| Size | Price | |
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| 10mg | ||
| 25mg | ||
| 50mg | ||
| 100mg | ||
| 250mg |
Purity: ≥98%
AFN-1252 tosylate monohydrate (API-1252; AFN-12520000; Debio-1452) is a novel and potent inhibitor of enoyl-(acyl-carrier protein) reductase Fabl with the potential for the treatment of acute bacterial skin. AFN-1252 exhibits typical MIC(90) values of ≤0·015 μg/ml against diverse clinical isolates of S. aureus, orally bioavailable absorption, long elimination half-live and efficacy in animal models. AFN-1252 displays a Staphylococcus-specific spectrum of activity.
| Targets |
FabI/enoyl-acyl carrier protein reductase
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|---|---|
| ln Vitro |
AFN-1252, a potent inhibitor of enoyl-acyl carrier protein reductase (FabI), inhibited all clinical isolates of Staphylococcus aureus (n = 502) and Staphylococcus epidermidis (n = 51) tested, including methicillin (meticillin)-resistant isolates, at concentrations of 4 microg/ml) against clinical isolates of Streptococcus pneumoniae, beta-hemolytic streptococci, Enterococcus spp., Enterobacteriaceae, nonfermentative gram-negative bacilli, and Moraxella catarrhalis. These data support the continued development of AFN-1252 for the treatment of patients with resistant staphylococcal infections[1].
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| ln Vivo |
Melioidosis is a tropical bacterial infection caused by Burkholderia pseudomallei (B. pseudomallei; Bpm), a Gram-negative bacterium. Current therapeutic options are largely limited to trimethoprim-sulfamethoxazole and β-lactam drugs, and the treatment duration is about 4 months. Moreover, resistance has been reported to these drugs. Hence, there is a pressing need to develop new antibiotics for Melioidosis. Inhibition of enoyl-ACP reducatase (FabI), a key enzyme in the fatty acid biosynthesis pathway has shown significant promise for antibacterial drug development. FabI has been identified as the major enoyl-ACP reductase present in B. pseudomallei. In this study, we evaluated AFN-1252, a Staphylococcus aureus FabI inhibitor currently in clinical development, for its potential to bind to BpmFabI enzyme and inhibit B. pseudomallei bacterial growth. AFN-1252 stabilized BpmFabI and inhibited the enzyme activity with an IC50 of 9.6 nM. It showed good antibacterial activity against B. pseudomallei R15 strain, isolated from a melioidosis patient (MIC of 2.35 mg/L). X-ray structure of BpmFabI with AFN-1252 was determined at a resolution of 2.3 Å. Complex of BpmFabI with AFN-1252 formed a symmetrical tetrameric structure with one molecule of AFN-1252 bound to each monomeric subunit. The kinetic and thermal melting studies supported the finding that AFN-1252 can bind to BpmFabI independent of cofactor. The structural and mechanistic insights from these studies might help the rational design and development of new FabI inhibitors[2].
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| Enzyme Assay |
BpmFabI enzyme inhibition assay for AFN-1252[2]
AFN-1252 was synthesized in-house using a published synthetic scheme.38 The potency of AFN-1252 to inhibit BpmFabI was evaluated in a spectrophotometric assay by monitoring the oxidation of the cofactor NADH.33 Buffer used for the assay was 30 mM PIPES, pH 6.8, containing 150 mM NaCl, and 1 mM EDTA. 175 nM BpmFabI enzyme was used in the assay. Michaelis–Menton constant (Km) and Kcat were determined from the enzyme activity at increasing concentrations of crotonyl-CoA. The values of Km (257 µM) and Kcat (307 min−1) were slightly higher than the reported values (188 µM and 215 min−1, respectively). To determine the IC50, AFN-1252 was preincubated with BpmFabI for 30 min and the reaction was started by adding substrate mix containing crotonyl-CoA (300 µM) and NADH (375 µM). The oxidation of NADH was monitored by following the decrease of absorbance at 340 nm. IC50 value was determined by fitting the dose-response data to sigmoidal dose response (variable slope) curve using Graphpad Prism software V4. To determine the mechanism of binding, kinetic studies were carried out at different concentrations of inhibitor and varying the concentration of NADH at a fixed concentration of crotonoyl-CoA (300 µM) and also by varying the concentrations of crotonoyl-CoA keeping NADH concentration fixed at 375 µM. Lineweaver–Burk plots were subsequently generated to determine the mechanism of binding of AFN-1252 to BpmFabI.
