaction of nitrile with lower alkanol in the presence of a base. Typically, the reaction is carried out at 0 to 40 ° C in the presence of excess alcohol using a catalytic amount of alkali metal alkoxide for 15 minutes to 4 hours.

3) Reaction of the amide RCONHR 2 with an alkyl chloroformate such as methyl chloroformate at 25-45 ° C for 1-4 hours.

4) Reaction of the N-substituted amide RCONHR 1 or RCONR 1 R 2 with an equivalent of an alkylating agent such as triethyloxonium fluoroborate in an inert solvent such as ether or chloroform at 0-23 ° C for 10 min to 2 hours.

5) Conversion of the readily available imidoester RC (0R ") NH to the desired imidoester RC (OR") NR by reacting the imidoester with an alkylamine R'NH2 in a mixture of water and a water-immiscible solvent such as ether or chloroform at 0-23 ° C at 5 min - 1 hour.

5 62306 (b) Substituted imidohalides:

Chloropiperidinomethylumium chloride, chlorodimethylforminium chloride, chlorodiethylforminium chloride and the like.

Substituted imidohalides (b) are suitably prepared in a manner known per se, such as: 1 2 - at 40 ° C for 1 to 5 hours.

The reaction comprising reagents (a) can be represented by the following scheme: OR3 -SCH2CH2NH2 - N - COX 'R3' 0 Λ R ·

OF

it R-C-OR "

3 V ORJ

- sch2CH2N = C-NHR '

I | t R

0 sn - COX 1R3 'where OR "is the leaving group of the imidoester reagent and R, R', 3 '3 R, R and X' have the same meaning as above. This reaction is particularly suitable for embodiments where R and R are hydrogen and X1 is oxygen.

6 62306

The reaction comprising reagents (b) can be represented by the following scheme: | r "1r2n + = cx 'χ · Θ _ or + * 7„ -f ^ Y-SCH2CH2N-C-NR1R2 χ, 0 • —-- * / I o p

^ J_N - * - COX 1 RJ

_ o

OH

mild hvdrolvvsl ^ Λ-ο- - SCH2CH2N = C-NR1R2

pH 3 - 6 II

L- n - COOH

O

2 Λ- * where all symbols have the same meaning as above. If desired 3 3 'product 2 is R and R suitably trimethylsilyl and X' is oxygen.

The compounds (II) prepared according to the invention form a wide variety of pharmacologically acceptable salts, such as acid addition salts, e.g. with hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, toluene-p-sulfonic acid and methylsulfonic acid. These salts are pharmacologically acceptable non-toxic derivatives that can be used as the active ingredient in suitable unit dosage forms. They can also be combined with other drugs to prepare compositions with broad-spectrum activity.

Results of Pharmacological Experiments The lowest effective concentration of new thienamycins and reference compounds was determined using standard gram-positive and gram-negative bacteria. The results are shown in the following tables.

7 62306 p

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Λ S

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tJi r- σ> r— CN ID

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• P 3 PQ O O O O O

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-P - ro CM

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3 ^ rH

® m ε · * -p \> s -u σ> · * = 3 3.

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K 3 »- o in c U Cd) m in o oo r- Φ (N -H p« - - | * 2 S C 3 o o nr oo

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C rO

‘D M

• 'j 3 05 tn to cp -h tn tn tn co tn B M-ι Ö Ö 3 β 3 tn 3 10 W O ·

β O M -H p -H -H

2 ϊ * 5 -P O -P O O

*B

-p c 0) • n 3 -P n n n n ro ro + {s 33 33 K 33 ® 33 ω α> = υυυυυυ

S -P 05 I I I I I I

s tn oooooo

3 -H UUUUUU

E- · Ό Λ> 1 3 33 35 p ro ro pe aarcKooas

• H 05 O O O W W

y o o

P

3

EH

9 62306

Thus, the new compounds are at least as active as the known thienamycins.

As is known, thienamycin has an excellent antibacterial effect. Unfortunately, however, its chemical stability is poor and, in addition, it is inversely proportional to the concentration (Kropp, H. et al., Thienamycin, A new, β-lactam Antibiotic.

II. In vitro- and in vivo Evaluation ", 16th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, 111, 1976). Thienamycin amidine derivatives have 5-10 times better stability than thienamycin, and equally good γ-lactamase stability.

The new compounds are thus valuable antibiotics against various gram-positive and gram-negative bacteria and thus can be used as human and veterinary drugs. The compounds according to the invention can be used as antibacterial drugs for the treatment of infections caused by gram-positive or gram-negative bacteria, e.g. Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Serratia, Salmonella typhosa, Pseudomonas and Bacterium Proteus. They can also be used as aqueous compositions in concentrations of 0.1 to 100 parts of antibiotic per million parts of solution for the purpose of inhibiting the growth of harmful bacteria in medical and dental equipment as well as as bactericides in industrial use, e.g.

The compounds of the invention may be used alone or in combination as an active ingredient in a variety of pharmaceutical preparations. These antibiotics and their corresponding salts can be used as capsules or tablets, powders or liquid solutions or suspensions or elixirs. They can be administered orally, intravenously or intramuscularly.

The amount to be administered depends largely on the condition of the subject to be treated and the weight, dosage and frequency of the patient, with parenteral administration being preferred for conventional infections and oral administration for intestinal infections. Generally, a daily oral dose will comprise about 2 to 600 mg of active ingredient / kg of body weight to be administered in one or more doses per day. The preferred daily dose for an adult human is about 15 to 150 mg of active ingredient / kg of body weight.

The compounds of the invention may be administered in a variety of unit dosage forms, e.g., solid or liquid oral dosage forms. The unit dosage compositions, either liquid or solid, may contain from 0.1 to 99% of active ingredient, preferably from about 10 to 60%. The composition usually contains about 15 to 1500 mg of active ingredient; however, it is generally preferred to use a dosage amount of about 100 to 1000 mg. For parenteral administration, unit dosage forms will usually comprise a pure compound in a slightly acidified sterile aqueous solution or a soluble powder to be dissolved before use.

The overall synthesis of thienamycin production is described below.

OH

^ -sch2ch2nh2

-N-- COOH

Step A; Preparation of 4- (2-acetoxyvinyl) azetidin-2-one fi ~ C = CH-CH = CHOC-CH, + 0 = C = N-SO_Cl

O

°? CH = CHOCCH3 CHeCHOCHCH <-. r-r O SO2cl 11 62306

A solution of 1.0 mL of distilled chlorosulfonyl isocyanate (1.65 g; 11.7 mmol) in 2.5 mL of anhydrous diethyl ether is cooled under nitrogen in a -20 ° C bath.

A solution of 2.5 g of 1-acetoxybutadiene (22 mmol) in 2.5 ml of anhydrous ether is likewise cooled under a nitrogen blanket in a -20 ° C bath.

The chlorosulfonyl isocyanate solution is added dropwise over 10 minutes to the acetoxybutadiene solution using a Teflon tube immersed in the CSI solution and pressurized with nitrogen. The reaction mixture is stirred at -20 ° C for half an hour. The solution is clear and pale yellow in color.

A solution of 2 g of sodium and 5 g l ^ HPO ^ 20 mL of water, prepared in the above-mentioned half-hour reaction time and is cooled in an ice bath; 20 ml of ether are added and the mixture is stirred vigorously with an ice bath. After half an hour the reaction mixture was transferred using nitrogen pressure and the Teflon tube from the reaction flask, which is maintained at -20 ° C solution in a bath, to the vigorously stirring the hydrolysis. Rapid dropwise addition takes 5 minutes. The hydrolysis is allowed to continue for another 5 minutes. The pH of the hydrolysis mixture is 6 to 8, preferably 8.

The phases are separated, leaving a yellow-orange gummy substance with the aqueous phase. The ether phase is dried directly over magnesium sulfate. The aqueous phase and the rubber phase are extracted three times with 50 ml portions of ether, each of which is added to the original ether / magnesium sulphate.

The dried products are filtered and concentrated under a nitrogen blanket to a volume of 5 ml; some of the product is crystalline at this stage.

A column of 10 g of Baker silica gel is filled with ether, and the ether concentrate is passed to the top of the column and allowed to flow through the column. The solids in the flask are rinsed three times with 2 ml of ether, the rinsing liquid is pipetted off and applied to the column. It is then eluted with ether. The first 25 ml is mainly ether, the next five 10 ml fractions are collected and then three 50 ml fractions are collected. All fractions are evaporated under a stream of nitrogen; 12 62306 the product crystallizes from fractions 4 to 6, and traces are found in fractions 3 and 7. Fractions 1 to 3 contain a yellowish crystallizing substance which resins on standing. Yield: 10 mg of a mixture of cis and trans isomers.

Step B: Preparation of 4- (2-acetoxyethyl) -2-azetidinone

O O

Il H

^ 0CCH3 ^ OCCH3 I ->

NH NH

~ S

O o

A solution of 4- (2-acetoxyvinyl) -2-azetidinone (10.0 g, 0.065 mol) in 200 mL of ethyl acetate containing 100 mg of 10% Pd / C is hydrogenated on a Parr shaker at 25 ° C. at 2.8 kg / cm for 15 minutes. The mixture is filtered through a pad of "Supercel" and washed further with ethyl acetate. The combined filtrate is evaporated in vacuo to give 4- (2-acetoxyethyl) -2-azetidinone (10.0 g) as a crystalline solid. Uudelleenkiteytettä-

white crystals are obtained from ether, m.p. 44-47 ° C; IR

(CHCl 3) 5.66, 5.74 μm; NMR (CDCl 3) γ3.44 (broad s, 1, NH), 5.82 (m, 2, CH 2 OCOCH 3), 6.29 (m, 1, C-4H), 6.87 (1/2 AB model share) to four parts C-4H and NH, 1, J = 12.8 Hz, J = 4.5 HJ =

gem NH

1.9Hz, 7.38 (1/2 AB model is divided into four parts C-4H and NH, 1,

Jgem = 12'8Hz 'J = 2'3 Ez' JNH = 1 »0Hz), 7.93 and 8.02 (s, m, a total of 5, OCOCH2 and CH2Cl2 OCOC2, respectively).

Step C: Preparation of 4- (2-hydroxyethyl) -2-acetyldinone

O

II

^ OCCH3 OH

.__ NH * _NH

13 62306

A solution of 4- (2-acetoxyethyl) -2-azetidinone (2.24 g, 0.014 mol) in 25 mL of anhydrous methanol is treated with a solution of sodium methoxide (77 mg, 1.4 mmol) in nitrogen at 5 ° C in 5 mL. in anhydrous methanol. After stirring for one hour, neutralize the solution with glacial acetic acid. The ethanol is removed in vacuo to give crude 4- (2-hydroxyethyl) -2-azetidinone as an oil. The product is purified by chromatography on silica gel eluting with 10% MeOH / CHCl to give 1.55 g of alcohol? mp. 50 ° C; IR (CHCl 3) 5.67 uni; NMR (CDCl 3) Of 3.20 (broad s, 1, NH), 6.24 and 6.28 (m, t, total 3, C-4H and Cl 2 OH, respectively), 6.90 (broad s, 1, NH); / 2 AB in the model divided into four parts C-4H and NH, a total of 2, OH and C-3H, respectively, Jgem = 13, OHz, J. = 4.2Hz, J..IT = 1.6Hz), 7, 42 (1/2 AB model divided into four

vie NH J J

to C-4H and NH, 1, C-3H, Jgem = 13.0Hz, Jvic = 2.2Hz, JNH = 1.1Hz), 8.16 (m, 2, CH2CH2OH).

Step D: Preparation of O-oxo-2,2-dimethyl-3-oxa-1-azabicyclo [2.2.1] octane -rX__ γ-τί

NH N O

o ^ - 0 ^ X

A solution of 4- (2-hydroxyethyl) -2-azetidinone (1.87 g, 0.16 mol) and 2,2-dimethoxypropane (1.69 g, 0.016 mol) in 25 ml of anhydrous methylene chloride is treated with boron. -trifluoride etherate (0.201 mL, 0.002 mol) at 25 ° C. The resulting solution is stirred for 10 minutes and the solvent is removed under reduced pressure to give 2.5 g of an oil. The crude product is chromatographed on silica gel using ethyl acetate / benzene 2: 1 as eluent to give O-oxo-2,2-dimethyl-3-oxa-1-azabicyclo / 4.2.0 / octane (1.59 g) as a crystalline solid. . Upon recrystallization from ether / hexane, the product has a melting point of 60-61 ° C. IR (CHCl 3): 5.73 μm (β-lactam) NMR (CDCl 3 δ Z: 6.02-6.28, m, 2H, C-4 methylene 6.22-6.62, m, 1H , C-6 methine 6.90, ddm 1H, J_ = 14Hz, J1 = 4.5Hz 7, / 6.7 14 62306 C-7 proton cis to C-6H 7.47, dd, 1H , J_ _ = 14Hz, .J £ _ = 2Hz /// O / C-7 proton trans C-6H with respect to 7.82 - 8.68, m, 2H, C-5 methylene 8.23, s , 3ΗΛ c_2 methyls 8.57, s, 3HJ Step E; 8-oxo-2,2-dimethyl-7 Ct- and [S- (1-hydroxyethyl) -3-oxa-1-azabicyclo] -4.2, Q7 octane production

OH

_N 0 7 N 0

To a solution of 1.1 equivalents of freshly prepared lithium diisopropylamide in anhydrous tetrahydrofuran under a nitrogen blanket at -78 ° C is added a solution of O-oxo-2,2-dimethyl-3-oxa-1-azabicyclo [4.2.0 / octane in anhydrous in tetrahydrofuran cooled to -78 ° C. After 2 minutes, the lithium enolate obtained is treated with an excess of acetaldehyde. The solution is stirred for 30 minutes at -78 ° C, after which it is poured into water. The aqueous phase is saturated with sodium chloride and extracted with ethyl acetate and filtered; the filtrate is evaporated under reduced pressure to give a crude product. Purification by chromatography on silica gel using ethyl acetate / benzene as eluent gives 8-oxo-2,2-dimethyl-7β- and [3- (1-hydroxyethyl) -3-oxa-1-azabicyclo [T], 2.0 7oktaani.

