DK157401B - METHOD OF ANALOGY FOR THE PREPARATION OF SUBSTITUTED N-METHYLENDER DERIVATIVES OF THIENAMYCIN - Google Patents

Description

x DK 157401B

The present invention relates to an analogous process for the preparation of novel substituted N-methylene derivatives of thienamycin or pharmaceutically acceptable salts or esters thereof having antibiotic properties.

5 Thienamycin is the subject of U.S. Patent No. 3,950,357. Thienamycin has been found to have the following structure: Formula Top Page 1 (Norwegian text) 10

There is a continuing need for new antibiotics. Unfortunately, there is no static efficacy of a given anti-biotic because continued use of any such antibiotic selectively causes formation of resistant strains of pathogens. Furthermore, the known antibiotics suffer from the disadvantage of being effective only against certain types of microorganisms. Therefore, the search for novel antibiotics continues.

Unexpectedly, it has been found that the compounds of the present invention are broad-spectrum antibiotics useful in animal and human therapy and in non-living systems.

It is an object of the present invention to provide a novel group of antibiotics which have the basic ring structure of thienamycin but are designated as 30 substituted N-methylene derivatives thereof. These antibiotics are effective against a wide range of pathogens which represent both gram-positive bacteria such as S. aureus, S. pyogenes and B. subtilis, as well as gram-negative bacteria such as E. coli, Proteus 35 morganii, Klebsiella, Serratia and Pseudomonas. That

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2, the invention further aims to prepare non-toxic pharmaceutically acceptable salts and esters thereof.

The substituted N-methylenthienamycin derivatives which can be prepared by the process of the invention have the following structural formula: OH R1 / V SCB2CT2N1 <R2

R II

And pt-butylbenzyl, 3-methyl-2-buten-1-yl and pivaloxymethyl esters thereof, and pharmaceutically acceptable salts thereof, wherein R 1 is hydrogen, alkyl of 1-6 carbon atoms, Alkenyl of 2-6 carbon atoms, cyano, cycloalkyl or cycloalkenyl of 3-6 carbon atoms, alkylthioalkyl, carboxyalkyl or dialkylamino of 1-6 carbon atoms, 2 1

trifluoroethyl or benzyl and R is hydrogen or R

12 and R are alkyl of 1-6 carbon atoms, or R and R are together with the nitrogen atom piperidyl and R is hydrogen, phenyl, pyridyl, thiazolyl, amino, aminoalkyl, azidoalkyl, alkyl, trifluoromethyl, alkoxyalkyl, thioalkyl or alkyloxalimide with 1-6 carbon atoms in the alkyl groups and alkenyl having 2-6 carbon atoms or where R is -ORa or -SRa wherein Ra is methylbenzyl, ethylallyl, 2-thienylmethallyl, p-nitrobenzyl or CH CH-O-CH CH.

The present compounds can be prepared from thienamycin I or a suitable carboxyl derivative thereof.

Such carboxyl derivatives of thienamycin have the formula:

OH

/ V_ / N-SCH2CH2 * H2 _N 'L COOR · 5' 35 0 le 3 'wherein the group R comprises conventional protective

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3 or carboxyl blocking groups. In this connection, reference is made to U.S. Patent No. 3,697,515.

The starting materials Ic may be prepared as set forth below, but it should be noted that direct esterification without protection of the amino group is also possible.

• OH OH OH

afblok.

1 o Th · -NR1 'R1' -Th · -NR1 'R1' -Th- -NH2 COOH COX'R2 'COX'R2' 1 Id Ic 15 »1 '2' wherein X = O, R and R are acyl and Th denotes the thiena-3 'mycine nucleus. R has the meaning previously stated.

In general, the reaction 1- * Ic is carried out in a conventional manner. Such methods include 1) Reacting 1 (or I) with a diazoalkane such as diazomethane, phenyldiazomethane and diphenyldiazomethane, in a solvent such as dioxane, ethyl acetate and acetonitrile, at a temperature of 0 ° C to reflux temperature for a few minutes to 2 hours.

Reaction of an alkali metal salt of 1 with an activated alkyl halide such as methyl iodide, methyl bromide or 30 m-phenoxybenzyl bromide, p-t-butylbenzyl bromide or pivaloyloxymethyl chloride. The reaction may conveniently be carried out in a solvent such as hexamethylphosphoramide at a temperature of 0 to 60 ° C for a few minutes to 4 hours.

35 2

Reaction of 1 with an alcohol such as methanol, ethanol or benzyl alcohol. This reaction can be carried out in 4

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the presence of a carbodiimide condensing agent such as dicyclohexylcarbodiimide. The reaction may conveniently be carried out at a temperature of 0 ° C to the reflux temperature for 15 minutes to 18 hours in a solvent such as chloroform, methyl chloride or methylene chloride.

4) Reaction of an N-acylated anhydride of 1 prepared by reacting the free acid 1 with an acid chloride, such as ethyl chloroformate or benzyl chloroformate, with an alcohol as indicated in 3) under the same reaction conditions as set out in 3). The anhydride is prepared by reacting 1 and the acid chloride in a solvent such as tetrahydrofuran 15 or methylene chloride at a temperature of 25 ° C to the reflux temperature for 15 minutes to 10 hours.

5) Reaction of labile esters of 1, such as the trimethylsilyl ester or dimethyl-t-butylsilyl ester, with X °, where X ° is halogen, such as bromine or chlorine, and 3 'R is as above, in a solvent , such as tetrahydrofuran, methylene chloride or the like, at a temperature of 0 ° C to the reflux temperature for 15 minutes to 16 hours.

The reaction can be illustrated by the following scheme:

• —OTMS i — OTMS r-ATMS

Th-NHTMS N-acylation. Th - NR1 TMS esterification ^ τίΐ._Μρ1 · 'φΜ <; 3 '

L COOTMS Λ-COOTMS L COOR

- OH

mild hydrolysis ^ Th- - NHR1 '35 LcOOR3' wherein TMS is triorganosilyl such as trimethylsilyl and the other symbols are as set forth above.

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5

The analogous method of the invention is characterized by the characterizing part of the claim.

Suitable solvents depending on the thienamycin substrate and reagent are water, dioxane, tetrahydrofuran, dimethylformamide, chloroform, acetone and acetonitrile or mixtures thereof. The reaction is carried out at a temperature of 0 ° C to approx. 25 ° C for 1 to approx. 6 hours.

These reaction conditions are not critical as long as the reaction agent is inert or substantially inert to the intended reaction course. Suitable reagents include the following: a) Imido esters: Rv

® N

R-C-X R ° "

X ° = 0 or S

20 Methylformimidate, ethylformimidate, methylacetimidate, ethylacetimidate, methylbenzimidate, ethyl 4-pyridyl carboxymidate, methylphenylacetimidate, methyl 3-thienylcarboxyimidate, methylaccharide acetate, methyl chloroacetimidate methyl 2,4-dimethoxybenzimidate, ethyl N-methylformimidate, methyl N-methylformimidate and methyl N-isopropyl formimidate and the like.

Such imidoester reagents a) are suitably prepared by any of a number of known methods, such as 1) Reaction of a nitrile, RCN, with a lower alkanol in the presence of HCl according to the well-known Pinner synthesis. 1 2) Reaction of a nitrile, RCN, with a lower alkanol in the presence of a base. The reaction is typically carried out at 0-40 ° C in the presence of an excess of the alcohol by one

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6 catalytic amount of an alkali metal oxide in 15 minutes to 4 hours.

