N-PHENYL-3-MERCAPTOPROPANAMIDE DERIVATIVES AS METALLO-BETA-LACTAMASE INHIBITORS FOR THE TREATMENT OF BACTERIAL INFECTIONS

Abstract

The present invention related to novel inhibitors of metallo-β3-lactamases of formula (I)

##STR00001##

wherein R.sup.1 is an optionally substituted aryl group of an optionally substituted heteroaryl group, and the use thereof in the treatment of bacterial infections, especially in combination with β-lactam antibiotics.

Claims

1. A compound of formula (I): ##STR00010## wherein R.sup.1 is an optionally substituted aryl group or an optionally substituted heteroaryl group, or a pharmaceutically acceptable salt thereof.

2. A compound according to claim 1, wherein R.sup.1 is an optionally substituted phenyl group, an optionally substituted naphthyl group or an optionally substituted heteroaryl group containing from 5 to 10 ring atoms selected from C, O, N and S.

3. A compound according to claim 1, wherein R.sup.1 is an optionally substituted phenyl group or an optionally substituted heteroaryl group containing 5 or 6 ring atoms selected from C, O, N and S.

4. A compound according to claim 1, wherein R.sup.1 is an optionally substituted phenyl group.

5. A compound according to claim 1, wherein group R.sup.1 is substituted by one, two or three substituents.

6. A compound according to claim 1, wherein group R.sup.1 is substituted by one or two substituents.

7. A compound according to claim 1, wherein the substituents are independently selected from fluorine, chlorine, bromine and iodine and OH, SH, NH.sub.2, —SO.sub.3H, —SO.sub.2NH.sub.2, —COOH, —COOMe, —COMe, —NHSO.sub.2Me, —SO.sub.2NMe.sub.2, —CH.sub.2NH.sub.2, NHAc, —SO.sub.2-N(CH.sub.2CH.sub.2).sub.2O, —SO.sub.2Me, —SO.sub.2NCNH.sub.2NH.sub.2, —CH.sub.2N(CH.sub.2CH.sub.3).sub.2, —SO.sub.2NHCONH.sub.2, SO.sub.2NHC(NH)NH.sub.2, —CH.sub.2N(CH.sub.2CH.sub.2).sub.2NCH.sub.3, —CONH.sub.2, —CN, —NHCONH.sub.2, N.sub.3 and NO.sub.2 groups and C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.10 alkenyl, C.sub.2-C.sub.10 alkynyl, heteroalkyl, C.sub.3-C.sub.18 cycloalkyl, C.sub.2-C.sub.17 heterocycloalkyl, C.sub.4-C.sub.20 alkylcycloalkyl, C.sub.2-C.sub.19 heteroalkylcycloalkyl, C.sub.6-C.sub.18 aryl, C.sub.1-C.sub.17 heteroaryl, C.sub.7-C.sub.20 aralkyl and C.sub.2-C.sub.19 heteroaralkyl groups.

8. A compound according to claim 1, wherein the substituents are independently selected from fluorine, chlorine, bromine and iodine and OH, SH, NH.sub.2, —SO.sub.3H, —SO.sub.2NH.sub.2, —COOH, —COOMe, —COMe, —NHSO.sub.2Me, —SO.sub.2NMe.sub.2, —CH.sub.2NH.sub.2, NHAc, —SO.sub.2-N(CH.sub.2CH.sub.2).sub.2O, —SO.sub.2Me, —SO.sub.2NCNH.sub.2NH.sub.2, —CH.sub.2N(CH.sub.2CH.sub.3).sub.2, —SO.sub.2NHCONH.sub.2, SO.sub.2NHC(NH)NH.sub.2, —CH.sub.2N(CH.sub.2CH.sub.2).sub.2NCH.sub.3, —CONH.sub.2, —CN, —NHCONH.sub.2, N.sub.3 and NO.sub.2 groups and C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.1-C.sub.6 heteroalkyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.2-C.sub.9 heterocycloalkyl, C.sub.7-C.sub.12 alkylcycloalkyl, C.sub.2-C.sub.11 heteroalkylcycloalkyl, C.sub.6-C.sub.10 aryl, C.sub.1-C.sub.9 heteroaryl, C.sub.7-C.sub.12 aralkyl and C.sub.2—Cii heteroaralkyl groups.