8-oxo-2,2-dimethyl-7- (1-hydroxyethyl) -3-oxa-1-azabicyclo [4.2.2.0 octane: IR (CH2Cl2): 5.52 (β-lactam) NMR (CDCl3) Z ': 5.53-6.43, m, 4H, C-4 methylene + C-6 methine + C-9 methine 6.90, dd with broad s, 2H, J = g = 9Hz 15 62306

J, n = 5.5 Hz, C-7 methine + OH

of /

7.70 - 8.83, m, 2H, C-5 methylene 8.27, s, 3Hj c_2 methyl 8.60, s, 3HJ

8.78, d, 3H, Jg = 6.5Hz, C-10 methyl 8-oxo-2,2-dimethyl-7β- (1-hydroxyethyl) -3-oxa-1-azabicyclo / 4.2.07 octane :

IR (CHCl 3): 2.9 μια broad O-H

5.73 β-lactam

NMR (acetone - d 6) δ Z: 4.23 - 3.33, m, C-9 methine + C-4 methylene + C-6 methine 3.33, broad s, OH

2.83, dd, J = 2Hz, 6Hz) n. ...

C C-7 methine 2.67, dd, J = 2Hz, 8Hz and 1.93-1.63, m, C-5 methylene 1.63, c_2 methyl

1.40, sS

1.23, d, J = 6.5 Hz, C-10 methyl

Step F: Preparation of 8-oxo-2,2-dimethyl-7Cγ- (1-p-nitrobenzylcarbonyldioxyethyl) -3-oxa-1-azabicyclo [4.2.0] octane

OH OR

_N 0 'J_N O

<T X o X

R = -COCHo — X N00 II 2 X, - X 2 0

A solution of 8-oxo-2,2-dimethyl-7β- (1-hydroxyethyl) -3-oxa-1-azabicyclo [4.2.0-7] octane (60 mg, 302 mmol) in 0.5 mL: The ether is treated with anhydrous potassium hydroxide (19 mg, 0.332 mmol) at 0 ° C under anhydrous conditions. After 15 minutes, 1-nitrobenzyl chloroformate (65 mg, 0.302 mmol) is added to the reaction mixture. Stirring is continued at 25 ° C for another 15 hours.

The mixture is extracted with 1 M phosphate buffer (pH 7) and ether.

16 62306

The ether phase is washed with water and brine, dried over magnesium sulfate and filtered. Evaporation of the filtrate under reduced pressure gives 67 mg of a colorless oil. Purify by preparative thin layer chromatography using silica gel and eluting with ethyl acetate / benzene (1: 9) to give 8-oxo-2,2-dimethyl-7β- (1-p-nitrobenzylcarbonyloxyethyl) -3-oxa-1-azabicyclo / 4 , 2.07 octane (40 mg) as a mixture of diastereomers.

IR (CH 2 Cl 2): 5.68 μm (β-lactam and carbonate), 6.19 and 6.54 μm (nitro)

N MR (CDCl 3: 1.67, d, 2H, ArH

2.37: d: 2H: ArH

4.67, s, 2H, ArCH 2

4.67 - 5.22, m, CH-jCH

5.98-6.25, m, 2H, C-4 methylene 6.25-6.62, m, 1H, C-6 methine 6.75-7.12, m, 1H, C-7 methine 7.75-8, 83, m, 2H, C-5 methylene 8.22, s, 3H, C-2 methyl 8.50-8.58, m, 5H, C-2 methyl + CH3CH7 / N-diastereoisomers or 7O1 and / or 3-mixtures are obtained in an analogous manner.

Step G:

Preparation of cis- and trans-3- (1-p-nitrobenzylcarbonyldioxyethyl) -4- (2-hydroxyethyl) -2-azetidinone

, 0R OR

J ^ I OH

_N O __NH

S X <f ~

O

R = -CQ-CH2 N2-> NO2 17 6 2 3 0 6 8-oxo-2,2-dimethyl-7OC- (1-p-nitrobenzylcarbonyl-hydroxyethyl) -2-oxa-1-azabicyclo [ , 2.0-7 octane (1/0 g) is dissolved in 8 ml of acetic acid and 2 ml of water and heated at 65 ° C for 1.25 hours. Acetic acid and water are removed under reduced pressure and the residue is dissolved in benzene and evaporated to give trans-3- (1-p-nitrobenzylcarbonyldioxyethyl) -4- (2-hydroxyethyl) -2-azetidinone as a mixture of diastereoisomers.

IR (CH 2 Cl 2): 5.67 μm (fo-lactam), 5.72 μm, 6.20 and 6.57 μm (nitro)

NMR (CDCl 3, T: 1.73, d, 2 H, J = 8.5 Hz, ArH 2.43, d, 2H, J = 8.5 Hz, ArH

3.63, broad s, 1H, NH 4.37 - 5.13, m, 1H, CH 3 CH 4.72, s, 2H, Ar CH 2 6.07 - 6.53, m, 1H, C-4 methine

6.23, t, 2H, J = 5.5 Hz, CH 2 OH

6.73 - 6.93, m, 1H, C-3 methine 7.63 - 8.97, m, 3H, CH 2 CH 2 OH 8.53, d, J = 6.5 Hz, CH 3 CH

The cis-diastereoisomers or cis-trans mixture are obtained in an analogous manner.

Steps D ', E1, F' and G 'as alternatives to steps D, E, F and G for the preparation of 3- (1-p-nitrobenzylcarbonyldioxyethyl) -4- (2-hydroxyethyl) azetidinone

OR

_NH

(j> R = -COCH 2 - ^> - NO 2 18 62306

Steps D1, E1 »F1 and G1 Preparation of 1- (2-tetrahydropyranyl) -4- [2- (2-tetrahydropyranyl) oxyethyl] -2-azetidinone

A solution of 4- (2-hydroxyethyl) -2-azetidinone (62 mg, 0.538 mmol) in 0.5 mL of anhydrous p-dioxane was treated under nitrogen at 25 ° C with 2,3-dihydropyran (0.98 mL, 1.08 mmol) and p-toluenesulfonic acid monohydrate (19 mg, 0.10 mmol). The resulting solution is stirred for 60 minutes, then extracted with a mixture of 10 ml of 0.5 M phosphate buffer (pH 7) and 10 ml of ethyl acetate. The aqueous phase is extracted a second time with ethyl acetate. The combined ethyl acetate extracts are washed with brine, dried over magnesium sulfate and filtered. The filtrate is evaporated under reduced pressure to give 216 mg of crude product. Purification by preparative thin layer chromatography and development with ethyl acetate gives 80 mg of 1- (2-tetrahydropyranyl) -4- [2- (2-tetrahydropyranyl) oxyethyl] -2-azetidinone as an oil.

NMR (CDCl 3) δ δ 5.13 - 5.60, m, OCH

5.83 - 85, m, C-4 H + OCH 2 6.95, dd, J = 5Hz and 15Hz Ϊ c_3 methylene

7.35, dd, J = 3Hz and 15Hz J

7.62 - 8.95, m, CHCH 2 CH 2 CH 2 CH 2 + CHCH 2 CH 2 O.

19 62306

Preparation of cis- and trans-1- (2-tetrahydropyranyl) -3- (1-hydroxyethyl) -4- [2- (2-tetrahydropyranyl) oxyethyl] -2-azetidinone ° H.xv.

I-X

° YD

By the same method described for the preparation of 8-oxo-2,2-dimethyl-TQ · and / h- (1-hydroxyethyl) -3-oxa-1-azabicyclo [3.2.0] octane, 8-oxo-2 From 2-dimethyl-3-oxa-1-azabicyclo [3.2.1] octane and using 1- (2-tetrahydropyranyl) -4- [2-tetrahydropyranyloxyethyl] -2-azetidinone to give both cis- and trans-1 - (2-tetrahydropyranyl) -3- (1-hydroxyethyl) -4- [2- (2-tetrahydropyranyl) oxyethyl] -2-azetidinone diastereomeric mixture.

Preparation of cis- and trans-1- (2-tetrahydropyranyl) -3- (1-p-nitrobenzylcarbonyldioxyethyl) -4- [2- (2-tetrahydropyranyl) oxyethyl] -2-azetidinone

0H OR

° Y ° x ~~ ^ y ° Ί o R = -coch2 —— no2 20 62 3 0 6

By the same method described for 8-oxo-2,2-dimethyl-.

For the preparation of 7β- (1-p-nitrobenzylcarbonyldioxyethyl) -3-oxa-1-azabicyclo [4.2.0] octane 8-oxo-2,2-dimethyl-7 '' - (1-hydroxyethyl) - From 3-oxa-1-azabicyclo [4.2.07] octane and using trans-1- (2-tetrahydropyranyl) -3- (1-hydroxyethyl) -4- [2- (2-tetrahydropyranyl) oxyethyl] -2-azetidinone to give trans -1- (2-tetrahydropyranyl) -3- (1-p-nitrobenzylcarbonyl-dioksietyyli) -4- / 2 "(2-tetrahydropyranyl) oxyethyl / -2-azetidin-methanone. The cis-diastereoisomers are obtained in an analogous manner.

Preparation of cis- and trans-3- (1-p-nitrobenzylcarbonyldioxyethyl) -4- (2-hydroxyethyl) -2-azetidinone

OR OR

Λ-xL_

J Y °. * oJ-NH

o R = -COCH2 -------- - NO2

A solution of trans-1- (2-tetrahydropyranyl) -3- (1-p-nitrobenzylcarbonyldioxyethyl) -4- [2- (2-tetrahydropyranyl) oxyethyl] -2-azetidinone in methanol at 25 ° C is treated with 0.1 M equivalent of p-toluenesulfonic acid monohydrate. The solution is stirred for 2 hours, then neutralized with 1 M phosphate buffer (pH 7). The product is extracted with ethyl acetate, the ethyl acetate solution is washed with brine, dried over magnesium sulfate and filtered. The filtrate is evaporated under reduced pressure to give trans-3- (1-p-nitrobenzylcarbonyldioxyethyl) -4- (2-hydroxyethyl) -2-azetidinone. The cis-diastereoisomers are obtained in an analogous manner.

21 62306

Step H:

Preparation of cis- and trans-3- (1-p-nitrobenzylcarbonyldioxyethyl) -4- [2,2-bis- (2-hydroxyethyl] -2-azetidinone OR r OR - A-A,

I CHC

X-NH \ NH

O 0 ^ -

OR L J

Λ-γγ ^ · _

_NH S

O * ^ OH

8 A

r = -coch2 y— no2

A mixture of anhydrous pyridine (0.146 mL, 1.81 mmol) and anhydrous powdered chromium trioxide (92 mg, 0.916 mmol) in 8 mL of anhydrous acetonitrile is stirred for 30 minutes under nitrogen at 25 ° C. To the resulting dark brown solution are added 250 mg of dry Supercel and a solution of trans-3- (1-p-nitrobenzylcarbonyldioxyethyl) -4- (2-hydroxyethyl) -2-azetidinone (186 mg, 550 mmol) in 1 mL of anhydrous acetonitrile. After stirring for 1 hour, the reaction mixture is filtered through a pad of 2 g of silica gel and 2 g of magnesium sulfate. The layer is washed several times with a total of 30 ml of acetonitrile. The filtrate is concentrated under reduced pressure at 25 ° C to a volume of 3 ml. By thin layer chromatography (silica gel; ethyl acetate / benzene 2: 1) · this solution contains a product (Rf = 0.38) which is less polar than the starting material (Rf = 0.21).

A solution of the trans-3- (1-p-nitrobenzylcarbonyldioxyethyl) -4- (2-oxoethyl) -2-azetidinone prepared above in acetonitrile is treated with 2-mercaptoethanol (0.386 mL, 5.5 mmol) under nitrogen at 0 ° C and immediately thereafter with boron trifluoride etherate (0.176 mL, 1.43 mmol). After stirring for 5 minutes, the solution is extracted with a mixture of dipotassium hydrogen phosphate solution (1.5 g of salt in 4 ml of water) and ethyl acetate (12 ml). The aqueous phase is extracted a second time with ethyl acetate. The combined ethyl acetate extracts are washed with brine, dried over magnesium sulfate and evaporated. The filtrate is evaporated under reduced pressure to give 229 mg of an oil. The product is purified by preparative thick layer chromatography using silica gel and eluting with ethyl acetate to give 118 mg of trans-3- (1-p-nitrobenzylcarbonyldioxyethyl) -4- [2,2-bis (2-hydroxyethyl) thioethyl] -2 -azetidinone as a colorless oil.