ISLAND

II

3) Reaction of an amide, RCNHR 3, with an alkyl chloroformate, such as methyl chloroformate, at 25-45 ° C for 1 to 4 hours.

4) Reaction of an N-substituted amide,

10 O O

II II

RCNHR1 or RCNR1 R2, with an equivalent of an alkylating agent, such as triethyloxonium fluoroborate, in an inert solvent, such as ether or chloroform, at 0 to 23 ° C for 10 minutes to 2 hours.

5) Transfer of a readily available imidoester 20 25 1 35

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7 RCNR 'in OR ", 5 wherein R may be hydrogen, to the desired imido ester, RCNR1 in OR" 10 by reacting the former with an alkylamine, R'NH2, in a mixture of water and an immiscible solvent such as ether or chloroform, at 0 to 23 ° C for 5 minutes to 1 hour.

B) Substituted imidohalides:

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

Such imidohalide reagents (b) are suitably prepared by any of a number of known methods, such as 1) reacting a Ν, Ν-disubstituted amide, 0 RCNR 35, such as chloroform or methylene chloride, at 0 to 40 ° C for 1 to 5 hours.

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C) -OR pseudoreabs and -SR pseudothioureas: X "R °

R1R2N = NR

O-methylpseudourea, S-methylpseudothiourea, S-methylpseu-dothionitrourea, 0-2,4-dichlorophenyl-pseudourea, S-p-nitrophenyl-pseudothiourea, Ο-Ν, Ν-trimethylpseudourea and the like.

The reaction involving the reagents (a) can be represented by the following reaction scheme: 15 or 3 _A \ _SCH-CH-NH- I 3 'J_Λ_LU cox * o 20 1 R'

K

u R-C-OR "25 ψ OR3 Λ — A- SCH-CH, N = C-NHR 'II 2 J k __N_L COX'R3

Where OR "is the leaving group of the imidoester reagent, R, R, R and X 'are as previously described and R' is H or R. This reaction is particularly 3 3 'suitable for embodiments wherein R and R are hydrogen.

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9 gene, and X 'is oxygen.

The reaction involving reagents (b) can be represented by the following scheme: 5 R Q r- 3 1 2 + 1. OR.

R1 «N = CX'_ Λ Y H i 2" ✓ /% Y_Y / \ _ SCH2CH2N = C-NR R X, (S> 10 1 -: -> FZL1cox'r3 'k _ O -'

OH

mild hydrolysis. / \ -

16 .iJJicooH

Ό 2 20 where all symbols are as above. When product 2 3 3 is desired, appropriate meanings of R and R are trimethylsilyl and X 'is oxygen.

25

The reaction involving reagents (c) can be represented by the following scheme: 1 35

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10

OH

- / \ - SQImCH ^ NH,,, ·,

I i il 1 λ rVnW

J_N —- i — cox'r3 i, „o

Oy 5 OH ^ i / V / nr1r2

/ Y _ / \ - SCH 2 C "rcXNRlH

Wherein X 1, R, R and R are as indicated above, X "is oxygen or sulfur and R 1 is as indicated and preferably lower alkyl.

15

Suitable solvents for such reactions include water and buffered aqueous polar organic solvent mixtures at pH 7 to 9 or anhydrous polar organic solvents such as dimethylformamide or hexamethylphosphoramide at a temperature of 0 to 40 ° C for 1 to 24 hours.

The compounds of formula II form a wide variety of pharmacologically acceptable salts, such as 25 acid addition salts, e.g. with hydrochloric acid, hydrogen bromide, sulfuric acid, nitric acid, toluene-p-sulfonic acid and methane sulfonic acid. Salts of the compounds are pharmacologically acceptable non-toxic derivatives which can be used as the active ingredient in appropriate pharmaceutical unit dosage forms. In addition, they can be combined with other drugs to obtain preparations with a broad spectrum of activity.

The novel compounds prepared by the method of the invention are valuable antibiotics which are active against various Gram-positive and Gram-negative bacteria and which consequently are useful in

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human medicine and veterinary medicine. Compounds II can therefore be used as antibacterial drugs to treat infections caused by gram-positive or gram-negative bacteria, e.g. against Staphylococcus aureus, Escherishia coli, Klebsiella pneumoniae,

Serratia, Salmonella typhosa, Pseudomonas and Bacterium proteus. Furthermore, the antibacterial agents can be used as additives to animal feed to preserve the feed and as disinfectants.

The antibacterial activity of a variety of the compounds is set forth below.

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O 'in vo 5 CO VO CO C' 'vo OJ (0 co - · * *' _k (dramorHcscoo co 2 o c

E -H

OtD m Ό C co cn i «

Oh oo vo oo o- m n Φ Φ o4 - * · - v ca CO CQ O H 04 00 o tU 2

VO

h Ίφ Η σ> ιΗ Γ4 vo

φ VO 00 N 00 'ί H

4-) Φ 04 - - -> 'omcQooooo - (0 O 2 Λ (0 o o

U H IS

φ O rji H IN oo N

4J VO Η H 04 04 H

g 04 «. ·. * ··> * λ ω cQ o o o o o H 2 E \ O) = i w" i vo 00 IN 00 OJ CT>

. OV 04 H 04 00 O

Oh. C4 '

g CQ O O O O O

O 2 04 X Ή

t-1 H

0) 0 04 _ ^ TJ Q 00 H CO CJi 00

g. ^ jitJOO ^ VOH

φ H 04 * · ** · * * u m o o o o o Φ 2 Λ r | Δ

C

H

T 'tf (0 r-l i-l r4 r4 t-l 04

g 00 O O O O O

ri ffl 04 * * - * *

g g CQ O O O O O

• Η Φ 2 E 3 Φ in • 00 00 'tf 04 <4 * 04

CQ CTi O O O O O

04 V V K ». '.

CQ O O O O O

2 ^ ps k. w æ o k oo

1-4 ttJ

tu ta as w o w d co Π m oi s S k z <&

TABLE II

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13 OH λ

, / \ Λ-SOh CH 2N = C-N / R

5 o'clock v

_ N J - COOH

ISLAND

Minimal inhibition concentration µg / ml

Compound - 1 2 (Ex. 37) RRR S.aureus E. coli 1.) H -CH2-C = CH2 H 0.16 0.62 CH3 2.) HH 0.05 0.3 20 /> 3. H) / H H 0.06 0.56 4.) H 3 H 3 O 02 ° 38 25 5.) H -CH3 -CH (CH3) 2 0.09 1.1 6.) -CH3 -CH ( CH3) 2H 0.09 0.89 7.) -CH = CH2 Η H 0.18 0.98 8.) SCH3 CH3 H - 0.29 9.) H -C = NH 0.07 0.06

The compounds of formula II can be used alone or in combination as an active ingredient of any of 14

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series of pharmaceutical preparations. These antibiotics and their corresponding salts can be used in capsule form or as tablets, powders or liquid solutions or as suspensions or elixirs. They can be administered orally, intravenously or intramuscularly.

The process of the invention is further illustrated by the following Examples wherein the thienamycin nucleus (I, above) is represented by the following symbol:

-OH

Th - NH 2 L-cooh in which the secondary alcohol group, the amino group and the carboxyl group are illustrated. Thus, the compounds of the invention can be illustrated by the formula 20 -OH R1 /

Th - N = C-N

I \ 2

25 R R

-COOH

II

1 2 30 wherein R, R and R have the above meaning.

