 Chemical Structure.png)
Physicochemical Properties
| Molecular Formula | C12H14BNO5 |
| Molecular Weight | 263.05 |
| Exact Mass | 263.096 |
| Elemental Analysis | C, 54.79; H, 5.36; B, 4.11; N, 5.32; O, 30.41 |
| CAS # | 1842397-36-7 |
| Related CAS # | 1842397-36-7 (free);1842399-68-1 (etzadroxil); |
| PubChem CID | 138455079 |
| Appearance | White to off-white solid powder |
| Density | 1.4±0.1 g/cm3 |
| Index of Refraction | 1.579 |
| Hydrogen Bond Donor Count | 3 |
| Hydrogen Bond Acceptor Count | 5 |
| Rotatable Bond Count | 3 |
| Heavy Atom Count | 19 |
| Complexity | 364 |
| Defined Atom Stereocenter Count | 1 |
| SMILES | O1B([C@H](CC2C=CC=C(C(=O)O)C1=2)NC(CC)=O)O |
| InChi Key | QAGGLFCWUVSERJ-VIFPVBQESA-N |
| InChi Code | InChI=1S/C12H14BNO5/c1-2-10(15)14-9-6-7-4-3-5-8(12(16)17)11(7)19-13(9)18/h3-5,9,18H,2,6H2,1H3,(H,14,15)(H,16,17)/t9-/m0/s1 |
| Chemical Name | (3R)-2-hydroxy-3-(propanoylamino)-3,4-dihydro-1,2-benzoxaborinine-8-carboxylic acid |
| Synonyms | Ledaborbactam; UNII-G2WGD69BLI; VNRX-7145 acid; 1842397-36-7; G2WGD69BLI; VNRX-5236; VNRX-7145 active metabolite; LEDABORBACTAM [INN]; |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets | beta-lactamase[1] |
| ln Vitro |
Compound 36 (Ledaborbactam etzadroxil (VNRX-7145)) Demonstrated Nearly Complete Hydrolysis to 5 [Ledaborbactam (VNRX-5236)] in Various Matrixes across Species [1] With the goal of creating a prodrug that allowed for complete biological conversion upon absorption to the active BLI, the metabolic stability of 36 was assessed in vitro in intestinal S9, liver S9, and plasma from CD-1 mice, Sprague-Dawley rats, beagle dogs, cynomolgus monkeys, and humans. [1] In intestinal S9, 36 was rapidly cleaved with short half-lives in all species with the exception of beagle dogs (Table 7). Very short half-lives were also observed in liver S9 across all species. No effect on the half-life of 36 was observed when including or excluding NADPH in either intestinal S9 or liver S9, indicating that cytochrome P450 enzymes do not play a role in the hydrolysis. The half-life in human plasma was short at about 11 min, which was closer to what was observed in the rodent species compared to the longer half-lives in dogs (43.9 min) and monkeys (22.0 min). In addition, 5 [Ledaborbactam (VNRX-5236)] demonstrated long half-lives (>120 min) in intestinal S9, liver S9, and plasma across all species (data not shown). Overall, 36 was nearly completely hydrolyzed to 5 in all tested matrixes from all species. Compound 36 (Ledaborbactam etzadroxil (VNRX-7145)) Yielded Only One Metabolite, 5 [Ledaborbactam (VNRX-5236)], in Hepatocytes across Species [1] An in vitro study to investigate the biotransformation of 36 in cryopreserved hepatocytes from mice, rats, rabbits, beagle dogs, cynomolgus monkeys, and humans was performed. After incubation, one metabolite was detected resulting from hydrolysis of 36. The metabolism was extensive in all species with 0–5% 36 remaining. The structure of the metabolite was positively identified as 5 [Ledaborbactam (VNRX-5236)] by comparing the observed accurate mass of the metabolite with the observed accurate mass of the authentic chemical standard for 5. No other metabolites were detected in any species tested. Compound 36 (Ledaborbactam etzadroxil (VNRX-7145)) Shows High Permeability through Caco-2 Monolayers [1] In an effort to gain insight into the absorption potential of 36 in humans, a Caco-2 cell permeability study was undertaken. The apparent permeability (Papp) for 36 averaged to 9.22 × 10–6 cm/s, which classifies it as having a high absorption potential in humans (Table 8). The active BLI (5/[Ledaborbactam (VNRX-5236)]) demonstrated poor absorption potential, which further emphasized the need for the prodrug approach for oral delivery. |
| ln Vivo |
