NO153298B - ANALOGY PROCEDURE FOR PREPARING THERAPYUTIC ACTIVE N-METHYLENDER DERIVATIVES OF THIENAMYCIN - Google Patents

Description

The present invention relates to an analogue method for the production of therapeutically active substituted N-methnylene derivatives of thienamycin. Such compounds and their pharmaceutically acceptable salts and esters are useful as antibiotics.

Thienamycin is mentioned and described in the US patent

No. 3 950 357.

Thienamycin has the following structure:

There is a continuing need for new antibiotics.

Unfortunately, there is no static effectiveness of any given antibiotic because continued widespread use of any such antibiotic selectively causes the formation of resistant strains of pathogens. In addition, the known antibiotics suffer from the disadvantage that they are effective only against certain types of microorganisms. Consequently, the search for new antibiotics continues.

It has unexpectedly turned out that the process compounds are broad-spectrum antibiotics that are useful in animal and human therapy, and in inanimate systems.

It is an aim of the present invention to produce a new group of antibiotics which have the basic ring structure of thienamycin (I), but which are characterized as substituted N-methylene derivatives thereof. These antibiotics are effective against a wide range of pathogens which representatively include both Gram-positive bacteria such as S. aureus, Strep. pyogenes and B. subtilis and Gram-negative bacteria such as E. coli, Proteus morganii, K.lebsiella, Serratia and Pseudomonas. A further aim is to produce non-toxic pharmaceutically acceptable salts and esters thereof.

The substituted N-methylene-thienamycin derivatives produced according to the present invention can be designated by the following structural formula:

and the p-t-butylbenzyl, 3-methyl-2-buten-l-yl and pivaloxymethyl esters thereof, and pharmaceutically acceptable salts thereof, wherein R^" is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-6 carbon atoms , cyano, cycloalkyl or cycloalkenyl with 3-6 carbon atoms, alkylthioalkyl, carboxyalkyl or 2dialkyl-amino with 1-6 carbon atoms, trifluoroethyl or benzyl, and R is hydrogen 12 12 or R and R is alkyl with 1-6 carbon atoms or R and R together with the nitrogen atom is 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 with 2-6 carbon atoms, or where R is -OR3 or -SR<a> wherein Ra is methylbenzyl, ethylallyl, 2-thienylmethallyl, p-nitrobenzyl or CH3~O-CH2.

The process compounds are conveniently prepared from thienamycin (I) or a suitable carboxyl derivative thereof.

Such carboxyl derivatives of thienamycin have the structural formula:

wherein the group (R 3') includes conventional protecting or carboxyl-blocking groups. The term "blocking groups" is used here in the same way and according to the teachings of US patent no. 3,697,515.

The starting materials Ic are produced according to the following scheme; but it should be noted that direct esterification, without protection of the amino group, is also possible.

11 2 1

where X' = 0, R and R are acyl and Th denotes the thienamycin skeleton. R 3 1 is as previously stated.

In general, the transfer (1—>Ic) is carried out by

known conventional methods. Such methods include:

1) Reaction of 1 (or I) with a diazoalkane such as diazomethane, phenyldiazomethane, ■ diphenyldiazomethane and the like,

in a solvent such as dioxane, ethyl acetate, acetonitrile and the like, at a temperature from 0° C to reflux temperature

pure for from a few minutes to 2 hours.

2) Reaction of an alkali metal salt of 1 with et

activated alkyl halide such as methyl iodide, benzyl bromide, or m-phenoxybenzyl bromide, p-t-butylbenzyl bromide, pivaloyloxymethyl chloride and the like. Suitable reaction conditions include solvents such as hexamethylphosphoramide and the like, at a temperature of from 0° to 60° C. for from a few minutes to 4 hours.

3) Reaction of 1 with an alcohol such as methanol,

ethanol, benzyl alcohol and the like. This reaction can perform

res in the presence of a carbodiimide condensing agent such as dicyclohexylcarbodiimide or the like. Suitable solvents

ler, at a temperature from 0° C to the reflux temperature for from 15 minutes to 18 hours, includes chloroform, methyl-

chloride, methylene chloride and the like.

4) Reaction of an N-acylated acid anhydride of 1 prepared by reacting the free acid 1 with an acid chloride such as ethyl chloroformate, benzyl chloroformate and the like, with an alcohol

hol as those indicated under 3) under the same reaction conditions as indicated for 3). The anhydride is produced by re-

put 1^ and the acid chloride in a solvent such as tetrahydro-

furan (THF), methylene chloride and the like, at a temperature from 25° C to the reflux temperature from 15 minutes to 10 hours.

5) Reaction of labile esters of 1 such as the trimethyl-silyl ester, the dimethyl-t-butyl silyl ester or the like, with R^ X° where X° is halogen such as bromine or chlorine, and R^ is as

above, in a solvent such as THF, methylene chloride or the like, at a temperature from 0° C. to the reflux temperature for from 15 minutes to 16 hours. For example, according to the following scheme:

where TMS is triorganosilyl such as trimethylsilyl, and the other symbols are as above.

The analog method according to the invention is characterized in that a compound with formula:

or a suitable carboxyl derivative thereof: is reacted with an imidoester: or an imidohalide: wherein -X°R" is a leaving group and X' is halogen, X° is 0 or S, and R ] , R 2 and R are as indicated above, or reacted with a compound of formula:

in which -X"R° is a removable group in which X" is oxygen or sulphur, and R <1> and R 2 are H or C-^-C8 alkyl, or that the compounds obtained are esterified or converted into salts in a known manner.