EXAMPLE 1

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15

-OTMS

5 Th - NHTMS or TH (TMS) 3

-COOTMS

TMS = trimethylsilyl 10

Preparation of silylated thienamyoin

Thienamycin (80.0 mg) is suspended in 40 ml of tetrahydrofuran (THF) under an atmosphere and concentrated to 10 ml; 15 hexane methyldisilazane (1.0 ml) and trimethylchlorosilane (300 aL) are added. The mixture is reacted for 20 minutes at 25 ° C with vigorous stirring. The suspension is then centrifuged to remove ammonium chloride. The above liquid is evaporated to an oil under a nitrogen stream for further reaction.

EXAMPLE 1a

CL®

25 \ ® / - \ r-OH

yP = N / \ H f

H> V_ / i:% Q

Thienamycin ---— * -) ^ _coow 30

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

Thienamycin (57 mg, 162 amol) is silylated by the above procedure. The silylated antibiotic TH (TMS) 3 is dissolved in methylene chloride (6 ml) in a partition closed flask under positive nitrogen pressure and cooled

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16 dry ice-acetone baths. To the solution stirred with magnetic stirrer is added a solution (100 µl) of triethylamine (644 µmol) in methylene chloride. This is followed by the addition of a solution of chloropiperidinomethylium chloride (67 5 mg, 405 µmol) in methylene chloride (465 µl). After one hour on a dry ice bath, the reaction solution is quickly added to a tetrahydrofuran solution at pH = 7 containing 0.1 N phosphate buffer (1: 1) (50 ml). The mixture is then evaporated in vacuo to give 10 ml of homogeneous solution. The solution is washed twice with ethyl acetate (2 x 5 ml) and ether (2 x 5 ml) and evacuated for a short time.

This aqueous solution is then chromatographed on XAD-2 resin column (60 ml layer). The product is eluted in 10% aqueous tetrahydrofuran (after elution with water) to give 12.9 mg (22% product) measured in solution assuming e s 8030 applicable to thienamycin. Paper chromatography R f = 0.42 (4: 1: 5, n-BuOH: EtOH: water).

EXAMPLE 2 20 °, Γ ° ΗΗ + NH,

HjC-O-C-Ø _l.rS '

Thienamycxn v L

“* O 25 Leo®

Preparation of N-benzimidoylthienamycin 30 Thienamycin (59 mg, 212 µmol) is dissolved in a 33% N, N-dimethylformamide pH = 7 phosphate buffer (0.05 N) solution (4.5 ml) and adjusted to pH = 9.5 with 2.5 N NaOH with an automatic dispensing burette. The solution is stirred with magnetic stirrer at 25 ° C and 35 methylbenzimidate is immediately added. HCl (340 mg, 1981 mmol). After 30 minutes, the solution is extracted twice with the same volume of chloroform and adjusted with dilute aqueous

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17 phosphoric acid to pH = 7.0. The buffered solution is chromatographed on XAD-2 resin (65 ml). The column is first eluted with water followed by 10% aqueous tetrahydrofuran which elutes the product. This fraction 5 is evaporated to half its volume and lyophilized to give 50 mg of the product. Electrophoretic mobility (50 V / cm, 20 min, pH = 7, 0.1 N phosphorus buffer) is 1.5 cm toward the anode.

UV kmax = 300 nm (e = 6960) pH = 7 0.1 N phosphate buffer.

EXAMPLE 3 Γ0Η NH / -λ pOH ©

Th mBrCHr- <0} ~ C (CH-J _ ^ -> ThT-NHC <@ LC02H-CO2C? 2 20 C (CH3) 3 3γΘ

Preparation of N-benzimidoylthienamycin, p-tert-butylbenzyl ester 25

Benzimidoylthienamycin (3.2 mg) is suspended in hexamethyl phosphoramide (75 µl) containing p-tert-butylbenzyl bromide (3.8 µl) and magnetically stirred at 22 ° C. After 45 minutes, a solution is obtained, which is stirred for a further 1 30 hours. The product is then precipitated by the solution with ether and the crude product is chromatographed on a 250 µm silica gel plate developed in 7: 3 chloroform: ethanol. The band with Rf = 0.6 is isolated and eluted with ethanol to give N-benzimidoyl-thienamycin, p-tert-butylbenzyl ester hydrobromide. Mass spectrum m / e 521 (M +), 487, 444, 418, 341, 323, 297, 226, 147.

EXAMPLE 4

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18 5 i-OH ©

Th - I? HC

* -COOCHo CH-, -t / 3 C = C / \ 10 H CH ^ Br

Preparation of N-benzimidoylthienamycin, 3-methyl-2-butene-1-yl ester 15

Benzimidoylthienamycin (5.9 mg) is dissolved in hexamethylphosphoramide (100 to) containing 1-bromo-3-methyl-2-butene (4.8 µl) and triethylamine (0.5 µΐ) and stirred with magnetic stirrer at 22 ° C. After 1 hour, 20 the crude reaction product is chromatographed on a 250 μm silica gel plate developed in 8: 2 chloroform: ethanol. The band with R f = 0.1 - R f = 0.3 is isolated and eluted with ethanol. Benzimidoylthienamycin, 3-methyl-2-buten-1-yl-ester hydrobromide is isolated as a solid after precipitation of an ethanol / chloroform solution with hexane.

EXAMPLE 5

Preparation of N-formimidoylthienamycin 30

OH

A -SCH 2CH 2N = C-NH 2

\ _TJ 1_COOH

6

Thienamycin (517 mg) is dissolved in pH = 7 0.1 N phosphorus

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19 buffer (25 ml) and cool in ice bath under magnetic stirring. The solution is adjusted to pH = 8.5 using 2.5 N sodium hydroxide solution from an automatic burette.

While maintaining a pH of 8.5, 5 methyl formimidate hydrochloride (711 mg) is added portionwise over 2-3 minutes, after a further 10 minutes the pH of 7.0 is adjusted with 2.5 N hydrochloric acid. The solution is chromatographed on a column of XAD-2 resin (150 ml) which is eluted with water. The N-formimidoylthienamycin derivative is eluted in 1.5 - 2.0 column volumes (200-300 ml) and lyophilized to a white solid (217 mg).

UV (pH = 7 0.1 N phosphate buffer) λ = 297 nm (8590).

IiloK

IR (Nujol mull) 1767 cm (ε-lactam).

NMR (D20) δ 1.37 (d, J = 6H, CH3-CH), 3.0-3.75 (m, -CH2-), 4.2-4.8 (m, CgH, ΟβΗ, C7H ), 20

NH

II

7.86 (s, - C-H).

