Metal beta-lactamase inhibitors

Abstract

There is provided a metal beta-lactamase (MBL) inhibitor C-A-L (I) having the general formula (I) wherein: C is a functionalized aza-cycloalkane zinc chelating moiety, L is a beta-lactam moiety that may be functionalized, and A is a C1-C10 linear or cyclic linker that may be functionalized. C-A-L (I)

Description

Technical Field

[0001] The present invention relates to inhibitors of metallo-β-lactamase (MBL). Background Art

[0002] β-lactam antibiotics are some of the most widely used antibiotics, and their successful use in the treatment of infectious diseases is well documented. However, bacterial resistance to all known β-lactam antibiotics is escalating worldwide, and the resulting loss of treatment options for infectious diseases poses a threat to public health. One of the main causes of bacterial resistance to β-lactam antibiotics is the production of metallo-β-lactamases by bacteria. These enzymes hydrolyze the β-lactam ring of the antibiotic, thereby inactivating the molecule. Metallo-β-lactamases (MBLs) are produced by pathogens such as Enterobacteriaceae and other Gram-negative bacteria.

[0003] Among carbapenem-resistant Enterobacteriaceae (CRE), MBL-producing Enterobacteriaceae are the least susceptible to antibiotics (Meletis G. Carbapenem resistance: overview of the problem and future perspectives. Ther Adv Infect Dis. 2016;3(1):15-21). MBL have been identified worldwide and have emerged across Africa (Gupta N, Limbago BM, Patel JB et al. Carbapenem-resistant Enterobacteriaceae: epidemiology and prevention. Clinical infectious diseases 2011;53:60-7). Carbapenem β-lactam antibiotics are reserved as agents of last resort due to their resistance to the action of MBL, however, enzymes that can hydrolyze carbapenem antibiotics have emerged in Gram-negative bacteria, leading to rapid resistance to this class of drugs. In previous studies of MBL inhibitors, many compounds showed good in vitro activity but were rejected in clinical trials due to cytotoxic effects, poor pharmacological properties, or adverse effects on human metalloenzymes (Tooke CL, Hinchliffe P, Bragginton EC, Colenso CK, Hirvonen VHA, Takebayashi Y, et al., β-Lactamases and β-Lactamase Inhibitors in the 21st Century. Journal of Molecular Biology. 2019; 431(18): 3472-500).

[0004] There are currently essentially no clinically available inhibitors for inhibiting MBL, and the development of MBL inhibitors is currently receiving attention worldwide (Palacios AR, Rossi MA, Mahler GS, Vila AJ. Metallo-β-Lactamase Inhibitors Inspired on Snapshots from the Catalytic Mechanism. Biomolecules. 2020; 10(6): 854).

[0005] Carbapenem hydrolases, also known as carbapenemases, are classified into Ambler classes A, B, or D based on the reaction site of the enzyme. Class A and D are serine carbapenemases, such as KPC-2, OXA-48, and SME-1, which attack the β-lactam ring covalently, and class B are MBLs, such as NDM-1, VIM-1, and IMP-1, which utilize Zn ++ The ions activate the nucleophilic water molecules to open the β-lactam ring of the antibiotic.

[0006] Various β-lactamase inhibitor combinations such as amoxicillin / clavulanate, ampicillin / sulbactam, piperacillin / tazobactam, as well as ceftazidime and avibactam have been used in clinical practice, and new inhibitors are currently undergoing clinical trials. Cyclic boronates, namely Taniborbactam (NCT03840148) and QPX7728 (NCT04380207), show good MBL inhibitor activity against type B β-lactamases. These two compounds are currently in phase III and phase I clinical trials, respectively, but their inhibitory mechanisms are not yet fully understood. Docquier et al. (Journal of Antimicrobial Agents and Chemotherapy (JAntimicrobe Chemother.) 2003 February; 51 (2): 257-66) have also found that VIM-2MBL is easily inactivated by chelators, indicating that the zinc cation of the enzyme may be loosely bound. 。

[0007] Several classes of non-toxic MBL inhibitors have been identified, such as Aspergillomarasmine A (King AM, Reid-Yu SA, Wang W et al. Aspergillomarasmine A overcomes metallo-[bgr]-lactamase antibiotic resistance. Nature 2014; 510: 503-6), which is a rapid and effective inhibitor of NDM-1 and VIM-2 MBL. This agent removes Zn by losing 1.8 Zn equivalents in NDM-1. 2+ ions, thereby acting as in vitro chelators that interact with the B1 subclass MBL. NOTA, DOTA, and DPA (Somboro AM, Tiwari D, Bester LA, et al. NOTA: a potent metallo-β-lactamase inhibitor. Journal of Antimicrobial Chemotherapy 2015;70:1594-6; Azumah R, Dutta J, Somboro AM, et al. In vitro evaluation of metal chelators as potential metallo-β-lactamase inhibitors. Journal of applied microbiology 2016) have also been reported to inhibit MBL, and in a recent PCT publication, Rongved et al. reported new MBL inhibitors (Rongved P, OAH, Bayer A et al. Inhibitors of Metallo-Beta-Lactamase (MBL) Comprising a Zinc Chelating Moiety. WO 2015 / 049546). In vitro and some in vivo effects of these inhibitors have also been reported (King et al.; von Nussbaum F, Schiffer G. Aspergillomarasmine A, an Inhibitor of Bacterial Metallo-β-Lactamases Conferring blaNDM and blaVIM Resistance. Angewandte Chemie International Edition 2014; 53: 11696-8; Rongved et al.).

[0008] The use of “stand-alone” zinc chelators, such as 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7,10-tetraazacyclononane-1,4,7,10-tetraacetic acid (DOTA), and dimethylpyridinium amine (DPA), have also been reported, all of which are metal chelators that are effective inhibitors of MBL-producing Enterobacteriaceae and are neither toxic nor hemolytic at effective concentrations in vitro (Somboro et al.; Azumah et al.). However, such stand-alone chelators were found not to have the desired pharmacokinetic characteristics for use in combination with β-lactam antibiotics.

[0009] Therefore, a major goal of antimicrobial chemotherapy (Craig WA, Welling P. Protein binding of antimicrobials: clinical alpharmacokinetic and therapeutic implications. Clinic alpharmacokinetics 1977; 2: 252-68) is to develop MBL inhibitors that can achieve and maintain effective concentrations for treating infection. One of the factors that influence the physiological and pharmacological properties of MBL inhibitors is the interaction of these agents with serum and tissue proteins. This makes in vitro assays to determine these properties very important as they can provide information about the drug's mode of action in vivo (Arhin FF, McKay GA, Beaulieu S, et al. Impact of human serum albumin on oritavancin invitro activity against Staphylococcus aureus. Diagnostic microbiology and infectious disease 2009;65:207-10; McKay GA, Beaulieu S, Sarmiento I, et al. Impact of human serum albumin on oritavancin in vitro activity against enterococci. Antimicrobial agents and chemotherapy 2009;53:2687-9).