Ledaborbactam etzadroxil (5–10 mg/kg) exhibits the most consistent oral bioavailability across species (F = 61–82%) when dosed in mice, dogs, and monkeys (VNRX-7145)[1]. Ledaborbactam etzadroxil is quickly cleaved in intestine S9, with brief half-lives in all species except beagle dogs. Human plasma has a short half-life of approximately 11 minutes, which is comparable to what was shown in rodent species as opposed to the greater half-lives of dogs (43.9 min) and monkeys (22.0 min)[1]. Ledaborbactam etzadroxil is administered orally in combination with Ceftibuten to demonstrate in vivo efficacy in a deadly mouse septicemia model. The 12.9 mg/kg ED50 value[1]. In vivo efficacy was demonstrated in a lethal murine septicemia model by dosing 5 [Ledaborbactam (VNRX-5236)] (subcutaneously) and 36 (Ledaborbactam etzadroxil (VNRX-7145), orally) with ceftibuten (dosed both subcutaneously with 5 [Ledaborbactam (VNRX-5236)] and orally with 36). A comparison of ceftibuten/5[Ledaborbactam (VNRX-5236)] dosed subcutaneously and ceftibuten/36 dosed orally demonstrated similar activity with ED50 (median effective dose) values of 13.5 and 12.9 mg/kg, respectively. Additionally, the ability to demonstrate in vivo efficacy in a UTI model which more closely mimics the target cUTI indication in humans was explored. A PK study was performed to guide selection of dosing regimens for the cUTI efficacy study. Oral dosing of 10 and 90 mg/kg of 36 in mice resulted in F = 72% and F = 38% of 5 [Ledaborbactam (VNRX-5236)], respectively. The lack of dose proportionality of 5 [Ledaborbactam (VNRX-5236)] by oral dosing prompted a transition to subcutaneous dosing for the in vivo efficacy study. A PK study utilizing subcutaneous injections of 1.2, 12, and 38 mg/kg of 5 [Ledaborbactam (VNRX-5236)] showed dose proportionality of AUC (7.8, 76, and 250 mg∗h/L, respectively). These results would allow for more control over the exposure of 5 [Ledaborbactam (VNRX-5236)] in the efficacy study[1]. |
| Enzyme Assay |
Inhibition Assay Method. [1] To determine the level of inhibition of β-lactamase enzymes, compounds were diluted in PBS at pH 7.4 to yield concentrations ranging from 100 to 0.00005 µM in 96-well microtiter plates. An equal volume of diluted enzyme stock was added, and the plates were incubated at 37 o C for 15 min. Substrates were: 1) nitrocefin for p99 AmpC and OXA-48; 2) cefotaxime for SHV-5; 3) imipenem for KPC-2. Substrates were dispensed into each well at a final concentration of 100 µM. Hydrolysis of substrates were monitored at 486 nm (nitrocefin), 300 nm (imipenem), or 260 nm (cefotaxime) for 10 min using a Biotek Powerwave XS2 microplate spectrophotometer using the GEN5 software package. Initial rates of hydrolysis were compared to those in control wells (without inhibitors), and percentages of enzyme inhibition were calculated for each concentration of inhibitor. The concentration of inhibitor needed to reduce the initial rate of hydrolysis of substrate by 50% (IC50) was calculated from the residual activity of β-lactamase at 486 nm using GraFit version 7 kinetics software package. Method for metabolic stability determination. [1] Compounds (3 µM) were incubated with intestinal S9, liver S9, and plasma from human, cynomolgus monkey, beagle dog, SpragueDawley rat, and CD-1 mouse for 2 h at 37 o C. In addition, NADPH (nicotinamide adenine dinucleotide phosphate) was included and excluded to examine the involvement of cytochrome P450 in the hydrolysis. Duplicate samples were taken throughout the incubation period. After protein precipitation, the supernatants were concentrated to dryness under N2 at 40 o C. Samples were reconstituted in 300 µL of 0.1% formic acid in water and analyzed by Ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS). |
| Cell Assay |
Method for metabolite profiling in various species. [1] Cryopreserved hepatocytes from mice, rats, rabbits, beagle dogs, cynomolgus monkeys, and humans were used. Hepatocyte suspensions were incubated with 2 µM and 20 µM of 36 in triplicate at 37 o C for 240 min. S5 Total incubation volume was 1.0 mL containing 1 x 106 cells. Incubations with a positive control, verapamil (2 µM), were performed concurrently to assess phase I and phase II metabolic activities. UPLC-HR-MS/MS (high resolution tandem mass spectrometry) with reverse phase chromatography was used for metabolite identification using both positive and negative ionization. Putative metabolites of the test compound were identified by analysis of full scan data using extracted ion chromatograms and fractional mass filtering to determine related components versus false positives. Method for bidirectional permeability through Caco-2 monolayers. [1] A 5 µM solution of the test article in assay buffer (Hanks’ balanced salt solution containing 10 mM HEPES and 15 mM glucose at pH 7.4) was dosed on the apical side (A-to-B) or basolateral side (B-to-A) of the Caco-2 cell monolayers and incubated at 37 o C with 5% CO2 in a humidified incubator. Samples were taken from the donor and receiver chambers at 120 minutes and assayed by LCMS/MS for 36 and 5 [Ledaborbactam (VNRX-5236)]. Antimicrobial Susceptibility Testing Method. [1] To determine the ability of test compounds to potentiate the inhibition of the growth of bacterial strains that produce β-lactamase enzymes, classic cell-based broth microdilution MIC assays were employed. The assay was conducted in cation adjusted Mueller Hinton broth. Bacteria strains were grown for 3-5 hours in CAMHB. Test compounds were added to a 96-well microtiter plate in two-fold serial dilutions in CAMHB at 3x the final concentration range of 32 μg/mL to 0.002 μg/mL. An overlay of CAMHB containing ceftibuten S3 was added at 3x the final static concentration of 1 μg/mL. Titration of test compounds with MIC readout indicates the concentration of test article needed to sufficiently inhibit β-lactamase enzyme activity and protect the intrinsic antibacterial activity of the β-lactam. In addition to the titration of test compounds, the MICs of a panel of control β-lactamase inhibitors was also tested to ensure the strains are behaving consistently between experiments. Once the test compounds and antibiotic were added, the plates were inoculated according to CLSI broth microdilution method so that the final bacteria concentration was 5 x 105 CFU/mL. After inoculation, the plates were incubated for 16-20 hours at 37 °C in ambient air. The MIC of the test compound was determined visually. |
| Animal Protocol |
Method for determination of oral bioavailability for PK screening. [1] The parent BLIs were dissolved in DMSO (diluted to a concentration of 5% in the final formulation) and formulated in a sodium acetate/acetic acid buffer with a final pH of approximately 5.0 and dosed intravenously (IV). The alkyl- and acyloxy-esters were similarly dissolved in DMSO and formulated in 0.5% Tween 80 (polysorbate 80) in sodium acetate/acetic acid buffer with a final pH of approximately 5.0 and dosed via oral gavage (PO). Dosing groups consisted of three rats for the initial PK screens. For the PK comparison between 20 and 36, dosing groups consisted of ten rats, three to four mice, five to ten dogs, and five to ten monkeys. Blood samples were taken at various time points post-dose and analyzed for both parent and ester by UPLC-MS/MS. Oral bioavailability was calculated by dividing AUC0-t of parent (PO) by AUC0-t of parent (IV). Method for determination of oral bioavailability in mice. [1] The IV dosing formulation for 5 [Ledaborbactam (VNRX-5236)] was prepared by solubilizing in dimethylacetamide (DMA) then diluting with CMC/Tween 80 until a 2.5 mg/mL concentration was achieved [final formulation was 5% DMA:95% (0.5%CMC/0.5% Tween 80)]. The PO dosing formulation of 36 was prepared by solubilizing in DMA then diluting with propylene glycol (PG) followed by CMC/Tween 80 until the desired 1.25 mg/mL (10 mg/kg dose) and 11.25 mg/mL (90 mg/kg dose) concentrations were achieved S4 [final formulation was 5%DMA:45% PG (0.5%CMC/0.5%Tween 80)]. Compound 5 was administered by intravenous bolus via the tail vein while 36 was administered via oral gavage using a metal gavage needle. Three mice were dosed for the IV group and four mice per group for PO administration. Blood samples were taken at various time points post-dose and analyzed for both parent and ester by LC-MS/MS. Oral bioavailability was calculated by dividing AUC0- t of parent (PO) by AUC0-t of parent (IV). Method for determination of pharmacokinetics in mice after subcutaneous (SC) injections. [1] Compound 5 [Ledaborbactam (VNRX-5236)] was formulated in a sodium phosphate buffer (50 mM, pH 7) at concentrations ranging from 0.3 to 3.5 mg/mL depending on intended dose. Final concentrations to deliver the intended doses of 1.2, 12, and 38 mg/kg based on the mean weight of the study mice population were attained by the necessary dilutions with buffer. Compound 5 [Ledaborbactam (VNRX-5236)] was administered via SC injections of 0.1-0.2 mL at each dose level to groups of 6 mice each. Blood samples were taken at various time points post-dose and analyzed for both parent and ester and drug plasma concentrations were determined by UPLC-MS/MS. Method for the mouse murine UTI model. [1] To establish the ascending UTI model, groups of 5 female C3H/HeJ mice were placed on 5% glucose water for 6 days and then transurethrally infected with approximately 9 log10 CFU/mouse of E. coli UNT167-1, UNT204-1, and UNT057-1. Mice were anesthetized with ketamine HCl (40 mg/kg) and xylazine (6 mg/kg) in 0.15 