25 1 35

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EXAMPLE 6

-OH

5 ^ NH

Th - NIIC "^

Lco2k 10 Preparation of N-guanylthienamycin

Thienamycin (8.9 mg) is dissolved in pH = 7 0.1 N phosphate buffer (0.7 ml) and N, N-dimethylformamide (0.3 ml) and the solution is adjusted to pH = 9.5 by adding 2.5 N sodium hydroxide solution. To the solution is added, under magnetic stirring, O-methylisohydrogen hydrogen sulfate (43 mg), which causes a slight decrease in the pH. More sodium hydroxide solution is added to bring the pH 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 the solution containing a mixture of thienamycin and N-guanylthienamycin exhibits two bioactive zones after electronforesis (50 V / cm, 20 minutes, 0.5 N pH = 7 phosphate buffer) and bioautography on 25 S. aureus plates. EXAMPLE 7

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21

Cl

Cljft \ -Q-cf * -HCl OH

5 Thienamycin Λ! ^ ~ 0 \ NHc ^ 'C1 2. ^ ΝΗλ --7 2 _co2h

Preparation of N-guanylthienamycin 10

Thienamycin (11 mg) dissolves pH = 7 0.1 N phosphate buffer (1 ml) and adjusted to pH = 8.3 with 0.1 N sodium hydroxide by means of an automatic dispersion burette.

To the magnetically stirred solution, 0-2,4,5-15 trichlorophenylisohydrochloride (76 mg) aliquots are added to allow the autoburn to maintain an approximately constant pH value. The reaction proceeds for 4 hours at 22 ° C and the mixture is then reset to pH = 7.0 by adding dilute acid. A sample 20 of this solution containing thienamycin and N-guanylthienamycin is electrophoresed (50 V / cm, 25 min, pH = 7 0.1 N phospha tpuf fer) and a positive Sakaguchi spray zone is detected at 2.0 cm toward the anode and a positive ninhydrin spray zone at 1.5 cm in the same direction.

25 1 35

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EXAMPLE 8

Cl © © p0H CH-, 5 (CH3) -R = CHC1 η 5_2η,

Th (TMS) o --- 7 Th il-C 3

* XH

-cof 10

Preparation of N-dimethylaminomethylenthienamycin

Thienamycin (16.5 mg) is silylated with hexamethyldisilazane (200 μ.1) and trimethylchlorosilane (60 μΐ) in known manner.

The silylated thienamycin is suspended in ethanol-free chloroform (1 ml) under magnetic stirring in a nitrogen atmosphere. The mixture is cooled to -45 ° C and a solution of triethylamine (21 µl) in chloroform (21 µl) is added followed by a solution of (chloromethylene) -20 dimethylammonium chloride (11.5 mg) in chloroform (50 µl). The mixture is heated to -25 ° C over one hour and 0.1 N pH * 7 phosphate buffer (5 ml) is added. The mixture is stirred vigorously for 15 minutes. The aqueous phase is separated and contains N-dimethylaminomethylenthienamycin, which has an electrophoretic mobility (50 V / cm, 1 hour, pH = 7 buffer) of 3.6 cm towards the cathode. EXAMPLE 9

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23

r-r ° H O - OH

+ ^ NH2] | + / NH Br

Th - NHC BrCH ^ OCC (CH-) _ Th - NHC ^

5 \ H -2 - JLL - \ H

CO 2 _§-0-CH 2 O C (CH 3) 3

Preparation of N-formimidoylthienamycin-pivaloxymethyl-10-ter-hydrobromide N-aminomethylenthienamycin (10 mg) is dissolved in hexamethylphosphoramide (200 μΐ) containing bromomethyl pivalate (10 nl) and triethylamine (1 al) and magnetically stirred at 22 ° C. After 2 hours, the hexamethylphosphoramide solution is dissolved in 2 ml of methylene chloride and the product is precipitated with a 50% hexane-ether solution. The precipitate is dissolved in aqueous 10% tetrahydrofuran solution and chromatographed on an XAD-2 resin packed column. N-formimidoyl-thienamycin-pivaloxymethyl ester is isolated as a solid after elution of the tetrahydrofuran column and lyophilization.

EXAMPLE 10 25

Preparation of N-trifluoroacetimidoyl-thienamycin OH

y'V— sch2ch2n = 9-nh2 in CF3

_tj COOH

OT

Thienamycin (199 mg) is dissolved in pH = 7 0.1 N phosphate buffer (7 ml) and adjusted to pH = 8.5 with 1 N sodium hydroxide solution. While maintaining this pH with an automatic burette, a solution of methyl trifluoroacetimidate (355 aL) in dioxane (2.5 ml) is added to

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24 once. After 30 minutes, the pH is adjusted by the addition of 1N hydrochloric acid. The solution is then chromatographed on Dowex 50-X4® resin (200 ml, Na + cycle, 200-400 mesh) and eluted with water. The N-trifluoroacet-imidoylthienamycin derivative is eluted in the first half column volume. This eluate is similarly chromatographed on Dowax 50-X4 (100 ml, Na + cycle, 200-400 mesh) and the first column volume is evaporated and chromatographed on XAD-2 resin (30 ml). The N-fluoroacetimidoylthienamine derivative is eluted in 2.5 - 5.0 column volumes, which is lyophilized to a white solid (15 mg).

UV (pH = 7, 1N phosphate buffer) λ = 302 nm (e = 4450).

ΙΠ3.Χ 15 IR (Nujol mull) 1750 cm-1 (--lactam).

Electrophoresis: (50 V / cm, 20 min, pH = 7, 0.1 N phosphate buffer) mobility 2.0 cm towards the cathode.

EXAMPLE 11

Preparation of N-acetimidoylthienamyoin

OH

- / V- SCH, CH, N = C-NH0 ok I z 2 i 2 25 CHo

£ _N_LI_COOH J

Thienamycin (190 mg) is dissolved in pH = 7 0.1 N phosphate buffer (13 ml) and cooled in an ice bath under magnetic stirring. The solution is adjusted to pH = 8.5 with 2.5 N sodium hydroxide solution delivered from an automatic burette. While maintaining a pH of 8.5, portion of ethyl acetimidate hydrochloride (400 mg) is added portionwise over a few minutes. After a further 40 35 minutes the solution is adjusted to pH = 7.0 with 2.5 N hydrochloric acid. The solution is then chromatographed on Dowex 50-X8 resin (250 ml, Na + cycle, 100-200 mesh) and

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25 is eluted with water. The N-acetimidoyl derivative is eluted in 1-2 column volumes (240-520 ml) and lyophilized to a white solid (88 mg).

UV (pH = 7, 0.1 N phosphate buffer) = 297 nm (e = 7620).

IR (Nujol mull) 1774 cm 2 (O-lactam).

10 NH

IV

NMR (D 2 O) δ 1.27 (d, J = 6 Hz, CHg-CH), 2.24 (s, -C-CH3) 3.2 - 3.5 (m, -CH₂), 3.5 - 3, 9 (m, -CH 2 -) δ 4.2 - 4.6 (m, C 5 H, C 6 r, C 7 h).

EXAMPLE 12

Preparation of N - [(4-pyridyl) (imino) methyl] thienamycin OH

X \ __— SCH_CHoN = C-NH0 [ir 2 2 i 2

. „_Ti, _JJL COOH

"" V

Thienamycin (80 mg, 0.294 mmol) is dissolved in aqueous sodium bicarbonate (24.7 mg, 0.294 mmol in 2.0 ml) at 30 ° C. Methyl isonicotin imidate (80 mg, 0.588 mmol) is dissolved in the solution and the reaction is monitored by periodic sampling, subjected to high pressure liquid chromatography (HPLC): Water instrument; 0.2 x 61 cm. C ^ g Bondapak Reverse Column; 1.5 ml / min, aqueous 10% THF; UV (254 nm) and R.I. monitors. The reaction is substantially complete over 40 minutes and the reaction solution is chromatographed directly over an 18.4 x 270 mm XAD resin.