9. A compound according to claim 1, wherein the substituents are independently selected from fluorine, chlorine, bromine and iodine and groups of formula —OH, —O—C.sub.1-6 alkyl, —NH.sub.2, —NHC.sub.1-6 alkyl, —N(C.sub.1-6 alkyl).sub.2, —COOH, —COOMe, —COMe, —NHSO.sub.2Me, —SO.sub.2NMe.sub.2, —SO.sub.2NCNH.sub.2NH.sub.2, —CH.sub.2N(CH.sub.2CH.sub.3).sub.2, —SO.sub.2NHCONH.sub.2, SO.sub.2NHC(NH)NH.sub.2, —CH.sub.2N(CH.sub.2CH.sub.2).sub.2NCH.sub.3, —SO.sub.3H, —SO.sub.2NH.sub.2, —CONH.sub.2, —CH.sub.2NH.sub.2, —CN, —C.sub.1-6 alkyl, —SH, —S—C.sub.1-6 alkyl, NHAc, —SO.sub.2—N(CH.sub.2CH.sub.2).sub.2O, —NO.sub.2, —NHCONH.sub.2, —SO.sub.2Me and cyclopropyl.

10. A compound according to claim 1, wherein the substituents are independently selected from Cl, —OH, —NH.sub.2, —COOH, —COOMe, —COMe, —NHSO.sub.2Me, —SO.sub.2NMe.sub.2, —CH.sub.2NH.sub.2, —NO.sub.2, —SO.sub.2—N(CH.sub.2CH.sub.2).sub.2O, —SO.sub.3H, —CH.sub.2N(CH.sub.2CH.sub.3).sub.2, —SO.sub.2NHCONH.sub.2, —SO.sub.2NHC(NH)NH.sub.2, —CH.sub.2N(CH.sub.2CH.sub.2).sub.2NCH.sub.3 and —SO.sub.2NCNH.sub.2NH.sub.2.

11. A compound according to claim 1, wherein group R.sup.1 is substituted by a heteroalkyl group containing from 1 to 6 carbon atoms and from 1 to 6 heteroatoms that are independently selected from O, S and N.

12. A compound according to claim 1, wherein group R.sup.1 is substituted by a group of formula —X—R.sup.1a, wherein X is O, S, SO, SO.sub.2, NH, NHSO.sub.2, CH.sub.2 or CO and R.sup.1a is an optionally substituted C.sub.3-8 cycloalkyl group or an optionally substituted heterocycloalkyl group containing from 3 to 8 ring atoms that are independently selected from C, O, S and N; preferably, X is SO.sub.2 or CH.sub.2; further preferably, R.sup.1a is unsubstituted or substituted by a methyl group.

13. A compound according to claim 1, wherein group R.sup.1 is substituted by a group of formula —Y—R.sup.1b, wherein Y is SO.sub.2, SO.sub.2NH or NHSO.sub.2 and R.sup.1b is a hydroxy group, a C.sub.1-6 alkyl group or a C.sub.1-6 heteroalkyl group.

14. A compound selected from the following compounds, or a salt thereof: ##STR00011## wherein R.sup.2 is —COOH, —NHSO.sub.2Me, —OH or —COOMe; R.sup.3 is —OH, —COOH, —NHSO.sub.2Me, —CH.sub.2NH.sub.2, —NO.sub.2, —SO.sub.3H or —CH.sub.2N(CH.sub.2CH.sub.3).sub.2; and R.sup.4 is —OH, —NH.sub.2, —COOH, —COOMe, —COMe, —SO.sub.2NMe.sub.2, —SO.sub.2—N(CH.sub.2CH.sub.2).sub.2O, —SO.sub.2NHCONH.sub.2, —SO.sub.2NHC(NH)NH.sub.2 or —CH.sub.2N(CH.sub.2CH.sub.2).sub.2NCH.sub.3.

15. Pharmaceutical composition comprising a compound according to claim 1 and optionally one or more carrier substances and/or one or more adjuvants.

16. Pharmaceutical composition according to claim 15, further comprising a β-lactam antibiotic.

17. Compound according to claim 1, or pharmaceutical composition according to claim 15 for use as a medicament.

18. Compound according to claim 1 or pharmaceutical composition according to claim 15 for use in the treatment of bacterial infections.

19. Compound or pharmaceutical composition for use according to claim 18, wherein a β-lactam antibiotic is co-administered with the compound of formula (I).

20. Use of a compound according to claim 1 or a pharmaceutical composition according to claim 15 for the preparation of a medicament for use in the treatment of bacterial infections.

21. A method for treating a bacterial infection which comprises administering to a subject in need of such treatment a therapeutically effective amount of a compound according to claim 1, or a pharmaceutically acceptable salt thereof, in combination with a β-lactam antibiotic.

22. A method for treating a bacterial infection which comprises administering to a subject in need of such treatment a pharmaceutical composition according to claim 15, and a β-lactam antibiotic.