[0010] The inventors have now developed a new class of MBL inhibitors that have improved pharmacological properties by attaching a β-lactam group to a bifunctional cyclic zinc chelator, enabling effective combination therapy when used in combination with β-lactam antibiotics. Several studies have well documented that simple metal chelators are rapidly cleared by the kidneys (Prata MI, Santos Ac Fau-Geraldes CF, Geraldes Cf Fau-de Lima JJ, de Lima JJ. 67Ga of triazamacrocyclic ligands 3+Characterization of the complex: biodistribution and clearance studies 3+ complexes of triaza macrocyclic ligands: biodistribution and clearance studies). Nucl Med Biol. 1999; 26(6): 707-10; Reddy N, Shungube M, Arvidsson PI, Baijnath S, Kruger HG, Govender T et al. 2018–2019 patent review of metallo beta-lactamase inhibitors. Expert Opinion on Therapeutic Patents. 2020: 1-15), which does not match the clearance rate of the concomitant β-lactam drug. Summary of the invention

[0011] According to one aspect of the present invention, there is provided a metallo-β-lactamase (MBL) inhibitor having the general formula (I):

[0012] CAL

[0013] (I)

[0014] in:

[0015] C is a functionalized aza-cycloalkane zinc chelating moiety,

[0016] L is a β-lactam moiety which may be functionalized, and

[0017] A is a C1-C 10 Straight chain or circular linker.

[0018] The functionalized aza-cycloalkane zinc chelating moiety C may be a functionalized aza-cyclononane moiety.

[0019] The functionalized aza-cycloalkane zinc chelating moieties C useful in the present invention typically have a ring nitrogen atom and a carboxylic acid substituent, or a ring nitrogen atom and a pyridyl substituent, and these zinc chelating moieties are also referred to as "bifunctional zinc chelating moieties" to indicate the two types of coordination substructures, i.e., a ring nitrogen atom and a carboxyl or pyridyl group that complexes with zinc.

[0020] Some examples of bifunctional cyclic chelators useful in the present invention are listed in Scheme 1.

[0021]

[0022] Solution 1

[0023] The functionalized aza-cycloalkane zinc chelating moiety C may be a substituted 1,4,7-triazacyclononane moiety.

[0024] The substituted 1,4,7-triazacyclononane moiety can be selected from the group consisting of a 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) moiety, a 1,4,7-triazacyclononane-1,4,7-tripentanedioic acid (NOTGA) moiety, a 1,4,7-triazacyclononane-1-succinic acid-4,7-diacetic acid (NODASA) moiety, a 1,4,7-triazacyclononane-1-pentanedioic acid-4,7-diacetic acid (NOD The substituted 1,4,7-triazacyclononane moiety is preferably a 1,4,7-triazacyclononane-1,4-diacetic acid-7-p-hydroxyphenyl-acetic acid (NODAPA) moiety, a 1,4,7-tris(2-pyridylmethyl)-1,4,7-triazacyclononane moiety, a 4,7-bis(2-pyridylmethyl)-1,4,7-triazacyclononane-1-acetic acid, and a 1-[(5-carboxy-2-methylpyridyl)]-4,7-bis(2-methylpyridyl)-1,4,7-triazacyclononane moiety. However, it should be understood that the substituted 1,4,7-triazacyclononane moiety is not necessarily limited to the moieties of the above-mentioned groups.

[0025] The linker A can be connected to the ring nitrogen atom of the functionalized aza-cycloalkane zinc chelating moiety C. For example, the linker can be formed by the aza Michael addition of the nitrogen atom of the functionalized aza-cycloalkane zinc chelating moiety C with an α, β-unsaturated carbonyl compound such as an α, β-unsaturated ester. The ester can be, for example, an α, β-unsaturated C4-C 10 Esters of monobasic or dibasic acids.

[0026] Thus, the linker A may be attached to the nitrogen atom of the substituted 1,4,7-triazacyclononane moiety.

[0027] The linker A may be a moiety selected from the group consisting of a saturated monoacid moiety, an unsaturated monoacid moiety, a saturated diacid moiety, an unsaturated diacid moiety, and esters thereof.

[0028] The linker A may be derived, for example, from a saturated or unsaturated monoacid or a saturated or unsaturated diacid, such as acetic acid, glutaric acid, maleic acid, fumaric acid, succinic acid or p-hydroxyphenylacetic acid or an ester thereof.

[0029] In one embodiment of the invention, said linker A is thus a moiety selected from the group consisting of an acetic acid moiety, a glutaric acid moiety, a maleic acid moiety, a fumaric acid moiety, a succinic acid moiety, a p-hydroxyphenylacetic acid moiety and esters thereof.

[0030] The β-lactam part L of the metallo-β-lactamase (MBL) inhibitor of the present invention can be derived from, but not limited to, cephalosporin, penem or carbapenem. Therefore, in one embodiment of the present invention, the β-lactam part L is selected from the group consisting of: a functionalized cephalosporin part, a penem part and a carbapenem part.

[0031] Examples of cephalosporins, penems or carbapenems from which moieties may be derived or selected are listed in Scheme 2.

[0032]

[0033]

[0034] Solution 2

[0035] The linker A can be connected to the β-lactam ring of the cephalosporin, penem or carbapenem substructure or part through an amide bond located at the alpha position of the carbonyl group of the β-lactam ring. In other words, the linker A can be connected to the β-lactam part L through an amide bond located at the alpha position of the carbonyl group of the β-lactam ring.

[0036] Scheme 3 shows an example of a metallo-β-lactamase (MBL) inhibitor of the present invention comprising a functionalized aza-cycloalkane zinc chelating moiety coupled to a cephalosporin substructure or moiety via a linker.

[0037]

[0038]

[0039] Solution 3

[0040] The present invention extends to a pharmaceutical composition comprising a metallo-β-lactamase (MBL) inhibitor having the general formula (I) and one or more pharmaceutically acceptable carriers or excipients.

[0041] According to another aspect of the present invention, there is provided a use of a metallo-β-lactamase (MBL) inhibitor having the general formula (I) in the preparation of a medicament for treating or preventing bacterial infection in a human or non-human mammal.

[0042] According to another aspect of the present invention, there is provided a metallo-β-lactamase (MBL) inhibitor or pharmaceutical composition having the general formula (I) as described above, which is used in a method for treating or preventing bacterial infection in a human or non-human mammal.

[0043] According to yet another aspect of the present invention, there is provided a method for treating or preventing bacterial infection in a human or non-human mammal, the method comprising the step of administering to the human or non-human mammal an effective amount of a metallo-β-lactamase (MBL) inhibitor having the general formula (I).

[0044] The method may further comprise administering one or more β-lactam antibiotics to the human or non-human mammal. Thus, the method may comprise the step of administering a metallo-β-lactamase (MBL) inhibitor of general formula (I) or a pharmaceutical composition comprising a metallo-β-lactamase (MBL) inhibitor of general formula (I) simultaneously, separately or sequentially with a β-lactam antibiotic.

[0045] The β-lactam antibiotic may be selected from the group consisting of meropenem, imipenem, doripenem, ertapenem and combinations thereof.

[0046] According to yet another aspect of the present invention, a kit for treating or preventing bacterial infection in humans or non-human mammals is provided, the kit comprising at least one of the following: a metallo-β-lactamase (MBL) inhibitor or a pharmaceutical composition having the general formula (I) as described above.

[0047] The kit may further comprise one or more β-lactam antibiotics.

[0048] The one or more β-lactam antibiotics may be selected from the group consisting of meropenem, imipenem, doripenem, ertapenem, and combinations thereof.

[0049] The bacterial infection may be an infection caused by bacterial strains such as Escherichia coli IMP-1, Escherichia coli NDM-1, Escherichia coli VIM-1, Enterobacter cloacae VIM-1, Enterobacter cloacae IMP-1, Klebsiella pneumoniae IMP-8, Klebsiella pneumoniae VIM-1, Klebsiella pneumoniae IMP-1, Klebsiella pneumoniae NDM or Providencia rettgere NDM, as well as other strains containing or producing metallo-β-lactamases.

