LFM-A13

Alias: LFM-A13; LFM A13; LFM A13

Cat No.:V0647 Purity: ≥98%

COA Product Data Instructions for use SDS

LFM-A13 (LFM-A1-3) is a novel, potent and specific Brutons tyrosine kinase (BTK) inhibitor with potential anticancer activity.

LFM-A13 Chemical Structure CAS No.: 244240-24-2
Product category: BTK

This product is for research use only, not for human use. We do not sell to patients.

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LFM-A13

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Top Publications Citing lnvivochem Products

Nature 2021, 597(7874):119-125.

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Immunity 2023,56(3):620-634.e11.

J Am Chem Soc 2022,144:21831-21836.

Cell 2020,183:1202-1218.e25.

Science Adv 2022:8,eabm9427.

Nature 2021,597(7874):119-125.

Cell 2020,183:1202-1218.e25.

PNAS Nexus 2022,1(3):pgac063.

ACS Nano 2023,17(10):9110-9125.

Biomaterial 2023 Sep16:302:122333.

Purity & Quality Control Documentation

Current batch：

Purity: ≥98%

COA

MSDS

Instructions

Product Description
Bioactivity & Protocols
Physicochemical Properties
Solubility Data
Calculators
Biological Data

Product Description

LFM-A13 (LFM-A1-3) is a novel, potent and specific Bruton's tyrosine kinase (BTK) inhibitor with potential anticancer activity. It inhibits BTK with an IC50 of 2.5 μM, and shows >100-fold selectivity over other protein kinases such as JAK1, JAK2, HCK, EGFR,and IRK. LFM-A13 inhibited recombinant BTK expressed in a baculovirus expression vector system. Besides its remarkable potency in BTK kinase assays, LFM-A13 was also found to be a highly specific inhibitor of Polo-like kinases.LFM-A13shows high in vivo anticancer efficacy in BALB/c mice bearing BCL-1 leukemia.

Biological Activity I Assay Protocols (From Reference)

ln Vitro	At an IC50 of 6.2 ± 0.3 μg/mL (= 17.2 ± 0.8 μM), LFM-A13 strongly suppresses BTK activity. The LFM-A13 estimated Kis values for BTK, JAK1, JAK3, IRK, EGFR, and HCK are 1.4, 110, 148, 31.6, 166, and 214 μM. Ceramide-induced apoptosis in ALL-1 cells is chemosensitive to LFM-A13 (200 μM)[1]. Epo-induced phosphorylation of EpoR, Jak2, Btk, Stat5, and Erk1/2 in R10 cells is suppressed by LFM-A13 (100 μM). In COS cells, LFM-A13 (100 μM) suppresses auto-phosphorylation of Jak2, Tec, and Btk, but not Lyn kinase auto-phosphorylation[2]. Potently inhibiting Plx1 at an IC50 of 10 μM, LFM-A13 also inhibits BRK, BMX, FYN, and has IC50s of 267, 281, 240, and 215 μM[4]. Z)-
ln Vivo	For rats, LFM-A13 at 25, 50, and 100 mg/kg does not appear to be harmful. In mice, LFM-A13 (50 mg/kg, i.p., three times a week) reduces the development of malignant tumors. In BALB/c mice, LFM-A13 either by itself or in conjunction with paclitaxel exhibits a significant impact on the incidence, mean number, weight, and size of breast tumors. In mice, LFM-A13 (50 mg/kg, i.p., three times a week) dramatically reduces the expression of PLK1, cyclin D1, CDK -4, P53, and Bcl-2, but enhances the expression of p21, IκB, Bax, and caspase 3[3]. Rats exposed to 200 mg/kg of LFM-A13 do not experience hematologic toxicity. The MMTV/Neu transgenic mouse model of breast cancer shows dose-dependent anti-tumor effects when treated with LFM-A13 (10 or 50 mg/kg, ip)[4].
Animal Protocol	BALB/c micebearing BCL-1 leukemia; 50 mg/kg/day i.p.
References	[1]. Mahajan S, et al. Rational design and synthesis of a novel anti-leukemic agent targeting Bruton's tyrosine kinase (BTK), LFM-A13 [alpha-cyano-beta-hydroxy-beta-methyl-N-(2, 5-dibromophenyl)propenamide]. J Biol Chem. 1999 Apr 2;274(14):9587-99. [2]. van den Akker E, et al. The Btk inhibitor LFM-A13 is a potent inhibitor of Jak2 kinase activity. Biol Chem. 2004 May;385(5):409-13. [3]. "Sahin K, et al. LFM-A13, a potent inhibitor of polo-like kinase, inhibits breast carcinogenesis by suppressing proliferation activity and inducing apoptosis in breast tumors of mice. Invest New Drugs. 2017 Nov 15. " [4]. Uckun FM, et al. Anti-breast cancer activity of LFM-A13, a potent inhibitor of Polo-like kinase (PLK). Bioorg Med Chem. 2007 Jan 15;15(2):800-14. Epub 2006 Oct 26

