Fluorescent carbapenems

Abstract

Chromogenic or fluorescent carbapenems according to formula I, wherein Ar is a mono or disubstituted carbocyclic aromatic group or an optionally mono or disubstituted heterocyclic aromatic group, are useful compounds for the detection of bacterial carbapenemase. ##STR00001##

Claims

1. A method for detection of bacterial carbapenemase, the method comprising: contacting a fluorescent or chromogenic carbapenem of formula I ##STR00009## and/or its salts, wherein R.sup.1 is selected from hydrogen or alkyl and acyl, each with 1 to 6 carbon atoms and wherein Ar is a mono or disubstituted carbocyclic aromatic or an optionally mono or disubstituted heterocyclic aromatic group wherein the carbocyclic aromatic moiety is monocyclic, bicyclic or tricyclic with 6 to 14 carbon atoms and the heterocyclic aromatic moiety is monocyclic, bicyclic or tricyclic and contains 1 to 13 carbon atoms and contains 1 to 5 hetero atoms, which are selected, independently from each other, from oxygen, nitrogen or sulphur and wherein the substituents R.sup.2 and R.sup.3 of the aromatic parts Ar are selected, independently from each other, from hydrogen, amino, hydroxy, oxo, fluoro, chloro, bromo, nitro, cyano, carboxy, carbamoyl, sulphamoyl, amidino, guanidino, sulfo, or alkyl, alkoxy, acyl, acylamino, monoalkylamino, dialkylamino, trialkylammonium, N,N-dialkylcarbamoyl, N-alkylcarbamoyl and alkoxycarbonyl, each with 1 to 6 carbon atoms; wherein at least one of R.sup.2 and R.sup.3 is an electron withdrawing group when Ar is a carbocylic aromatic group; and wherein R.sup.2 and R.sup.3 are not both hydrogen when Ar is a carbocylic aromatic group with a bacterium or a preparation obtained by lysis of at least one bacterial species; and detecting a change in color or fluorescence; wherein the change in color or fluorescence is indicative of the presence of bacterial carbapenemase.

2. The method of claim 1, wherein R.sup.1 is selected from hydrogen or alkyl and acyl, each with 1 to 6 carbon atoms and wherein Ar is a mono or disubstituted carbocyclic aromatic or an optionally mono or disubstituted heterocyclic aromatic group wherein the carbocyclic aromatic group Ar is selected from phenyl, 1-biphenyl-2-yl or 1-biphenyl-4-yl and wherein the heterocyclic aromatic group is selected from 2-pyridyl, 4-pyridyl or 2-pyrimidyl and wherein the substituents R.sup.2 and R.sup.3 of the aromatic parts Ar are selected, independently from each other, from hydrogen, amino, hydroxy, oxo, fluoro, chloro, bromo, nitro, cyano, carboxy, carbamoyl, sulphamoyl, amidino, guanidino, sulfo, or alkyl, alkoxy, acyl, acylamino, monoalkylamino, dialkylamino, trialkylammonium, N,N-dialkylcarbamoyl, N-alkylcarbamoyl and alkoxycarbonyl, each with 1 to 6 carbon atoms.

3. The method of claim 1, wherein R.sup.1 is selected from hydrogen or acetyl and wherein Ar is phenyl or 1-biphenyl-4-yl, R.sup.2 is hydrogen and R.sup.3 is 4-acetyl or 4-acetyl.

4. The method of claim 1, wherein microbial carbapenem resistance or the presence of bacterial carbapenemase is detected by monitoring the disappearance of fluorescence upon -lactam hydrolysis.

5. The method of claim 1, wherein the compound of Formula I, a nutrition medium, and at least one bacterial species, are incorporated in a diluent.

6. The method of claim 1, wherein the compound of Formula I, an ionic or non-ionic detergent, and at least one bacterial species, are incorporated in a diluent.

7. The method of claim 1, wherein the compound of Formula I and a preparation obtained by lysis of at least one bacterial species are incorporated in a diluent.

8. The method of claim 1, wherein the intensity of color or fluorescence of a compound is monitored in a diluent by vision or by photoelectronic devices.