[0050] The synthesis of compound BP1 (chelator attached to cephalosporin, compound 4 of Scheme 4) is outlined in Scheme 4.

[0051]

[0052] Option 4 - BP1( Connect to Synthesis of cephalosporin chelating agents

[0053] BP1 (compound 4 of Scheme 4) was synthesized in two steps using the peptide coupling agent COMU from 1,4,7-triazacyclononane (1) in the presence of diethylamine to produce the coupling product (3), and subsequent removal of the protecting groups using TFA to produce the deprotected final product (4).

[0054] Compound BP2 (chelator attached to cephalosporin, compound 7 of Scheme 5) and compound BP3 (chelator of penem, compound 10 of Scheme 6) were prepared similarly as shown in Schemes 5 and 6 below.

[0055]

[0056] Option 5 - BP2( Connect to Synthesis of cephalosporin chelating agents

[0057]

[0058] Option 6 - BP3( Connect to Synthesis of penicillin chelators

[0059]

[0060]

[0061] Scheme 7 - Hno1a2py (8) and Hno1pa2py (10) cyclic chelators and metalloproteins with cephalosporin moieties Synthesis of β-lactamase (MBL) inhibitors (11) and (12)

[0062] Other BP compounds disclosed herein but whose synthesis is not specifically described are prepared analogously to the BP compounds whose synthesis is specifically described herein. DETAILED DESCRIPTION

[0063] The present invention will now be described by way of example with reference to the following examples, drawings and tables, in which:

[0064] Figure 1 The time-dependent traces of the time-kill kinetics of Klebsiella pneumoniae IMP-8 at different concentrations of meropenem and BP1 are shown;

[0065] Figure 1A The time-dependent traces of the killing kinetics of Klebsiella pneumoniae NDM at different concentrations of meropenem and BP1 are shown;

[0066] Figure 1BThe time-dependent traces of the killing kinetics of Klebsiella pneumoniae NDM at different concentrations of meropenem and BP6 are shown;

[0067] Figure 1C The time-dependent traces of the killing kinetics of Klebsiella pneumoniae NDM at different concentrations of meropenem and BP10 are shown;

[0068] Figure 1D The time-dependent traces of the killing kinetics of Klebsiella pneumoniae NDM at different concentrations of meropenem and BP14 are shown;

[0069] Figure 1E Shown is a trace of the time-dependent killing kinetics of Klebsiella pneumoniae NDM at different concentrations of meropenem and BP1, BP6, BP10, and BP14. This figure shows a fixed concentration of each BP compound (32 mg / L), and the lowest concentration of meropenem (0.5 mg / L) was studied;

[0070] Figure 2 The time-dependent traces of the time-killing kinetics of E. coli NDM-1 at different concentrations of meropenem and BP1 are shown;

[0071] Figure 3 Two bar graphs showing HepG2 cell viability as a function of BP1 dose and lactate dehydrogenase as a function of BP1 dose are shown;

[0072] Figure 3A Two additional bar graphs are shown for HepG2 cell viability as a function of BP1 dose and lactate dehydrogenase as a function of BP1 dose. Nonlinear fitting of BP1 on HepG2 cells yielded an IC of 59.96 μg / ml 50 Values. A dose-dependent decrease in cell viability was observed, with significant decreases at 100 and 200 μg / ml. *p<0.5 and **p<0.01 relative to control. Extracellular lactate dehydrogenase (LDH) was significantly reduced at all concentrations, indicating that BP1 does not induce necrosis after exposure. ***p<0.0001 relative to control;

[0073] Figure 3B Two bar graphs showing HepG2 cell viability as a function of BP6 dose and lactate dehydrogenase as a function of BP6 dose are shown. BP6 induced a dose-dependent increase in HepG2 cell viability, however, cell viability was significantly altered only at 200 μg / ml. *p<0.05 relative to control. LDH was significantly reduced at all concentrations, indicating that BP6 does not induce necrosis after exposure. ***p<0.0001 relative to control;

[0074] Figure 3C Two bar graphs showing HepG2 cell viability as a function of BP10 dose and lactate dehydrogenase as a function of BP10 dose are shown. Cell viability was not significantly altered at 50-100 μg / ml, however, it was significantly reduced at 200 μg / ml. *p<0.5 and **p<0.01 relative to control. LDH levels remained unaffected at 1 μg / ml and were significantly reduced at 8-200 μg / ml, indicating that BP10 does not induce HepG2 cell necrosis after exposure. **p<0.01 and **p<0.001 relative to control;

[0075] Figure 3D Two bar graphs showing HepG2 cell viability as a function of BP14 dose and lactate dehydrogenase as a function of BP14 dose are shown. BP-14 reduced cell viability at 1 μg / ml and 10-200 μg / ml; however, proliferation occurred at 8 μg / ml. *p<0.5 and **p<0.01 relative to control. LDH levels were significantly reduced at 8 μg / ml and remained unaffected at 1 μg / ml and 10-200 μg / ml, indicating that BP-14 does not induce necrosis in HepG2 cells after exposure. **p<0.001 relative to control;

[0076] Figure 4 The pharmacokinetics of BP1 and meropenem in a healthy mouse model are shown, and the data are presented as mean ± SD (n = 3);

[0077] Figure 5 shows the change in thigh CFU counts after treatment with BP1 over a 24 hour period, with data presented as mean ± SD (n = 3); and

[0078] Figure 6 Plasma BP1 concentrations over the 8-hour treatment period are shown, and data are presented as mean±SD (n=3).

[0079] discuss

[0080] In the discussion below, "BP1" refers to compound 4 of Scheme 4, and "TG1" refers to 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA).

[0081] Material

[0082] Meropenem and human serum were purchased from Sigma-Aldrich.

[0083] Bacterial strains

[0084] The CRE reference strain producing MBL was purchased from Nordmann (Nordmann et al., Rapid Detection of Carbapenemase-Producing Enterobacteriaceae - Vol. 18, No. 9 - September 2012 - Journal of Emerging Infectious Diseases - CDC. 2012) at the Institut National de la Santé et de la Recherche Médicale, Paris, France. The reference strain used for quality control was Escherichia coli ATCC 25922.

[0085] Synthesis of compounds 4, 7 and 10

[0086] General information:

[0087] All chemicals were purchased from commercial sources and used without further purification. Monomethyl fumarate, N,N-diisopropylethylamine (DIEA), tetrahydrofuran (THF), and dichloromethane (CH2Cl2) were purchased from Sigma-Aldrich.

[0088] All compounds were analyzed by RP-HPLC (Agilent 1100, USA). Experiments were performed using a linear gradient of solvent A (0.1% TFA in H2O) and solvent B (0.1% TFA in ACN); wherein the gradient was from 5% B to 95% B in 15 minutes at a flow rate of 1 ml / min. The corresponding masses of all synthetic steps were further characterized using LC / MS (Shimadzu 2020UFLC-MS, Japan) loaded with a YMC-Triart C18 (5 μm, 4.6×150 mm) column. NMR spectra were recorded on a Bruker AVANCE III 400 MHz spectrometer using deuterated methanol as a solvent ( 1 H NMR, 13 C NMR, HMBC, and HSQC). HRMS was performed on a Bruker MicroTOF-QII.