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26 column, first eluting with deionized distilled water, then changing to aqueous 10% tetrahydrofuran. The UV and HPLC controlled eluate is used to locate the pure product.

5 Correct fractions are combined and lyophilized to give a colorless fluffy powder (80 mg, 73%).

h20 UV Kmax = 298 nm (e = 7800).

IR (Nujol mull) 1762 cm -1 (β-lactam).

NMR (60 MHz, D 2 O) δ 1.27, 3H (d, J = 7 Hz, CH 3 CH (OH)), 7.75 and 8.80, 4H (m, m, 4-pyridyl).

HPLC, 1.58 x retention of thienamycin, conditions as above.

EXAMPLE 13 20

In the procedure of Example 12, the reagent is replaced with methyl picolinimidate to give: N - [(2-pyridyl) (imino) methyl] thienamycin (85 mg, 77%) H 2 O = 267, 300 nm (6 = 8150, 7600).

max IR (Nujol mull) 1764 cm ”'(Ø-lactam).

NMR (60 MHz, D 2 O) δ 1.24, 3H (d, J = 7 Hz, CH 3 (QH), 7.80, 8.07, 8.80, 4H (m, m, m, 2-pyridyl) ).

HPLC, 1.8 x retention of thienamycin.

EXAMPLE 14

DK 157401B

27

In the procedure of Example 12, the reagent is replaced with methylnicotin imidate to give: 5 N - [(3-pyridyl) (imino) methyl] thienamycin (77 mg, 70%): «2 ° UV λ = 264, 299 nm (e = 5570 , 6120). max 10 IR (Nujol mull) 1766 cm-1 (β-lactam).

NMR (60 MHz, D 2 O) δ 1.24, 3H (d, J = 7 Hz, CH 3 CH (OH)), HPLC, 1.57 x retention of thienamycin.

EXAMPLE 15

In the procedure of Example 12, the reagent is replaced with 20 methyl 4-thiazole carboxximidate to give: N - [(4-thiazolyl-imino) methyl] -thienamycin (99 mg, 89%) H2 O UV λ = 300 nm (e = 7530). max IR (Nujol mull) 1764 cm-1 (β-lactam).

NMR (60 MHz, D 2 O) δ 1.23, 3Η (3, J = 7 Hz, CH 3 CH (OH)), HPLC, 1.8 x retention of thienamycin.

35

Preparation of N * - (2-methylthioethyl) -N-formimidoylthienamycin EXAMPLE 16

DK 157401 B

28

-OH

n-ch2ch2sch3

II

Th - NHC

10 \

H

-COOH

Thienamycin (105 mg) is dissolved in pH = 7, 0.1 N phosphate buffer (5 ml) and to this is added a solution of ethyl N-2-methylthioethylformimidate (300 µl) in tetrahydrofuran (2 ml). The pH of the solution is adjusted and maintained at 8.5 by means of an autoburette which delivers 1 N sodium hydroxide. After 30 minutes, adjust the pH to 7.0 with 2.5 N HCl. The solution is chromatographed on an ice-water-cooled column of Dowex 50 ® + X4 resin (53 ml. Na-cycle, 200-400 mesh), eluting with deionized water. The Ν '- [2-methylthioethyl] -N-formimidoyl derivative is eluted in 2-4 column volumes and lyophilized to give a white solid.

UV (pH = 7, 0.1 N phosphate buffer) λ = 298 nm (e =

IUaX

7760). IR (Nujol mull) 1760 cm cm ”(5-lactam).

DK 157401B

EXAMPLE 17 29

Preparation of Ν'-tert-butyl-N-formimidoylthienamycin 5

N ”C ^CH3) 3 ir

Th - NHC

\

10 H

-COOH

Thienamycin (105 mg) is dissolved in pH = 7, 0.1 N phosphate buffer (5 ml) and to this is added a solution of 15 N-tert-butylformimidate (290 mg) in tetrahydrofuran (1 ml). The pH of the solution is adjusted to and maintained at 8.5 by means of an autoburette which delivers 1 N sodium hydroxide. After 30 minutes, adjust the pH to 7.0 with 2.5 N HCl. The solution is chromatographed on an ice-cooled column of Dowax 50-X4® resin (53 ml, Na + cycle, 200-400 mesh) eluted with deionized water. The fractions containing the aforementioned product are combined and lyo f in lyses.

25 1 35

Preparation of N1- [1-methyl-2-propenyl] N-formimidoylthienamycin EXAMPLE 18

DK 157401 B

30

i-OH

N "C (CH3) 3! 11

Th— —NH-C-H

10 \

H

-COOH

Thienamycin (126 mg) is dissolved in pH = 7 0.1 N phosphate buffer (6 ml) and the pH of the solution is adjusted to 8.5 with an automatic burette which delivers 1 N NaOH. To this solution is added ethyl N-1-methyl-2-propenylformimidate hydrochloride (300 μΐ) with stirring while maintaining the pH of 8.5. After 30 minutes 20, the pH of the solution is adjusted to 7.0 with 2.5 N hydrochloric acid and the solution is chromatographed on a column of ice-cooled Dowex 50-X4® resin (49 ml, Na + cycle, 200-400 mesh). , eluting with deionized water. The Ν '- [1-methyl-2-propenyl] -N-formimidoyl derivative is eluted in 2-4 column volumes and lyophilized to a white solid (59 mg).

UV (pH = 7, 0.1 N phospha tpuf fer) λ = 299 nm (e = Π13Χ 7820).

IR (Nujol mull) 1760 cm ~ ((β-lactam).

35

Preparation of N * -dimethylamino-N-formimidoyl-thienamycin Example 19

DK 157401B

31 CH3 5 j-OH /

N-N

"\

Th - NHC CH3 \

10 H

-COOH

Thienamycin (115 mg) is dissolved in pH = 7, 0.1 N phosphate buffer (7 ml) and the pH of solution 15 is adjusted to 8.5 with an automatic burette which delivers 1 N NaOH. To this solution is added methyl-N-dimethylaminoformimidate hydrochloride (284 mg) with stirring while maintaining the pH at 8.5. After 20 minutes, the pH of the solution is adjusted to 7.0 with 2.5 N HCl and the €> 20 solution is chromatographed on Dowex 50-X4 resin (53 ml, Na + cycle, 200-400 mesh) eluting with deionized water. The chromatography is carried out in a water jacket column at 3 ° C. The Ν'-dimethylamino-N-formimidoyl derivative is eluted in 2 column volumes and lyophilized to a white solid (40 mg).

UV (pH * 7, 0.1 N phosphate buffer) k ~ 298 nm (e = ΧοΧ 6910).

IR (Nujol mull) 1760 cm -1 (β-lactam).

NMR (D 2 O) δ 1.29 (d, J = 6 Hz, CH 2 -CH), 2.59 (s, N- (CH 3) 2), 7.76 (s, NHC).

35

Preparation of Methyloxalimidoylthienamycin Example 20

DK 157401 B

32

5 i-OH

NH NH

II II

Th - NHC-C-C-OCH3 10

-COOH

Thienamycin (105 mg) is dissolved in pH = 7, 0.1 N phosphate buffer (5 ml) and the pH of the solution is adjusted to 8.5 with an automatic burette giving 1 N NaOH. To this solution is added methyloxalimidate (200 µl) while maintaining the pH of 8.5. After 30 minutes, adjust the pH to 7.0 with 2.5 N HCl and chromatograph the solution q + on Dowex 50-X4 resin (53 ml, Na-20 cycle, 200-400 mesh), eluting with deionized water . The chromatography is carried out in a water jacket column at 3 ° C. The methyloxalimidoyl derivative is eluted 2 column by volume and lyophilized to a white solid (44 mg). UV (pH = 7, 0.1 N phosphate buffer) λ = 298 nm (s =

luoX

6230).