9. The method of claim 1, the method comprising: contacting a fluorescent or chromogenic carbapenem of formula I ##STR00010## and/or its salts, wherein R.sup.1 is selected from hydrogen or alkyl and acyl, each with 1 to 6 carbon atoms and wherein Ar is a mono or disubstituted carbocyclic aromatic or an optionally mono or disubstituted heterocyclic aromatic group wherein the carbocyclic aromatic moiety is monocyclic, bicyclic or tricyclic with 6 to 14 carbon atoms and the heterocyclic aromatic moiety is monocyclic, bicyclic or tricyclic and contains 1 to 13 carbon atoms and contains 1 to 5 hetero atoms, which are selected, independently from each other, from oxygen, nitrogen or sulphur and wherein the substituents R.sup.2 and R.sup.3 of the aromatic parts Ar are selected, independently from each other, from hydrogen, amino, hydroxy, oxo, fluoro, chloro, bromo, nitro, cyano, carboxy, carbamoyl, sulphamoyl, amidino, guanidino, sulfo, or alkyl, alkoxy, acyl, acylamino, monoalkylamino, dialkylamino, trialkylammonium, N,N-dialkylcarbamoyl, N-alkylcarbamoyl and alkoxycarbonyl, each with 1 to 6 carbon atoms; wherein at least one of R.sup.2 and R.sup.3 is an electron withdrawing group when Ar is a carbocylic aromatic group; and wherein R.sup.2 and R.sup.3 are not both hydrogen when Ar is a carbocyclic aromatic group with a known or suspected carbapenemase inhibitor in the presence of (i) at least one carbapenemase producing bacterial species, or (ii) a preparation obtained by lysis of a carbapenemase producing bacterial species; and detecting a change in color or fluorescence; wherein the change in color or fluorescence is indicative of inhibition of carbapenemase by the known or suspected carbapenemase inhibitor.

10. The method of claim 9, wherein R.sup.1 is selected from hydrogen or alkyl and acyl, each with 1 to 6 carbon atoms and wherein Ar is a mono or disubstituted carbocyclic aromatic or an optionally mono or disubstituted heterocyclic aromatic group wherein the carbocyclic aromatic group Ar is selected from phenyl, 1-biphenyl-2-yl or 1-biphenyl-4-yl and wherein the heterocyclic aromatic group is selected from 2-pyridyl, 4-pyridyl or 2-pyrimidyl and wherein the substituents R.sup.2 and R.sup.3 of the aromatic parts Ar are selected, independently from each other, from hydrogen, amino, hydroxy, oxo, fluoro, chloro, bromo, nitro, cyano, carboxy, carbamoyl, sulphamoyl, amidino, guanidino, sulfo, or alkyl, alkoxy, acyl, acylamino, monoalkylamino, dialkylamino, trialkylammonium, N,N-dialkylcarbamoyl, N-alkylcarbamoyl and alkoxycarbonyl, each with 1 to 6 carbon atoms.

11. The method of claim 9, wherein R.sup.1 is selected from hydrogen or acetyl and wherein Ar is phenyl or 1-biphenyl-4-yl, R.sup.2 is hydrogen and R.sup.3 is 4-acetyl or 4-acetyl.

12. The method of claim 9, wherein the known or suspected inhibitor, a compound of Formula I, a nutrition medium, and at least one carbapenemase producing bacterial species are incorporated in a diluent.

13. The method of claim 9, wherein the known or suspected inhibitor, a compound of Formula I, and a preparation obtained by lysis of a carbapenemase producing bacterial species are incorporated in a diluent.