Suitable solvents depending on the identity of the thienamycin substrate and reagent include water, dioxane, tetrahydrofuran (THF), dimethylformamide (DMF, chloroform, acetone, acetonitrile or mixtures thereof. The reaction is carried out at a temperature of from 0°C to about 25°C for from 1 to about 6 hours.There is nothing critical with regard to the exact identity of the reaction solvent or the variables of the reaction within the limits described above, provided only that the reactant is inert or substantially inert to the intended course of reaction. Suitable reagents include representatively:

a) Imido esters:

Methyl formimidate, ethyl formimidate, methyl acetimidate, ethyl acetimidate, methyl benzimidate, ethyl 4-pyridyl carboximidate, methyl phenylacetimidate, methyl 3-thienyl carboximidate, methyl azidoacetimidate, methyl chloroacetimidate, methyl- cyclohexylcarboximidate, methyl-2-furylcarboximidate, methyl-1-p-nitrobenzimidate, methyl-2,4-dimethoxybenzimidate, ethyl-N-methyl-formimidate, methyl-N-methyl-formimidate, methyl-1-N-isopropyl-formimidate and the like.

Such imidoester reagents (a) are conveniently prepared by any of a number of known methods, such as: 1) The reaction of a nitrile, RCN, with a lower alkanol in the presence of HCl according to the well-known Pinner synthesis. 2) The reaction of a nitrile, RCN, with a lower alkanol in the presence of a base. Typically, the reaction is carried out at 0-40°C in the presence of an excess of the alcohol with a catalytic amount of an alkali metal alkoxide for from 15 minutes to 4 hours. ^ 3) The reaction of an amide, RC" NHR , with an alkyl chloroformate, such as methyl chloroformate at 25 - 45° C for 1 - 4 hours.

4) The conversion of an N-substituted amide,

OE OE

"1 "12

RCNHR or RCNR R , with one equivalent of an alkylating agent such as triethyloxonium fluoroborate in an inert solvent such as ether, chloroform or the like, at 0 - 23°C for from 10 minutes to 2 hours. 5) The conversion of a readily available imidoester, RCNR' (R<1> can be hydrogen), to the desired imidoester, OR" RCNR"<1>", by reacting the former with an alkylamine, R'NH2, OR" 1 a mixture of water and an immiscible solvent such as ether or chloroform, at 0 - 23° C for from 5 minutes to 1 hour. b) Substituted imidohalides: Chloropiperidino-methylium chloride, chlorodimethylforminium-

chloride, chlordiethylforminium chloride and the like.

Such imidohalide reagents (b) are prepared be-

by any of a number of known methods such as:

1) The conversion of an N,N-disubstituted amide,

0

"12

RCNR R , with a halogenating agent such as thionyl chloride, phosgene, phosphorus pentachloride or the like, in an inert solvent such as chloroform, methylene chloride and the like, at 0 - 40° C in 1 -

5 hours.

X"R°

12I

(b) - OR- pseudoureas and - SR- pseudothioureas: R R N =NR

O-methyl-pseudourea, S-methylpseudothiourea, S-methyl-pseudothionitrourea, 0-2,4-dichlorophenyl-pseudourea, S-p-nitro-phenyl-pseudothiourea, O-N,N-trimethylpseudourea and the like.

The reaction involving the reagents (a) can represent

tative is shown by the following diagram:

where OR" is the leaving group of the imidoester reagent, and

3<1> 3 3'

R, R , R and X' are as indicated above and R' is H or R

3 3'

This reaction is particularly suitable for embodiments where R and R are hydrogen, and X<1> is oxygen.

The reaction involving the reagents (b) can be represented representatively by the following diagram:

where all symbols are as above. When product 2 is desired

3 31

are suitably R and R trimethylsilyl and X<1> is oxygen.

The reaction involving the reagents (c) can be represented representatively by the following diagram:

3<1> 1 2

where X<1>, R , R and R are as above; X" is oxygen or sulphur, and R is as indicated and is preferably lower alkyl.

Suitable solvents for such reactions include

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

The process compounds (II) form a wide variety of different pharmacologically acceptable salts such as acid addition salts, e.g. with hydrochloric acid, hydrogen bromide, svo-

velic acid, nitric acid, toluene-p-sulphonic acid and methane-sulphonic acid.

The salts of the process compounds are pharmacologically acceptable non-toxic derivatives which can be used as the active ingredient in suitable forms of pharmaceutical unit doses. They can also be combined with other drugs to give preparations with a broad spectrum of activity.

The new process compounds are valuable anti-

biotica that are active against various gram-positive and gram-

negative bacteria, and consequently find use in human and veterinary medicine. The method compounds can therefore an-

are used as antibacterial drugs for the treatment of infections caused by gram-positive or gram-negative bacteria, e.g.

against Staphylococcus aureus, Escherichia coli, Klebsiella pneumo-

niae, Serratia, Salmonella typhosa, Pseudomonas and Bacterium proteus. The antibacterial agents can also be used as additives to animal feed to concentrate the feed and as disinfectants.

The antibacterial activity of some of the method compounds is as follows:

The method compounds can be used alone or in combination as an active ingredient of any of a number 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 following examples illustrate the invention. In the examples, the thienamycin ring system (I) is denoted by the following symbol: where it. secondary alcohol group, the amino group and the carboxyl group are illustrated. Compounds produced according to the invention can thus be conveniently stated as follows:

where R, R 1 and R 2 are as indicated above.

Example 1

Preparation of silylated thienamycin

80.0 mg of thienamycin is suspended in 40 ml of tetrahydrofuran (THF) under a nitrogen atmosphere and evaporated to 10 ml;

1.0 ml of hexamethyldisilazane and 300 µl of trimethylchlorosilane 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 remaining liquid is evaporated to an oil under a stream of nitrogen for further reaction.