IR (CH 2 Cl 2): 5.75 μm (5.79 μm shoulder), β-lactam and carbonate; 6.20 and 6.55 pm (nitro)

NMR (acetone-d 6) T: 1.70, d, J = 8.5Hz, 2H, ArH

2.28, d, J = 8.5 Hz, 2H, ArH 2.48 - 2.88, m, 1H, NH

4.63, s, ArCH 2 3H

4.63 - 5.12, m, CH 3 CH

5.80, t, J = 7Hz, CH CH 2 Cl 2

1 ^ S I 13H

5.80 - 7.45, m, C-4H + C-3H + SCH2CH2OH

7.63 - 8.33, m, 2H, CH 2 CH 8.53, d, J = 6.5 Hz 3H, CH 3 CH

The cis diastereoisomer is obtained in an analogous manner.

Alternatively, mixed diastereoisomers are obtained when the starting material comprises a mixture of diastereomers.

23 62 30 6

Step I: Trans-3- (1-p-nitrobenzylcarbonyldioxyethyl) -4-

Preparation of Fl, 2-bis (2-azidoethyl) thioethyl] -2-azetidinone

<y00PNB 0H

j + 2CH3SO2Cl2Et3N NaN3

NH THF DMSO

O

fOORNB / \ ^ N3

NH

Q>

To a solution of 211 mg (molecular weight = 474; 0.445 mmol) of trans-3- (1-nitrobenzylcarbonyldioxyethyl) -4- [2,2-bis- (2-hydroxyethyl) thioethyl] -2-azetidinone in 5 mL of tetra- hydrofuran (distilled from lithium aluminum hydride) at 0 ° C, 103 mg of mesyl chloride (molecular weight = 114; 0.904 mmol) in 1 ml of tetrahydrofuran are added, followed immediately by 134 μΐ of triethylamine (molecular weight = 101; = 0.729; 0.967 mmol). The reaction mixture is stirred for 1 hour under a nitrogen blanket. The triethylamine hydrochloride is filtered under a nitrogen blanket and washed with a few milliliters of tetrahydrofuran. The clear colorless filtrate is concentrated under a stream of nitrogen and then pumped under high vacuum for 10 minutes. The dimethylate is immediately dissolved in 5 ml of dimethyl sulfoxide (distilled from Cat at 8 mm and stored in the presence of a 4A Linde molecular sieve) containing 347 mg of NaN3 (molecular weight = 65; 5.34 mmol). Stir overnight under a nitrogen blanket and add 10 ml of water and 20 ml of ethyl acetate. The layers are separated and the aqueous phase is washed three times with 10 ml of ethyl acetate, washing each organic layer with a further 10 ml of water and 10 ml of brine. The combined ethyl acetate extracts are dried over anhydrous magnesium sulfate, filtered and concentrated under a stream of nitrogen to give crude diazide. Preparative thin layer chromatography using silica gel gives trans-3- (1-p-nitrobenzyl-24,630,6 carbonyldioxyethyl) -4H-2,2-bis- (2-azidoethyl) thioethyl] -2-azetidinone.

The cis diastereomers or the cis trans mixture are obtained in an analogous manner.

Step J: co2h co2pnb C (OH) 2 + 2NO2 CHN2 E ^ Et2 ° E (0H> 2 CO2H co2pnb +

OCOOPNB OCOOPNB

<SCH2CHoN-s C

A -aa * Ay \ / SCiW3 ANH '®CH2CH2E3 .A, ^ «= 2 ° * 2» 3 0 2 o' γ '(CO2PNB) 2 1 A »PNB = -CH2 - <^ - NO2 p-nitrophenyldiazomethane ( 29 mmol) of freshly prepared solution (H. Davies and M. Schwarz, JOC 1242 (1965)) in 150 ml of ether is added with stirring to a solution of 1.0 g of oxomalonic acid monohydrate (molecular weight = 136; 735 ramol). ml of ethyl acetate at 0 ° C. After 2.5 hours, the yellow solution is concentrated on a rotary evaporator with gentle heating to about half volume, dried over anhydrous sodium sulfate, filtered and concentrated as before to an oil. To the crude p-nitrobenzyl ester in 50 ml of toluene is added 3.54 g of trans-3- (1-p-nitrobenzylcarbonyldioxyethyl) -4- [5,2-bis (2-azidoethyl) thioethyl] -2-azetidinone (molecular weight = 524; 6.75 mmol). The reaction mixture is heated with stirring in an oil bath and about 1/3 of the toluene is allowed to boil off. Toluene is re-added (dried with a 3A 1/16 "Linde molecular sieve) to give a volume of 25 62306 50 ml and the aze-drying process is repeated 3 more times. a solution in a stream of nitrogen gives crude compound 1. The crude product is chromatographed on silica gel to give compound 1. The cis-diastereoisomers or cis-trans mixture is obtained in an analogous manner.

False K:

OCOOPNB

\ SCH.CH.N, A— «I \ sch2ch2n3" J-Ν · \> ° Η + SOCl „-—->

> r 2 THF

(C02PNB)

J "OCOOPNB

I Γ \ SCH2CH2N3 + 0jP ag. DMF _ q / Γ (co2pnb) 2 «a

OCOOPNB

/ Αη — sch2ch2n3 I ^ sch2ch2n3 <co2pnb) 2 2 26 62306

To a solution of 2.80 g of compound 1 (molecular weight = 912; 3.07 mmol) in 35 ml of tetrahydrofuran (distilled from lithium aluminum hydride) at -20 ° C is added 0.3 ml of pyridine (molecular weight * 79; 9 = 0.983; 3.73 mmol) (distilled from NaH and stored on a 4A Linde molecular sieve). While stirring under nitrogen, 0.438 g of thionyl chloride (molecular weight = 119; 3.68 mmol) in 1 ml of tetrahydrofuran are added dropwise. The reaction solution is stirred under a nitrogen blanket for 5 minutes at -20 ° C, then for half an hour at 0 ° C and finally for 1 hour at 25 ° C. The pyridine hydrochloride is filtered under a nitrogen blanket and washed twice with benzene (dried on a 3A 1/16 "Linde molecular sieve). The combined filtrate and washings are concentrated under a stream of nitrogen. To the resulting chlorine compound, 0.885 g of triphenylphosphine (molecular weight = 262; 3.38 mmol) in 66 ml of dimethylformamide / water 9: 1 and 550 mg of 1 H-HPO 2 (molecular weight = 174; 3.16 mmol) are added with stirring. The reaction mixture is stirred at 25 DEG C. for 35 minutes, diluted with ethyl acetate, the layers separated by brine and the aqueous layer extracted three times with ethyl acetate. using silica gel to give compound 2.

The cis-diastereoisomers or cis-trans mixture are obtained in an analogous manner.

OCOOPNB

Step L: 27 6230 6 SCH2CH2N3 j rV ^ eCH2CH2N3 + Br2 -Et20 / C5H12 - - 'γ'? (CO2PNB) 2 2

/ V

F- - 1 OCOOPNB Br “Br

I SCH.CH.N, L JJ

'' N- / 2 2 3 (1)

JNXsCH2CH2N3 (2) NaH, DMF

V

(C02pnb) 2

OCOOPNB

^ scH3 +

/-OF. AND

(CO 2 PNN) 2 3 / N / 28 62306 To 7.8 ml of pentane (dried on a 4A Linde molecular sieve) is added 0.2 ml of Br 2 (molecular weight = 160; 9 = 3.12; 3.9 mmol). To a solution of 950 mg of compound 2 (molecular weight = 896; 1.06 mmol) in 15 mL of E 2 Orta (dried with a 3A 1/16 "Linde molecular sieve) at 0 ° C under a nitrogen blanket is added dropwise, with stirring, 2.3 mL the above-mentioned 0.49-m solution of Br 2 (1.13 mmol) After stirring for 10 min at 0 ° C, 114 μl of cyclohexene (molecular weight = 82; Q = 0.81; 1.13 mmol) are added. After 5 min at 0 ° C, 53 mg of 57% NaH (57% of 53 mg = 30.2 mg, molecular weight = 24; 1.26 mmol) in mineral oil are added to the stirred reaction mixture, followed by the immediate addition of 14 mL of ice-cold dimethylformamide. (Distilled in the presence of anhydrous CaSO 4 at 40 mm and stored in the presence of a 4A Linde molecular sieve.) Stirring at 0 ° C under nitrogen is continued for 3 hours. and 75 ml of ethyl acetate, after separation of the layers, the aqueous phase is saturated with NaCl and the ethyl acetate is re-extracted. with. The combined organic phases are washed once with brine, dried over anhydrous magnesium sulfate, filtered and concentrated under a stream of nitrogen and then kept under high vacuum to give crude compound 3. Preparative thin layer chromatography on silica gel gives compound 3.

The cis-diastereoisomers or cis-trans mixture are obtained in an analogous manner.

Step M; 29 6 2 3 0 6

OCOOPNB

A ^ ^ ch2ch2h3 Γ Et2O / C5H12___ + Br2 -> °> ('CO2 CO2N2) 2 3 -

OCOOPNB

J Γ / *

e CHCH CT

SCH-CH N

I 2 2-5 DMF

+ NaH —-> (CO 2 PNB) 2 OCOOPNB Br ΛτΛ 1 V ^ wSCH 2 CH 2 N 3

/ "V

(CO 2 pnb) 2 4 rv * 30 62306 To 9.16 ml of pentane (dried on a 4A Linde molecular sieve) is added 0.2 ml of Br 2 (molecular weight = 160; 3.9 mmol).

474 mg of compound 3 (molecular weight = 793; 0.598 mmol) in 13 ml of ether (dried on a 3A 1/16 "Linde molecular sieve) at 0 ° C under a nitrogen blanket are added dropwise with stirring 1.52 ml of the 0.42 M solution obtained above. Br2 solution (0.63 mmol).

After 15 minutes at 0 ° C, 33 mg of 57% NaH (57% of 33 mg = 18.8 mg; molecular weight = 24; 0.78 mmol) are added, followed immediately by 6.3 ml of ice-cold dimethylformamide (distilled anhydrous CaSO at 40 mm and stored in the presence of a 4A Linde molecular sieve). The reaction mixture is stirred for 1.5 hours at 0 [deg.] C., then poured into an ice-cold mixture of 1.6 ml of 1 M HCl, 20 ml of water and 20 ml of ethyl acetate. After separation of the layers, the aqueous phase is saturated with sodium chloride and re-extracted with ethyl acetate. The combined organic extracts are washed once with brine, dried over anhydrous magnesium sulfate, filtered and concentrated under a stream of nitrogen and then kept under high vacuum to give crude compound 4. Preparative thin layer chromatography using silica gel gives compound 4.

The cis and diastereoisomers and the cis-trans mixture are obtained in an analogous manner.

Step N; OCOOPNB ar, __ 31 62 3 Q 6

O

I CH N + \ ^ ^

I | / ^ ^ "2 ^ 2 w3 N

Λ- V

(CO2PNB) 2 4 rv ^

OCOOPNB

^ "RTV

DMSO / SCH2CH2N3 * (CO2PNB) 2 A »To 210 mg of compound 4 (molacyl weight = 871; 0.241 mmol) dissolved in 0.5 ml of diraethyl sulfoxide (distilled from CaH2 at 8 mm and stored in the presence of a 4A Linde molecular sieve) is added at 25 ° C. while stirring 40 mg of 1,5-diazobicyclo [5.4.0] undec-5-ene (distilled at 80 ° C / 2 mm) (molecular weight 152; 0.263 mmol) in 0.7 ml of dimethyl sulfoxide. The solution is stirred under a nitrogen blanket for 4 hours, then added to a stirred ice-cold mixture of 0.48 ml of 1 M KH 2 PO 4, 7 ml of water and 10 ml of ethyl acetate. After separation of the layers, the aqueous layer is saturated with NaCl and re-extracted with ethyl acetate. The combined organic layers are washed once with brine, dried over anhydrous magnesium sulfate, filtered, concentrated under a stream of nitrogen, and then kept under high vacuum to give crude compound 5. Preparative thin layer chromatography on silica gel gives compound 5.

The cis-diastereoisomers or cis-trans mixture are obtained in an analogous manner.

OCOOPNB

Step O: 62306 1 / SCH CH N + Li - amcollidine

λ ....... NV

0 I

(co2pnb) 2 5

'V

OCOOPNB

Arrv | V— sch2ch2n3

s-: X

H CO2PNB

6

Λ-J

A solution of 187 mg of compound 5 (molecular weight = 791; 0.236 mmol) in 2.5 ml of s-collidine (distilled from powdery KOH at 30 mm) is added to 45 mg of anhydrous LiJ (dried for a few hours at 100 ° C). P2O2 in vacuo) (molecular weight = 134; 0.336 mmol). While stirring under nitrogen, the reaction mixture is heated in an oil bath at 120 ° C. After 25 minutes, the reaction mixture is cooled to 25 ° C, diluted with CH 2 Cl 2 and transferred to a round bottom flask for concentration under a stream of nitrogen and then in vacuo. The residue is extracted with a mixture of 10 mL of ethyl acetate, 1.8 mL of 1 M KH 2 PO 4 and 10 mL. ml of water, and then the aqueous phase is extracted twice with ethyl acetate. The combined organic phases are extracted with brine, dried over anhydrous magnesium sulfate, filtered and concentrated under a stream of nitrogen to give crude compound 6. Preparative thin layer chromatography using silica gel gives compound 6.