mL PBS via intraperitoneal injection. Using a dissecting scope with 10x magnification, the urethral orifice was located and a tapered PE10 catheter was inserted and 50 µL of inoculum slowly injected. Treatments of ceftibuten alone (1-300 mg/kg), ceftibuten with Compound 5 [Ledaborbactam (VNRX-5236)] 1:1 (1-300 mg/kg), and amoxicillin-clavulanate 2:1 (10-300 mg/kg) were initiated 4 days postinfection and administered subcutaneously every 12 hours for 3 days. At the time treatment began, an untreated control cohort of mice was sacrificed to measure bacterial burden at the start of therapy. Seven days post infection, all mice were euthanized approximately 18 hours after the last administered dose. Kidneys and bladders were collected into sterile PBS, S6 homogenized, diluted, and plated for the determination of bacterial CFU titers. Urine was expressed manually and directly plated. |
| ADME/Pharmacokinetics | Upon dosing the pivoxyl- (20) and 3-pentylacyloxy- (36 or Ledaborbactam etzadroxil (VNRX-7145)) prodrugs in mice, dogs, and monkeys, 36 demonstrated the most consistent oral bioavailability across species (Table 6). While 20 exhibited excellent oral bioavailability in rats (F = 99%), bioavailability was considerably lower in the other three tested species. In addition, prodrugs that liberate pivalate (trimethylacetic acid) upon hydrolysis have resulted in the generation of pivaloylcarnitine which, upon elimination in the urine, leads to depletion of the limited carnitine pool in the body. While the potential toxicity resulting from carnitine depletion is dependent on the amount of pivalate dosed/generated, 36 does not have this liability and as a result, based on these data and its consistent and high oral bioavailability data across species, was selected as the candidate for further development.[1] |
| References |
[1]. Discovery of VNRX-7145 (VNRX-5236 Etzadroxil): An Orally Bioavailable β-Lactamase Inhibitor for Enterobacterales Expressing Ambler Class A, C, and D Enzymes. J Med Chem. 2021 Jul 22;64(14):10155-10166. [2]. WO2015191907. |
| Additional Infomation |
A major antimicrobial resistance mechanism in Gram-negative bacteria is the production of β-lactamase enzymes. The increasing emergence of β-lactamase-producing multi-drug-resistant "superbugs" has resulted in increases in costly hospital Emergency Department (ED) visits and hospitalizations due to the requirement for parenteral antibiotic therapy for infections caused by these difficult-to-treat bacteria. To address the lack of outpatient treatment, we initiated an iterative program combining medicinal chemistry, biochemical testing, microbiological profiling, and evaluation of oral pharmacokinetics. Lead optimization focusing on multiple smaller, more lipophilic active compounds, followed by an exploration of oral bioavailability of a variety of their respective prodrugs, provided 36 (VNRX-7145/VNRX-5236 etzadroxil), the prodrug of the boronic acid-containing β-lactamase inhibitor 5 (VNRX-5236). In vitro and in vivo studies demonstrated that 5 restored the activity of the oral cephalosporin antibiotic ceftibuten against Enterobacterales expressing Ambler class A extended-spectrum β-lactamases, class A carbapenemases, class C cephalosporinases, and class D oxacillinases.[1] Starting from a cyclic boronate template, we discovered highly potent inhibitors of Ambler class A, C, and D β-lactamase enzymes that rescued the activity of an oral cephalosporin, ceftibuten, against ceftibuten-resistant E. coli and K. pneumoniae, two frequently isolated species of Enterobacterales causing clinical infections. The synthesis of prodrugs of these active BLIs, followed by comparisons of oral bioavailability in rats, led to the selection of 36. Compound 36 demonstrated excellent oral bioavailability in rats, dogs, and monkeys. Hydrolysis of the prodrug ester to release the active BLI, 5, was demonstrated in multiple species. Compound 5 restored ceftibuten activity in a mouse model of UTI due to ESBL- and KPC-carbapenemase-producing strains of E. coli and K. pneumoniae. Compound 36 is in Phase 1 clinical studies (ClinicalTrials.gov Identifier: NCT04243863).[1] |
Solubility Data
| Solubility (In Vitro) | May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples |
| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.8016 mL | 19.0078 mL | 38.0156 mL | |
| 5 mM | 0.7603 mL | 3.8016 mL | 7.6031 mL | |
| 10 mM | 0.3802 mL | 1.9008 mL | 3.8016 mL |