IR (Nujol mull) 1760 cm -1 (β-lactam).

NMR (D 2 O) δ 1.27 (d, J = 6 Hz, CHg-CH), 3.87 (s, -OCHg).

Preparation of N1-propionimidoylthienamycin Example 21

DK 157401B

33

5 r-OH

NH

II

Th - NHC

\ 10 CH2CH3

-COOH

Thienamycin (114 mg) is dissolved in pH = 7, 0.1 N phosphate buffer (10 ml) and the pH of solution 15 is adjusted to 8.5 with automatic burette giving 1 N NaOH. Solid ethyl propionimidate hydrochloride (231 mg) is added portionwise as soon as possible to keep the pH close to 8.5. After 30 minutes, the pH is adjusted to 7.0 with 2.5 N HCl and the solution of chromatography is dissolved on A 4 * 20 Dowex 50-X4 resin (72 ml, Na-cycle, 200-400 mesh), eluted with deionized water. The N-propionimidoyl derivative is eluted in 2 column volumes and lyophilized to a white solid (76 mg).

UV (pH = 7, 0.1 N phosphate buffer) k = 298 nm (e =

IUcLX

7830).

NMR (D 2 O) δ 1.28 (d, J = 6 Hz, CH 3 CH (OH)), 1.23 (t, J = 8 Hz -CH 2 -CH 3), 2.50 (q, J - 8 Hz, CH 2 CH 3) ).

30 35 34

Preparation of N'-methyl-N-formimidoylthienamycin

5 j-OH

EXAMPLE 22 n-gh3

II

Th - NHC

\

10 H

-COOH

Thienamycin (140 mg) is dissolved in pH = 7, 0.1 N phosphate buffer (10 ml) and the pH of solution 15 is adjusted to 8.5 with an automatic burette that delivers 1 N NaOH. To this solution is added methyl N-methyl formimidate hydrochloride (200 nl) while adjusting the pH to 8.5. After 40 minutes, adjust the pH to 7.0 with 2.5 N HCl and chromatograph the solution on Dowex 50-X4® resin (53 mL, Na + cycle, 200-400 mesh) eluting with deionized water. The N 'methyl-N-formimidoyl derivative is eluted in 2 column volumes and lyophilized to a white solid (43 mg). UV (pH = 7, 0.1 N phosphate buffer) λ = 298 nm (e = max 7250).

IR (Nujol mull) 1765 cm 2 (? Lactam).

NMR (D 2 O) δ 1.29 (d, J = 6 Hz, CTg-CH), 2.92 (s, N-CHg).

DK 157401 B

EXAMPLE 23

Preparation of Ν'-benzyl-N-formimidoylthienamycin 5 r-OH i-i N-CH2f? "\ ~ rr /

Th - NHC

\

10 H

-COOH

Thienamycin (110 mg) is dissolved in pH = 7, 0.1 N phosphate buffer (7 ml) and the pH of the solution is adjusted to 8.5 with an automatic burette giving 1 N NaOH. A solution of ethyl N-benzylformimidate fluoroborate (572 mg) in p-dioxane (2 ml) is added to the buffered solution while maintaining the pH of 8.5. After 20 minutes, the pH of the solution was adjusted to 7.0 with 2.5 N 20 HCl and chromatographed on Dowex 50-X4 resin (53 ml, Na + cycle, 200-400 mesh) eluting with deionized water . The chromatography is carried out in a water jacket column at 3 ° C. The Ν'-benzyl-N-formimidoyl derivative is eluted in 2 column volumes and lyophilized to a white solid (5 mg).

UV (pH = 7, 0.1 N phosphate buffer) λ = 295 nm (e = ΠΙαΚ 3980).

IR (Nujol mull) 1765 cm (ε-lactam).

NMR (D 2 O) δ 1.29 (d, J = 6 Hz, CHg-CH), 4.44 (s, CH₂-Ar), 7.37 (s, aryl), 8.14 (s, NCH).

EXAMPLE · 24

DK 157401 B

36

Preparation of N * -isopropyl-N-formimidoylthienamycin 5 in -OH

NCH (CH 3) 2

II

Th - NHC

\

10 H

-COOH

Thienamycin (110 mg) is dissolved in pH = 7, 0.1 N phosphate buffer (7 ml) and the pH of the solution is adjusted to 8.5 with an automatic burette giving 1 N NaOH. A solution of methyl N-isopropylformimidate hydrochloride (300 mg) in p-dioxane (1 ml) is added to the solution under magnetic stirring while maintaining the pH of 8.5.

After 25 minutes, the pH of the solution was adjusted to 7.0 with 2.5 N HCl and chromatographed on Dowex 50-X4® resin (53 ml, Na + cycle, 200-400 mesh) eluting with deionized water. The chromatography is carried out in a water jacket column at 3 ° C. The N * -isopropyl-N-formimoyl derivative is eluted in 2 column volumes and lyophilized to a white solid (12 mg).

UV (pH = 7, 0.1 N phosphate buffer) * max = 299 nm (e = 8130).

IR (Nujol mull) 1760 can (i) -lactam).

NMR (D 2 O) δ 1.26 (d, J = 6 Hz, CH 2 CH 2 OH)), 1.29 (d, J = 6 Hz CH (CH 2 (3), 7.89 (s, NHCH), 7.96 ( s, NHCH).

f & 35

DK 157401 B

EXAMPLE 25

Preparation of N (Nr-allylformimidoyl) thienamycin 5 p-OH

/ ch2ch = ch2 n-Ch3 tr

Th - NH-C

10 \

H

-COOH

To a pre-cooled sample of thienamycin (123 mg, 0.452 15 mmol) is added 13 ml of cold 0.1 N phosphate buffer. The solution is adjusted to a pH of 9 with 1N sodium hydroxide.

To this basic solution at 2 ° C is added 0.3 g of ethyl N-allylformimidate hydrochloride at one time. The pH drops to 7.3 and is brought back to 8.5 with more sodium hydroxide. The reaction mixture is stirred at 2 ° C for an additional 30 minutes and the pH is adjusted to 7 with cold 0.1 N sulfuric acid. The reaction mixture is analyzed by high-pressure liquid chromatography on a C ^ g-Porosil column developed with 10% aqueous tetrahydrofuran and found to contain only traces of thienamycin (retention time 5 minutes) and virtually pure product (retention time 10.5 minutes) ). The reaction mixture is chromatographed on a Dowex 50 x 4 column (6 ml, 200-400 mesh), eluting with water at a flow rate of 0.5 ml / cm resin. After discarding the first 400 ml of eluate, the next 150 ml are lyophilized to obtain the product.

Yield: 96 mg (63%).

UV λ = 298 nm, 24.6 O.D. units / mg. (NHoOH max <5 off, 90% purity).

35

DK 157401 B

38 IR Nujol exhibits C = 0 at 5.67 u and 5.90 u.

NMR 100 MHz D 2 O shows that it is a 1: 1 mixture of syn- and anti-N (N'-allylformimidoyl) thienamycin.