The cis-diastereoisomers or cis-trans mixture are obtained in an analogous manner.

Step P; 33 6 2 3 0 6

O

il

OCOPNB

+ (j \ -N -LcOjPNB \ f ^ Sl rv

O

II

OCOPNB

DMSO -T ^> - SCH2CH2N3 -

J_N - C02PNB

Q

7 'Λ.'

To a solution of the mixed diastereoisomers of compound 6 (34 mg; · molecular weight = 612; 0.055 mmol) in 0.2 ml of dimethyl sulfoxide (distilled from CaH2 at 8 mm and stored in the presence of a 4A Linde molecular sieve) is added with stirring 9.5 μΐl. 5-diazobicyclo / S, 4, Q7undek-5-ene (distilled at 80 ° C / 2 torr) (molecular weight = 152; 0.0625 mmol). The solution is stirred under a nitrogen blanket for 15 minutes and diluted to a total volume of 1 ml with CHCl 3, after which it is immediately transferred to a 2 to 1000 μπι silica gel plate. The product strip gives compound 7 as a mixture of cis and trans diastereomers.

OCOOPNB 0H

Step Q: 34 62306 ^ N - r ^ VSCH.CH.N-, -f ^ V-SCH CHONH.

! 1 2 2 3 H / 2.8 kg / cm2 I 222

A-N --- CO_PNB ---> A - N-1-CO-H

O 1 o 7 8 Λ, O '61 mg of compound 7 (molecular weight = 612; 0.1 mmol) in 6 ml of dioxane, 6 ml of tetrahydrofuran and 3 ml of water are hydrogenated 2 in the presence of 61 mg of PtO 2 at a pressure of 2.8 kg / cm of hydrogen for 4 hours. The reaction mixture is then filtered through Celite and washed with 2 mL of 0.1 N phosphate buffer (pH 7). Concentrate in vacuo to cloud point and extract the aqueous mixture with ethyl acetate. The aqueous layer is concentrated to a small volume and applied to a column containing 100 g of XAD-2 resin. Elute with water, discard the initial fractions, and recover the product-containing fractions, which are lyophilized to give compound 8 as a mixture of cis- and trans-diastereoisomers.

The following method for the enzymatic N-deacylation of thienamycin is suitable for use with all isomers of thienamycin, especially the separate N-acetyl isomers 890A, and 890A, which will be described later.

Deacetylation of N-acetyl-thienamycin

A 1% (w / v) suspension of fertile grass peat is prepared by suspending 1 g of grass peat in 100 ml of sterile phosphate buffered saline, in which the phosphate buffered saline solution has the following composition:

NaCl 8.8 g 1-m phosphate buffer, pH 7.5 * 10 ml distilled water 1000 mc * 1-m phosphate buffer (pH 7.5) is prepared by mixing 16 ml 1-m KH-PO. With 84 ml of 1-m K ^ HPO .. The pH of the solution is adjusted to 2 4 2 4 7.5 by adding small amounts of either 1-m I ^ HPCl 2 or 1-m KH 2 PO 4 · From this 1% herb peat suspension 10 x 100 x, and 1000 x dilutions.

3530306 1 ml of suspension or 1 ml of 10 x, 100 x and 1000 x dilutions is added to 2 ml of sterile 1% agar solution at 48 ° C. The mixtures are rapidly poured onto the surface of 85 mm sterile petri dishes containing 20 ml of medium A. Medium A has the following composition: KH2PO4 3.0 g K2HPO4 7.0 g

MgSO 4 0.1 g distilled water 1000 ml N-acetylethanolamine solution * 8.5 ml * N-acetylethanolamine solution is prepared by diluting N-acetylethanolamine with water 10 times and membrane sterilizing, followed by autoclaving. The pH is adjusted to 6 with NaOH. For solid media, add 20 g of agar.

Petri dishes are cultured for 18 days at 28 ° C. The well-isolated column is recovered and brushed into a petri dish containing medium B. Medium B has the following composition;

Tomato paste 40 g

Oatmeal 15 g

Distilled water 1000 ml pH is adjusted to 6 using NaOH.

For solid media, add 20 g of agar.

A separate column is selected and cultured for 2 days at 28 ° C as an oblique culture in medium B. Part of this growth is applied to the surface of six oblique cultures prepared from medium B. These oblique cultures are cultured for 2 days at 28 ° C; resulting in a culture of Protaminobacter ruber (deposited with the Agricultural Research Service, U.S. Department of Agriculture under the designation NRRL B-8143).

Part of the growth of Protaminobacter ruber MB-3528 oblique culture is used to inoculate a 250 ml Erlenmeyer flask containing 50 ml of medium C. Medium C has the following composition:

Dextrose 20 g

Farmamedia 8 g

Corn soaking liquid (calculated wet) 5 g

Distilled water 1000 ml 36 6 2 3 0 6 The pH is adjusted to 7 using NaOH or HCl N-acetylethanolamine solution 8/5 ml 8.5 ml N-acetylethanolamine solution N-acetylethanolamine is diluted 10-fold with water and membrane sterilized. This solution is added after treatment with an autoclave.

The flask is shaken at 28 ° C (220 rpm, swing 5 cm) for 4 days. A 25 ml aliquot of the flask is centrifuged for 15 minutes at 8000 rpm. The supernatant is removed and the cells on the medium are scraped off into 0.5 ml of 0.05 M potassium phosphate buffer, pH 7.4. The resulting suspension is subjected to ultrasonic digestion (Branson Instrument Model LS-75 Sonifier) with a probe of 12.5 mm at setting 4, using intervals of 4 and 15 seconds, with the suspension being cooled with ice water between digests. 10 μΐ of the sonicate is mixed with 25 μl of a solution containing 840 pg / ml N-acetylthienamycin in 10 mM potassium phosphate buffer, pH 7, and cultured overnight at 28 ° C. Control experiments containing only antibiotic and buffer are also performed; as well as control experiments involving sound-treated cells and buffer without antibiotic. After culturing overnight at 28 ° C, 2 μΐ volumes are applied to cellulose-coated TLC plates developed with a mixture of ethanol and water (70:30). The TLC plate is air dried, then placed on a STaphylococcuc aureus ATCC 6538P assay plate for 5 minutes.

The assay plates are prepared as follows: Overnight growth of the test organism Staphylococcus aureus ATCC 6538P in nutrient broth containing 0.2% yeast extract is diluted with nutrient broth containing 0.2% yeast extract to a suspension having a transmittance of 60% at 660 nm. This suspension is added to Difco nutrient agar supplemented with 2.0 g per liter of Difco yeast extract at 37-48 ° C to obtain a composition containing 33.2 ml of suspension per liter of agar. 40 ml of this suspension is poured onto 22.5 cm x 22.5 cm petroleum plates and these plates are cooled and kept at 4 ° C until use (maximum 5 days).

The TLC plate is removed and the analysis plate is cultured overnight at 37 ° C. Except for the spot of unreacted bioactive N-acetyl-thienamycin at = 0.7 to 0.89, a bioactive spot due to thienamycin at 37 62306 is found at Rf = 0.44 to 0.47. Culture mixtures prepared for the control of antibiotic and buffer, cell sonicate and buffer, and antibiotic and buffer to which the latter cell sonicate has been added just before the addition of TLC do not produce any bioactive substance at Rf = 0.44 to 0.47.

Preparation of the separate isomer 890A ^ OH

JL. ^ n ^ -s' \ -SCH2CH2NHCCH3

^ N - J-COOH

Tubes of lyophilized culture of Streptomyces flavogriseus Ma-4434 (NRR1 8139) are opened aseptically and the contents are suspended in a tube containing 0.8 ml of sterile Davis salts having the following composition:

Sodium citrate 0.5 g K2HPO4 7.0 g KH2PO4 3.0 g (NH4) 2SO4 1.0 g

MgSO 4 · 7H 2 O 0.1 g

Distilled water 1000 ml This suspension is used to inoculate an oblique culture of medium A 4 having the following composition:

Glycerol 20.0 g

Primary yeast 5.0 g

Fishmeal 15.0 g

Distilled water 1000 ml

Agar 20.0 g The pH is adjusted to 7.2 using NaOH.

Inoculated slants are cultured for 1 week at 27-28 ° C and stored at 4-6 ° C until use.

38 62 3 0 6 1/3 The growth of the above 3 oblique cultures is used to inoculate a 250 ml Erlenmeyr flask with 9 partitions containing 50 ml of medium B having the following composition: Yeast autolysate ("Ardamine") * 10.0 g

Glucose 10.0 g

Phosphate buffer ** 2.0 ml

MgSO4.7H2O 50 mg

Distilled water 1000 ml

The pH is adjusted to 6.5 using HCl or NaOH

* Yeast Products Corporation ** Phosphate buffer solution: KH2PO4 91.0 g

Na 2 HPO 4 95.0 g

Distilled water 1000 ml

The seed flask is shaken for 1 day at 27-28 ° C on a shaker (220 rpm, swing 5 cm). The flask and its contents are stored for 1 day at 4 ° C.

33 2-liter Erlenmeyer production flasks, each containing 250 ml of medium C, are inoculated into 8 ml flasks per growth from the seed flask. Medium C has the following composition:

Tomato paste 20.0 g

Primary yeast 10.0 g

Dextrin (amidex) 20.0 g

CoCl 2 .6H 2 O 5.0 mg

Distilled water The pH of 1000 ml is adjusted to 7.2 to 7.4 using NaOH.

After inoculation, the production flasks are cultured at 24 ° C with stirring on a shaker (212 rpm, swing 5 cm) for 4 days. The flask is harvested and analyzed for activity (standard Salmonella gallinarum MB1287 and Vibrio percolans ATCC 846 assay plates) using 12.5 mm assay plates dipped in centrifuged broth samples. Samples were diluted with 0.02 M phosphate buffer (pH 7, θ ') as needed. The results are shown in the following table: 39 62306

Table

Culture time 96 hours pH 7.2

Zone:

Salmonella gallinarum 29.5 mm

Vibrio percolans (1/10-Dil.) 31 mm 890 test 40 units 7 liters of fermentation broth are cooled to 3 ° C and centrifuged in 200 ml portions (9000 rpm, 15 minutes).

To the combined surface materials, 7 ml of 0.1 M neutral EDTA is added and the total sample is adsorbed using a Dowex® x 2 (Cl), 50-100 mesh column with a bed size of 5.1 x 25 cm and a flow rate of 40 ml per hour. The column is washed with 500 ml of deionized water containing 5 ml of 1 M Tris-hydrochloride buffer (pH 7.0) and 20 pmol of neutral EDTA. The antibiotic is eluted with 1 liter of deionized water containing 50 g of sodium chloride and the column is then washed with 300 ml of deionized water. Collect 100 ml fractions starting from the fraction first containing salt at the removal of the column. Bioactivity occurs in fractions 1-10 with a peak in fraction 2. Fractions 2-5 containing a 17% increase in activity are recovered.

The recovered fractions are concentrated to a volume of 110 ml on a rotary evaporator under reduced pressure and the pH is adjusted to 5.8 by adding 4.2 ml of 1M hydrochloric acid. The adjusted concentrate is added to the column with a size of 3.8 x 50 cm XAD-2 layer, which has been pre-washed with 3 liters of 60% aqueous acetone (v / v) and then with 3 liters of deionized water, 3 liters of 5-% (w / v). volume) of sodium chloride and 1 liter of 25% (w / v) sodium chloride in deionized water. The added concentrate is allowed to drain to the bed. The antibiotic is eluted with deionized water at a flow rate of 15 ml per minute. Collect 22 fractions of 100 ml starting from the first fraction of the sample. Bioactivity occurs in fractions 6-22 with a peak in fractions 9 and 10. Fractions 9-20 are pooled for further processing. Fractions of 40 62306 prepared in the same manner are combined and concentrated to a volume of 70 ml on a rotary evaporator under reduced pressure.

The concentrate is adsorbed onto a Dowex-1 x 4 (Cl ”) 400 mesh column with a layer size of 2.2 x 27 cm. The column is washed with 50 ml of deionized water and the antibiotic is eluted with 2 liters of 0.070-m NaCl + 0.005-m NH4Cl + 0.0001-m NH4 in deionized water at a flow rate of 2 ml per minute. 9.5 ml fractions are collected.

The major antibiotic peak is eluted in fractions 142-163. Fractions 146-157 are pooled for further processing. The combined fractions 146 to 157 are concentrated to a volume of 3 ml on a rotary evaporator under reduced pressure and the concentrate is adjusted to pH 6.5 by the addition of 5 .mu.l of 1 m NH3. The concentrate is applied to a column (2.2 x 70 cm) - 400 mesh) washed with 20 ml of 5-m NaCl and 500 ml of deionized water. After the concentrate has been run into the bed, rinse twice with 1 ml of deionized water and allow to drain into the bed, then elute the column with deionized water at a flow rate of 0.6 ml per minute. 2.9 ml fractions are collected.