Example la

Preparation of thienamycin-N-piperidir-l-yT-methylene derivative

57 mg (162 µmol) of thienamycin is silylated by the method described above. The silylated antibiotic Th-(TMS)3 is dissolved in 6 ml of methylene chloride in a septum-closed flask under positive nitrogen pressure and cooled in a dry ice-acetone bath. 180 μl of a solution of 644 μmol of triethylamine in methylene chloride is added to the magnetically stirred solution. This is followed by the addition of a solution of 67 mg (405 µmol) of chloropiperidinomethylium chloride in 465 µl of methylene chloride. After 1 hour in the dry ice bath, the reaction solution is quickly added to 50 ml tetrahydrofuran - pH 7, 0.1 N phosphate buffer (1:1) solution. The mixture is then evaporated under vacuum to 10 ml to obtain a homogeneous solution. The solution is washed with 2 x 5 ml ethyl acetate and 2 x 5 ml ether and pumped briefly under vacuum. This aqueous solution is then chromatographed on an "XAD-2" resin column (60 ml layer). The product is eluted in 10% aqueous tetrahydrofuran (after water elution) whereby 12.9 mg (22%) product is obtained (measured in solution assuming e = 8030, the same as thienamycin. Paper chromatography Rf 0.42 (4:1:5 , n-butanol:ethanol:water).

Example 2

Preparation of N-benzimidoyl-thienamycin

59 mg (212 μmol) of thienamycin is dissolved in 4.5 ml of a 33% N,N-dimethylformamide - pH 7 phosphate buffer (0.05 N) solution and adjusted to pH 9.5 using 2.5 N sodium hydroxide with an automatic burette. The solution is magnetically stirred at 25° C. and 340 mg (1981 µmol) of methyl benzimidate hydrochloride is added once. After 30 minutes, the solution is extracted twice with an equal volume of chloroform and adjusted to pH 7.0 with dilute aqueous phosphoric acid. The buffered solution is chromatographed on 65 ml "XAD-2" resin. The column is first eluted with water followed by 10% aqueous tetrahydrofuran which elutes the product. This fraction is evaporated to half the volume and freeze-dried, whereby 50 mg of the product is obtained. Electrophoretic mobility (50 V/cm, 20 min, pH 7 0.1 N phosphate buffer) is 1.5 cm towards the anode. UV ^max = 300 nm (e 6,960) pH 7 0.IN phosphate buffer.

Example 3

Preparation of N-benzimidoyl-thienamycin, p-t-butylbenzyl ester 3.2 mg of benzimidoyl-thienamycin is suspended in 75 l of hexamethylphosphoramide containing 3.8 yl p-t-butyl-benzyl bromide and magnetically stirred at 22° C. After 45 minutes, a solution which is stirred for a further 1 hour. The product is then separated from the solution with ether and the crude product is chromatographed on a 250 µm thick silica gel plate developed in 7:3, chloroform:ethanol. The band at R 0.6 is removed and eluted with ethanol, whereby N-benzimidoyl-thienamycin, p-t-butylbenzyl ester hydrobromide is obtained. Mass Spectr. m/e 521 (M<+>), 487, 444, 418, 341, 323, 297, 226, 147. Example 4

Preparation of N-benzimidoyl-thienamycin-3-methyl-2-buten-1-yl ester

Dissolve 5.9 mg of benzimidoyl-thienamycin in 100 µl of hexamethylphosphoramide containing 4.8 µl of l-bromo-3-methyl-2-butene and 0.5 µl of triethylamine and stir magnetically at 22° C. After 1 hour, chromatograph the crude reaction mixture on a 250 µm thick silica gel plate developed in 8:2, chloroform:ethanol. The band for R f 0.1 - R^ 0.3 is removed and eluted with ethanol. Benzimidoyl-thienamycin-3-methyl-2-buten-1-yl-ester hydrobromide is isolated as a solid after precipitation from an ethanol-chloroform solution with hexane.

Example 5

Preparation of N-formimidoyl-thienamycin

517 mg of thienamycin are dissolved in 25 ml of pH 7 0.1N phosphate buffer and cooled in an ice bath under magnetic stirring. The solution is adjusted to pH 8.5 with 2.5N sodium hydroxide solution dispensed from an automatic burette. While maintaining a pH of

8.5, 711 mg of methyl formimidate hydrochloride is added in portions over the course of 2-3 minutes. After a further 10 minutes, the pH of the solution is brought to 7.0 using 2.5N hydrochloric acid.

The solution is chromatographed on a column of 150 ml "XAD-2" resin eluted with water. The N-formimidoyl thienamycin derivative is eluted in 1.5 - 2.0 column volume (200 - 300 ml) and lyophilized to a white solid (217 mg).

UV (pH 7 0.1N phosphate buffer) X . 297 nm (8,590).

_, max

ir (Nujol porridge) 1767 cm (B-lactam)

nmr (D2O) <fl, 37 (d, J=6Hz, CH3-CH) , 3.0 - 3.75

(m, -CH3-), 4.2 - 4.8 (m, <C>5H, CgH, C?H), 7.86

NH

(s, -C-H)

Example 6

Preparation of N-guanyl-thienamycin

8.9 mg thienamycin is dissolved in 0.7 ml pH 7 0.1N phosphate buffer and 0.3 ml N,N-dimethylformamide, and the solution is brought to pH 9.5 by adding 2.5N sodium hydroxide solution. 43 mg of O-methyl-isourea-hydrogensulphate is added to the magnetically stirred solution, which causes a slight drop in pH. Additional 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-guanyl-thienamycin shows two biotically active zones after electrophoresis (50 V/cm, 20 minutes, 0.05N pH 7 phosphate buffer) and biotautography on S. aureus plates .

Example 7

Preparation of N-guanyl-thienamycin

11 mg of thienamycin is dissolved in 1 ml of pH 7 0.1N phosphate buffer and adjusted to pH 8.3 with 0.1N sodium hydroxide using an automatic burette. To the magnetically stirred solution, 76 mg of 0-2,4,5-trichlorophenylisourea hydrochloride is added portionwise to allow the automatic burette to maintain a nearly constant pH. The reaction is allowed to proceed for 4 hours at 22° C. and the mixture is then adjusted again to pH 7.0 by adding dilute acid. A sample of this solution containing thienamycin and N-guanyl-thienamycin is subjected to electrophoresis (50 VOcm, 25 minutes, pH 7 0.1N phosphate buffer) and shows a positive Sakaguchi spray zone at 2.0 cm towards the anode and a positive ninhydrin spray zone at 1.5 cm in the same direction.