14. The method according to claim 9, wherein the inhibitor is a protease or a metallo protease inhibitor.

Description

EXAMPLE 1

Preparation of potassium-(4S,5R,6S)-3-(4-acetyl-phenyl)-6-((1R)-hydroxy-ethyl)-4-methyl-7-oxo-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylate

(1) ##STR00005##

(4S,5R,6S)-3-(4-acetyl-phenyl)-4-methyl-7-oxo-6-((1R)-triethylsilanyloxy-ethyl)-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylic acid 4-nitro-benzyl-ester

(2) In a 25 ml Schlenk flask fitted with a rubber septum, magnetic stirrer and a balloon filled with dry nitrogen at 78 C. to a solution of (4R,5S,6S)-4-methyl-3,7-dioxo-6-((1R)-triethylsilanyloxy-ethyl)-1-aza-bicyclo[3.2.0]heptane-2-carboxylic acid 4-nitro-benzyl-ester (526 mg, 1.10 mmol) in dry dichloromethane (4.3 ml) was added triethylamine (153 l, 1.10 mmol). After 15 min, trifluoromethanesulfonic anhydride (187 l, 1.10 mmol) was added to the resulting orange-yellow solution. After 30 min at 78 C., Pd(dba).sub.2 (33 mg, 0.057 mmol, 5 mol %), a solution of 4-acetyl-phenyl-boronic acid (158 mg, 0.96 mmol) in tetrahydrofuran (7.2 ml), and K.sub.3PO.sub.4 (712 mg, 3.35 mmol) were added sequentially. The dry-ice/acetone bath was removed and the mixture was allowed to warm to ambient temperature. Upon reaction completion, the wine-red solution was diluted in toluene (200 ml), washed three times with portions (50 ml) of water and once with brine (50 ml) and dried over magnesium sulfate. After filtration, the solvent was removed in a vacuum rotary evaporator, leaving a brownish oil. The crude product was purified by silica gel column chromatography using toluene-ethyl acetate (19:1) yielding a yellow oil (200 mg, 36%). IR-spectrum (ATR): 2956, 2876, 2359, 2344, 1779, 1730, 1684, 1605, 1523, 1457, 1431, 1403, 1376, 1346, 1264, 1228, 1190, 1148, 1108, 1015, 958, 847, 804, 736, 697 cm.sup.1.

(4S,5R,6S)-3-(4-acetyl-phenyl)-4-methyl-7-oxo-6-((1R)-hydroxy-ethyl)-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylic acid 4-nitro-benzyl-ester

(3) In a 50 ml round-bottom flask fitted with a rubber septum and a magnetic stirrer at room temperature to a solution of (4S,5R,6S)-3-(4-acetyl-phenyl)-4-methyl-7-oxo-6-((1R)-triethylsilanyloxy-ethyl)-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylic acid 4-nitro-benzyl-ester (190 mg, 0.328 mmol) in tetrahydrofuran (48 ml) and water (9.5 ml) was added aqueous hydrochloric acid (1.0 M, to pH 2.3). After stirring for 1 h, the yellow solution was diluted in dichloromethane (180 ml), washed once with 10% aqueous NaHCO.sub.3 (60 ml), twice with portions (50 ml) of water and dried over magnesium sulfate. After filtration, the solvent was removed in a vacuum rotary evaporator. A yellow oil (195 mg) was obtained. IR-spectrum (ATR): 3444, 2957, 2875, 1773, 1725, 1682, 1601, 1521, 1456, 1432, 1403, 1378, 1346, 1265, 1195, 1107, 1014, 959, 911, 845, 774, 733, 696 cm.sup.1.

Potassium-(4S,5R,6S)-3-(4-acetyl-phenyl)-6-((1R)-hydroxy-ethyl)-4-methyl-7-oxo-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylate

(4) In a 25 ml Schlenk flask fitted with a magnetic stirrer, hydrogenation burette and a balloon filled with hydrogen at 0 C. to a suspension of palladium on activated carbon (25 mg, 10%) in tetrahydrofuran (1.8 ml) was added a solution of (4S,5R,6S)-4-methyl-3,7-dioxo-6-((1R)-hydroxy-ethyl)-1-aza-bicyclo[3.2.0]heptane-2-carboxylic acid 4-nitro-benzyl-ester (50 mg, 0.11 mmol) in tetrahydrofuran (0.7 ml) and aqueous KHCO.sub.3 (1.1 ml, 0.1 M). The reaction suspension was stirred under hydrogen atmosphere. After 20 min, the catalyst was centrifuged off and the supernatant liquid was decanted off. The black residue was washed once with a mixture (5 ml) of tetrahydrofuran and water (2:1). The combined aqueous layers were washed twice with portions (4 ml) of ethyl acetate and the aqueous layer was concentrated to small volume under vacuum. After sterile filtration and lyophilization at 25 C., a yellow foam (32 mg, 80%) was obtained. IR-spectrum (ATR): 3283, 2926, 2541, 1750, 1677, 1602, 1394, 1265, 1141, 1015, 838, 690 cm.sup.1.