The major peak of the antibiotic is eluted in fractions 63-70.

Fractions 64 and 65 are recovered for further processing. The combined fractions 64-65 are evaporated on a rotary evaporator under reduced pressure to a volume of 2 ml, cooled and lyophilized in a 14 ml screw-flask for 8 hours to give 2.25 mg of essentially pure antibiotic 890 Å. The N-weapon style group is cleaved as described above to give the free base:

OH

Y ^ JT SCHjCHjNHj

0 f-N - I - COOH

Preparation of separate isomer 890A3 OH

O-SCH 2 CH 2 NH 2 CH 3 O - N-Hcooh o

Tubes of lyophilized culture of Streptomyces flavogriseus MA-4434 (NRR1 8139) are opened aseptically and the contents are suspended in a tube containing 0.8 ml of sterile Davis salts having the following composition:

Sodium citrate 0.5 g K2HPO4 7.0 g KH2PO4 3.0 g (NH4) 2SO 1.0 g

MgSO4.7H2O 0.1 g

Distilled water 1000 ml This suspension is used to inoculate an oblique culture of medium A 4 having the following composition:

Glycerol 20.0 g

Primary yeast 5.0 g

Fishmeal 15.0 g

Distilled water 1000 ml

Agar 20.0 g The pH is adjusted to 7.2 using NaOH.

Inoculated slants are cultured for 1 week at 27 to 28 ° C and then stored at 4 to 6 ° C until use (maximum 21 days).

A third part of the growth of the above oblique culture is used to inoculate a 250 ml Erlenmeyer flask with 12 partitions containing 50 ml of medium B having the following composition: Yeast autolysate ("Ardamine") * 10.0 g

Glucose 10.0 g

Phosphate buffer ** 2.0 g

MgSO4.7H2O 50 mg

Distilled water 1000 ml of pH is adjusted to 6.5 using HCl or NaOH.

* Yeast Products Corporation 42 62306 ** Phosphate buffer solution: KH2PO4 91.0 g

Na 2 HPO 4 95.0 g

Distilled water 1000 ml

The seed flask is shaken for 1 day at 27 to 28 ° C on a shaker (220 rpm, swing 5 cm). The flask and its contents are stored for 1 day at 4 ° C.

44 2-liter Erlenmeyer production flasks, each containing 200 ml of medium C, are inoculated with 8 ml per flask of growth obtained from the seed flask. Medium C has the following composition:

Tomato paste 20.0 g

Primary yeast 10.0 g

Dectrin (Amidex) 20.0 g

CoCl 2 -5H 2 O 5.0 mg

Distilled water The pH of 1000 ml is adjusted to 7.2 to 7.4 using NaOH.

After inoculation, the production flasks are cultured at 24 ° C with stirring on a shaker (212 rpm, swing 5 cm) for 4 days and 5 hours. The crop is recovered from the bottle. and analyzed for activity (standard Salmonella gallinarum MB 1287 and Vibrio percolans ATCC 8461 assay plates) using 12.5 mm assay plates dipped in centrifuged broth samples. Samples are diluted with 0.02 M phosphate buffer (pH 7.0) as needed. The results are shown in the following table:

Culture time 101 hours pH 7.2

Zone:

Salmonella gallinarum 35 mm

Vibrio percolans (1/10-Dil.) 34 mm 890 test 121 units * 3 62306 Fermentation gives 7.0 liters of fermentation broth, which is cooled to 30 ° C and centrifuged in 200 ml batches (9000 rpm for 15 minutes). . 1.7 ml of 0.1 M neutral EDTA is added to the combined surface liquids and the batch is kept at 3 ° C.

The fermentation described above is repeated under identical conditions, except that 44 2-liter Erlenmeyer production flasks are inoculated with 7 ml per flask of growth obtained from the seed flask. The pH and analytical results are as follows:

Culture time 101 hours pH 7.3

Zone:

Salmonella gallinarum 38 mm

Vibrio percolans (1/10-Dil.) 39 mm 890 test 92.8 units

The resulting 7.4 liters of fermentation broth is cooled to 3 ° C and centrifuged in 200 ml aliquots (9000 rpm for 15 minutes). To the combined supernatant fluids is added 1.8 mL of 0.1 N EDTA.

The supernatant obtained from the centrifuged broth obtained from the above fermentations is combined to give a total volume of 13 liters.

The combined supernatants are applied to a Dowex® 2 x (Cl) column, 50 to 100 mesh with a bed size of 4.7 x 50 cm and a flow rate of 60 ml per minute. The column is washed with 1 liter of deionized water and the antibiotic is eluted with 5 liters of 5% (w / v) NaCl solution containing 0.01 M Tris-hydrochloride buffer (pH 7.0) and 20 pmol of neutral EDTA. Collect 220 ml fractions at a flow rate of 50 ml per minute and analyze the fractions on Salmonella gallinarum MB1287 plates.

Antibiotic activity occurs in fractions 3-26 with a peak in fractions 5 and 6. Fractions 5-9 are pooled for further processing. The pooled fractions have a pH of 7.8 and contain 24% of the original bioactivity measured on Salmonella gallinarum MB1287 plates.

6 2 3 0 6 44

The combined fractions 5-9 are concentrated to a volume of 150 ml on a rotary evaporator under reduced pressure and applied to a column (layer size 4.9 x 47 cm) of XAD-2 washed with 5 liters of 60% (v / v) aqueous acetone and then with 5 liters of NaCl. solution (50 g / l) in deionized water. The sample is added in 20 ml portions, running the column to the bed level each time. At the end of the addition, three 20 ral aliquots of deionized water are fed and allowed to run to the bed level each time. The sample is eluted with deionized water at a flow rate of 20 ml / min. All treatments using an XAD column are performed at room temperature (24 ° C) and the eluted fractions are rapidly cooled in an ice bath immediately after recovery. Fractions 95 to 230 ml are collected.

Antibiotic activity is present in fractions 2-21 (measured by analysis on Salmonella gallinarum MB1287 plates) with a peak in fractions 5-7 (comprising 510-895 ml of eluted volume calculated from the first addition of deionized water). Fractions 6-21 are collected and concentrated to a volume of 28 ml on a rotary evaporator under reduced pressure and then diluted to a volume of 112 ml by adding deionized water. The concentrate is applied to a column (2.2 x 21 cm layer size) Dowex® x 4 (Cl), 400 mesh. The column is washed with 20 ml of deionized water and the antibiotic is eluted with 2 liters of 0.07-m NaCl + 0.005-m NH4 Cl + 0.0001-m NH4 deionized water at a flow rate of 1.6 ml / min. Collect 8 ml fractions. Fractions 157 to 179 with the highest activity are pooled. These fractions are concentrated on a rotary evaporator under reduced pressure to a volume of 7 ml and the pH is adjusted to 7.5 by adding 20 μΐ of 1-m sodium hydroxide.

The solution is further concentrated to a volume of 5 ml and applied to a column (2.2 x 75 cm) of Bio-Gel P-2, 200-400 mesh. The sample is washed on the column bed by rinsing twice with 1 ml of deionized water and eluted with deionized water at a flow rate of 0.6 ml per minute. Collect 10 3.3 ml fractions, 60 2.65 ml fractions and 10 2.0 ml fractions.

The fractions are adjusted to pH 7.5 to 8.0 by adding 1.5 to 2.5 of 0.1 M sodium hydroxide. Fractions 62, 63, 64 and 65 are cooled and lyophilized separately in 14 ml glass vials and stored at -20 ° C under vacuum.

62306 45

To isolate antibiotic 890A ^ free from antibiotic 890A.J, the relatively higher resistance of antibiotic 890A ^ to penicillinase-induced degradation is utilized as follows:

Bio-Gel fraction 63 is combined with fractions 61, 66 and 67 obtained from the Bio-Gel column. To these four combined fractions are added 0.2 ml of 1 M tris-hydrochloride buffer (pH 7.5) and 0.2 ml of penicillinase (Difco "Bacto-Penase"). Hold for 113 minutes at 23 ° C and add a further 0.2 ml of penicillinase. After 7 hours at room temperature, a further 0.2 ml of penicillinase is added and after 2 hours the reaction mixture is cooled in an ice bath, after which the reaction mixture is diluted to a volume of 15 ml by adding 5 ml of deionized water.

The reaction mixture is adsorbed onto a Dowex x 4 (Cl), 400 mesh column with a bed size of 2.15 x 40 cm. The antibiotic 890Al is eluted using 0.07-m NaCl + 0.005-m NH 4 Cl + 0.0001-m NH 4 in deionized water at a flow rate of 3 ml / min. Collect 13.8 ml fractions, combine fractions 187-200.

The combined fractions are concentrated to a volume of 4 ml on a rotary evaporator under reduced pressure and the concentrate is applied to a column (2.2 x 70 cm) of Bio-Gel P-2, 200-400 mesh. Antibiotic 890A2 is eluted with deionized water at a flow rate of 0.6 ml / min. Collect 30 fractions, 3.3 ml in size, followed by 50 fractions, 2.65 ml in size.

Fractions 66-70 are combined and the combined samples are concentrated to a volume of 1.5 ml on a rotary evaporator under reduced pressure, and the concentrate is cooled and lyophilized to give 5.4 mg of a solid containing the antibiotic δΟΟΑ ^ and the remaining salts. The N-acyl group is cleaved to the free base as described above

OH

- - sch2ch2nh2

I- N - C00H

To obtain 0 ^.

46 62306

The following examples illustrate the method of the invention.

Example 1 ci® clx ΘΑ- \ Γοη

H / C W h H

Th - N = C

Θ \ ^ \ --- * - CO (0

Preparation of Thienamycin-N-piperidin-1-yl-methylene derivative

Thienamycin (57 mg, 162 pmol) is silylated according to the method described above. The silylated antibiotic is dissolved in methylene chloride (6 mL) in a sealed flask under nitrogen and cooled in a dry ice-acetone bath. To the magnetically stirred solution is added a solution (180 μl) of triethylamine (660 pmol) in methylene chloride. A solution of chloropiperidinomethylumium chloride (67 mg, 405 pmol) in methylene chloride (465 pmol) is then added. After 1 hour in a dry ice bath, the reaction mixture is added rapidly to a mixture of tetrahydrofuran and 0.1 N (pH 7.0) phosphate buffer (1: 1) (50 ml), and the mixture is concentrated in vacuo to a volume of 10 ml to obtain a homogeneous solution. The solution is washed twice with ethyl acetate (2x5 ml) and then with ether (2x5 ml) and evaporated in vacuo. This aqueous solution is chromatographed using an XAD-2 resin column (60 mL, once). The product is eluted with 10% aqueous tetrahydrofuran (followed by elution with water) to give 12.9 mg (22%) of product (measured in solution, assuming a value of € 8,030 for thienamycin). Paper chromatographic analysis: = 0.42 (4: 1: 5, n-BuOH: EtOH: water).

47 6 2 3 0 6 Preparation of the starting material, silylated thienamycin

- OTMS

Th - NHTMS

-COOTMS

TMS = trimethylsilyl

Thienamycin (80.0 mg) is suspended in 40 ml of tetrahydrofuran under a nitrogen blanket and concentrated to a volume of 10 ml, and hexamethyldisilazane (1.0 ml) and trimethylchlorosilane (300 μΐ) are added. The mixture is allowed to react for 20 minutes at 25 ° C with vigorous stirring. The suspension is then centrifuged to remove ammonium chloride. The supernatant is evaporated to an oil in a stream of nitrogen for reaction.

Example 2

"OH

Preparation of N "benzimidoyl-thienamycin

Thienamycin (59 mg, 212 pmol) is dissolved in a solution of 33% N, N-dimethylformamide and 0.05 N (pH 7.0) in phosphate buffer (4.5 mL) and the pH is adjusted to 9.5 using 2.5 sodium hydroxide and an automatic dosing burette. The solution is stirred magnetically at 25 ° C and methyl benzimidate hydrochloride (340 mg, 1981 pmol) is added in one portion. After 30 minutes, the solution is extracted twice with an equal volume of chloroform and the pH is adjusted to 7.0 with dilute phosphoric acid solution. The buffered solution is chromatographed using XAD-2 resin (65 mL). The column is eluted first with water and then with 10% aqueous tetrahydrofuran. This fraction is concentrated to half volume and freeze-dried to give 50 mg of product. Electrophoretic mobility (50 V / cm, 20 min, pH 7 / 0.1 phosphate buffer) is 1.5 cm per anode. uvJt mafcs = 300 11111 (£ 6960), pH 7 / 0.1 phosphate buffer.

Example 3 Preparation of N-formimidoyl-thienamycin

OH

SCH CH N = C-NH_ li 2 2 L 2

¢¢ 7-N COOH

Thienamycin (517 mg) is dissolved in 0.1 N phosphate buffer (pH 7, 25 mL) and cooled in an ice bath with magnetic stirring. The solution is adjusted to pH 8.5 using 2.5 N sodium hydroxide solution (automatic burette). While maintaining the pH at 8.5, methyl formimidate hydrochloride (711 mg) is added portionwise over 2-3 minutes. After 10 minutes, adjust the pH of the solution to 7.0 using 2.5 N hydrochloric acid. The solution is chromatographed on an XAD-2 resin column (150 ml) eluting with water. The N-formimidoyl-thienamycin derivative is eluted in 1.5 to 2.0 column volumes (200 to 300 ml) and lyophilized to a white solid (217 mg). UV (pH 7, 0.1 n phosphate buffer) λ λ J \ maJcs = 297 nm (8590). IR (Nujol) 1767 cm (β-lactam).