[0089] Preparation of Compounds 2 (Scheme 4), 6 (Scheme 5) and 9 (Scheme 6)

[0090] Compound 1 (Scheme 4), 5 (Scheme 5) or 8 (Scheme 6) (1.0 equivalent) was dissolved in anhydrous ACN:THF (30 mL / mmol), then N, N-diisopropylethylamine (3.0 equivalent) was added once, followed by DIC (2.0 equivalent). After 5 minutes, 7-amino-3-chloromethyl-3-cephem-4-carboxylic acid p-methoxybenzyl ester hydrochloride (Scheme 1, 1→2), 7-aminocephalosporanic acid (Scheme 5, 5→6) or ampicillin (Scheme 6, 8→9) (1.0 equivalent) was added, and the progress of the reaction was then monitored by LC / MS. After 24 hours, the reaction was complete. The purification of compound 2 of Scheme 4 was carried out by eluting with EtOAc: hexane (1:1) through an alumina column with a yield of 83%. Compound 6 (Scheme 5) and compound 9 (Scheme 6) were purified using preparative SFC with the following parameters: sample concentration = 8 mg / mL (ACN, 0.3% Et3N), injection volume = 100 μL, column = ethylpyridine (250×10 mm, ), at 40°C, mobile phase = 10–50% MeOH:ACN:Et3N (99.85:99.85:0.3) as modifier, industrial grade wet CO2 as equilibrium mobile phase, time 10 minutes, flow rate = 10 ml / min, stacked injection program with two minutes equilibrium time, BPR setting = 150 bar, detection and collection at 220 nm. Yields were 50-70%.

[0091] Preparation of Compound 3 of Scheme 4

[0092] The synthesis method is adapted from the method of Long et al. (D.Long, J.B. Aggen, J.Chinn, S.-K.Choi, B.G. Christensen, P.R. Fatheree, D.Green, S.S. H. Segde, J.K. Judice, K.Kaniga, K.M. K. Rause, M.Leadbetter, M.S. Linsell, D.G. Marquess, E.J. Moran, M.B. Nodwell, J.L. Pace, S.G. Trapp and S.D. Turner, Journal of Antibiotics (J. Antibiot.), 2008, 61, 603-614). Compound 2 (1.0 equivalent) of Scheme 4 was dissolved in anhydrous acetone (4 mL / mmol relative to compound 2 of Scheme 4), and the flask was covered with aluminum foil to avoid light reaction. Sodium iodide (1.0 equivalent) was then added at room temperature once, and the reaction was stirred for 1.5 hours, and then pyridine (1.1 equivalent) was added. The reaction was stirred for another 1.5 hours (the progress of this step was monitored by LC / MS). A quick check revealed that the solution had turned red / purple. The solvent was then removed, producing a red / purple foam. The foam was sonicated (5 min) in Et2O (5 mL / mmol relative to compound 2 of Scheme 4) and the washes were decanted, this was repeated twice. The washes were >90% pure, however, further purification was performed using preparative SFC with the following parameters: sample concentration = 16 mg / mL (ACN, 0.3% Et3N), injection volume = 100 μL, column = ethylpyridine (250×10 mm, ), at 40°C, mobile phase = 10–50% MeOH:ACN:Et3N (99.85:99.85:0.3) as a modifier, industrial grade wet CO2 as an equilibrium mobile phase, time 10 minutes, flow rate = 10 ml / min, stacked injection program with two minutes equilibrium time, BPR setting = 150 bar, detection and collection at 220 nm. Compound 7 of Scheme 5 was obtained as a light yellow solid in a 45% yield based on compound 3.Et3N salt.

[0093] Preparation of compounds 4, 7 and 10

[0094] Triisopropylsilane (relative to 7,4 mL / mmol) was added to compound 3 (Scheme 4), compound 6 (Scheme 5) or compound 9 (Scheme 6) (1.0 equivalents), and then TFA (relative to compound 3, 6 or 9, 76 mL / mmol) was added. The reaction was stirred at room temperature for 20 minutes, and then volatiles were removed by passing a gentle stream of nitrogen through the reaction until a pale yellow residue was left. The residue was ultrasonically treated (10 minutes) in Et2O (relative to compound 3, 6 or 9, 5 mL / mmol), and the washings were decanted to obtain a fine precipitate. The aforementioned steps were repeated twice with Et2O to produce 85-95% yields of pure compounds 4 (Scheme 4), compound 7 (Scheme 5) and compound 10 (Scheme 6) as white solids.

[0095] Synthesis of compounds 2, 3, 4, 5 and 6 of Scheme 7

[0096] General information:

[0097] All chemicals were purchased from commercial sources and used without further purification. Monomethyl fumarate, N,N-diisopropylethylamine (DIEA), tetrahydrofuran (THF), and dichloromethane (CH2Cl2) were purchased from Sigma-Aldrich.

[0098] All compounds were analyzed by RP-HPLC (Agilent 1100, USA). The quality of all synthetic steps was further characterized using LC / MS (Shimadzu 2020UFLC-MS, Japan) loaded with a YMC-Triart C18 (5 μm, 4.6×150 mm) column. NMR spectra were recorded on a Bruker AVANCE III 400 MHz spectrometer using deuterated chloroform or methanol as solvent ( 1 HNMR, 13 C NMR, HMBC, and HSQC). HRMS was performed on a Bruker MicroTOF-QII.

[0099] Compound 2 of Scheme 7 was prepared using a method adapted from Roger, M. et al. (Monopicolinate-dipicolyl Derivative of Triazacyclononane for Stable Complexation of Cu2+ and 64Cu2+). Inorganic Chemistry, 2013. 52(9): pp. 5246-5259).

[0100] (1→2). 1,4,7-Triazacyclononane (compound 1 of Scheme 7) (1.0 equivalent) was dissolved in a mixture of toluene and chloroform (1.3 and 0.3 ml / mmol, respectively). Dimethoxymethyl-N,N-dimethylamine (1.0 equivalent) was added once and the mixture was refluxed. NMR was used to monitor the progress of the reaction, and the reaction took 2 hours to complete. The solvent was then removed to produce pure compound 2 of Scheme 7 in a yellow oily form in 85-95% yield.

[0101] Compound 3 of Scheme 7 was prepared using a method adapted from Gasser, G. et al. (Synthesis, Copper(II) Complexation, 64Cu-Labeling, and Bioconjugation of a New Bis(2-pyridylmethyl) Derivative of 1,4,7-Triazacyclononane. Bioconjugate Chem., 2008. 19(3): 719-730).

[0102] (2→3). A solution of chloromethylpyridine (1.0 equivalent) in THF was carefully added dropwise to compound 2 (1.0 equivalent) of Scheme 7 dissolved in anhydrous THF (1 ml / mmol). The reaction was stirred overnight at room temperature to obtain an orange-red precipitate. The liquid was decanted, and the precipitate was sonicated in THF (5 mL / mmol relative to compound 2 of Scheme 7) (5 minutes), and the washings were decanted. The aforementioned steps were repeated twice. The precipitate was dried to produce compound 3 of Scheme 7 in 75-86% yield.

[0103] Compound 4 of Scheme 7 was prepared using a method adapted from Gasser, G. et al. (Synthesis, copper(II) complexation, 64Cu labeling and bioconjugation of new bis(2-pyridylmethyl) derivatives of 1,4,7-triazacyclononane. Bioconjugate Chemistry, 2008. 19(3): pp. 719-730).

[0104] Compound 3 was dissolved in milli Q water (10 ml / mmol) and refluxed. The reaction was monitored by LC / MS. After 4 hours, the reaction was complete. The pH of the mixture was adjusted to about 12 and extracted 3 times with CHCl. ​​The organic extract was dried and condensed with MgSO to obtain compound 4 of Scheme 7 in 45-60% yield.