EXAMPLE 2

Preparation of potassium-(4S,5R,6S)-6-[(1R)-hydroxy-ethyl]-4-methyl-7-oxo-3-(4-amino-phenyl)-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylate

(5) ##STR00006##

(4S,5R,6S)-4-methyl-3-(4-nitro-phenyl)-7-oxo-6-((1R)-triethylsilanyloxy-ethyl)-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylic acid 4-nitro-benzyl-ester

(6) In a 25 ml Schlenk flask fitted with a rubber septum, magnetic stirrer and a balloon filled with dry nitrogen at 78 C. to a solution of (4R,5S,6S)-4-methyl-3,7-dioxo-6-((1R)-triethylsilanyloxy-ethyl)-1-aza-bicyclo[3.2.0]heptane-2-carboxylic acid 4-nitro-benzyl-ester (440 mg, 0.92 mmol) in dry dichloromethane (3.6 ml) was added triethylamine (128 l, 0.92 mmol). After 15 min, trifluoromethanesulfonic anhydride (156 l, 0.92 mmol) was added to the resulting orange-yellow solution. After 30 min at 78 C., Pd(dba).sub.2 (28 mg, 0.048 mmol, 5 mol %), a solution of 4-nitro-phenyl-boronic acid (132 mg, 0.80 mmol) in tetrahydrofuran (6.0 ml), and aqueous potassium hydroxide (520 l, 2.28 mmol, 5.4 M) were added sequentially. The dry-ice/acetone bath was removed and the mixture was allowed to warm to ambient temperature. Upon reaction completion, the brown solution was diluted in toluene (200 ml), washed three times with portions (50 ml) of water and once with brine (50 ml) and dried over magnesium sulfate. After filtration, the solvent was removed in a vacuum rotary evaporator, leaving a brownish oil. The crude product was purified by silica gel column chromatography using toluene-ethyl acetate (19:1) yielding a yellow oil (100 mg, 21%). IR-spectrum (ATR): 3079, 2955, 2910, 2875, 1776, 1725, 1601, 1517, 1456, 1413, 1376, 1345, 1290, 1271, 1192, 1145, 1105, 1088, 1052, 1002, 962, 850, 805, 770, 736, 697, 616 cm.sup.1.

(4S,5R,6S)-6-((1R)-Hydroxyethyl)-4-methyl-3-(4-nitro-phenyl)-7-oxo-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylic acid 4-nitro-benzyl-ester

(7) In a 50 ml round-bottom flask fitted with a rubber septum and a magnetic stirrer at room temperature to a solution of (4S,5R,6S)-4-methyl-3-(4-nitro-phenyl)-7-oxo-6-((1R)-triethylsilanyloxy-ethyl)-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylic acid 4-nitro-benzyl-ester (100 mg, 0.17 mmol) in tetrahydrofuran (25 ml) and water (5 ml) was added aqueous hydrochloric acid (1.0 M, to pH 2.3). After stirring for 2 h, the yellow solution was diluted in dichloromethane (180 ml), washed three times with portions (60 ml) of water and dried over magnesium sulfate. After filtration, the solvent was removed in a vacuum rotary evaporator. A yellow oil was obtained, which was purified by silica gel column chromatography using toluene-ethyl acetate (2:1) yielding a yellow oil (60 mg, 75%). IR-spectrum (ATR): 3502, 3112, 3080, 2968, 2930, 2873, 2856, 1769, 1722, 1602, 1600, 1514, 1454, 1379, 1344, 1274, 1192, 1139, 1103, 1037, 1014, 936, 910, 850, 806, 772, 751, 731, 697, 648 cm.sup.1.