NMR (D 2 O) 61.37 (d, J = 6Hz, CH 2 CH), 3.0 - 3.75 (m, -CH 2 -), 4.2 - 4.8 (m, C 5 R, C 6 R, C R), 7.86

NH

II

(S, -C-H)

Example 4 Preparation of -OH-NH Th - NHCl 2 -CO 2 H NH 2 N-guanyl-thienamycin

Thienamycin (8.9 mg) is dissolved in a mixture of 0.7 ml of 0.1 N phosphate buffer (pH 7) and 0.3 ml of N, N-dimethylformamide and the solution is adjusted to pH 9.5 by adding 2, 5-n 49 62306 sodium hydroxide solution. To the magnetically stirred solution is added O-methyl isourea hydrogen sulfate (43 mg), whereupon the pH drops slightly. Sodium hydroxide solution is then added to bring the ρΗ back to 9.5 and the solution is stirred for 30 minutes at 23 ° C. The solution is then acidified to pH 7.0. A sample of a solution containing a mixture of thienamycin and N-guanyl-thienamycin shows two bioactive zones according to electrophoresis (50 V / cm, 20 minutes, 0.05 N phosphate buffer, pH 7) and bioautography on a plate of S. aureus.

Example 5

C1 OH

N_NH pOH

ci- <öy-o-cf * hci ^ nh

\ _ / ^ NH2 Th - NHC

thienamycin ^ NH

^ 2

- CO2H

Preparation of N-guanyl-thienamycin

Thienamycin (11 mg) is dissolved in 0.1 N phosphate buffer (pH 7, 1 mL) and adjusted to pH 8.3 using 0.1 N sodium hydroxide and an automatic dosing burette. To the magnetically stirred solution is added 0-2,4,5-trichlorophenylisourea hydrochloride (76 mg) portionwise by means of an automatic burette, keeping the pH almost constant. The reaction is allowed to proceed for 4 hours at 22 ° C and then the pH is readjusted to 7.0 with dilute acid. A sample of this solution containing thienamycin and N-guanyl-thienamycin is subjected to electrophoresis (50 V / cm, 25 minutes, phosphate buffer pH 7 / 0.1) showing a positive Sakaguchi spray zone at 2.0 cm per anode and a positive ninhydrin shower zone at 1.5 cm in the same direction.

so 62 3 0 6 Preparation of the starting material, 0-2,4,5-trichlorophenylisourea hydrochloride ~ - <C 1 H 2 -HCl

Cl cl

A solution of cyanamide (0.28 mg) in ether (0.50 ml) is mixed with 2,4,5-trichlorophenol (12.5 g); the mixture is heated to 70 ° C and the melt is stirred magnetically while purging the reaction flask with nitrogen. Dry hydrogen chloride gas is then slowly bubbled into the melt and the reaction is allowed to cool to 22 ° C. The resulting solid is washed well with ether and filtered to give O-2,4,4-trichlorophenyl isourea hydrochloride as a white solid, m.p. 205-206 ° C.

Example 6

Cl © r0H ί "3

0 H

(CH) N = CHCl 3 /

Th (TMS) -—- * in W

Preparation of -CO 2 N-dimethylaminomethylene-thienamycin

Thienamycin (16.5 mg) is silylated with hexamethyldisilazane (200 μΐ) and trimethylchlorosilane (60 μΐ). The silylated thienamycin is suspended (from ethanol) in free chloroform (1 ml) with magnetic stirring under a nitrogen blanket. The mixture is cooled to -45 ° C and a solution of triethylamine (21 μΐ) in chloroform (21 μΐ) and a solution of (chloromethylene) -dimethylammonium chloride (11.5 mg) in chloroform (50 μΐ) are added. The mixture is warmed to -25 ° C over 1 hour and 0.1 N phosphate buffer (pH 7.5 mL) is added. The mixture is stirred vigorously for 15 minutes. Separate the aqueous phase containing N-dimethylaminomethylenetienamycin with electrophoretic mobility (50 V / cm, 1 hour, pH 8 buffer) 3.6 cm per cathode.

51 62306

Example 7 OH

__- SCH CH_N = C-NH_ | 2 2) 2 I CF3

J- N - COOH

Preparation of O'N-trifluoroacetimidoyl-thienamycin

Thienamycin (199 mg) is dissolved in 0.1 N phosphate buffer (pH 7, 7 mL) and adjusted to pH 8.5 using 1 N sodium hydroxide solution. While maintaining the pH with an automatic burette, a solution of methyl trifluoroacetimidate (355 μΐ) in dioxane (2.5 ml) is added. After 30 minutes, the pH is readjusted to 7.0 by the addition of 1N hydrochloric acid. The solution is chromatographed using Dowex 50-X4 resin (200 cc, Na + cycle, 200-400 mesh) and eluted with water.

The N-trifluoroacetimidoyl-thienamycin derivative elutes in the first half of the column volume. The eluate is rechromatographed in the same manner using Dowex 50-X4 (100 cc, Na + cycle, 200-400 mesh) and the first column volume is concentrated and chromatographed using XAD-2 resin (30 mL). The N-trifluoroacetimidoyl-thienamycin derivative is eluted in 2.5 to 5.0 column volumes and lyophilized to give a white solid (15 mg).

UV (pH 7 / 0.1 phosphate buffer). 30 g at 302 nm (£ 4450).

IR (Nujol) 1750 cm-1 lactam).

Electrophoresis: (50 V / cm, 20 min, pH 7 / 0.1 phosphate buffer) Mobility 2.0 cm (per cathode).

62306

Example 8 OH

- SCH2CH2N = C-NH2 I | ch3

J-N --COOH

O

Preparation of N-acetimidoyl-thienamycin

Thienamycin (190 mg) is dissolved in 0.1 N phosphate buffer (pH 7, 13 mL) and cooled in an ice bath with magnetic stirring. The pH of the solution is adjusted to 8.5 using 2.5 N sodium hydroxide solution dispensed from an automatic burette. Maintain the pH at · 8.5, and add ethyl acetimidate hydrochloride (400 mg) portionwise over a few minutes.

After 40 minutes, the solution is adjusted to pH 7.0 using 2.5 N hydrochloric acid. The solution is chromatographed using Dowex 50-X8 resin (250 cc, Na + cycle, 100-200 mesh) and eluted with water. The N-acetimidoyl derivative is eluted in 1-2 column volumes (240-520 cc) and lyophilized to give a white solid (88 mg).

UV (pH 7 / 0.1 phosphate buffer) λmax 297 nm (£ 7620).

IR (Nujoi) 1774 cm -1 (β-lactam). NH

NMR (D 2 O) δ 1.27 (d, J = 6Hz, CH 3 -CH) 2.24 (S, -C-CH 3 3.2 - 3.5 (m, -CH 2), 3.5 - 3.9 (m, - CH 2 -) 4.2 - 4.6 (m, C 5 H, C 6 H, C 2 H).

Example 9 Preparation of N-isobutyrimidoyl-thienmycin

According to the procedure of Example 8, but substituting isobutyrimidate hydrochloride for ethyl acetimidate hydrochloride and allowing the reaction to proceed at 20 ° C and pH 8.2, N-isobutyrimidoyl-thienamycin (14%) is obtained.

UV (pH 7 / 0.1 phosphate buffer) λ max 298 nm (δ 8290) NMR (D 2 O) β 1.27 (d, J = 7Hz, CH (CH 3) 2, 1.29 (d, J = 6Hz) , CH 2 CH (OH), 2.79 (heptette, J = 7Hz, ch (ch 3) 2).

53 6 2 3 0 6

Example 10 Preparation of N1-methyl-N-acetimidoyl-thienamycin According to the procedure of Example 8, but substituting methyl N-methylacetimidate for ethyl acetimidate hydrochloride, N-methyl-N'-acetimidoyl-thienamycin (10%) is obtained.

UV (pH 7 / 0.1 phosphate buffer) Amax 298 nm (£ 6700) IR (Nujol) 1750 cm 1 (β-lactam), 1660 cm 1 (c = nch 3) NMR (D 2 O) δ 1/27 (d , J = 6Hz, CH 3 CH (OH), 2.22 and 2.25

OF

(S, N-CCH3), 2.97 (S, NCH3).

Example 11 Preparation of N1-methyl-N-formimidoyl-thienamycin According to the procedure of Example 8, but substituting ethyl N-methylformimidate hydrochloride for ethyl acymididate hydrochloride, N'-methyl-N-formimidoyl-thienamycin (10% ). UV (pH 7 / 0.1 phosphate buffer) λmax 198 nm NMR (D 2 O) δ 1/30 (d, J = 6Hz, CH 3 CH (OH), 2.92 (S, N-CH 3), ti 7.78 (S, -CH)

Example 12 Preparation of N- (methoxyacetimidoyl) -thienamycin According to the method of Example 8, but substituting an equivalent amount of methyl methoxyacetimidate for the reagent, N- (methoxyacetyrimidoyl) thienamycin is obtained (34%); UV. CH (OH) 03.50, 3H, (S, CH3.O.CH2) 04.35, 2H, (S, CH3.O.CH2) HPLC, 1.50 x thienamycin reaction time.

S4 62306

Example 13 X ^ ^ -p η— sch2ch2n = c-nh2 J- N - COOH jl

o I

Preparation of N-Z (pyridyl) imino) methyl-thienamycin Thienamycin (80 mg, 0294 mmol) is dissolved in sodium carbonate solution (24.7 mg, 0.294 mmol NaCO 2 in 2 mL water) at 25 ° C. Methyl isonicotine imidate (80 mg, 0.588 mmol) is added with stirring and the progress of the reaction is monitored using high performance liquid chromatography, HPLC (Water's instrument) 0.2 x 61 cm, C1Q Bondapak reverse phase column? 1.5 ml / min 10%

I O

tetrahydrofuran; UV (254 nm) and IR monitors. The reaction is practically complete in 40 minutes. The reaction solution is chromatographed directly using an 18.4 x 270 mm XAD resin column; eluted first with deionized distilled water and then with aqueous 10% tetrahydrofuran. The eluate is monitored by UV and HPLC is used to locate the pure product. The product-containing fractions were combined and lyophilized to give a colorless fluffy substance (80 mg = 73%).

UV λmax 2M m »(S 7800) IR (Nujol) 1762 cm (? 4 -actam) NMR (60 MHz, D 2 O), δ 1.27, 3H (d, J = 7Hz, CH 3 CH (OH)) )? 7.75 and 8.80, 4H, (m, m, 4-pyridyl) HPLC, 1.58 x thienamycin retention time.

Example 14 Preparation of NZ- (2-pyridyl) - (imino) methyl] thienamycin Following the procedure of Example 13, but substituting methyl picoline imidate for the reagent, N - [(2-pyridyl) - (imino) methyl] thienamycin (85 mg, 77%) is obtained. VH, δ UV λ 267, 300 nm (ε 8150, 7600) λ (OH)) 07.80, 8.07, 8.80, 4H, (m, m, m, 2-pyridyl) HPLC, 1.8 x retention time of thienamycin.

55 6 2 3 0 6

Example 15 Preparation of N- [3-pyridyl (imino) methyl] thienamycin According to the method of Example 1 3, but substituting methyl nicotine imidate for the reagent, N- [13-pyridyl- (imino) methyl] thienamycin (77 mg, 70%) is obtained, <2 ° UV λ max 264 '299 nm' (δ 5570, 6120) IR (Nujol) 1766 cm -1 (γ-lactam) NMR (60 mHz, D 2 O) 61.24, 3 H, (d, J = 7Hz, CH 3 .CH.CH (OH)) δ 7.6, 8.2, 8.9, 4H (m, m, m, 3-pyridyl) HPLC, 1.57 x retention time of thienamycin.

Example 16 Preparation of N- [4- (4-thiazolylimino) methyl] thienamycin Following the procedure of Example 13, but substituting methyl 4-thiazolecarboxyimidate for the reagent, N - [(4-thiazolylimino) methyl] thienamycin (99 mg, 89%) is obtained. UV λmax 300 nm, (δ 7530) IR (Nujol) 1764 cm (γ-lactam) NMR (60 MHz, D 2 O) δ 1.23, 3H, (d, J = 7Hz, CH 3 .CH (OH)) δ 8.60, 9.17, 2H, (d, d, J = 2Hz 4-thiazolyl) HPLC, 1.8 x thienamycin retention time.

Example 17

i-OH

^ N-CH, CH, SCH

Th - NH <

^ H

-COOH

Preparation of N1- (2-methylthioethyl) -N-formimidoyl-thienmycin

Thienamycin (105 mg) is dissolved in 0.1 N phosphate buffer (pH 7.5 ml) and a solution of ethyl N-2-methylthioethylformimidate (300) in tetrahydrofuran (2 ml) is added. The pH of the solution is adjusted to 8.5 and maintained there using an automatic dosing burette and 0.1 N NaOH. After 30 minutes, the pH is adjusted to 7.0 using 2.5 N HCl.