23. A method for treating a bacterial infection which comprises administering to a subject in need of such treatment a pharmaceutical composition according to claim 16.

Description

EXAMPLES

[0071] General. All reagents were used from commercial suppliers without further purification. Procedures were not optimized regarding yield. NMR spectra were recorded on a Bruker AV 500 (500 MHz) spectrometer. Chemical shifts are given in parts per million (ppm) and referenced against the residual proton, .sup.1H, or carbon, .sup.13C, resonances of the >99% deuterated solvents as internal reference. Coupling constants (J) are given in Hertz. Data are reported as follows: chemical shift, multiplicity (s=singlet, d=doublet, t=triplet, m=multiplet, dd=doublet of doublets, br=broad and combinations of these), coupling constants, and integration. Liquid chromatography-mass spectrometry was performed on a LC-MS system, consisting of a Dionex UltiMate 3000 pump, autosampler, column compartment and detector (Thermo Fisher Scientific, Dreieich, Germany) and ESI quadrupole MS (MSQ Plus or ISQ EC, Thermo Fisher Scientific, Dreieich, Germany). High-resolution mass was determined by LC-MS/MS using Thermo Scientific Q Exactive Focus Orbitrap LC-MS/MS system. Purity of compounds synthesized by us was determined by LC-MS using the area percentage method on the UV trace recorded at a wavelength of 254 nm and found to be >95%. For purification of compounds further referred to as “purified by flash chromatorgaphy”, the crude product was adsorbed on silica (Macherey-Nagel, 60 M, 0.04-0.063 mm) and was purified using a CombiFlash Rf 150 (Teledyne Isco) System equipped with RediSepRf silica columns. For normal phase flash chromatography, a Redisnap Rf 4 g or 12 g silica column was used with a solvent system of either hexane/EtOAc (gradient 0% to 100%) or DCM/MeOH (gradient 0 to 5%). For reversed phase chromatography, a Redisnap Rf C.sub.18 4 g column was used with a solvent system of H2O+0.1%FA/MeCN+0.1%FA (gradient 0% to 100%).

[0072] Synthesis of N-aryl mercaptopropionamides

[0073] The overall synthetic route for N-aryl mercaptopropionamide derivatives is outlined in Scheme 1. Following route A (a), neat reaction of the corresponding anilines with commercially available 3-mercaptopropionic acid at 120° C. afforded the corresponding compounds. For synthesis of further compounds, route B (b) was followed. For the activation of 3-(acetylthio) mercaptopropionic acid, three different coupling reagents were used. A coupling reaction using HATU in presence of diisopropylethylamine furnished compounds JMK-313 and JMK-320, while activation of 3-(acetylthio) mercaptopropionic acid with ethylchloroformate in presence of trimethylamine gave compounds JMK-292 and JMK-307. Activation with EDC.Math.HCl, followed by reaction with the corresponding aniline afforded the other compounds. Hydrolysis of thioacetate using sodium hydroxide in methanol at room temperature provided the final corn pounds.

##STR00005## ##STR00006##

[0074] a) (Route A)

[0075] i. 3-mercaptopropionic acid, neat, overnight, 120° C.

[0076] iii. 2 M NaOH, MeOH, 2-3 h, r.t. or 2 M NaOH, 1,4-dioxane, 2-3 h, r.t b) (Route B)

[0077] ii. 3-(acetylthio) mercaptopropionic acid, EDC.Math.HCl, DCM, overnight, r.t.; or 3-(acetylthio) mercaptopropionic acid, ClCO.sub.2Et, Et.sub.3N, THF, overnight, 0° C. to r.t.; or 3-(acetylthio) mercaptopropionic acid, HATU, DIEA, DCM, overnight, r.t.

[0078] iii. 2 M NaOH, MeOH, 2-3 h, r.t.

[0079] iv. TFA, DCM, overnight, r.t.

[0080] General Procedure A-1: Synthesis of thioacetate derivatives using EDC.Math.HCl as coupling reagent (Scheme 1, Reaction ii)

[0081] 3-(Acetylthio) propionic acid (1.2-1.5 eq) was placed in a vial and dissolved in DCM. To this solution, EDC-HCl (1.2-1.5 eq) was added and left to form a cloudy solution. Then, the corresponding amine (1.0 eq) was added, and the reaction was stirred at room temperature (r.t.). The reaction was monitored using TLC or LC-MS. The reaction was quenched with 1 M HCl until pH═1. The organic phase was washed with saturated aqueous NaCl solution (1×) and then dried over anh. Na.sub.2SO.sub.4. The product was filtered and concentrated. Purification was done by column chromatography.