Potassium-(4S,5R,6S)-6-[(1R)-hydroxy-ethyl]-4-methyl-7-oxo-3-(4-amino-phenyl)-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylate

(8) In a 25 ml Schlenk flask fitted with a magnetic stirrer, hydrogenation burette and a balloon filled with hydrogen at 0 C. to a suspension of palladium on activated carbon (30 mg, 10%) in tetrahydrofuran (1.8 ml) was added a solution of (4S,5R,6S)-6-((1R)-Hydroxyethyl)-4-methyl-3-(4-nitro-phenyl)-7-oxo-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylic acid 4-nitro-benzyl-ester (30 mg, 0.064 mmol) in tetrahydrofuran (0.7 ml) and aqueous KHCO.sub.3 (0.64 ml, 0.1 M). The reaction suspension was stirred under hydrogen atmosphere. After 10 min, the catalyst was centrifuged off and the supernatant liquid was decanted off. The black residue was washed once with a mixture (5 ml) of tetrahydrofuran and water (2:1). The combined aqueous layers were washed twice with portions (4 ml) of ethyl acetate and the aqueous layer was concentrated to small volume under vacuum. After sterile filtration and lyophilization at 25 C., a yellow foam (9 mg, 42%) was obtained. IR-spectrum (ATR): 3342, 2958, 1733, 1606, 1513, 1393, 1296, 1263, 1218, 1179, 1137, 841 cm.sup.1.

EXAMPLE 3

Preparation of potassium-(4S,5R,6S)-3-(4-acetyl-biphenyl-4-yl)-6-[(1R)-hydroxyethyl]-4-methyl-7-oxo-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylate

(9) ##STR00007##

(4S,5R,6S)-3-(4-Acetyl-biphenyl-4-yl)-4-methyl-7-oxo-6-((1R)-triethylsilanyloxy-ethyl)-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylic acid 4-nitro-benzyl-ester

(10) In a 25 ml Schlenk flask fitted with a rubber septum, magnetic stirrer and a balloon filled with dry nitrogen at 78 C. to a solution of (4R,5S,6S)-4-methyl-3,7-dioxo-6-((1R)-triethylsilanyloxy-ethyl)-1-aza-bicyclo[3.2.0]heptane-2-carboxylic acid 4-nitro-benzyl-ester (110 mg, 0.23 mmol) in dry dichloromethane (0.9 ml) was added triethylamine (32 l, 0.23 mmol). After 15 min, trifluoromethanesulfonic anhydride (39 l, 0.23 mmol) was added to the resulting orange-yellow solution. After 30 min at 78 C., Pd(dba).sub.2 (7 mg, 0.012 mmol, 5 mol %), a solution of 4-acetyl-biphenyl boronic acid (48 mg, 0.20 mmol) in tetrahydrofuran (2.5 ml), and aqueous potassium hydroxide (130 l, 0.7 mmol, 5.4 M) were added sequentially. The dry-ice/acetone bath was removed and the mixture was allowed to warm to ambient temperature. Upon reaction completion, the wine-red solution was diluted in toluene (170 ml), washed three times with portions (40 ml) of water and once with brine (40 ml) and dried over magnesium sulfate. After filtration, the solvent was removed in a vacuum rotary evaporator, leaving a brownish oil. The crude product was purified by silica gel column chromatography using toluene-ethyl acetate (19:1) yielding a yellow oil (23 mg, 18%). IR-spectrum (ATR): 2957, 2876, 1773, 1724, 1681, 1603, 1521, 1496, 1457, 1415, 1376, 1346, 1264, 1228, 1190, 1148, 1053, 1004, 958, 818, 736 cm.sup.1.