Example 8

Preparation of N-dimethylaminomethylene-thienamycin

16.5 mg of thienamycin is silylated with 200 µl of hexamethyldisilazane and 60 µl of trimethylchlorosilane in the usual way. The silylated thienamycin is suspended in 1 ml of ethanol-free chloroform under magnetic stirring under a nitrogen atmosphere. The mixture is cooled to -45° C. and a solution of 21 µl of triethylamine in 21 µl

chloroform is added followed by a solution of 11.5 mg of (chloromethylene)-dimethylammonium chloride in 50 µl of chloroform. The mixture is heated to -25° C within 1 hour and 5 ml of Q,IN pH 7 phosphate buffer is added. The mixture is stirred vigorously for 15 minutes. The water phase is separated and contains N-dimethylaminomethylene-thienamycin which has an electrophoretic mobility (50 V/cm,

1 hour, pH 7 buffer) of 3.6 cm towards the cathode.

Example 9

Preparation of N-formimidoyl - thienamycin-pivaloxymethyl-ester hydrobromide 10 mg of N-aminomethylene-thienamycin is dissolved in 200 μl of hexamethylphosphoramide containing 10 μl of bromomethyl pivalate and 1 μl of triethylamine and stirred magnetically at 22° C. After 2 hours, the hexamethylphosphoramide solution is dissolved in 2 ml of methylene chloride and the product is combined with a 50% hexane-ether solution. The precipitate is dissolved in an aqueous 10% tetrahydrofuran solution and chromatographed on an "XAD-2" resin packed column. N-formimidoyl-thienamycin-<p>ivaloxymethyl ester is isolated as a solid after tetrahydrofuran elution of the column and lyophilized.

Example 10

Production of N-trifluoroacetimidoyl-thienamycin

19 9 mg of thienamycin is dissolved in 7 ml of pH 7 Q.1N phosphate buffer and adjusted to pH 8.5 with IN sodium hydroxide solution. While maintaining this pH with an automatic burette, a solution of 355 µl of methyl trifluoroacetimidate in 2.5 ml of dioxane is added all at once. After 30 minutes, the pH is reset to 7.0 by adding 1N hydrochloric acid. The solution is then chromatographed on Dowex 50-X4 resin (200 ml, Na<+> form, 200 - 400 mesh) and eluted with water. The N-trifluoroacetimidoyl thienamycin derivative is eluted in the first half column volume. This eluate is similarly rechromatographed on "Dowex 50-X4"

(100 ml, Na<+> form, 200 - 400 mesh) and the first column volume is evaporated and chromatographed on 30 ml "XAD-2" resin. The N-trifluoroacetimidoyl thienamycin derivative is eluted in 2.5 - 5.0 column volume which is lyophilized to a white solid (15 mg).

UV (pH 7 0.1N phosphate buffer) X . 302 nm (e 4,450).

_-in ms Isa

ir (Nujol porridge) 1750 cm (B-lactam).

Electrophoresis: (50 V/cm, 20 min, pH 7, 0.1N phosphate buffer)

mobility 2.0 cm (toward the cathode)

Example H Preparation of N-acetimidoyl-thienamycin

190 mg of thienamycin is dissolved in 13 ml of pH 7 0.1N phosphate buffer and cooled in an ice bath under magnetic stirring. The solution is adjusted to pH 8.5 with 2.5N sodium hydroxide solution dispensed from an automatic burette. While maintaining a pH of 8.5, 400 mg of ethyl acetimidate hydrochloride is added portionwise over the course of a few minutes. After a further 40 minutes, the solution is adjusted to pH 7.0 with 2.5N hydrochloric acid. The solution is then chromatographed on Dowex 50-X8® resin (250 ml, Na<+> form, 100 - 200 mesh) and 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 0.1N phosphate buffer) *max 297 nm (e 7 620).

ir (Nujol porridge) 1774 cm ^, 3-lactam. viti nmr (D20)<fl,27 (d, J=6 Hz, CH3-CH) 2.24 (S, -C-CH3)

3.2 - 3.5 (m, -CH2), 3.5 - 3.9 (m, -CH2~)

4.2 - 4.6 (m; <C>5H<,>C6R,<C>?H).

Example 12

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

80 mg (0.294 mmol) of thienamycin is dissolved in 24.7 mg (0.294 mmol in 2.0 ml) of aqueous sodium bicarbonate at 25° C. 8Q

mg (0.588 mmol) methyl-isonicotinimidate is dissolved in the solution and reaction progress is followed with samples taken at certain times using high precision liquid chromatography (HPL): Waters instrument; 0.2 x 61 cm Clg "Bondapak reverse phase column"; 1.5 mL/min aqueous 10% THF; UV (254 nm) and R.I. monitors. The reaction is substantially complete within 40 minutes, and the reaction solution is chromatographed directly on an 18.4 x 270 mm "XAD" resin column, eluting first with deionized, distilled water, and then switching to aqueous 10% THF. The eluate is monitored by UV and HPLC is used to determine the pure product. Correct fractions are combined and lyophilized, whereby a colourless, voluminous powder is obtained (80 mg 73%).

UV X ^2° 298 nm (e 7 888); IR (Nujol porridge) 1762 cm"1 (B-lactam) ; NMR (60 MHz, D2O) , (fl, 27, 3H (d, J=7Hz, CH3H(CH)); éi .75 and 8.80, 4H, (m,m,4-pyridyl);EPLC, 1.58x retention of thienamycin, conditions as above.