[0105] (4→5). Compound 5 of Scheme 7 was prepared using a method adapted from Gasser, G. et al. (Synthesis, copper(II) complexation, 64Cu labeling and bioconjugation of new bis(2-pyridylmethyl) derivatives of 1,4,7-triazacyclononane. Bioconjugate Chemistry, 2008. 19(3): pp. 719-730).

[0106] Compound 4 of scheme 7 is dissolved in anhydrous ACN (1.4mL / mmol), K2CO3 (4.0 equivalents) and KI (16.0 equivalents). The mixture is stirred at room temperature. Thereafter, an ACN solution (1.4ml / mmol) of chloromethyl pyridine hydrochloride is added dropwise in 10-15 minutes. The reaction is further stirred at room temperature for one hour, and then refluxed overnight. Thereafter, the mixture is cooled and filtered. The aliquot is concentrated to obtain compound 5 of scheme 7 in 50-70% yield.

[0107] Compound 6 of Scheme 7 was prepared using a method adapted from Gasser, G. et al. (Synthesis, copper(II) complexation, 64Cu labeling and bioconjugation of new bis(2-pyridylmethyl) derivatives of 1,4,7-triazacyclononane. Bioconjugate Chemistry, 2008. 19(3): pp. 719-730).

[0108] Compound 5 was dissolved in HCl 6M (10 ml / mmol) and refluxed. The reaction was monitored by LC / MS. After 4 hours, the reaction was complete.

[0109] Compound 6 of Scheme 7 was isolated in analogy to compound 4 of Scheme 7 with a yield of 55-65%.

[0110] Preparation of compounds 8 (Hno1a2py) and 10 (Hno1pa2py) of Scheme 7

[0111] The improved synthesis method is adapted from the method of Gasser, G. et al. (Synthesis, copper (II) complexation, 64Cu labeling and bioconjugation of new bis(2-pyridylmethyl) derivatives of 1,4,7-triazacyclononane. Bioconjugate Chemistry, 2008.19(3): pp. 719-730).

[0112] Compound 6 (1.0 equivalent) of scheme 7 is dissolved in anhydrous ACN (1mL / mmol), K2CO3 (0.4 equivalent) and KI (0.2 equivalent). The mixture is stirred at room temperature. Thereafter, the ACN solution (1ml / mmol) of chloromethyl pyridine hydrochloride (scheme 7, 6→7) or 6-chloromethyl pyridine-2-carboxylic acid methyl ester (scheme 7, 6→9) is added dropwise in 10-15 minutes. The reaction is further stirred for one hour at room temperature, and then refluxed overnight. Thereafter, the mixture is cooled and filtered. The aliquot is concentrated to obtain a yield of 50-70% of compound 7 of scheme 7 or compound 9 of scheme 7. Compound 7 (7→8) (1 equivalent) of scheme 7 or compound 9 (9→10) (1 equivalent) of scheme 7 is diluted with 20-30ml / mmol of Milli Q water, and 6M HCl (3 equivalents) is added. The reaction mixture is set to reflux at 100°C in a microwave, and the progress of the reaction is monitored by LC / MS. After 40 minutes, the reaction was complete. The mixture was further diluted five times with water, frozen and the water was removed by freeze drying to obtain a quantitative yield. 6-Chloromethylpyridine-2-carboxylic acid methyl ester was prepared using a method adapted from Mato-Iglesias, M. et al. (Lanthanide Complexes Based on a 1,7-Diaza-12-crown-4 Platform Containing Picolinate Pendants: A New Structural Entry for the Design of Magnetic Resonance Imaging Contrast Agents. Inorganic Chemistry, 2008. 47 (17): pp. 7840-7851).

[0113] Preparation of compounds 11 and 12 (Scheme 7)

[0114] Compound 8 or 10 (1.0 eq.) of Scheme 7 was dissolved in anhydrous DMF (1-2 mL / mmol). N,N-diisopropylethylamine (5.0 eq.) was then added all at once, followed by HOBt (1.2 eq.) and DIC (1.2 eq.). After 5 minutes, 7-aminocephalosporanic acid (5→6) was added, and the progress of the reaction was then monitored by LC / MS. After overnight, the reaction was complete. Compounds 11 and 12 of Scheme 7 were purified using preparative SFC with the following parameters: sample concentration = 10 mg / mL (ACN, 0.3% Et3N), injection volume = 100 μL, column = ethylpyridine (250×10 mm, ), at 40 °C, mobile phase = 10–50% MeOH:ACN:Et3N (49.85:49.85:0.3) as modifier, industrial grade wet CO2 as equilibrium mobile phase, time 10 min, flow rate = 10 ml / min, stacked injection program with two minutes equilibrium time, BPR setting = 150 bar, detection and collection at 220 nm.

[0115] Minimum inhibitory concentration (MIC)

[0116] Minimum inhibitory concentration (MIC) determinations were performed according to CLSI guidelines and Keepers et al. (Keepers TR, Gomez M, Celeri C, et al. Bactericidal activity, absence of serum effect, and time-kill kinetics of ceftazidime-avibactam against β-lactamase-producing Enterobacteriaceae and Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 2014: AAC.02894-14). Briefly, each meropenem / BP or meropenem / TG was diluted twofold (0.015 to 16 μg / mL and 1 to 64 μg / mL) in cation-adjusted Miller-Hayden broth (CAMHB) in 96-well microtiter plates using the checkerboard method (King et al.). A 0.5 McFarland turbidimetric standardized bacterial inoculum is added to a total volume of 100 μl per microtiter well. The initial inoculum density is determined by plating separate MHA plates for the MBC test. The plates are then incubated at 37°C for 18-22 hours under aerobic conditions. The MIC is determined as the lowest concentration with no visible growth. Control wells contain the amount of solvent used to dissolve the candidate drug.

[0117] Serum effect in MIC

[0118] The effect of serum on the combined MIC was similar to the MIC method described above, however, in this case, 100% human serum was added to the broth to make the final broth 50% human serum. Two CRE strains (E. coli NDM-1 and K. pneumoniae IMP-8) were used in this experiment.

[0119] Time-kill assay

[0120] Time-kill studies were performed according to previously published methods, including the methods described in CLSI document M26-A. Escherichia coli NDM-1, Klebsiella pneumoniae IMP-8, and Klebsiella pneumoniae NDM were selected for this assay. Briefly, freshly prepared colonies were resuspended in 10 mL of CAMHB and incubated in a shaking incubator (37° C., 180 rpm) for 1 to 2 hours. The cultures were then diluted to 0.5 McFarland turbidimetric standards (approximately 10 8 CFU / mL) and further diluted 1:20 in CAMHB to give a starting inoculum of approximately 5 × 10 6 CFU / mL. Meropenem was added to the prepared bacterial suspension so that the final meropenem concentration was 2, 4, or 8 times the MIC of meropenem-BP, and BP was added to a final concentration of 64 μg / mL. A growth control without antibiotics was also included. The starting inoculum was determined from the growth control tube immediately after dilution and recorded as the count at time zero. After the addition of antibiotics, the starting inoculum was approximately 1×10 6 Up to 5×10 6 CFU / mL. Incubate the tubes at 37°C, 180 rpm in a shaking incubator and perform viability counts at 1, 2, 4, 6, 8, and 24 hours by removing 100 μL of the culture, diluting appropriately, and plating 100 μL on MHA. Incubate the MHA plates at 37°C for at least 18 hours. Count the colonies and record the results as CFU / mL. A ≥3-log decrease in CFU / mL 10 Considered antiseptic.