[0082] General Procedure A-2: Synthesis of thioacetate derivatives using ethyichloroformate as coupling reagent (Scheme 1, Reaction ii)

[0083] 3-(Acetylthio) propionic acid (1.2 eq) was dissolved in THF and cooled in an ice-bath. Et.sub.3N (1.2 eq) was added, followed by addition of CICO2Et (1.3 eq). After 5 minutes, the ice-bath was removed and the reaction was stirred at r.t. for 30 minutes. The corresponding amine (1.0 eq) was slowly added. The reaction was monitored using TLC or LC-MS. After the reaction was completed, volatiles were evaporated under reduced pressure and the crude product was purified using column chromatography or preparative HPLC (H.sub.2O+0.05%FA/ACN+0.05%FA 95/5.fwdarw.5/95).

[0084] General Procedure A-3: Synthesis of thioacetate derivatives using HATU as coupling reagent (Scheme 1, Reaction ii)

[0085] 3-(Acetylthio) propionic acid (1.5 eq), HATU (1.5 eq) and DIEA (1.5 eq) were dissolved in DCM. The corresponding amine (1.0 eq) was added and the reaction was stirred at r.t. for 2 days. Volatiles were evaporated under reduced pressure and the crude product was purified using preparative HPLC (H.sub.2O+0.05%FA/ACN+0.05%FA 95/5.fwdarw.5/95).

[0086] General Procedure B: Hydrolysis of thioacetate (Scheme 1, Reaction iii) Thioacetate (1.0 eq) was dissolved in methanol or compound HIPS1685 (1.0 eq) was dissolved in 1,4-dioxane under Ar atmosphere and 2 M aq. solution of NaOH (2.0-3.0 eq) or solid NaOH (3.0-4.0 eq) was added. The reaction was stirred 1-2 h at r.t. After quenching with 1 M HCl, the product was extracted 3 times with EtOAc. The combined organic extracts were washed with saturated aqueous NaCl solution, dried over anh. Na.sub.2SO.sub.4 and filtered. The solvent was removed under reduced pressure. Purification was done via flash chromatography. In case of more polar compounds, instead of quenching the reaction with 1 M HCl, pH was adjusted to acidic using Amberlite IR-120. After filtration, Amberlite was washed with MeOH (3x), solvent was evaporated and the product was purified using preparative HPLC (H.sub.20+0.05%FA/ACN+0.05%FA 95/5.fwdarw.5/95).

[0087] General Procedure C: Synthesis of free thiols (Scheme 1, Reaction i)

[0088] Aniline (1.0 eq) was placed in a crimp vial. The vial was evacuated and flushed with Ar followed by addition of 3-mercaptopropionic acid (1.2-1.5 eq). The vial was flushed with Ar again and heated to 120° C. for 5 h. The crude product was purified using reverse phase flash chromatography (H.sub.2O+0.1%FA/ACN+0.1%FA 95/5.fwdarw.5/95).

[0089] General Procedure D: Deprotection of the tert-butyloxycarbonyl group (Scheme 1, Reaction iv)

[0090] To a solution of the corresponding Boc-protected compound (1.0 eq) in DCM, a concentrated solution of TFA (5.0-7.0 eq) was added and the reaction was stirred overnight at r.t. Volatiles were evaporated and the crude product was used in the next step without further purification.

[0091] N-(3,4-Dichlorophenyl)-3-mercaptopropanamide (HIPS1447)

[0092] Compound HIPS1447 was synthesized according to general procedure C, using 3,4-dichloroaniline (150 mg, 1.25 mmol) and 3-mercaptopropionic acid (330 μL, 3.75 mmol). The product was purified by reverse flash chromatography (5% ACN to 100% ACN). Final product was obtained as white solid (120 mg, 51%). .sup.1H NMR (500 MHz, DMSO-d.sub.6) 6 ppm: 10.27 (s, 1H), 8.00 (d, J=2.4 Hz, 1H), 7.56 (d, J=8.8 Hz, 1H), 7.47 (dd, J=8.8, 2.4 Hz, 1H), 2.77-2.71 (m, 2H), 2.66-2.62 (m, 2H), 2.45-2.40 (m, 1H). .sup.13C NMR (126 MHz, DMSO) δ 169.91, 139.14, 130.96, 130.68, 124.53, 120.24, 119.08, 40.33,19.47.HRMS (ESI.sup.− m/z calcd. for C.sub.9H.sub.8Cl.sub.2NOS [M−H].sup.− 247.97091 found