Example 13

By following the procedure in example 12, but by replacing the reagent with methyl picolinimidate, the following is obtained: N-[(2-pyridyl)-(imino)-methyl]-thienamycin (85 mg 77%)

UV A<H>2° 267, 300 nm (e 8 150), 7 600); IR (Nujol porridge)

rn 9. Tv p

1764 cm (B-lactam); NMR (60 MHz, D 2 O), <f 1.24, 3H (d, J=7 Hz, CH 3 -CH(OH)); 'cf 7 .80 , 8.07, 8.80, 4H (m,m,m, 2-pyridyl); KPLC, 1.8 x retention of thienamycin.

Example 14

By following the procedure in example 12, but by replacing the reagent with methyl nicotinimidate, the following is obtained: N-[(3-pyridyl-(imino)-methyl]-thienamycin (77 mg, 70%) : UV XH2° 264, 299 nm, (e 5570, 6120); IR, (Nujol porridge),

mci.K.s

1766 cm (B-lactam); NMR, (60 MHz, D 2 O), cf 1.24, 3H (d, J= 7Hz, CH 3 -CH(OH)); cf 7.6, 8.2, 8.9, 4H, (m,m,m, 3-pyridyl); HPLC, 1.57 x retention of thienamycin.

Example 15

Following the procedure in Example 12, but replacing the reagent with methyl-4-thiazolecarboximidate, gives: N-[(4-thiazolylimino)-methyl]-thienamycin (99 mg, 89%): UV X H2? 300 nm, (e 7530); IR (nujol porridge) 1764 cm"1 (B- max

lactam); NMR (60 MHz, D 2 O), cf 1.23, 3H, (d, J=7Hz, CH_3'CH(OH)); cf 8.60, 9.17, 2H (d, d, J=2Hz, 4-thiazolyl); HPLC, 1.8 x retention time of thienamycin.

Example JL6

Preparation of N'-(2-methylthioethyl)-N-formimidoyl-thienamycin

105 mg of thienamycin is dissolved in 5 ml of pH 7 0.1N phosphate buffer and to this is added a solution of 300 μl of ethyl-N-2-methylthioethyl-formimidate in 2 ml of tetrahydrofuran. The pH of the solution is adjusted to and maintained at 8.5 using an automatic burette which emits 1N sodium hydroxide. After 30

minutes, the pH is adjusted to 7.0 with 2.5N hydrochloric acid. The solution is chromatographed on a column with Dowex 50-X4® resin (53 ml, Na form, 200 - 400 mesh) washed with an ice water jacket, eluting with deionized water. The N'-[2-methylthioethyl]-N-formimidoyl derivative is eluted in 2 - 4 column volumes and lyophilized, whereby a white solid is obtained.

u.v. (pH 7 0.1N phosphate buffer) Xmax 298 (e 7760)

i.r. (Nujol porridge) 1760 cm<-1> (B-lactam).

Example 17

Preparation of N'-t-butyl-N-formimidoyl-thienamycin

105 mg thienamycin is dissolved in 5 ml pH 7 0.1N phosphate buffer and to this is added a solution of 290 mg ethyl-N-t-butyl-formimidate in 1 ml tetrahydrofuran. The pH of the solution is adjusted to and maintained at 8.5 using an automatic burette that dispenses 1N sodium hydroxide. After 30 minutes, the pH is adjusted to 7.0 with 2.5N hydrochloric acid. The solution is chromatographed on a column of Dowex 50 resin (53 ml, Na+ form, 200 - 400 mesh) provided with an ice water jacket, and eluted with deionized water. The fractions containing the title product are combined and lyophilized.

Example 18

Preparation of N1 -[l-Methyl-2-propenyl]-N-formimidoyl-thienamycin

126 mg of thienamycin is dissolved in 6 ml of pH 7 0.1N phosphate buffer and the pH of the solution is adjusted to 8.5 using an automatic burette that emits 1N sodium hydroxide. To this stirred solution, 300 l of ethyl-N-1-methyl-2-propenyl-formimidate hydrochloride are added while the pH is kept at 8.5. After 30 minutes, the pH of the solution is adjusted to 7.0 with 2.5N hydrochloric acid and the solution is chromatographed on a column of Dowex 50-X4® resin (49 ml, Na<+> form, 200 - 400 mesh) equipped with an ice-water jacket, and eluted with deionized water. The N'-[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. 1N phosphate buffer<r>) X 1113. K, S 29-.9 nm (e = 7820)

1. r. (Nujol porridge) 1760 cm" (3-lactam).

Example 19

Preparation of N'-dimethylamino-N-formimidoyl-thienamycin

115 mg of thienamycin is dissolved in 7 ml of pH 7 0.1N phosphate buffer and the pH of the solution is adjusted to 8.5 with an automatic burette that emits IN sodium hydroxide. 284 mg of methyl-N-dimethylamino-formimidate hydrochloride is added to this stirred solution while maintaining the pH at 8.5. After 20 minutes, the pH is adjusted to 7.0 using 2.5N hydrochloric acid and the solution

chromatographed on Dowex 50-X4® resin (53 ml, Na<+> form,

200 - 400 mesh) eluted with deionized water. The chromatography is carried out in a column with a water jacket at 3° C. The N'-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) X 298 nm (e 6 910) ir (Nujol porridge) 1760 cm-1 (B-lactam) nmr (D2O) 6 1.29 (d, J=6 Hz, CH3"CH), 2.59 (s, N (CH3)2), 7,<7>6 (s,

NCH).

Example 20

Preparation of methyl-oxalimidoyl-thienamycin

105 mg of thienamycin is dissolved in 5 ml of pH 7 0.1 N phosphate buffer and the pH of the solution is adjusted to 8.5 with an automatic burette that emits 1 N sodium hydroxide. 200 µl of methyl oxalimidate is added to this solution while the pH is kept at 8.5. After 30 minutes, the pH is now adjusted to 7.0 using 2.5 N

(R)

hydrochloric acid and the solution is chromatographed on Dowex 50-X4 har<p>iks (53 ml, Na<+> form, 200 - 400 mesh) eluted with deionized water. The chromatography is carried out in a column with a water jacket at 3° C. The methyl oxalimidoyl derivative is eluted in 2 column volumes and lyophilized to a white solid (44 mg) uv (pH 7 0.1 N phosphate buffer) Xmax 298 nm (e 6 230) ir (Nujol porridge) 1760 cm"<1> (3-lactam); nmr (D 2 O) 51.27 (d, J=6 Hz, CH 3 -CH), 3.87 (s, -OHCH 3 ).