EXAMPLE 26

Preparation of N (N * -trifluoroethylformimidoyl) thienamycin 10-OH

N-CH2CF3

II

Th - NH-C

\

15 H

-C00H

To a pre-cooled sample of thienamycin (123 mg, 0.452 mmol) is added 15 ml of cold 0.1 N phosphate buffer. The solution 20 is adjusted to a pH of 9 with 1N sodium hydroxide.

To this basic solution at 0-2 ° C, 0.3 g of ethyl N-trifluoroethylformimidate in 2 ml of dioxane is added portionwise over 30 minutes. The pH of the reaction mixture is maintained at 8.5 - 9 during the addition. The reaction mixture is stirred for a few minutes. After the addition of imidate is complete and the pH is adjusted to 7 with cold 0.1 N H2SO4, HPLC, C1-6 Porosil reverse phase, with 10% aqueous tetrahydrofuran exhibits a new maximum at 12.2 nm. there is the desired product.

The mixture is chromatographed on a Dowex 50 x 4 column (60 ml, 200-400 mesh). The column is eluted with water at a flow rate of 0.5 ml min / cm of the resin layer.

A process is discarded and the fractions containing the product are combined and lyophilized to give a hydroscopic substance, 10.2 mg, * max “302.

DK 157401B

EXAMPLE 27 39

Preparation of N (N1-carboxymethylformimidoyl) thienamycin sodium salt 5

-OH

n-ch2cooh I »

Th - NHC

10 \

H

-COONa

Thienamycin (130 mg) is dissolved in pH = 7, 0.1 N phosphate buffer (4 ml) and a solid sodium ethyl N-carboxymethylformimidate (500 mg) is added at once. The pH of the solution is adjusted to 8.5 with an automatic burette that delivers 1 N sodium hydroxide. After 25 minutes at a pH of 8.5, the solution 20 is adjusted to pH = 7.0 with 2.5 N hydrochloric acid. The solution is then chromatographed on an ice-cooled column of ® +

Dowex 50 x 4 resin (51 ml. Na-cycle, 200-400 mesh) eluting with deionized water. The eluate of the first column volume is combined and evaporated to 7 ml.

This solution is then chromatographed on an ice-cooled column of XAD-2 resin (53 ml), eluting with ionized water. The two to four column volumes are collected and combined and lyophilized to give sodium N ('- carboxymethylformimidoyl) thienamycin (25 mg).

UV (pH = 7, 0.1 N phosphate buffer) λ * 300 nm (ε-6390). 1 IR (Nujol mull) 1755 cm -1 (β-lactam).

DK 157401 B

NMR (D 2 O) δ 1.29 (d, J = 6 Hz, CH 3 -CH), 7.85 (s, NCH).

EXAMPLE 28 Preparation of N (3-azidopropionimidoyl) thienamycin

-OH

NH

II

10 Th - NHCCH2CH3N3

-COOH

To a solution of thienamycin (133 mg) in 10 ml of 0.1 M

To pH = 7.0 phosphate buffer 1.2 g of 0-ethyl-3-azido acid is added.

propionimidate * HCl while maintaining the solution at pH

= 8.5 with 2.5 N NaOH. The mixture is stirred at 0 ° C for 0.5 hours and then neutralized with 2.5 N HCl to pH = 7.0, evaporated to 5 ml and chromatographed on a Dowex €> 20 50W x 8 (Na form) column (3 , 8 x 30 cm), which is eluted with

water to form the desired product. The product exhibits UV

h2 ° λ__ = 300 nm; High Pressure Liquid Chromatography (HPLC) 25 retention time of 10 minutes compared to 4.8 minutes for the starting material under the same conditions (3 mm x 60 cm Bondapak C ^ g reverse phase column eluted with 10% THF in water at a flow rate of 1.5 ml /mine.); electrophoretic mobility 5 mm to the cathode at 30 50 V / cm for 20 minutes in 0.05 M pH - 7.0 phosphate buffer.

EXAMPLE 29 41

DK 157401 B

Preparation of N (3-aminopropionimidoyl) thienamycin

OH r OH

5 Γ ° H ΐ® ί? Η H? / Pd v. Th-r NHCCH0CH-NH,

Th-NHCCH 2 CH 2 N 3 - 7 * λ Z in L CO 2 - CO 2 g of Pd catalyst (10% palladium on activated carbon) for 30 minutes Electrophoresis of the resulting mixture exhibits a new bioactive product moving 30 mm toward the cathode (50 V / cm for 20 minutes at 0.05 M pH = 7.0 phosphate buffer) in addition to the starting material I which moves 5 mm toward the cathode The electrophoretic mobility of the product is consistent with the mobility of the expected product II. The reaction mixture formed from the hydrogenation reaction is neutralized with 2.5 N HCl and filtered from the catalyst. The filtrate is evaporated to 10 ml and chromatographed on XAD-2 resin (2.3 x 16 cm column). The column is eluted with water to give the desired product II as a hydrochloride after lyophilization, 23 mg, N- (3). -aminopropionimidoyl) -thienamycin hydrochloride.

H 2 ° 30 UV kmax = 301 nm (e = 7080). IR: Nujol mull 1765 citf1 * NMR 60 MHz (D 2 O) δ 1.30 ppm, (3), 2.60 - 3.72 ppm, (11) , 4.18 ppm (2).

DK 157401 B

EXAMPLE 30

Preparation of N-nitroguanylthienamycin 5 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-thiopseudourine (0, 3 g). The solution is heated in a water bath at 45 ° C while a stream of nitrogen is bubbled vigorously through the solution. After 50 minutes 10, the solution is evaporated under high vacuum to 1.0 ml and dissolved in 0.05 N pH = 7 phosphate buffer (7 ml). The unreacted thiopseudourse precipitate is precipitated and filtered.

The solution is then chromatographed on Dowez 50-X4 resin 3+ (53 cm, 200-400 mesh, Na cycle) and eluted with water.

The N-nitroguanyl derivative is eluted in the first column volume and lyophilized to a solid (23%).

UV (pH = 7, 0.1 N phosphate buffer) λ = 269 nm (e = 11000) 20

Electrophoresis (40 V / cm, pH = 7, 0.1 N phosphate buffer, 20 min) 3.0 cm toward the cathode.

EXAMPLE 31 25

Preparation of N-isobutyrimidoylthienamycin

The procedure of Example 11 is followed, replacing ethyl acetimidate hydrochloride with isobutyrimidate hydrochloride and reacting at 20 ° C and pH = 8.2 to give N-isobutyrimidoylthienamycin (14%) UV (pH = 7, 0.1 N phosphate buffer) * max = 298 nm (s = 35 8290)

DK 157401 B

43 NMR (D 2 O δ 1.27 (d, J = 7 Hz, CH (CH 3) 2, 1.29 (d, J = 6 Hz, CH 3 CH (OH), 2.79 (heptet, J = 7 Hz, CH (CH 3) ) 2).

EXAMPLE 32

Preparation of Ν'-methyl-N-acetimidoylthienamycin

The procedure of Example 11 is repeated, replacing ethyl acetate-imidate hydrochloride with ethyl N-methylformimidate hydrochloride to give Ν'-methyl-N-formimidoyl-thienamycin (10%).

UV (pH = 7, 0.1 N phosphate buffer) kmax = 298 nm.