[0093] Synthesis of 3-(3-(acetylthio)propanamido)benzoic acid (HIPS5670). Compound HIPS5670 was synthesized according to general procedure A-1, using 3-aminobenzoic acid (130 mg, 0.95 mmol), 3-(acetylthio) propionic acid (210 mg, 1.42 mmol) and EDC HCl (272 mg, 1.42 mmol) in DCM (10 mL). The product was purified using preparative HPLC. The final product was obtained as white powder (62 mg, 25%) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ ppm: 12.96 (br s, 1H), 8.21 (s, 1H), 7.79 (d, J=6.6 Hz, 1H), 7.61 (d, J=7.9 Hz, 1H), 7.41 (t, J=7.9 Hz, 1H), 3.09 (t, J=6.8 Hz, 2H), 2.65 (t, J=6.8 Hz, 2H), 2.33 (s, 3H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ ppm: 195.50, 169.45, 167.23, 139.23, 131.49, 129.06, 124.06, 123.08, 119.80, 35.30, 30.61, 24.25. HRMS (ESI.sup.+) m/z calcd. for C.sub.12H.sub.14NO.sub.4S [M+H].sup.+268.06380, found 268.06381.

[0094] Synthesis of 3-(3-mercaptopropanamido)benzoic acid (HIPS5694). Compound HIPS5694 was synthesized according to general procedure B, using compound HIPS5670 (62 mg, 0.23 mmol) and NaOH (37 mg, 0.92 mmol) in MeOH (2 mL). The product was purified using preparative HPLC. Final product was obtained as white powder (35 mg, 67%). .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ ppm: 10.15 (s, 1H), 8.23 (t, J=2.0 Hz, 1H), 7.84-7.78 (m, 1H), 7.60 (m, 1H), 7.41 (t, J=7.9 Hz, 1H), 2.75 (m, 2H), 2.63 (t, J=6.7 Hz, 2H), 2.40 (t, J=7.8 Hz, 1H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ ppm: 169.61, 167.22, 139.26, 128.93, 123.96, 123.06, 119.85, 40.29, 19.63. HRMS (ESI.sup.−) m/z calcd. for C.sub.10H.sub.10NO.sub.3S [M−H].sup.− 224.03868, found 224.03814.

[0095] Further compounds shown in Scheme 1:

[0096] The other compounds shown in Scheme 1 have been prepared and characterized according to the procedures described above.

[0097] Biological Assays

[0098] Protein expression and purification. The expression of IMP-7, VIM-1, and NDM-1 was performed according to the protocol previously published by Klingler et al. (Klingler, F. M.; Wichelhaus, T. A.; Frank, D.; Cuesta-Bernal, J.; El-Delik, J.; Müller, H. F.; Sjuts, H.; Gottig, S.; Koenigs, A.; Pos, K. M.; et al. Approved Drugs Containing Thiols as Inhibitors of Metallo-β-Lactamases: Strategy to Combat Multidrug-Resistant Bacteria. J. Med. Chem. 2015, 58 (8), 3626-3630. https://doi.org/10.1021/jm501844d) with slight modifications.

[0099] In vitro inhibition assay. Activity assays for NDM-1, VIM-1, VIM-2 and IMP-7 were carried out, as described by Klingler et al. Final protein concentrations were 0.5 nM (NDM-1), 4.0 nM (VIM-1), and 0.1 nM (IMP-7) in a 50 mM HEPES buffer (pH 7.5, 0.01% Triton X-100) Substrate (Fluorocillin™ (Invitrogen, Darmstadt, Germany) was dissolved in assay buffer to a final concentration of 888 nM. Test compounds were dissolved and pre-diluted in DMSO (final concentration: 1%). In a black polystyrol 96-well plate (Corning) an amount of 1 pL of the respective inhibitor solution at different concentrations was incubated with 89 pL of respective protein containing buffer for 30 minutes at room temperature. 10 pL of substrate solution was added. The readout of the emitted fluorescence was started immediately (45 s for 30 cycles) using a Tecan Infinite F200Pro (Tecan Group Ltd; excitation at 495 nm and emission at 525 nm). Blank controls were performed without enzyme. Positive controls were performed with enzyme but without inhibitor. The inhibitory activity of each test compound was measured in three independent experiments. For calculation of IC.sub.50 values, data obtained from measurements with eight different inhibitor concentrations were used. For the evaluation of the sigmoidal dose response equation (variable slope with four parameters) GraphPad Prism 5 (GraphPad Software, La Jolla, Calif., USA) was used.

[0100] Cytotoxicity assays and Selectivity assays. Cytotoxicity assays on HepG2, HEK293 and A549 cells and selectivity assays on various MMPs were performed as described previously (Haupenthal, J.; Baehr, C.; Zeuzem, S.; Piiper, A. RNAse A-like Enzymes in Serum Inhibit the Anti-Neoplastic Activity of SiRNA Targeting Polo-like Kinase 1. Int. J. Cancer 2007, 121 (1), 206-210. https://doi.org/10.1002/ijc.22665).