Example 21

Preparation of N-propionimidoyl-thienamycin

114 mg of thienamycin is dissolved in 10 ml of pH 7 0.1N phosphate buffer and the pH of the solution is adjusted to 8.5 with an automatic burette that emits 1 N sodium hydroxide. 231 mg of solid ethyl propionimidate hydrochloride are added in portions as quickly as possible, keeping the pH close to 8.5. After 30 minutes, the pH is adjusted to 7.0 with 2.5 N hydrochloric acid, and the solution is chromatographed on Dowex 50-X4<®> resin (72 ml, Na<+> form, 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) Xmax 298 nm (e 7 830) nmr (D20) 61.28 (d, j =6 Hz, CH3 CH(OH)), 1.23 (t, J= 8 Hz, -CH2"<CH>3), 2.50 (q, J= 8 Hz, CH2CH3).

Example 2 2

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

140 mg of thienamycin is dissolved in 10 ml of pH 7 0.1N phosphate buffer and the pH of the solution is set to 8.5 with an automatic burette that emits 1 N sodium hydroxide. To this solution, 200 µl of methyl-N-methyl-formimidate hydrochloride is added while the pH is kept at 8.5. After 40 minutes, the pH is adjusted to 7.0 with 2.5

'" " (s)

N hydrochloric acid, and the solution is chromatographed on Dowex 50-X4^ resin (72 ml, Na+ form, 200 - 400 mesh) eluted with deionized water. The N'-methyl-N-formimidoyl derivative is eluted in 2 column volumes and lyophilized to a white solid (43 mg). uv (pli 7 0.1 N phosphate buffer) Xmax 298 nm (e 7 250) ir (Nujol porridge) 1765 cm<-1> (B-lactam). no. (D2O) & 1.29 (d, J=Hz, CH3~CH), 2.92 (s, N-CH3) 7._80 (s, N-CH).

Example 2 3

Preparation of N'-benzyl-N-formimidoyl-thienamycin

110 mg of thienamycin is dissolved in 7 ml of pH 7 0.1 N phosphate buffer and the pH of the solution is adjusted to 8.5 with an automatic burette that emits 1 N sodium hydroxide. A solution of 572 mg of ethyl-N-benzyl-formimidate-fluoroborate in 2 ml of p<->dioxane is added to the buffered solution while maintaining the pH at 8.5. After 20

minutes, the dH of the solution is adjusted to 7.0 with 2.5 N hydrochloric acid and chromatographed on Dowex 50-X4® resin (53 ml, Na<+> form, 200 - 400 mesh) eluted with deionized water. The chromatography is carried out in a column with a water 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 N phosphate buffer) Xmax 295 nm (e 3,980) ir (Nujol porridge) 1765 cm"<1> (B-lactam) nmr (D20) 51.29 (d, J= 6 Hz, CH3CH ), 4.44 (s, CH2~Ar), 7.37 (s, aryl), 8.14 (s, NCH).

Example 24

Preparation of N'-isopropyl-N-formimidoyl-thienamycin

110 mg of thienamycin is dissolved in 7 ml of pH 7 0.1N phosphate buffer and the solution's pH is set to 8.5 with an automatic burette that emits 1N sodium hydroxide. A solution of 300 mg of methyl-N-isopropyl-formimidate hydrochloride in 1 ml of p-dioxane to-

is added to the magnetically stirred buffered solution while the pH

is kept at 8.5. After 25 minutes, the pH of the solution is adjusted

of 7.0 with 2.5N hydrochloric acid and chromatographed on Dowex 50-X4®

resin (53 ml, Na<+> form, 200 - 400 mesh) eluted with avionic

hard water. The chromatography is carried out in a column with a water jacket at 3° C. The N<1->isopropyl-N-formimidoyl derivative is eluted in 2 column volumes and lyophilized to a white solid (12 mg).

UV (pH 7 0.1N phosphate buffer) Xmax 299 nm (e 8 130) ir (Nujol

porridge) 1760 cm"<1> (3-lactam) nmr (D2O) 6 1.26 (d, J=6Hz, CH3CH(OH)),

1.29 (d, J=6Hz, CH(CH3)3), 7.89 (s, NHCH), 7.96 (s, NHCH).

Example 25

Preparation of N(N'-allyl-formimidoyl)-thienamycin

To a pre-cooled sample of 123 mg (0.452 mmol) thienamycin, 13 ml of cold 0.1N phosphate buffer is added. The solution is adjusted to pH 9 with IN sodium hydroxide. To this basic solution at 2° C., 0.3 g of ethyl N-allyl formimidate hydrochloride is added at once. The pH drops to 7.3 and is brought back to 8.5 with additional sodium hydroxide. The reaction mixture is stirred at 2° C. for a further 30 minutes and the pH is adjusted to 7 with cold 0.1N sulfuric acid. The reaction mixture is analyzed using high pressure liquid chromatography on a C-^g-"Porosil" column, developed with 10% aqueous tetrahydrofuran and found to exhibit only trace amounts of thienamycin (retention time 5 min) and essentially pure product (retention time 10.5 minutes). The reaction mixture is chromatographed on a Dowex-50x4^ column (60 ml, Na<+> form, 200 - 400 mesh) eluting with water at a flow rate of 0.5 ml/min.cm 2 resin layer. After discarding the first 400 ml of eluate, the next 150 ml are lyophilized to obtain the product. Yield 96 mg (63%). UV Amax 301 nm, 24.6 optical density units/mg. (NE^OH quenched) 90% purity. IR Nujol shows C=0 at 5.67 y and 5.90 y. NMR 100 MHz D2O shows that it is a 1:1 mixture of syn- and anti-N(N'-allylformimidoyl)-thienamycin.