IR (Nujol mull) 1750 cnT 2 (ε-lactam), 1660 cm- ^ (C = NCH 3).

NMR (D 2 O) δ 1.27 (d, J = 6Hz, CH 3 CH (OH), 2.22 and 2.25 N

II

(s, N-CCH 3, 2.97 (S, NCH g).

EXAMPLE 33 25

Preparation of N'-methyl-N-formimidoylthienamycin

The procedure of Example 11 is repeated, replacing ethyl acetimidate hydrochloride with ethyl N-methylformimidate hydrochloride to give Ν'-methyl-N-formimidoyl-thienamycin (10%).

UV (pH = 7, 0.1 N phosphate buffer) λ = 298 nm.

NMαΧ 1 NMR (D20) δ 1.30 (d, J = 6Hz, CH 3 CH (OH), 2.92 (s, n-ch 3),

DK 157401 B

44

N

II

7.78 (s, -CH) EXAMPLE 34

The procedure of Example 11 is followed, replacing the reagent with an equivalent amount of methyl methoxyacetimidate to give N (methocyacetimidoyl) thienamycin 10 (34%).

H2 ° UV k = 198, 301 nm (e = 16180, 8700). max 15 IR (Nujol mull) 1760 cm -1 (ε-lactam).

NMR (60 MHz, D 2 O) δ 1.28, 3H, (d, J = 6Hz, CH 3 CH (OH), 3.50, 3 H (s, CH 3 CH 2) 4.35, 2H, (s, CH 3 HPLC, 1.50 x retention time for thienamycin.

EXAMPLE 35

Preparation of N- [Ν'-ethylformimidoyl] thienamycin 25

-OH

r

Th - NHC-H.

_C0, H

30 z

Thienamycin (100 mg) in 10 ml of 0.1 M pH = 7.0 phosphate buffer is adjusted and maintained at pH = 8.5 - 9.0 with 2.5 N sodium hydroxide. To the solution is added 300 mg of ethyl N-ethylformimidate hydrochloride. The mixture is stirred at 23 ° C for 20 minutes, then neutralized at pH = 45

DK 157401B

7.0 with 2.5 N HCl and chromatographed on a Dowex 50 X 8 (Na form) ion exchange resin column (3.8 x 25 cm). The column is eluted with water, taking 6.7 ml fractions. Fractions 40-90 are combined, evaporated and freeze-dried to give 15 mg of the solid product.

Electrophoresis of the product at 50 V / cm for 20 minutes in 0.1 M, pH = 7.0 phosphate buffer shows a single bioactive zone moving 2 mm toward the cathode.

10 h2o UV λ __ »301 nm.

max NMR (100 MHz, D 2 O) δ 7.77 (s) and 7.82 (s) (formimidoyl CH).

EXAMPLE 36

Preparation of η- [N'-coclopropylformimidoyl] thienamycin 20 PK]

1¾ - NHC

XH

-COOH 1 2 3 4 5 6 7 8 9 10 11 2

Thienamycin (100 mg) in 10 ml of 0.1 M pH = 7.0 phosphate buffer 3 is adjusted and maintained at pH = 8.5 - 9.0 while 4 300 mg of ethyl N-ethylformimidate hydrochloride is added dropwise to the solution. . The mixture is stirred at 23 ° C for 40 minutes, then neutralized and chromatographed on a 7β8

Dowex 50 X 8 (Na form) ion exchange resin column (3.8 x 9 25 cm). The column is eluted with water, taking 6.7 ml of 10 fractions. Fractions 40 - 95 are combined, evaporated and lyophilized to give 54 mg of solid product. Electrophoresis of the product exhibits a single bioactive zone moving 10 mm toward the cathode (50 V / cm, 1 hour in 0.05 M pH = 7.0 phosphate buffer).

DK 157401 B

46 h2 ° UV λ = 301 ran. max 5 NMR (100 MHz, D 2 O): 60 - 1.30 ppm (m cyclopropyl) and 7.80 ppm (formimidoyl CH) EXAMPLE 37 10 In the same way as in the previous examples, the following compounds are prepared.

15 20 25 30 35

DK 157401B

47

Hp h '_L-λ ^^ r1 H T> ^ Vr2 5

o C07K

Connect. R 2 07 IR NMR

10 1. H CHnCH = CH2 H 300 (8500) no åi3 2. H j = \ H Mujoi 10GL MHz neH 1750 στΓ1 (0.0): 1.25 (d, 301 nm 15 j = 6.2 Hz) 2.40 - 2.80 (n) 2.90 - 3.20 (:: l) 3.33 (q, J = 2.8, J = 6.2 Hz) 4.1 - 4.4 ( m),> 5.73 (m), 6.16 (n), 7.76 (s) 3. H iv Η ρΗ, Ο Nujol 100'MHz U ma5c 3150 (br), (D, 0) S 1.3 (d, 29S niti 1760, 1700, CF + CKOH, J = 6Hz), 3.38 C = 6560 1690, (dd, H6, j = 3.6 Hz) (Ηο0 / ΝΗ20Η, 1585 cm 1 7.68 (s, NHCH = N) hci / k ^ ipo4) λ max 298 nm £ * 6400 4. Η Ι ^ 'Ί)' H Nujol no 293 nm 1765 cm -1 £ = 7674 35

DK 157401B

48 5 For-

Vol. R R * R * UV m NMR

CH λΗ O

5. H-ζ CH 3 'idk no 100 Mfe ^ 3 £ = 5878 tough »1'30 (d, 9H, J = 6.2Hz, 10 N ^ 0 * 3, 2.97> v æ3 (s, 3H, N-CH 3) 3.05-3.15 (m), 3.20 (q, 1H, j = 6.2, J = 3.0 Hz) 3.50 - 4.40 (n) 7.90 (s, CH-N) 6. h - * C ^ 3 η λ ^. Nujol ioo: mz 3 298 nm 3200 (br) r (D 2 O) S 1.38 - 1.40 20 8010 1760, (ra, CH₂CHDH, (H₂O / NH₂OH, 1660, CH (CH3) 2), 2.42 HCl / K₂HPO0> 1590 cm "1 (s, NC (CH3) = N-), * max 2.45 (s, KC (CH3) ) = N-) 298 ran 25 f - 7850 7. -CH „-CH„ Η H AH2? Nujol none * z irax _i 300 ran 1760 can £ 8500- 8. 8. SCH-, -CH-, Η λΗ2? Nujol no 3 3 ϊκβχ J, 302 ran 1760 on ^ £ = 9000 9. H-CN Η λΗ2? Nujol 2185 none ΠΒΧ 302 ran 1757 cm £ 6930 35

Claims (2)

A / x X \ - / V- SCH-CH-NH-ll 222 X_Nl COOH T in 1 L or a suitable carboxyl derivative thereof is reacted with an imido ester of the formula: DK 157401 B NR1R2 J-xV formula: 10 AA & c-x · n | R · Θ v R 15 wherein -X ° R "is a leaving group, X 'is halogen, and X ° is 0 or S and R1, Rz and R have the meaning given above, or are reacted with a compound having the formula: 20 x "r ° rVn-on-r1 VI 25 where X" is O or S and -X "R ° is a leaving group, 1 2 and R and R are H or C 1 -C 6 alkyl on which they obtained compounds, if desired, are esterified or converted into salts thereof in a manner known per se. 2 35 2