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| Cell Assay |
Minimum inhibitory concentration (MIC) determination was carried out using the microdilution technique described by Wiegand et al. with some modifications.39 A twofold serial dilution of the compound was prepared in Brain Heart infusion broth (BHIB) and dispensed into 96-well plate. An inoculum of 1 × 108 cfu/mL of BpR15 was added into each well and incubated at 37°C for 24 h. Growth control (bacterial inoculum only), sterility control (broth only) and positive control (bacterial inoculum with Triclosan) wells were prepared and incubated simultaneously. The MIC, defined as the lowest concentration of AFN-1252 that inhibited visible growth of BpR15, was recorded. The MIC results are average of n = 2[2].
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| Animal Protocol |
Burkholderia pseudomallei strain R15 (herein referred to as BpR15) was isolated from an individual who succumbed to melioidosis at the Kuala Lumpur Hospital in Malaysia.40 BpR15 was routinely cultured on Ashdown agar at 37°C and overnight bacterial cultures were prepared in BHIB. AFN-1252 was dissolved in dimethyl sulfoxide (DMSO) and stored at −20°C until use[2].
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| References |
[5]. Yao J, Ericson ME, Frank MW, Rock CO. Enoyl-Acyl Carrier Protein Reductase I (FabI) is Essential for the Intracellular Growth of Listeria monocytogenes. Infect Immun. 2016 Oct 10. pii: IAI.00647-16. PubMed PMID: 27736774. |
| Molecular Formula |
C29H31N3O7S
|
|---|---|
| Molecular Weight |
565.637346506119
|
| Exact Mass |
565.188
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| Elemental Analysis |
C, 61.58; H, 5.52; N, 7.43; O, 19.80; S, 5.67
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| CAS # |
1047981-30-5
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| Related CAS # |
1047981-31-6;620175-39-5;1047981-30-5 (tosylate hydrate);
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| PubChem CID |
25029351
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| Appearance |
Typically exists as solids (or liquids in special cases) at room temperature
|
| LogP |
6.04
|
| tPSA |
150.91
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| SMILES |
Cc1ccc(cc1)S(=O)(=O)O.Cc1c2ccccc2oc1CN(C)C(=O)/C=C/c3cc4c(nc3)NC(=O)CC4.O
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| InChi Key |
OUSNFNLWEQZRCQ-WRQJSNHTSA-N
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| InChi Code |
1S/C22H21N3O3.C7H8O3S.H2O/c1-14-17-5-3-4-6-18(17)28-19(14)13-25(2)21(27)10-7-15-11-16-8-9-20(26)24-22(16)23-12-151-6-2-4-7(5-3-6)11(8,9)10/h3-7,10-12H,8-9,13H2,1-2H3,(H,23,24,26)2-5H,1H3,(H,8,9,10)1H2/b10-7+
|
| Chemical Name |
(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamide
4-methylbenzenesulfonate hydrate
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| Synonyms |
AFN-1252 tosylate monohydrate; AFN-1252 tosylate; AFN1252; AFN-12520000; API-1252 tosylate; AFN-1252; Debio 1452; AFN 1252; API-1252; AFN-1252 tosylate monohydrate; 1047981-30-5; API-1252 tosylate monohydrate; UNII-004HXS4MNN; 004HXS4MNN; 2-Propenamide, N-methyl-N-((3-methyl-2-benzofuranyl)methyl)-3-(5,6,7,8-tetrahydro-7-oxo-1,8-naphthyridin-3-yl)-, (2E)-, 4-methylbenzenesulfonate, hydrate (1:1:1); (E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamide 4-methylbenzenesulfonate hydrate; SCHEMBL726863;
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| HS Tariff Code |
2934.99.9001
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| 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)
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| 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
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|---|---|
| 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
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] 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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.7679 mL | 8.8395 mL | 17.6791 mL | |
| 5 mM | 0.3536 mL | 1.7679 mL | 3.5358 mL | |
| 10 mM | 0.1768 mL | 0.8840 mL | 1.7679 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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