Example 26

Preparation of N-(N'-trifluoroethyl-formimidoyl)-thienamycin

To 123 mg (0.452 mmol) of a pre-cooled sample of thienamycin, 15 ml of cold 0.1N phosphate buffer is added. The solution is adjusted to pH 9 with 1N sodium hydroxide. To the alkaline solution at 0 - 2° C, 0.2 ml of ethyl N-trifluoroethyl formimidate in 2 ml of dioxane is added in portions over the course of 30 minutes. The pH of the reaction mixture is kept 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 brought to 7 with cold 0.1 N sulfuric acid, HPLC, C1g-"Porosil" reverse phase using 10% aqueous tetrahydrofuran shows a new peak at 12.2 nm attributed to the desired product. The mixture chromato-(R)

is carried out on a Dowex 50x4^ column (60 ml, 200 - 400 mesh). The column is eluted with water at a flow rate of 0.5 ml/

2

min.cm resin layer. The process is discarded and fractions containing the product are combined and lyophilized, whereby a hygroscopic solid is obtained, 10.2 mg, Xmax 302 nm.

Example 27

Preparation of N-(N'-carboxymethyl-formimidoyl)-thienamycin sodium salt

130 mg of thienamycin is dissolved in 4 ml of nH 7 0.1N phosphate buffer and 5QQ mg of solid sodium ethyl-N-carboxymethyl-formimidate is added at once. The pH of the solution is adjusted to 8.5 by an automatic burette that emits 1N sodium hydroxide. After 25 minutes at pH 8.5, the solution is adjusted to pH 7.0 with 2.5N hydrochloric acid. The solution is then chromatographed on a column of

Dowex 50-X4<®>resin (51 ml, Na<+> form, 200 - 400 mesh) provided with an ice-water jacket, and eluted with deionized water. The eluate of the first column volume was combined and evaporated to 7 ml. This solution was then chromatographed on a column of "XAD-2" resin (53 mL) with an ice-water jacket, eluting with deionized water. The second, third and fourth column volumes were collected and combined and lyophilized to give sodium N-(N'-carboxy-methylformimidoyl)-thienamycin (25 mg). UV (pH 7 0, IN phosphate buffer) X max 300 nm (e 6 390) ir (Nujol porridge) 1755 cm"<1> (B-lactam) nmr (D20) 6 1.29 (d, J=6 Hz, CH 3 CH ), 7.85 (s, NCH).

Example 28

h

Preparation of N-(3-azidopropionimidoyl)-thienamycin

To a solution of 133 mg of thienamycin in 10 ml of 0.1 M pH 7.0 phosphate buffer, 1.2 g of 0-ethyl-3-azidopropionimidate hydrochloride is added while the solution is kept at pH 8.5 with 2.5 N sodium hydroxide. The mixture is stirred at 0° C for 0.5 hour, then neutralized with 2.5N hydrochloric acid to pH 7.0, evaporated to 5 ml and chromatographed on a Dowex 50W x 8w(r) column (the Na form)

38 mm x 30.5 cm) which is eluted with water, thereby obtaining 30 mg of

HO

the desired product. The product shows UV X 2,300 nm; high-performance liquid chromatography (HPLC) retention time of 10 min compared to that of 4.8 min for the starting material under the same conditions (3.18 mm x 61 cm, "Bondapak C^g" reverse phase column eluted with 10% THF in water at a flow rate of 1.5 mL/min); electrophoretic mobility 5 mm towards the cathode at 50V/cm for 20 minutes in 0.05M pH 7.0 phosphate buffer.

Example 29

Preparation of N-(3-aminopropionimidoyl)-thienamycin

43 mg of N-(3-azidopropionimidosyl)-thienamycin (I) in 40 ml of water is hydrogenated under 1 atm of hydrogen in the presence of 0.1 g of 10% palladium-on-charcoal for 30 minutes. Electrophoresis of the resulting mixture shows a new bioactive product moving 30 mm towards the cathode (50 V/cm for 20 minutes in 0.05 M pH 7.0 phosphate buffer) in addition to the starting material (I) moving 5 mm towards the cathode. The electrophoretic mobility of the product agrees with that of the expected product (II). The resulting reaction mixture from the hydrogenation reaction is neutralized with 2.5 N hydrochloric acid and the catalyst is filtered off. The filtrate is evaporated to 10 ml and chromatographed on "XAD-2" resin (2.3 cm x 16 cm column). The column is eluted with water to obtain the desired product (II) as the hydrochloride after lyo-

philization (23 mg, N-(3-aminopropionimidoyl)-thienamycin-

HO

hydrochloride). UV A 2, = 301 nm ( = 7080), IR: nujol mull

, max

1765 cm; NMR: 609MHz, D2O & 1.30 ppm, (doublet, 3), 6 2.60 -

3.72 ppm (multiplet, 11) and 6 4.18 ppm (multiplet, 2).

Example 30 Preparation of N-nitroguanyl-thienamycin

131 mg of thienamycin is dissolved in a solution of 10 ml of dimethylsulfoxide, 0.30 ml of tri-n-butylamine and 0.3 g of 2-methyl-1-nitro-2-thiopseudourea. The solution is heated in a water bath at 45° C while a stream of nitrogen is vigorously bubbled through the solution. After 50 minutes, the solution is evaporated under high

vacuum to 1.0 ml and dissolve in 7 ml 0.05N pH 7 phosphate buffer.

The unreacted thiopseudourea is collected and removed by filtration. The solution is then chromatographed on Dowex 50-X4<®> har<p>iks (53 ml,

200 - 400 mesh, the Na+ form) and eluted with water. The N-nitroguanyl derivative is eluted in the first column volume and lyophilized to a solid (23%).