[0101] MIC assays. Minimal inhibitory concentrations (MICs) of imipenem and the compounds of the present invention against MBL-positive and MBL-negative clinical isolates and/or against transformed E. coli strains producing different recombinant metallo-β-lactamases were determined according to the microdilution method established by Clinical and Laboratory Standards Institute (CLSI) (Wayne, P. A. Clinical and Laboratory Standards Institute Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically Standard, Approval CDM-A.; Clin. Lab. Stand. Inst. 2018, 91).

[0102] Fractional inhibitory concentration index (FICI). The checkerboard assay was performed to test for synergy in vitro. The microtiter-plates were set up with serial doubling dilutions of the test compounds (16-1024 mg/L) and imipenem (1-1024 mg/L). FICIs≤0.5 were defined as synergistic; FICIs from >0.5 to 51 were defined as additive; FICIs from 1 to ≤4 were defined as indifferent and FICIs>4 were defined as antagonistic (Doern, C. D. When Does 2 plus 2 Equal 5? A Review of Antimicrobial Synergy Testing. J. Clin. Microbial. 2014, 52 (12), 4124-4128. https://doi.org/10.1128/JCM.01121-14).

[0103] Zebrafish Toxicity. Toxicity testing was performed according to the procedure described in the literature (Maes, J.; Verlooy, L.; Buenafe, 0. E.; de Witte, P. A. M.; Esguerra, C. V.; Crawford, A. D. Evaluation of 14 Organic Solvents and Carriers for Screening Applications in Zebrafish Embryos and Larvae. PLoS One 2012, 7 (10), 1-9. https://doi.org/10.1371/journal.pone.0043850) with minor modifications using zebrafish embryos of the AB wild-type line at 1 day post-fertilization. Embryos were collected and kept in a Petri dish at 28° C. until the next day in 0.3x Danieau's medium (17 mM NaCl, 2 mM KCl, 1.8 mM Ca(NO.sub.3).sub.2, 1.5 mM HEPES (pH 7.1-7.3), 0.12 mM MgSO.sub.4, and 1.2 μM methylene blue). The toxicity assay was performed using a 96-well plate with one embryo per well and 10 embryos per condition. To obtain compound concentrations between 30 μM and 150 μM, solutions of the compounds were prepared freshly using 0.3× Danieau's medium with a final DMSO concentration of 1% (v/v). Single zebrafish embryos were placed in wells and directly incubated in the corresponding compound solutions. Monitoring of developmental defects, heart rate, touch-evoked locomotion response, and survival rate was done daily (up to 120 hpf) via microscopy (Table x). All of the described experiments were performed with zebrafish embryos <120 h post-fertilization (hpf) and are not classified as animal experiments according to EU Directive 2010/63/EU. Protocols for husbandry and care of adult animals are in accordance with the German Animal Welfare Act (§11 Abs. One TierSchG).

[0104] Table 1 shows the in vitro activity of the compounds of the present invention against three MBLs (IMP-7, VIM-1 and NDM-1) belonging to class B1 in a kinetic fluorescence-based activity assay.