The solution is chromatographed on an ice-cooled column using Dowex 50-X4 resin (53 cc, Na + cycle, 200-400 mesh) 56 62 30 6 and eluting with deionized water. The N '- / 2-methylthioethyl7N-formimidoyl derivative is eluted in 2-4 column volumes and lyophilized to give a white solid.

UV (pH 7 / 0.1 phosphate buffer) λ max 298 nm (≤ 7760) IR (Nujol) 1760 cm -1 (γS-lactam) Preparation of starting material, ethyl N-2-methylthioethyl formimidate, in a 60 ml separatory funnel Add ethyl formimidate hydrochloride (0.97 g, 8.8 mmol), a solution of N-methylthioethylamine (0.80 g, 8.8 mmol) in CH 2 Cl 2 (35 mL) and water (35 mL). The mixture is shaken vigorously for 5 minutes. The CH 2 Cl 2 layer is separated, washed with brine, dried over MgSO 4, filtered and evaporated under reduced pressure to give the crude imidate (0.59 g) as a pale yellow cloudy liquid.

IR 1660 and 1230 cm-1.

Example 18

- OH

N-CH (CH-) CH = CH- ^ 32

Th - NHC H

-COOH

Preparation of N '- (1-methyl-2-propenyl) -N-formimidoyl-thienamycin

Thienamycin (126 mg) is dissolved in 0.1 N phosphate buffer (pH 7, 6 mL) and the pH of the solution is adjusted to 8.5 and maintained there using an automatic dosing burette and 1 N sodium hydroxide. To this stirred solution is added ethyl N-1-methyl-2-propenylformimidate hydrochloride (300).

After 30 minutes, the pH is adjusted to 7.0 using 2.5 N HCl and the solution is chromatographed on an ice-cooled column using Dowex 50-X4 resin (49 cc, Na + cycle, 200-400 mesh) and eluted with deionized water. The N'-E-methyl-2-propenyl-7-N-formimidoyl derivative is eluted in 2-4 column volumes and lyophilized to give a white solid (59 mg).

UV (pH 7 / 0.1 phosphate buffer)? 299 nm (€ 7820) IR (Nujol) 1760 cm-2 (β-lactam) 62306

Example 19 —OH

rc (cH 3 »3

Th - NHC

\

-COOH

Preparation of N'-tert-butyl-N-formimidoyl-thienamycin

Thienamycin (105 mg) is dissolved in 0.1 N phosphate buffer (pH 7.5 ml) and a solution of ethyl N-tert-butylformimidate (290 mg) in tetrahydrofuran (1 ml) is added. The pH of the solution is adjusted to 8.5 and maintained there by the addition of 1 N sodium hydroxide from the dosing burette. After 30 minutes, the pH is adjusted to 7.0 using Dowex 50-X4 resin (53 mL, Na + cycle, 200-400 mesh) and eluted with deionized water. Fractions containing the title product are combined and lyophilized.

Example 20

i— OH

r N <CH33

Th - NHC J

-COOH

Preparation of N1-dimethylamino-N-formimidoyl-thienamycin

Thienamycin (115 mg) is dissolved in 0.1 N phosphate buffer (pH 7, 7 mL) and the pH of the solution is adjusted to 8.5 by adding 1 N sodium hydroxide from an automatic burette. To this stirred solution is added methyl N-dimethylaminoformimidate hydrochloride (284 mg) while maintaining the pH at 8.5. After 20 minutes, the pH of the solution is adjusted to 7.0 with 2.5 N hydrochloric acid and the solution is chromatographed using Dowex 50-X4 resin (53 mL, Na + cycle, 200-400 mesh) and eluted with deionized water. Chromatography is performed on a column surrounded by an aqueous jacket at 3 ° C. The N'-dimethylamino-N-formimidoyl derivative is eluted in 2 column volumes and lyophilized to give a white solid (40 mg).

58 6 2 3 0 6 UV (pH 7 / 0.1 phosphate buffer) λ max 298 nm (ε 6910) IR (Nujol) 1760 cm-1 lactam) NMR (D 2 O) δ 1/29 (d, J = 6Hz , CH 2 CH 2 2.59 (s, N (CH 3) 2, 7.76 (s, NCH).

Preparation of the starting material, methyl N-dimethylaminoformimidate

To a stirred solution of 0.22 g of N, N-dimethylformhydrazide in 2.0 ml of chloroform is added methyl chloroformate (0.5 ml) under a nitrogen blanket. The mixture is heated at 40 ° C for 3 hours, then evaporated under a nitrogen blanket. The mixture is triturated with anhydrous ether, the supernatant is decanted and the residue is dried under a stream of nitrogen. Yield: 284 mg. NMR CDCl 3 δ, 9.13 (CH), 3.80 (OCH 3), 3.01 (N (CH 3) 2).

Example 21 “OH

NH NH

ii i

Th - NHC - C - OCH3

-COOH

Preparation of methyl-oxalimidoyl-thienamycin Thienamycin (105 mg) is dissolved in 0.1 N phosphate buffer (pH 7.5 ml) and the pH of the solution is adjusted to 8.5 by adding 1 N sodium hydroxide from an automatic burette. To this solution is added methyl oxalimidate (200 μΐ) while maintaining the pH at 8.5. After 30 minutes, the pH of the solution is adjusted to 7.0 with 2.5 N hydrochloric acid and the solution is chromatographed using Dowex 50-X4 resin (53 mL, Na + cycle, 200-400 mesh) and eluted with deionized water. Chromatography is performed on a column surrounded by an aqueous jacket at 3 ° C. The methyl oxalimido derivative is eluted in 2 column volumes and lyophilized to a white solid (44 mg).

UV (pH 7 / 0.1 phosphate buffer) 298 nm (£ 6230) IR (Nujol) 1760 cm-1 (β-lactam) NMR (D 2 O) δ 1.27 (d, J = 6Hz, CH 3 -CH ), 3.87 (s, -OCH 3) 59 62306

Example 22

-OH

NH

K

Th - NHC ^ οη2οη3

^ -COOH

Preparation of N-propionimidoyl-thienamycin Thienamycin (114 mg) is dissolved in 0.1 N phosphate buffer (pH 7, 10 mL) and the pH of the solution is adjusted to 8.5 by adding 1 N sodium hydroxide from an automatic burette. To this is added solid ethyl propionimidate hydrochloride (231 mg) while maintaining the pH at 8.5. After 30 minutes, the pH of the solution is adjusted to 7.0 with 2.5 N hydrochloric acid and the solution is chromatographed using Dowex 50-X4 resin (72 mL, Na + cycle, 200-400 mesh) and eluted with deionized water. Chromatography is performed on a column surrounded by an aqueous jacket at 3 ° C. The N-propionimidoyl derivative is eluted in 2 column volumes and lyophilized to a white solid (76 mg).

UV (pH 7 / 0.1 phosphate buffer) λmax 298 nm (€ 7830) NMR (D 2 O) 6.18 (d, J = 6Hz, CH 3 CH (OH)), 1.23 (t, J = 8Hz, -CH 2 -CH 3) 2.50 (q, J = 8Hz, CH 2 H 2).

Example 23 -OH

N-CH

Il 3

Th - NHC

OF

-COOH

Preparation of N'-methyl-N-formimidoyl-thienamycin

Thienamycin (140 mg) is dissolved in 0.1 N phosphate buffer (pH 7, 10 mL) and the pH of the solution is adjusted to 8.5 by adding 1 N sodium hydroxide from an automatic burette. To this solution is added methyl N-methylformimidate hydrochloride (200) while maintaining the pH at 8.5. After 40 minutes, the pH of the solution is adjusted to 7.0 with 2.5 N hydrochloric acid and the solution is chromatographed 62306 using Dowex 50-X4 resin (72 mL, Na + cycle, 200-400 mesh) and eluted with deionized water. The N'-methyl-N-formimidoyl derivative eluted in 2 column volumes and lyophilized to a white solid (43 mg).

UV (pH 7 / 0.1 phosphate buffer) λ max 298 nm (ε 7250) IR (Nujol) 1765 cm -1 (β-lactam) NMR (D 2 O) 61f29 (d, J = 6Hz, CH-j-CH) , 2.92 (s, N-CH 3), 7.80 (s, N-CH).

Example 24

- OH

Γ'Ό

Th - NHC

- COOH

Preparation of N * -benzyl-N-formimidoyl-thienamycin Thienamycin (110 mg) is dissolved in 0.1 N phosphate buffer (pH 7, 7 mL) and the pH of the solution is adjusted to 8.5 by adding 1 N sodium hydroxide from an automatic burette. To this buffered solution is added a solution of ethyl N-benzyl formimidate fluoroborate (572 mg) in p-dioxane (2 mL) maintaining the pH at 8.5.

After 20 minutes, the pH of the solution is adjusted to 7.0 with 2.5 N hydrochloric acid and the solution is chromatographed using Dowex 50-X4 resin (53 cc, Na + cycle, 200-400 mesh) and eluted with deionized water. Chromatography is performed on a column surrounded by an aqueous jacket at 3 ° C. The N'-benzyl-N-formimidoyl derivative is eluted in 2 column volumes and lyophilized to a white solid (5 mg).

UV (pH 7 / 0.1 phosphate buffer) λ 295 nm (ε 3980) IR (Nujol) 1765 cm -1 (β-lactam) NMR (D 2 O) δ 1.29 (d, J = 6Hz, CH 3 CH), 4.44 (s, CH 2 Ar), 7.37 (s, aryl), 8.14 (s, NCH).

61 62306 Preparation of the starting material, ethyl N-benzylformimidate

A solution of 690 mg (5.1 mmol) of N-benzylformamide in 5 ml of methylene chloride is cooled in an ice-water bath and placed under argon. The solution is stirred while adding dropwise 4.9 ml (4.9 mmol) of 1-m triethyloxonium fluoroborate in methylene chloride. After a reaction time of 45 minutes, the mixture is concentrated to dryness under reduced pressure at room temperature, and the residue is dried under reduced pressure using phosphorus pentoxide. The nuclear magnetic resonance spectrum of the product in deuterochloroform is completely consistent with the fluoroborate-etherate complex of ethyl N-benzylformimidate.

Example 25

-OH

NCH (CH _) _ (I 3 2

Th - NHC

"" H - COOH

Preparation of N-isopropyl-N-formimidoyl-thienamycin

Thienamycin (110 mg) is dissolved in 0.1 N phosphate buffer (pH 7, 7 mL) and the pH of the solution is adjusted to 8.5 by adding 1 N sodium hydroxide from an automatic burette. To this magnetically stirred buffered solution is added methyl N-isopropylformimidate hydrochloride (300 mg) in p-dioxane (1 mL) while maintaining the pH at 8.5. After 25 minutes, the pH of the solution is adjusted to 7.0 with 2.5 N hydrochloric acid and the solution is chromatographed using Dowex 50-X4 resin (53 mL, Na + cycle, 200-400 mesh) and eluted with deionized water. Chromatography is performed on a column surrounded by an aqueous jacket at 3 ° C.

The N'-isopropyl-N-formimidoyl derivative is eluted in 2 column volumes and lyophilized to give a white solid (12 mg).

UV (pH 7 / 0.1 phosphate buffer) λ max 299 nm (¢ 8130) IR (Nujol) 1760 cm-1 (γ3-lactam) NMR (D2 O) δ 1.26 (d, J = 6Hz, CH3CH (OH )), 1.29 (d, J = 6Hz, CH (CH 3) 3), 7.89 (s, NHCH), 7.96 (s, NHCH).

62 6 2 3 0 6 Preparation of the starting material, methyl N-isopropylformimidate Isopropylformamide (535 mg) is treated with an equivalent amount of ethyl chloroformate (440) for 2-3 hours under nitrogen at 40-45 ° C. The mixture is washed successively with petroleum ether, anhydrous ether and benzene to leave the product as an oil.

Example 26

- OH JZ H9CH = CH

* * il

Th - NH-C

- COOH

Preparation of N- (N'-allyl-formimidoyl) thianmycin To a cooled sample of thienamycin (123 mg, 0.452 mmol) is added 13 mL of cold 0.1 N phosphate buffer (pH 7).

The solution is adjusted to pH 9 using 1N sodium hydroxide. To this basic solution at 2 ° C is added ethyl N-allylformimidate hydrochloride (0.3 g) in one portion. The pH drops to 7.3 and is brought back to 8.5 by adding sodium hydroxide. The reaction mixture is stirred at 2 ° C for a further 30 minutes and the pH is adjusted to 7 with cold 0.1 N sulfuric acid. The reaction mixture is analyzed using high pressure liquid chromatography on a C18-Porosil column developed with 10% aqueous tetrahydrofuran, and the analysis shows only traces of thienamycin (retention time 5 minutes) and substantially pure product (retention time 10.5 minutes). The reaction mixture is chromatographed using a Dowex 50-X4 column (60 ml, Na + cycle, 200-400 mesh) and eluted with water at a flow rate of 0.5 ml / cm / min. Discard the first 400 ml of eluate and lyophilize the next 150 ml to obtain the product. Yield 96 mg (69%).