(4S,5R,6S)-3-(4-Acetyl-biphenyl-4-yl)-6-((1R)-hydroxy-ethyl)-4-methyl-7-oxo-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylic acid 4-nitro-benzyl-ester

(11) In a 50 ml round-bottom flask fitted with a rubber septum and a magnetic stirrer at room temperature to a solution of (4S,5R,6S)-3-(4-Acetyl-biphenyl-4-yl)-4-methyl-7-oxo-6-((1R)-triethylsilanyloxy-ethyl)-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylic acid 4-nitro-benzyl-ester (20 mg, 0.031 mmol) in tetrahydrofuran (5 ml) and water (1 ml) was added aqueous hydrochloric acid (1.0 M, to pH 2.3). After stirring for 40 min, the yellow solution was diluted in dichloromethane (180 ml), washed once with 10% aqueous NaHCO.sub.3 (60 ml), twice with portions (50 ml) of water and dried over magnesium sulfate. After filtration, the solvent was removed in a vacuum rotary evaporator. A yellow oil was obtained (23 mg). IR-spectrum (ATR): 3438, 2957, 2925, 2855, 1770, 1724, 1679, 1604, 1521, 1496, 1456, 1377, 1346, 1266, 1187, 1140, 1106, 1039, 1016, 1004, 959, 909, 819, 768, 731, 648 cm.sup.1.

Potassium-(4S,5R,6S)-3-(4-acetyl-biphenyl-4-yl)-6-[(1R)-hydroxyethyl]-4-methyl-7-oxo-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylate

(12) In a 25 ml Schlenk flask fitted with a magnetic stirrer, hydrogenation burette and a balloon filled with hydrogen at 0 C. to a suspension of palladium on activated carbon (9 mg, 10%) in tetrahydrofuran (1.8 ml) was added a solution of (4S,5R,6S)-3-(4-Acetyl-biphenyl-4-yl)-6-((1R)-hydroxy-ethyl)-4-methyl-7-oxo-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylic acid 4-nitro-benzyl-ester (17 mg, 0.031 mmol) in tetrahydrofuran (0.7 ml) and aqueous KHCO.sub.3 (0.31 ml, 0.1 M). The reaction suspension was stirred under hydrogen atmosphere. After 10 min, the catalyst was centrifuged off and the supernatant liquid was decanted off. The black residue was washed once with a mixture (5 ml) of tetrahydrofuran and water (2:1). The combined aqueous layers were washed twice with portions (4 ml) of ethyl acetate and the aqueous layer was concentrated to small volume under vacuum. After sterile filtration and lyophilization at 25 C., a yellow foam (4.4 mg, 59%) was obtained. IR-spectrum (ATR): 3364, 2925, 2727, 1743, 1674, 1631, 1602, 1400, 1267, 1112, 1007, 830, 704 cm.sup.1.

EXAMPLE 4

Preparation of potassium-(4S,5R,6S)-3-(4-acetyl-phenyl)-6-[(1R)-acetoxyethyl]-4-methyl-7-oxo-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylate

(13) ##STR00008##

(4S,5R,6S)-3-(4-Acetyl-biphenyl-4-yl)-6-((1R)-acetoxy-ethyl)-4-methyl-7-oxo-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylic acid 4-nitro-benzyl-ester

(14) In a 10 ml Schlenk flask fitted with a rubber septum, magnetic stirrer and a balloon filled with dry nitrogen at 0 C. to a solution of (4S,5R,6S)-4-methyl-3,7-dioxo-6-((1R)-hydroxy-ethyl)-1-aza-bicyclo[3.2.0]heptane-2-carboxylic acid 4-nitro-benzyl-ester (10 mg, 0.022 mmol) in dry dichloromethane (2.0 ml) was added a solution of dimethyl-pyridin-4-yl-amine (2.7 mg, 0.022 mmol) in dry dichloromethane (0.5 ml). Acetic acid chloride (1.6 l, 0.022 mmol) was added. After 1 h at 0 C., the opaque solution was diluted in toluene (20 ml), washed once with saturated aqueous NaHCO.sub.3 (10 ml), once with water (10 ml), once with brine (10 ml) and dried over magnesium sulfate. After filtration, the solvent was removed in a vacuum rotary evaporator. A yellow oil was obtained (10 mg). IR-spectrum (ATR): 3420, 2960, 2927, 2874, 1777, 1728, 1682, 1646, 1601, 1562, 1521, 1455, 1374, 1346, 1264, 1237, 1192, 1107, 1073, 1015, 957, 912, 847, 807, 775, 734, 696 cm.sup.1.

