KL-10 is a small-molecule ribosome rescue inhibitor that possesses a broad-spectrum of antimicrobial activity against bacteria. KKL-10 exhibited exceptional antimicrobial activity against both attenuated and fully virulent strains of F. tularensis in vitro and during ex vivo infection. Addition of KKL-10 to macrophages or liver cells at any time after infection by F. tularensis prevented further bacterial proliferation. When macrophages were stimulated with the proinflammatory cytokine gamma interferon before being infected by F. tularensis, addition of KKL-10 reduced intracellular bacteria by >99%, indicating that the combination of cytokine-induced stress and a nonfunctional ribosome rescue pathway is fatal to F. tularensis. KKL-10 was not cytotoxic to eukaryotic cells in culture. Therefore, KKL-10 is a good lead compound for antibiotic development.

Physicochemical Properties

Molecular Formula	C14H10BRN3O2S
Molecular Weight	364.22
Exact Mass	362.97
Elemental Analysis	C, 46.17; H, 2.77; Br, 21.94; N, 11.54; O, 8.79; S, 8.80
CAS #	952849-76-2
Related CAS #	952849-76-2
PubChem CID	16854524
Appearance	2934.99.9001
LogP	4
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	3
Heavy Atom Count	21
Complexity	379
Defined Atom Stereocenter Count	0
InChi Key	QKSDWSBGDVYRKQ-UHFFFAOYSA-N
InChi Code	InChI=1S/C14H10BrN3O2S/c1-8-2-4-9(5-3-8)13-17-18-14(20-13)16-12(19)10-6-7-11(15)21-10/h2-7H,1H3,(H,16,18,19)
Chemical Name	5-bromo-N-(5-(p-tolyl)-1,3,4-oxadiazol-2-yl)thiophene-2-carboxamide
Synonyms	KKL10; KKL 10; KKL-10
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

ln Vitro	KL-10 is a small-molecule ribosome rescue inhibitor that possesses a broad-spectrum of antimicrobial activity against bacteria. KKL-10 exhibited exceptional antimicrobial activity against both attenuated and fully virulent strains of F. tularensis in vitro and during ex vivo infection. Addition of KKL-10 to macrophages or liver cells at any time after infection by F. tularensis prevented further bacterial proliferation. When macrophages were stimulated with the proinflammatory cytokine gamma interferon before being infected by F. tularensis, addition of KKL-10 reduced intracellular bacteria by >99%, indicating that the combination of cytokine-induced stress and a nonfunctional ribosome rescue pathway is fatal to F. tularensis. KKL-10 was not cytotoxic to eukaryotic cells in culture. Therefore, KKL-10 is a good lead compound for antibiotic development.
ln Vivo	The hind legs of euthanized C57 mice were skinned and removed at the hip joint, and feet and excess muscle tissue were removed. Marrow was liberated by removing the femurs proximal to each joint and crushing them in a 70-μm nylon mesh filter in 5 ml phosphate-buffered saline (PBS) using a sterile pestle. The marrow was added to conical tubes and centrifuged at 400 × g for 10 min at room temperature. The supernatant was discarded, and the pellet was resuspended in Dulbeccos modified Eagle medium (DMEM) (ThermoFisher, USA). The cells were plated in 100-mm petri dishes at a density of 4 × 105 cells/ml in 10 ml complete macrophage differentiation medium (DMEM plus 20% L929 cell supernatant containing macrophage colony-stimulating factor [M-CSF]). The cells were supplemented with an additional 5 ml of medium on days 1 and 3, and bone marrow-derived macrophages (BMDMs) were harvested on day 7.
Enzyme Assay	For MIC assays, triplicate 2-fold serial dilutions of each compound were made in cation-adjusted Mueller-Hinton broth (CAMHB) and added to a 96-well microtiter plate. Stocks of each compound were prepared in 100% dimethyl sulfoxide (DMSO). Overnight cultures of LVS or Schu S4 were diluted to an OD600 of 0.05 in CAMHB to a final volume of 0.1 ml and added directly to the diluted compounds. The microtiter plates were incubated overnight (∼18 h) at 37°C in a humidified incubator with 5% CO2. Bacterial growth was monitored by measuring the optical density at 600 nm. The MIC was determined by observing the lowest concentration at which the compound prevented a significant increase in the optical density. To enumerate F. tularensis after exposure to various concentrations of KKL-10 or KKL-40, the contents of the MIC assay microtiter plate were removed and plated on chocolate agar at appropriate dilutions. After incubation for 48 h at 37°C and 5% CO2, colonies were counted to calculate CFU per milliliter.
Cell Assay	Cytotoxicity assays were performed using RAW 264.7 cells and a lactate dehydrogenase (LDH) release assay kit (Pierce Biochemicals, USA) following the manufacturers instructions.
Animal Protocol	C57 mice
References	[1]. Inhibitors of Ribosome Rescue Arrest Growth of Francisella tularensis at All Stages of Intracellular Replication. Antimicrob Agents Chemother. 2016 May 23;60(6):3276-82. [2]. Inhibitors of Ribosome Rescue Arrest Growth of Francisella tularensis at All Stages of Intracellular Replication. Antimicrob Agents Chemother. 2016 May 23;60(6):3276-82.

Solubility Data

Solubility (In Vitro)

DMSO : 2.78~3 mg/mL ( 7.63 ~8.23 mM)

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	2.7456 mL	13.7280 mL	27.4559 mL
	5 mM	0.5491 mL	2.7456 mL	5.4912 mL
	10 mM	0.2746 mL	1.3728 mL	2.7456 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.