UV λ max 3 ° 1 nm, 24.6 opt. tiheysyks. / mg, (N 2 OH quenching), 90% purity IR (Nujol) gives C = 0 at 5.67 μm and 5.90 μm NMR (100 MHz, D 2 O) shows the product to be syn- and anti-N ( N'-allylformimidoyl) thienamycin 1: 1.

63 62306 Preparation of the starting material, ethyl N-allyl-formimidate hydrochloride

Ethyl chloroformate (2.66 g, 24.47 mmol) is added via syringe to N-allylformamide (2.08 g, 24.47 mmol) in a dry flask under a nitrogen blanket. The resulting mixture is stirred at 25 ° C for 2 hours during which time CCl 2 is rapidly evolved. The reaction mixture is heated at 45 ° C until no more gas is evolved.

Example 27

f OH

| N-CH.CF-II 2 3

Th - NH-C ^

B

- COOH

Preparation of N- (N'-trifluoroethylformimidoyl) thienamycin To a cooled sample of thienamycin (123 mg, 0.452 mmol) is added 15 mL of cold 0.1 N phosphate buffer (pH 7). The solution is adjusted to pH 9 by adding 1N sodium hydroxide. To this basic solution at 0-2 ° C is added ethyl N- (2,2,2-trifluoroethyl) formimidate (0.3 ml) in dioxane (2 ml) portionwise over 30 minutes. The pH of the reaction mixture is maintained at 8.5 to 9 during the addition, and the mixture is stirred for a few minutes. Upon completion of the imidate addition and after adjusting the pH to 7 with cold 0.1 N sulfuric acid, HPLC g- “Porosil ,, reverse phase, 10% tetrahydrofuran shows a new peak at 12.2 min, corresponding to the desired product. The mixture is chromatographed using

Dowex 50x4 column (60 ml, 200-400 mesh). The column is eluted with 2 water at a flow rate of 0.5 ml / cm / min. The pre-run is discarded and the product-containing fractions are combined and lyophilized to give 10.2 mg of a hygroscopic solid.

UV X. 302 nm.

max Preparation of the starting material, ethyl N- (2,2,2-trifluoroethyl) formimidate

Ethyl formimidate hydrochloride (0.555 g, 5 mmol), 2,2,2-trifluoroethylamine hydrochloride (0.677 g, 5 mmol) and potassium carbonate (0.345 g, 2.5 mmol) are suspended in 20 mL of CH 2 Cl 2 and treated with 2 mL. : 11a of water. The mixture is shaken vigorously for 3 minutes. The organic phase is separated and the aqueous phase 64 62306 is extracted twice with 10 ml portions of CH2Cl2. The combined organic phase is dried and the CH 2 Cl 2 is distilled through a Vigraeux column to give ethyl N- (2,2,2-trifluoroethyl) formimidate.

NMR δ 1.33 X (CH 3 CH 2); 3.8 q (J = 10 Hz, CF 3 CH 2) 4.23 q (J = 7.5 Hz, CH 3 CH 2), 7.6 S (H-C = N).

Example 28 - OH

N-CH COOH

t! 2

Th - NH-C

Ή Preparation of COONa N- (N'-carboxymethyl-ibrmimidoyl) thienamycin sodium salt

Thienamycin (130 mg) is dissolved in 0.1 N phosphate buffer (pH 7, 4 mL) and solid sodium methyl N-carboxymethyl formimidate (500 mg) is added in one portion. The pH of the solution is adjusted to 8.5 using 1 N sodium hydroxide using an automatic Byret. After maintaining the pH at 8.5 for 25 minutes, it is adjusted to 7.0 using 2.5 N hydrochloric acid.

The solution is chromatographed on an ice-water cooled column of Dowex 50-X4 resin (51 ml, Na + cycle, 200-400 mesh) and eluted with deionized water. The first column volume of eluate is combined and concentrated to a volume of 7 ml. This solution is chromatographed on an ice-water cooled column of XAD-2 resin (53 ml).

Column volumes of 2-4 are collected, combined and lyophilized to give sodium N- (N'-carboxymethylformimidoyl) thienamycin (25 mg).

UV (pH 7 / 0.1 phosphate buffer) λ300 nm (ε -6390) IR (Nujol) 1755 cm -1 (β-lactam) NMR (D 2 O) δ 1.29 (d, J = 6Hz, CH 2 CH) , 7.85 (s, NCH) 65 62306

Example 29 -OH

NH

B

Th - nhcch2ch2n3

-COOH

Preparation of N - (- azidopropionimidoyl) thienamycin

To a solution of thienamycin (135 mg) in 10 ml of 0.1 M phosphate buffer (pH 7) is added 1.2 g of O-ethyl-3-azidopropionimidate hydrochloride while maintaining the pH of the solution at 8.5 by adding 2.5 g of sodium hydroxide. The mixture is stirred at 0 ° C for 0.5 h, and neutralized with 2.5 N hydrochloric acid to pH 7.0, concentrated to a volume of 5 mL and chromatographed on a Dowex 50W x 8 (Na + form) column (length 30 cm, diameter 3.8 cm).

Elute with water to give 30 mg of the desired product.

The product shows ΗτΟ UV A max 300 nm HPLC shows a retention time of 10 minutes compared to the starting material value of 4.8 minutes under the same conditions. (1.8 "x 2 ',' Bondapak 'C18 reverse phase column eluted with 10% tetrahydrofuran at a flow rate of 1.5 ml / min); electrophoretic mobility 5 mm per cathode at 50 V / cm, 20 min, 0.05-m phosphate buffer (pH 7).

Example 30

—Oh r0H

NH F

| l H iO)

Th - NHCCH2CH2N3 H2 / Pd TH - NHCCH2H ^ H ^ "->

Preparation of Leo® (I) (II) N- (3-aminopropionimidoyl) thienamycin N- (3-azidopropiomimidoyl) thianmycin (1) (49 mg in 40 ml of water) is hydrogenated with hydrogen (1 atm) using 10 as a catalyst. -% palladium on carbon for 40 minutes. Electrophoretic analysis of the resulting mixture showed a new product moving 30 mm towards the cathode (50 V / cm, 20 min, 0.05 m phosphate buffer, pH 7) with starting material (I) moving 5 mm towards the cathode. The electrophoretic mobility of the product corresponds to the mobility of the expected product (II).

The reaction mixture obtained from the hydrogenation is neutralized with 2.5 N hydrochloric acid and the catalyst is filtered off. The filtrate is concentrated to a volume of 20 mL and chromatographed using XAD-2 resin (2.3 x 16 cm). The column is eluted with water and lyophilized to give the product (II) as the hydrochloride, yield 23 mg.

UV λ max 301 n »(ε 7080) IR (Nujol) 1765 cm NMR (69 MHz, D 2 O) δ 1.30 ppm (doublet, δ), 2.60 to 3.72 ppm (multiplet, δ) and 4.18 ppm (multiplet, 2). Example 31 Preparation of N-nitroguanyl-thienamycin Thienamycin (131 mg) is dissolved in a solution of dimethyl sulfoxide (10 ml), tri-n-butylamine (0.30 ml) and 2-methyl-1-nitro-2-thiopseudourea (0.3 g). The solution is heated in a water bath at 45 ° C while allowing a stream of nitrogen to bubble vigorously into the solution. After 50 minutes, the solution is concentrated under high vacuum to a volume of 1.0 mL and dissolved in 0.05 N phosphate buffer (pH 7, 7 mL). Unreacted thiopseudoure is precipitated and removed by filtration. The solution is then chromatographed using Dowex 50-X4 resin (53 cm -1, 200-400 mesh, Na + cycle) and eluted with water. The N-nitroguanyl derivative elutes in the first column volume and lyophilized to give a solid (23%).

UV (pH 7 / 0.1 phosphate buffer) λ 269 nm (€ 1000)

Electrophoresis (40 V / cm, pH 7 / 0.1 phosphate buffer, 20 min) 3.0 cm per cathode.

Example 32

- OH

N-CH.-CH II 2 3

Th - NHC H

—Co2h Preparation of N- / N1-ethylformimidoyl] / / clenamycin Thienamycin (100 mg) is dissolved in 10 ml of 0.1 M phosphate buffer (pH 7.0) and the pH is adjusted to 8.5-9.0 2.5- n sodium 67 62306 hydroxide and keep the pH the same. To the solution is added 300 mg of ethyl N-ethyl formimidate hydrochloride. The mixture is stirred at 23 ° C for 10 minutes, then neutralized to pH 7.0 with 2.5 N hydrochloric acid and chromatographed on a Dowex 50-X8 (Na form) ion exchange column (length 25 cm, diameter 3.8). cm). The column is eluted with water to give 6.7 ml of fractions. Fractions 40-90 are combined, concentrated and freeze-dried to give 15 mg of solid product. Electrophoresis of the product at 50 V / cm, 20 min, 0.1 M phosphate buffer (pH 7) shows one bioactive zone moving 2 mm per cathode.

UV λ H2? 301 nm NMR (100 MHz, D 2 O) δ 7.77 (S) and 7.82 (S) formimidoyl-CH). Example 33

- OH

OF-

Th - J

Ti

- COOH

Preparation of N- / N1-cyclopropylformimidoyl-thienamycin Thienamycin (100 mg) is dissolved in 10 ml of 0.1 M phosphate buffer (pH 7) and adjusted to pH 8.5-9.0 while maintaining the pH at this value while adding dropwise 300 mg of ethyl N-cyclopropyl-liformimidaatti hydrochloride. The mixture is stirred at 23 [deg.] C. for 40 minutes, after which it is neutralized and chromatographed on a Dowex 50x8 (Na + leach) ion exchange column (length 25 cm, diameter 3.8 cm). The column is eluted with water, and 6.5 ml fractions are taken. Fractions 43-95 are combined, concentrated and freeze-dried to give 54 mg of solid product. Electrophoresis of the product shows one bioactive zone moving 10 mm towards the cathode (50 V / cm, 1 hour, 0.05 m phosphate buffer, pH 7).

301 nm NMR (100 MHz, D 2 O) 0.60 to 1.30 ppm (m, cyclopropyl) and 7.80 ppm (formimidoyl-CH).

se 6230 6 Preparation of starting material, ethyl N-cyclopropylformimidate Ethyl chloroformoformate (4.078 g, 37.58 mmol) is added via syringe to N-cyclopropylformamide (3.194 g, 37.58 mmol) in a dry flask under a nitrogen blanket. After 30 seconds, strong gas evolution begins. The resulting reaction mixture is stirred at 25 ° C until no more gas evolution is observed (about 4 hours) and then the viscose is placed under vacuum (0.5 torr) for 1 hour to remove unreacted ethyl chloroformate.

NMR analysis of the product shows formyl proton at S 9.37 (broad singlet, CDCl 3 solution).

Claims (2)

69 6 2 3 0 6 Claim: Process for the preparation of substituted N-methylene derivatives of thienamycin useful as antibiotics of the formula OH_ / N_SCH-CH N = CR λ Δ I 1 2 NR R II JNJ-COOH O wherein R is hydrogen atom, lower alkyl group, phenyl, pyridyl, thiazolyl, trifluoromethyl, amino, azidoalkyl or aminoalkyl group or group, R 1 is a hydrogen atom, lower alkyl group, lower alkenyl group, lower alkylthioalkyl group, benzyl, dialkylamino, , a carboxy-2-methyl, alkoxy or nitro group, and R is a hydrogen atom or a lower 1 2 alkyl group, or R and R together with the nitrogen atom form a piperidyl ring, and for the preparation of pharmacologically acceptable acid addition salts of these compounds, characterized in that the compound of formula OH-f'N-SCH2CH2NH2 ^ -N-- COOH111 or a suitable O- and / or carboxyl derivative thereof is reacted with an imidoester of the formula NR1R2 H o RCX R '* IV in which X ° R "is a leaving group, wherein X ° is 0 or S and R" is lower alkyl or the same -12 va, and R, R and R are as defined above, or in formula 70 62306 R1R2N® = CX · fN | With an imidohalide of the formula X '^ VR, in which X' is halogen, 1 2 R, R and R are as defined above, or with a compound of the formula X ° R "1. f R RN-C = NR VI o 12, in which in the formula X, R ", R, R and R have the same meaning as above, after which the compound obtained is converted into an acid addition salt. 62306 71 For the preparation of an antibiotic containing an substituent of the N-methylene derivative of thienamycin, in the form of OH "^ --- rX ^ s> -SCH-CH_N = C-R Z Z I 12 II NR R 1 - n ------ i-COOH a group or a group, an alkyl group, an alkyl group, an alkenyl group, an alkylthioalkyl group, a benzyl, dialkylamino, trifluoroethyl, carboxymethyl, alkoxy 2 or a nitro group, and an alkyl group the alkyl group, 1 2 or R and R is represented by a quaternary atom with a piperidyl ring, and with the addition of a pharmacologically active compound of the formate salt, which can be treated with OH-f ^ N-SCH CH NH \ III JN-- i — COOH QT or with O- and / or carboxyl derivatives in the form of an imidoester with a formIn NR1R2 RCX ° R "IV for X ° R" is a group of colors, colors X ° is 0 or S is R 1 in the case of alkyl or hydrogenated, and R, R and R are selected from the group consisting of imidohalides and formula