UV (pH 7 0.1N phosphate buffer) A max <2>69 nm (E 11,000)

Electrophoresis (40 V/cm, pH 7 0.IN phosphate buffer, 20 min) 3.0 cm

towards the cathode.

Example 31

Preparation of N-isobutyrimidoyl-thienamycin """"

By following the procedure in example H, but by

replace ethyl acetimidate hydrochloride with isobutyrimidate hydrochloride

chloride and allowing the reaction to proceed at 20° C and pH 8.2, N-isobutyrimidoyl-thienamycin (14%) is obtained.

UV (pH 7 0.1N phosphate buffer) Amax 298 nm (e 8 290)

NMR (D2/S 1.27 (d, J=7Hz, CH(CH3)2, 1.29

(d, J=6HZ, CH3CH(OH), 2.79 (heptet, J=7Hz, CH(CH3)2).

Example 32

Preparation of N'-methyl-N-acetimidoyl-thienamycin

By following the procedure in example 11, but by

replace ethyl acetimidate hydrochloride with methyl-N-methy1-

acetimidate, N-methyl-N<1->acetimidoyl-thienamycin (10%) is obtained.

UV (pH 7 0.1 N phosphate buffer) Xmax 298 nm (e 6 700)

IR (Nujol porridge) 1750 cm"<1> (3-lactam), 1660 cm"1 (C=NCH3)

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

N

M

(S, N-C-CH3), 2.97 (S, NCH3')

Example 3 3

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

By following the procedure in example 11, but by replacing ethyl acetimidate hydrochloride with ethyl-N-methyl-formimidate hydrochloride, N'-methyl-N-formimidoyl-thienamycin (10%) is obtained.

UV (pH 7 0.1N phosphate buffer) A max- 298 nm

NMR (D2O) 6 1.30 (d, J=6Hz, CH_3 CH(OH), 2.92 (S, N-CH3),

N

.7.78, (S, -CH)

Example 34

By following the procedure in Example 11, but by replacing the reagent with an equivalent amount of methyl 1-methoxyacetimidate, there is obtained: N-(methoxyacetimidoyl)-thienamycin (34%);

UV X H2° 198, 301 nm (e 16,180, 8,700);

max ,

IR (Nujol porridge) 1760 cm (3-lactam);

NMR (60 MHz, D2<D) δ 1.28, 3H, (d, J=6Hz, CH3'CH(OH);

63.50, 3H, (S, CH3-O-CH2) 6 4.35, 2H, (S, CH3-O-CH2);

HPLC, 1.50 x the retention time of thienamycin.

E xample 3 5

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

100 mg thienamycin in 10 ml 0.1M pH 7.0 phosphate buffer

adjusted and maintained at pH 8.5 - 9.0 with 2.5N sodium hydroxide.

300 mg of ethyl-N-ethylformimidate is added to the solution

hydrochloride. The mixture is stirred at 23° C. for 20 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 (38 mm x 254

etc.). The column is eluted with water, taking 6.7 ml fractions. Fractions 40 - 90 are combined, evaporated and freeze-dried whereby

you get 15 mg of solid product. Electrophoresis of the product at 50 V/cm for 20 min in 0.1 M, pH 7.0 phosphate buffer shows a single bioactive zone moving 2 mm toward the cathode.

uv A<H>2° 301 nm; nmr (100 MH 2 , D 2 O) ; 6 7.77 (S) and 7.82 (S)

max

(formimidoyl CH).

Example 36

Preparation of N-[N'-cyclopropylformimidoyl]-thienamycin

100 mg of thienamycin in 10 ml of 0.1 M, pH 7.0 phosphate buffer is adjusted and maintained at pH 8.5 - 9.0, while 300 mg of ethyl-N-cyclopropylformimidate hydrochloride is added dropwise to

the solution. The mixture is stirred at 23° C for 40 minutes, neutral

then lysed and chromatographed on a Dowex-50X8 ( r) (Na form) ion exchange column (38 mm x 254 mm). The column is eluted with water,

whereby 6.5 ml fractions are collected. The fractions 43 - 95 for

combined, evaporated and freeze-dried, whereby 54 mg of the solid product is obtained. Electrophoresis of the product shows a single bioactive zone moving 10 mm towards the cathode (50 V/cm,

1 hour in 0.05 M pH 7.0 phosphate buffer).

uv X H2° 301 nm; Nmr (100 MHz, D20): 0.60 - 1.30 ppm

(m, cyclopropyl) and 7.80 ppm (formimidoyl CH).

Example 3 37

By proceeding as described in the previous examples, the following compounds were prepared:

For-

Claims (1)

Analogous method for the preparation of therapeutically active compounds of formula: and p-t-butylbenzyl, 3-methyl-2-buten-l-yl and pivaloxymethyl esters thereof, and pharmaceutically acceptable salts thereof, wherein R is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-6 carbon atoms, cyano , cycloalkyl or cycloalkenyl with 3-6 carbon atoms, alkylthioalkyl, carboxyalkyl or dialkyl-amino with 1-6 carbon atoms, trifluoroethyl or benzyl, and R 2 is hydrogen 12 12 or R . and R is alkyl with 1-6 carbon atoms or R and R together with the nitrogen atom is 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 with 2-6 carbon atoms, or where R is -0R<a> or -SRa in which Ra is methylbenzyl, ethylallyl, 2-thienylmethallyl, p-nitrobenzyl or CH3 -0-CH2, characterized in that a compound with formula: or a suitable carboxyl derivative thereof'. is reacted with an imidoester: or an imido halide: wherein -X°R" is a leaving group and X' is halogen, X is 0 or S, and R 1 , R 2 and R are as above, or reacted with a compound of formula: in which -X"R° is a leaving group in which X" is oxygen or sulphur, 12 and R and R are H or C-^-C8 alkyl, or that the compounds obtained are esterified or converted to salts in a known manner.