TABLE-US-00001 TABLE 1 Structures and MBL inhibitory activity of N-aryl mercaptopropionamides..sup.a [00007] IC.sub.50 (μM) Compound R NDM-1 IMP-7 VIM-1 HIPS1686 2-CO.sub.2H 3.4 ± 0.1 4.1 ± 0.3 2.2 ± 0.2 HIPS5654 2-NHSO.sub.2Me 7.0 ± 0.3 n.i. 1.9 ± 0.2 HIPS5661 2-OH 2.2 ± 0.4 3.3 ± 0.4 0.2 ± 0.0 HIPS1685 2-CO.sub.2Me 1.3 ± 0.2 1.0 ± 0.1 n.i. HIPS5640 3-CH.sub.2NH.sub.2 6.5 ± 0.4 5.5 ± 0.7 0.8 ± 0.1 HIPS1687 3-OH 2.7 ± 0.5 1.4 ± 0.2 0.1 ± 0.0 HIPS5662 3-NO.sub.2 2.5 ± 0.2 2.6 ± 0.1 0.1 ± 0.0 HIPS5664 3-CH.sub.2NEt.sub.2 6.5 ± 0.4 5.5 ± 0.7 0.8 ± 0.1 HIPS5672 3-SO.sub.3H 1.3 ± 0.0 3.6 ± 0.3 1.6 ± 0.1 HIPS5585 3-NHSO.sub.2Me 2.2 ± 0.3 1.3 ± 0.0 1.3 ± 0.0 HIPS5694 3-CO.sub.2H 4.0 ± 0.0 6.4 ± 0.6 1.2 ± 0.0 HIPS1447 3,4-di-Cl 1.3 ± 0.0 5.0 ± 0.6 0.1 ± 0.0 HIPS5586 4-CO.sub.2Me 2.8 ± 0.1 1.1 ± 0.7 0.1 ± 0.0 HIPS5188 4-COMe 1.5 ± 0.3 5.5 ± 0.1 0.4 ± 0.1 HIPS5190 4-OH 4.0 ± 1.1 3.4 ± 0.7 0.2 ± 0.0 HIPS5695 4-CO.sub.2H 1.0 ± 0.4 n.i. 0.6 ± 0.0 HIPS5665 4-SO.sub.2NMe.sub.2 2.8 ± 0.1 2.1 ± 0.2 0.8 ± 0.0 HIPS5666 4-NH.sub.2 6.5 ± 0.5 2.1 ± 0.3 0.5 ± 0.0 HIPS5682 4-SO.sub.2NHCONH.sub.2 8.6 ± 0.2 12.6 ± 2.8  2.0 ± 0.1 HIPS5683 4-SO.sub.2NHCNHNH.sub.2 2.1 ± 0.1 3.3 ± 0.4 0.5 ± 0.0 HIPS5659 [00008] 2.5 ± 0.2 7.1 ± 1.4 1.7 ± 0.3 HIPS5696 [00009] 6.4 ± 0.6 5.8 ± 0.4 1.5 ± 0.1 .sup.aMeans and SD of at least two independent experiments.

[0105] In addition to its potency, HIPS1447 is selective over six representative human matrix metalloproteases (MMPs) (Table 2a), and shows no significant toxicity when tested on three different human cell lines (Table 2b).

TABLE-US-00002 TABLE 2a Activity of HIPS1447 against human MMPs, n.i = <10% inhibition. % Inh. @ 100 μM of MMP-1 n.i. HIPS1447 MMP-2 15.8 ± 13.5 MMP-3 n.i. MMP-7 n.i. MMP-8 38.2 ± 24.8 MMP-14 17.9 ± 7.8 

TABLE-US-00003 TABLE 2b Cytotoxicity of HIPS1447 against human cell lines. IC.sub.50 [μM] HepG2 >100 HEK293 >100 A549 >100

[0106] Also other compounds of the present invention showed no cytotoxic effects at a concentration of 100 μM.

[0107] Antibacterial activities and minimum inhibitory concentrations (ICs). The minimum inhibitory concentration (MIC) for several compounds (HIPS1447, HIPS1686, HIPS5682, HIPS5683, HIPS5694, HIPS5695, HIPS540, HIPS5672) in different isolates from E. coli and P. aeruginosa has been evaluated. All compounds showed no intrinsic antibacterial activity.

[0108] Since the utility of carbapenems is threatened by MBLs, imipenem has been chosen as β-lactam antibiotic for combination. Imipenem is a broad-spectrum member of the carbapenem family of β-lactam antibiotics. A series of compounds has been selected to be tested in a synergistic checkerboard assay.

[0109] Compound HIPS1447 was tested in imipenem-resistant P. aeruginosa producing VIM-2 at a single concentration (1024 μg/mL), yielding a 64-fold decreased MIC value.

[0110] Table 3 shows that HIPS1686, bearing no intrinsic antibacterial activity, is able to maintain the effect of imipenem.

TABLE-US-00004 TABLE 3 MIC (μg/mL) of imipenem for E. coli producing NDM-1 in the presence of HIPS 1686. Substance MIC in mg/L Imipenem (IMI) 128 HIPS1686 >1024 IMI + HIPS1686 (constant 1024 μg/ml) 1 IMI + HIPS1686 (constant 256 μg/ml) 1 IMI + HIPS1686 (constant 64 μg/ml) 4

TABLE-US-00005 TABLE 4 Synergistic antimicrobial activity (checkerboard assay) of imipenem with HIPS1686 against E. coli NDM-1 (T2359) HIPS1686 Imipenem MIC * in mg/L − 128 +256 mg/L 2 +128 mg/L 2 +64 mg/L 8 +32 mg/L 16 +16 mg/L 64 +8 mg/L 64 +4 mg/L 64 * The median of three experiments is given.