(15) Potassium-(4S,5R,6S)-3-(4-acetyl-phenyl)-6-[(1R)-acetoxyethyl]-4-methyl-7-oxo-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylate was prepared according to example 1.

EXAMPLE 5

Detection of Klebsiella pneumoniae carbapenemases (KPC) by fluorogenic potassium-(4S,5R,6S)-3-(4-acetyl-phenyl)-6-((1R)-hydroxy-ethyl)-4-methyl-7-oxo-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylate after physical disruption by sonication

(16) Bacterial strain Klebsiella pneumoniae (producing KPC-2, Heidelberg) was grown over night at 37 C. (220 rpm) in Mueller-Hinton-Broth (10 ml) using a 45-ml-Falcon tube, harvested by centrifugation at 13.000 rpm (2 min, 1 ml for analytical studies, McFarland 10), washed once with PBS (1 ml), and resuspended in PBS (500 l). Subsequently the cells were lysed by sonication with a Bandelin Sonopuls (515 s), pulsed at 80% max. power. Further PBS (500 l) and fluorogenic carbapenem (100 g) were added. After storage at room temperature for 5 min, the disappearance of the green-yellow fluorescence colour (UV light =366 nm) could be observed, indicating the production of carbapenemases by Klebsiella pneumoniae.

EXAMPLE 6

Detection of Klebsiella pneumoniae carbapenemases (KPC) by fluorogenic potassium-(4S,5R,6S)-3-(4-acetyl-phenyl)-6-((1R)-hydroxy-ethyl)-4-methyl-7-oxo-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylate during physical disruption by sonication with glass beads

(17) Bacterial strain Klebsiella pneumoniae (producing KPC-2, Heidelberg) was grown over night at 37 C. in Mueller-Hinton-Broth (10 ml). To a sample (1 ml, McFarland 0.5) were added fluorogenic carbapenem (100 g) and glass beads (100 mg, Sigma-Aldrich, G9018, 150-212 m, unwashed) using a 5-ml-glass-tube with a plane bottom. The cells were lysed by sonication with a commercial available ultrasound bath. After treatment for 45 min, the disappearance of the green-yellow fluorescence colour (=366 nm) could be observed, indicating the production of carbapenemases by Klebsiella pneumoniae.

EXAMPLE 7

Detection of inducible (Oxa-type) carbapenemases of Escherichia coli by fluorogenic potassium-(4S,5R,6S)-3-(4-acetyl-phenyl)-6-((1R)-hydroxy-ethyl)-4-methyl-7-oxo-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylate after physical disruption by sonication

(18) Bacterial strain Escherichia cog (producing Oxa-48, Heidelberg) was grown over night at 37 C. (220 rpm) in Mueller-Hinton-Broth (10 ml) using a 45-ml-Falcon tube. A sample (1 ml, McFarland 10) was diluted in Mueller-Hinton-Broth (10 ml) and incubated with Imipenem (2 g/ml) for 4 h using a 45-ml-Falcon tube. After centrifugation at 13.000 rpm (2 min, 1 ml for analytical studies), the sample was washed once with PBS (1 ml) and resuspended in PBS (500 l). Subsequently the cells were lysed by sonication with a Bandelin Sonopuls (515 s), pulsed at 80% max. power. Further PBS (500 l) and fluorogenic carbapenem (100 g) were added. After storage at room temperature for 5 min, the disappearance of the green-yellow fluorescence colour (UV light =366 nm) could be observed, indicating the production of Oxa-48-carbapenemases by Escherichia coli. The corresponding non-induced sample did not show any change in fluorescence colour within 30 minutes.