[0121] Cell culture

[0122] Human hepatoma (HepG2) cells were cultured in Dulbecco's Minimum Essential Medium (Lonza Biowhittaker, Switzerland) supplemented with 1% pen / strep / amphotericin, 1% L-glutamine and 10% fetal bovine serum. The cells were maintained at 37° C. in an atmosphere of 5% CO 2 . Once 80% cell density was reached, the cells were enzymatically detached (trypsin) and used for cell viability assays.

[0123] Cell viability assay

[0124] The effect of MPR-SMN-05 on cell viability was determined using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay based on studies previously reported by Mosmann (Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of immunological methods 1983; 65: 55-63). Approximately 15,000 cells were seeded in 96-well microtiter plates and allowed to adhere overnight. Cells were then exposed to 0 to 250 mg / L MPR-SMN-05 (experiments were performed in triplicate). After incubation with MPR-SMN-05 for 24 hours, cells were washed twice with 0.1 M phosphate buffered saline (PBS) and incubated (37° C., 4 hours) with 20 μl MTT saline (5 mg / ml in 0.1 M PBS) and 100 μl complete culture medium (CCM). The MTT saline was then discarded, and 100 ml DMSO was added to each well and incubated (37° C., 1 hour) to dissolve the formazan crystals. Optical density was measured at 570 / 690 nm using a spectrophotometer (Bio-Tek uQuant, Winooski, VT). Results are expressed as percentage of cell viability versus logarithmic concentration of MPR-SMN-05, from which the half-maximal inhibitory concentration (IC 50 ). Control treatments consisted of an equal amount of DMSO solvent in CCM without test compounds.

[0125] Cytotoxicity assay

[0126] Lactate dehydrogenase (LDH) cytotoxicity assay kit (Roche, Mannheim, Germany) was used to measure cell damage and cytotoxicity in MPR-SMN-05 treated HepG2 cells. Extracellular LDH activity was quantified by incubating 100 ml of cell culture supernatant with 100 ml of substrate solution (diaphorase / NAD+; INT / sodium lactate) in a 96-well microtiter plate at room temperature for 30 minutes. The assay was performed three times. After incubation at 500 nm, the optical density (OD) was measured using a spectrophotometric plate reader (UQuant, Berten, Winooski, Vermont).

[0127] Statistical analysis

[0128] All determinations were performed at least in duplicate. Results are provided as mean ± standard deviation. Statistical analysis of the experiments was performed using a statistical one-way Anova test. p values ​​< 0.05 were considered statistically significant.

[0129] Bioanalytical method for determining the concentrations of meropenem, BP1, and meropenem-BP1 combination in mouse plasma

[0130] instrument

[0131] The liquid chromatography tandem mass spectrometry (LC-MS) system consisted of an Agilent series 1100 with an online degasser, gradient pump and autosampler, coupled to a time-of-flight mass spectrometer (TOF-MS) maXis 4G electrospray ionization (ESI) instrument (Bruker Daltonics, Bremen, Germany). All results were stored and analyzed using Data Analysis 4.0 SP 5 (Bruker Daltonics).

[0132] Preparation of standards and calibration curves

[0133] Separate stock solutions of meropenem, BP-1 and internal standard (IS) TG-1 were prepared by dissolving 10 mg of each substance in 10 mL of 50% methanol in distilled water. The solution was stored at refrigerated temperature (0 to 4 ° C). Calibration standards were prepared by spiking working standard solution and IS into 100 μL of blank plasma from untreated mice so that the analyte concentrations of the two compounds in plasma were 25, 50, 100, 200, 400, 800 and 1000 ng / mL, respectively. Throughout the experiment, both analytes and samples used 200 ng / mL of IS. Quality control (QC) samples (10; 75, 500 and 900 ng / mL, respectively) of the lower limit of quantification (LLQC), low concentration (LQC), medium concentration (MQC) and high concentration (HQC) were prepared in the same manner.

[0134] Chromatographic conditions

[0135] Use the YMC Triart C equipped with the corresponding guard column 18 HPLC separation was performed using a column (YMC Europe GmbH, Germany) with spherical hybrid silica particles (150 mm×3.0 mm, inner diameter S=3 μm). Two mobile phases were used, namely 0.1% FA in water and 0.1% FA in ACN. The flow rate was 0.3 mL min -1, and the temperature of the column oven was set to 25°C. The gradient profile was initially from 25% to 50% ACN in 10 minutes, then reached 75% in 2 minutes, after which the mobile phase returned to the initial condition (25% ACN) in 3 minutes and maintained for 5 minutes. The sample injection volume was 5 μL.

[0136] Mass spectrometry analysis

[0137] Accurate mass spectra of the target analytes were obtained using a Bruker Daltonics instrument maXis 4G ESI time-of-flight mass spectrometry (TOF-MS). The optimized MS conditions and acquisition parameters were as follows: source type, ESI; ion polarity, positive; nebulizer, 1.5 bar; capillary, 6000 V; dry heater, 180 °C; scan range: m / z 300-1200; end plate offset, -500 V; drying gas, 8.0 L / min; collision cell radio frequency, 3000 Vpp, collision energy, 7 eV. These conditions were suitable for both target compounds (meropenem and BP1). However, different MS / MS conditions were optimized for each analyte as follows: isolation mass was m / z; isolation width was set to 38, 25, respectively; isolation energy 21, 24; and acquisition factor 2, 2.

[0138] Plasma samples

[0139] Blank plasma samples were obtained from Life Technologies (Burlington, ON, Canada) and stored at -20°C prior to analysis.

[0140] After dosing with the analytes, blood samples were collected from mice by cardiac puncture, centrifuged at 3500 rpm for 10 min, and plasma was separated and stored at -80°C prior to bioanalysis. Samples were thawed at room temperature prior to any experiments.

[0141] Sample preparation

[0142] Aliquots of 100 μL blank plasma, calibration standards, QC samples and IS were added to 1 mL with methanol to induce plasma protein precipitation. Treated mouse plasma samples followed the same procedure. The mixture was vigorously mixed for 30 seconds and then centrifuged at 15000 rpm for 10 minutes at 4 ° C. The supernatant was filtered through a DSC-18-SPE column (50 mg / mL). The filtered sample (500 μL) was transferred to an automatic injection bottle for injection into the chromatographic system. Thawing of all frozen plasma samples was completed at room temperature.

[0143] stability

[0144] The stability of BP1 in plasma was evaluated by measuring three replicates of low, medium and high concentrations of QC samples under different conditions: short-term stability after 6 hours of storage at room temperature (25°C); freeze / thaw stability through three freeze / thaw cycles (-80°C to 25°C). Post-preparation stability was checked after 24 hours of maintenance at 25°C in an autosampler. Stock solution stability of BP1 was determined after storage at 4°C. Quantitative concentrations of stability were compared with theoretical concentrations.

[0145] result

[0146] MIC

[0147] To investigate the potential activity of BP1 and TG1 as MBL inhibitors, well-characterized carbapenem-resistant bacteria expressing known class B carbapenemases were used. According to the CLSI guidelines (Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Twenty-Fourth Informational Supplement M100-S24. CLSI, Wayne, PA, USA, 2014), MICs using broth microdilution were performed using cation-adjusted Miller-Hayden broth for meropenem alone (see Table 4) and in combination with metal chelators BP1 and TG1 at different concentrations (two-fold dilutions from 1-64 ug / mL). Escherichia coli ATCC 25922 was used as a control.