TABLE-US-00006 TABLE 5 Synergistic antimicrobial activity of imipenem with HIPS1686 against clinical isolates expressing various MBLs. MIC (mg/L) K. pneumoniae E. cloacae S. marcescens E. coli E. cloacae P. aeruginosa E. cloacae Substance NDM-1 (T2301) NDM-1 (T2311) NDM-1 (T2352) NDM-5 (T2351) VIM-1 (T2353) IMP-1 (T2325) GIM-1 (T2218) Imipenem 16 16 256 16 32 64 2 Imipenem 1 1 4 2 4 32 0.5 (+HIPS1686 256 mg/L)

TABLE-US-00007 TABLE 6 Synergistic antimicrobial activity of imipenem with several compounds against E. coli NDM-1 (T2359) Imipenem MIC in mg/L HIPSHIPS HIPS5640 HIPS5672 HIPS5682 HIPS5683 HIPS5694 HIPS5695 − 128 128 128 128 128 128 +128 mg/L 64 2 16 16 ≤0.5 8 +64 mg/L 128 32 64 64 2 32 +32 mg/L 128 128 128 128 8 128 +16 mg/L 128 128 128 128 64 128 +8 mg/L 128 128 128 128 64 128 +4 mg/L 128 128 128 128 128 128 +2 mg/L 128 128 128 128 128 128

TABLE-US-00008 TABLE 7 Synergistic antimicrobial activity of imipenem with several compounds against E. coli VIM-1 (T2544) Imipenem MIC in mg/L HIPS HIPS5640 HIPS5672 HIPS5682 HIPS5683 HIPS5694 HIPS5695 − 32 64 32 32 32 32 +128 mg/L 16 4 2 2 0.5 1 +64 mg/L 32 8 8 8 8 2 +32 mg/L 32 32 16 16 16 4 +16 mg/L 32 32 32 32 16 16 +8 mg/L 32 64 32 32 32 16 +4 mg/L 32 64 32 32 32 16 +2 mg/L 32 64 32 32 32 16

TABLE-US-00009 TABLE 8 Synergistic antimicrobial activity of imipenem with several compounds against E. coli IMP-1 (T2360) Imipenem MIC in mg/L HIPS HIPS5672 HIPS5682 HIPS5683 HIPS5694 HIPS5695 − 2 2 2 2 2 +128 mg/L 0.5 1 0.5 0.5 0.5 +64 mg/L 1 1 1 0.5 0.5 +32 mg/L 1 1 1 1 1 +16 mg/L 1 1 1 1 1 +8 mg/L 2 2 1 1 2 +4 mg/L 2 2 1 2 2 +2 mg/L 2 2 2 2 2

TABLE-US-00010 TABLE 9 Intrinsic antimicrobial activity of HIPS5694 Bacteria HIPS 5694 MIG (mg/L) E. coli NDM-7 (T2239) >256 K. pneumoniae NDM-1 (T2301) >256 E. cloacae NDM-1 (T2311) >256 S. marcescens NDM-1 (T2352) >256 E. coli NDM-5 (T2351) >256 E. cloacae VIM-1 (T2353) >256 P. aeruginosa VIM-1 (T2357) >256 P. aeruginosa IMP-1 (T2325) >256 E. cloacae GIM-1 (T2218) >256 A. baumannii OXA-23 (T2434) >256 E. coli KPC-2 (T2435) >256

TABLE-US-00011 TABLE 10 Synergistic antimicrobial activity of imipenem with HIPS5694 against clinical isolates expressing various MBLs and non-MBL carbapenemases. MIC (mg/L) Imipenem Bacteria Imipenem (+HIPS 5694 128 mg/L) E. coli NDM-7 (T2239) 32 2 K. pneumoniae NDM-1 (T2301) 16 0.5 E. cloacae NDM-1 (T2311) 8 1 S. marcescens NDM-1 (T2352) 256 4 E. coli NDM-5 (T2351) 8 2 E. cloacae VIM-1 (T2353) 4 2

[0111] In vivo toxicity in zebrafish model.

[0112] Owing to its in vitro activities against three MBLs, selectivity against human MMPs and the lack of cytotoxicity against three human cell lines, compounds HIPS1686, HIPS5682 and HIPS5683 were subjected to a toxicity study based on zebrafish larvae. This non-mammalian in vivo model has a high genetic homology to humans and provides follow-up information on the type of toxicity encountered (Chakraborty, C. et al, (2016). J. Nanobiotechnology, 14, 1-13.). In addition, it can also evaluate lethality and malformation during development of embryonic zebrafish (MacRae, C. A., Peterson, R. T. (2015). Nat. Rev. Drug Discov. 14, 721-731). The tested compounds showed a maximum tolerated concentration (MTC) of ≥150 μM.