EXAMPLE 8

Detection of metallo-carbapenemases by fluorogenic potassium-(4S,5R,6S)-3-(4-acetyl-phenyl)-6-((1R)-hydroxy-ethyl)-4-methyl-7-oxo-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylate in PBS during incubation

(19) Bacterial strains Klebsiella pneumoniae (VIM-1, Heidelberg) and Pseudomonas aeruginosa (IMP, Heidelberg) were grown over night at 37 C. in Mueller-Hinton-Broth (10 ml). To each PBS (1 ml) of fluorogenic carbapenem (100 g) were added five drops of corresponding inoculum (McFarland 0.5). After further incubation over night at 37 C., the disappearance of the green-yellow fluorescence colour (UV light =366 nm) could be observed, indicating the production of carbapenemases.

EXAMPLE 9

Detection of carbapenemases by fluorogenic potassium-(4S,5R,6S)-3-(4-acetyl-phenyl)-6-((1R)-hydroxy-ethyl)-4-methyl-7-oxo-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylate during incubation

(20) Bacterial strains Escherichia coli (ESBL-1, Heidelberg), Escherichia coli (ESBL-2, Heidelberg), Escherichia coli (Oxa-48), Klebsiella pneumoniae (KPC-2, Heidelberg) Klebsiella pneumoniae (VIM-1, Heidelberg), Pseudomonas aeruginosa (GES-2, Heidelberg), Pseudomonas aeruginosa (IMP, Heidelberg) and Pseudomonas aeruginosa (VIM-2, Heidelberg) were grown over night at 37 C. in Mueller-Hinton-Broth (10 ml). To each sample (1 ml, McFarland 0.5) was added fluorogenic carbapenem (100 g). After further incubation over night at 37 C., the disappearance of the green-yellow fluorescence colour (UV light =366 nm) could be observed for each carbapenemase-producing strain. The corresponding ESBL-producers did not show any change in colour.

EXAMPLE 10

Detection of carbapenemases by fluorogenic potassium-(4S,5R,6S)-3-(4-acetyl-phenyl)-6-((1R)-hydroxy-ethyl)-4-methyl-7-oxo-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylate during optimized incubation

(21) Bacterial strains Escherichia coli (ESBL-1, Heidelberg), Escherichia coli (ESBL-2, Heidelberg), Escherichia coli (Oxa-48), Klebsiella pneumoniae (KPC-2, Heidelberg) Klebsiella pneumoniae (VIM-1, Heidelberg), Pseudomonas aeruginosa (GES-2, Heidelberg), Pseudomonas aeruginosa (IMP, Heidelberg), Pseudomonas aeruginosa (VIM-2, Heidelberg) were incubated for 6 h at 37 C. (220 rpm) in Mueller-Hinton-Broth (10 ml) using 45-ml-Falcon-tubes. To each sample (1 ml) was added fluorogenic carbapenem (100 g) using 15-ml-Falcon-tubes. After further incubation over night at 37 C., the disappearance of the green-yellow fluorescence colour (=366 nm) could be observed for each carbapenemase-producing strain. The corresponding ESBL-producers did not show any change in fluorescence colour.

EXAMPLE 11

Detection of carbapenemases by fluorogenic potassium-(4S,5R,6S)-3-(4-acetyl-phenyl)-6-((1R)-hydroxy-ethyl)-4-methyl-7-oxo-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylate during chemical lysis

(22) Bacterial strains Pseudomonas aeruginosa DSM 1117 (no resistance), Pseudomonas aeruginosa (VIM-2, Heidelberg) and Klebsiella pneumoniae (KPC-2, Heidelberg) were incubated over night at 37 C. on Mueller-Hinton-Agar plates. A sample of each strain was suspended (McFarland 10) in primary-amine-free buffer solution (1 ml) of a detergent from Merck (Germany). Fluorogenic carbapenem (100 g) was added. After further incubation for 6 h at 37 C., the disappearance of the green-yellow fluorescence colour (=366 nm) could be observed for both carbapenemase-producing strains. The corresponding sample of Pseudomonas aeruginosa DSM 1117 did not show any change in fluorescence colour.