[0148] BP1 and TG1 were able to restore the activity of a carbapenem antibiotic (meropenem) against MBL-producing bacteria at concentrations as low as 0.03 mg / L. None of the serine β-lactamases were sensitive to the carbapenem / metal chelator combination, confirming the mode of action of these compounds.

[0149] Serum effect

[0150] The sera had no significant effect on the MICs of the meropenem / BP1 combination, as these values ​​remained within the sensitive range and varied only within 1 to 2 dilutions for both isolates studied (E. coli NDM-1 and K. pneumoniae IMP-8). This variation is acceptable according to CLSI guidelines when the same experiment is repeated.

[0151] Time-kill kinetics

[0152] Time-kill studies with meropenem / BP1 were performed on E. coli NDM-1 and K. pneumoniae IMP-8. For both strains, the combination of carbapenem and MBL inhibitor resulted in a decrease in CFU / mL relative to the initial bacterial density at increasing time points (0, 1, 2, 4, 6, 8, and 24 hours) at all different concentrations of the MIC tested (2x, 4x, 8x MIC) (see Figure 1 When treated with meropenem / BP1 at 4x and 8x MIC for 4 hours, a 3 log reduction in the number of CFU / ml was observed 10 6 hours after treatment with the combination molecule, a 3 log increase of 2x the MIC was observed 10 reduce.

[0153] The time-kill kinetic results indicated that the combination of the MBL inhibitor (BP1) and the carbapenem antibiotic (meropenem) had bactericidal activity against the tested strains of carbapenem-resistant Enterobacteriaceae.

[0154] Combination therapy of selected BP compounds with meropenem was compared with monotherapy of meropenem alone. Analysis of the results is given in Tables 1 and 2.

[0155] Table 1: Comparative analysis of combination therapy and monotherapy

[0156]

[0157] The lowest dose of 32 mg / L BP + 0.5 mg / L meropenem was analyzed

[0158] Table 2: Statistical analysis of the differences shown by combination therapy compared with monotherapy and no therapy

[0159]

[0160] * represents the average log of all BP compounds at each time point 10 cfu / ml.

[0161] The performance of BP compounds is very similar (P = 0.9986)

[0162] The lowest dose of 32 mg / L BP + 0.5 mg / L meropenem was analyzed

[0163] Cell viability

[0164] The results of cell viability assay are shown in Table 3.

[0165] Table 3: Half maximal inhibitory concentrations of selected metallo-β-lactamase inhibitors (MBLI) against HepG2 cells

[0166]

[0167] Cytotoxicity

[0168] In vitro studies demonstrated that compound BP1 is safe at active concentrations. Effects on cell lines were only seen at high concentrations, which were much higher than the MIC reported in this study.

[0169] Table 4

[0170] MICs (in μg / mL and μM) of a combination of a carbapenem antibiotic (meropenem), a β-lactam metallo-β-lactamase inhibitor (BP1), and a metal chelator (TG1).

[0171]

[0172]

[0173] *Combination of meropenem and metal chelator (TG1)

[0174] **Combination of meropenem and β-lactam MBL inhibitor (BP1)

[0175] ***Meropenem only

[0176] ****Metal Chelators (TG1) only

[0177] *****β-lactam MBL inhibitors only (BP1)

[0178] In vivo pharmacokinetics

[0179] This study was approved by the institutional Animal Research Ethics Committee of the University of KwaZulu-Natal (approval reference number AREC / 013 / 016D). Female Balb / c (6-8 weeks old) were purchased from the Biomedical Resources Unit (BRU). Experimental animals received a combination of meropenem (10 mg / kg body weight) and BP1 (10 mg / kg body weight) by intraperitoneal administration. Animals were then regularly sacrificed at 0, 5, 15, 30, 45, 60, 90 and 120 minutes after administration to determine the plasma pharmacokinetics of meropenem and BP1, respectively. Plasma was collected by cardiac puncture and stored at -80°C.

[0180] LC-MS quantification

[0181] An Agilent Series 1100 (Agilent Technologies, Waldbronn Germany) liquid chromatography system was coupled to a maXis 4G quadrupole time-of-flight mass spectrometry (Q-TOF-MS) instrument (Bruker Daltonics, Bremen, Germany).

[0182] use Chromatographic separation was achieved on a fast F5 column (5 cm × 2.1 mm, 2.7 μm particle size) with a gradient mobile phase consisting of Millipore water (0.1% v / v formic acid) (A) and acetonitrile (0.1% v / v formic acid) (B). The gradient started from 5.0% to 95.0% B in 8.0 min, then held at 95% for 2.0 min, and then returned to 5% in 1 min. The column equilibration time was 4 min with a flow rate of 0.4 mL min -1 , and the column oven temperature was 25° C. The injection volume was 10 μL, and the total run time of the method was 15 minutes.

[0183] Quantitative and qualitative studies were performed using the MS mode via the ESI interface with the following source parameters: end plate offset -500 V; capillary 5 kV; nitrogen nebulizer gas 1.5 bar; drying gas 8.0 L min -1 , and drying temperature 200°C. The optimized molecular ion of BP1 was m / z 325.1, the optimized molecular ion of meropenem was m / z 384.2, and the optimized molecular ion of ampicillin (IS) was m / z 350.1. The results were analyzed using Data Analysis 4.0SP 5 (Bruker Daltonics). All data are expressed as mean ± SD.

[0184] In vivo infection model

[0185] This study was approved by the Institutional Animal Research Ethics Committee of the University of KwaZulu-Natal (approval reference number AREC / 081 / 015D).Female Balb / c (6-8 weeks old) were purchased from the Biomedical Resources Unit (BRU).

[0186] All animal studies were approved by the Institutional Animal Research Ethics Committee of the University of KwaZulu-Natal (approval number AREC / 081 / 015D). The thigh infection protocol was performed as described by Michail et al. (2013). Briefly, six-week-old, pathogen-free male Bagg inbred albino c-line (BALB / c) mice weighing 20-25 g (n=40) were rendered neutropenic (neutrophils <100 / mm) by pretreatment with cyclophosphamide (ip) on day 4 (150 mg / kg) and day 1 (100 mg / kg).3 Two hours before the start of treatment with the combination of meropenem and BP1 (100 mg / kg body weight each), 10 7 -10 8 The left thigh was infected with a bacterial suspension of Klebsiella pneumoniae 449 (100 μL) at CFU / ml. The mice were randomly divided into two groups, an infection control group and a treatment group. At 2 hours, 4 hours, 6 hours and 8 hours after treatment, the mice were humanely euthanized by an overdose of halothane. The left thigh muscle was then aseptically removed and homogenized in 5 ml of phosphate buffered saline (PBS). The homogenate was serially diluted and plated on a Miller-Hayden agar plate without antibiotics and incubated at 35°C for 24 hours. After the incubation period, the growth of the plate was evaluated and quantitative counts were performed using colony forming units (CFU), and the titer was then expressed as log10 CFU / thigh muscle.

[0187] result

[0188] In vivo pharmacokinetics

[0189] The results are Figure 4 and are listed in Table 5 below.

[0190] Table 5

[0191] Pharmacokinetics of BP1 and meropenem in healthy mouse models. Data are presented as mean ± SD (n = 3).

[0192]

[0193] It was found that meropenem and BP1 showed complementary pharmacokinetic properties.

[0194] In vivo infection model

[0195] The results are as follows Figure 5 and 6 The graphs show changes in thigh CFU counts after treatment with BP1 over a 24-hour period (data presented as mean±SD (n=3)) and plasma BP1 concentrations over an 8-hour treatment period (data presented as mean±SD (n=3)).

[0196] Table 6 shows the minimum inhibitory concentration (MIC) of meropenem when co-administered with all synthesized and tested metallo-β-lactamase (MBL) inhibitors of general formula (I) against two different carbapenemase-producing microorganisms.

[0197] Table 6

[0198] Minimum inhibitory concentration (MIC) of meropenem co-administered with BP compounds against carbapenemase-producing microorganisms

[0199] For bacterial isolates (E. coli NDM-1 and K. pneumoniae 449), the minimum inhibitory concentration (MIC) of all BPs administered alone was >64 μg / ml, and the MIC of meropenem administered alone was >32 μg / ml

[0200] Co-administration of meropenem and BP inhibited the growth of bacterial isolates at different concentrations

[0201] a = concentration of meropenem (μg / ml)

[0202] b = BP concentration (μg / ml)

[0203] · 1 Concentrations of meropenem and BP that resulted in inhibition

[0204]

[0205]

[0206]

[0207]

[0208] The combination of the compound of formula (I) and a β-lactam antibiotic has been found to be effective in vitro at concentrations that have no cytotoxic effect on cell lines. The bactericidal activity and effect of the serum on the administered β-lactam antibiotic when used in combination with the compound of formula (I) against metallo-β-lactamase producing Enterobacteriaceae bacteria has been determined.

[0209] MIC, killing time kinetics and serum assays were performed using broth microdilution techniques according to CLSI guidelines. It was found that in the presence of the compound of formula (I), the β-lactam antibiotics restored their activity against CRE-producing MBL, with the minimum inhibitory concentration (MIC) values ​​reduced to concentrations as low as 0.03 mcg / mL. The combination has the ability to maintain its bactericidal activity against resistant Enterobacteriaceae. The presence of serum also had no significant effect on the combination.

Claims

1. A metallo-β-lactamase (MBL) inhibitor having the general formula (I) CAL (I) in: C is a functionalized aza-cycloalkane zinc chelating moiety, L is a β-lactam moiety which may be functionalized, and A is a C1-C 10 Straight or circular linkers.

2. The inhibitor according to claim 1, wherein the functionalized aza-cycloalkane zinc chelating moiety C is a functionalized aza-cyclononane moiety.

3. The inhibitor of claim 2, wherein the functionalized aza-cycloalkane zinc chelating moiety C is a substituted 1,4,7-triazacyclononane moiety.

4. The inhibitor of claim 3, wherein the substituted 1,4,7-triazacyclononane moiety is selected from the group consisting of a 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) moiety, a 1,4,7-triazacyclononane-1,4,7-triglutaric acid (NOTGA) moiety, a 1,4,7-triazacyclononane-1-succinic acid-4,7-diacetic acid (NODASA) moiety, a 1,4,7-triazacyclononane-1-glutaric acid-4,7 -diacetic acid (NODAGA) moiety, 1,4,7-triazacyclononane-1,4-diacetic acid-7-p-hydroxyphenyl-acetic acid (NODAPA) moiety, 1,4,7-tris(2-pyridylmethyl)-1,4,7-triazacyclononane moiety, 4,7-bis(2-pyridylmethyl)-1,4,7-triazacyclononane-1-acetic acid and 1-[(5-carboxy-2-methylpyridyl)]-4,7-bis(2-methylpyridyl)-1,4,7-triazacyclononane moiety.

5. The inhibitor of claim 3 or claim 4, wherein the linker A is attached to the nitrogen atom of the substituted 1,4,7-triazacyclononane moiety.

6. The inhibitor according to any one of claims 1 to 5, wherein the linker A is a moiety selected from the group consisting of a saturated monoacid moiety, an unsaturated monoacid moiety, a saturated diacid moiety, an unsaturated diacid moiety, and esters of these moieties.

7. The inhibitor according to claim 6, wherein the linker A is a moiety selected from the group consisting of an acetic acid moiety, a glutaric acid moiety, a maleic acid moiety, a fumaric acid moiety, a succinic acid moiety, a p-hydroxyphenylacetic acid moiety, and esters of these moieties.

8. The inhibitor according to any one of claims 1 to 7, wherein the β-lactam moiety L is selected from the group consisting of a cephalosporin moiety, a penem moiety and a carbapenem moiety.

9. The inhibitor according to claim 8, wherein the β-lactam moiety L is derived from a cephalosporin, a penem or a carbapenem selected from:

10. The inhibitor according to any one of claims 1 to 9, wherein the linker A is connected to the β-lactam moiety L via an amide bond located at the alpha position to the carbonyl group of the β-lactam ring.

11. The inhibitor according to claim 1, which is selected from the following compounds:

12. A pharmaceutical composition comprising the metallo-β-lactamase (MBL) inhibitor according to any one of claims 1 to 11 and one or more pharmaceutically acceptable carriers or excipients.

13. Use of a metallo-β-lactamase (MBL) inhibitor according to any one of claims 1 to 11 in the preparation of a medicament for treating or preventing bacterial infections in humans or non-human mammals.

14. A metallo-β-lactamase (MBL) inhibitor according to any one of claims 1 to 11 or a pharmaceutical composition according to claim 12 for use in a method of treating or preventing a bacterial infection in a human or non-human mammal.

15. The inhibitor for use or the pharmaceutical composition for use according to claim 14, wherein the inhibitor or the pharmaceutical composition is for simultaneous or sequential administration of the metallo-β-lactamase (MBL) inhibitor with a β-lactam antibiotic.

16. A kit for treating or preventing bacterial infection in a human or non-human mammal, the kit comprising at least one of: (i) a metallo-β-lactamase (MBL) inhibitor according to any one of claims 1 to 11; and (ii) a pharmaceutical composition according to claim 12.

17. The kit of claim 16, further comprising one or more β-lactam antibiotics.

18. The kit of claim 17, wherein the one or more β-lactam antibiotics are selected from the group consisting of meropenem, imipenem, doripenem, ertapenem, and combinations thereof.

19. A method for treating or preventing bacterial infection in a human or non-human mammal, the method comprising the step of administering to the human or non-human mammal an effective amount of a metallo-β-lactamase (MBL) inhibitor according to any one of claims 1 to 11 or a pharmaceutical composition according to claim 12.

20. The method of claim 19, further comprising administering one or more β-lactam antibiotics to the human or the non-human mammal.

21. The method of claim 20, wherein the one or more β-lactam antibiotics are selected from the group consisting of meropenem, imipenem, doripenem, ertapenem, and combinations thereof.

22. The use according to claim 13, the inhibitor for use according to claim 14 or claim 15, or the pharmaceutical composition for use, the kit according to any one of claims 16 to 18, or the method according to any one of claims 19 to 21, wherein the bacterial infection is caused by a bacterial strain selected from the group consisting of: Escherichia coli IMP-1, Escherichia coli NDM-1, Escherichia coli VIM-1, Enterobacter cloacae VIM-1, Enterobacter cloacae IMP-1, Klebsiella pneumoniae IMP-8, Klebsiella pneumoniae VIM-1, Klebsiella pneumoniae IMP-1, Klebsiella pneumoniae NDM and Providencia retgeri NDM and combinations thereof.

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