These protocols are for reference only. InvivoChem does not independently validate these methods.

Physicochemical Properties

Molecular Formula	C11H8BR2N2O2
Molecular Weight	360.001420974731
CAS #	244240-24-2
Related CAS #	LFM-A13;62004-35-7
SMILES	BrC1C(NC(/C(=C(/C)\O)/C#N)=O)=CC(=CC=1)Br
InChi Key	UVSVTDVJQAJIFG-VURMDHGXSA-N
InChi Code	InChI=1S/C11H8Br2N2O2/c1-6(16)8(5-14)11(17)15-10-4-7(12)2-3-9(10)13/h2-4,16H,1H3,(H,15,17)/b8-6-
Chemical Name	2-Cyano-N-(2,5-dibromophenyl)-3-hydroxy-2-butenamide
Synonyms	LFM-A13; LFM A13; LFM A13
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)

Solubility Data

Solubility (In Vitro)	DMSO: 72 mg/mL (200.0 mM) Water:<1 mg/mL Ethanol:<1 mg/mL
Solubility (In Vivo)	Solubility in Formulation 1: ≥ 2.5 mg/mL (6.94 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: 2.5 mg/mL (6.94 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. 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. View More Solubility in Formulation 3: ≥ 2.5 mg/mL (6.94 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.)

Solubility (In Vitro)

DMSO: 72 mg/mL (200.0 mM) Water:<1 mg/mL Ethanol:<1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.94 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

Solubility in Formulation 2: 2.5 mg/mL (6.94 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (6.94 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

(Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.7778 mL	13.8889 mL	27.7778 mL
	5 mM	0.5556 mL	2.7778 mL	5.5556 mL
	10 mM	0.2778 mL	1.3889 mL	2.7778 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.

Calculator

Mass

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Molarity Calculator allows you to calculate the mass, volume, and/or concentration required for a solution, as detailed below:

Calculate the Mass of a compound required to prepare a solution of known volume and concentration
Calculate the Volume of solution required to dissolve a compound of known mass to a desired concentration
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See Example

An example of molarity calculation using the molarity calculator is shown below:
What is the mass of compound required to make a 10 mM stock solution in 5 ml of DMSO given that the molecular weight of the compound is 350.26 g/mol?

Enter 350.26 in the Molecular Weight (MW) box
Enter 10 in the Concentration box and choose the correct unit (mM)
Enter 5 in the Volume box and choose the correct unit (mL)
Click the “Calculate” button
The answer of 17.513 mg appears in the Mass box. In a similar way, you may calculate the volume and concentration.

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Dilution Calculator allows you to calculate how to dilute a stock solution of known concentrations. For example, you may Enter C1, C2 & V2 to calculate V1, as detailed below:

What volume of a given 10 mM stock solution is required to make 25 ml of a 25 μM solution?
Using the equation C1V1 = C2V2, where C1=10 mM, C2=25 μM, V2=25 ml and V1 is the unknown:

Enter 10 into the Concentration (Start) box and choose the correct unit (mM)
Enter 25 into the Concentration (End) box and select the correct unit (mM)
Enter 25 into the Volume (End) box and choose the correct unit (mL)
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The answer of 62.5 μL (0.1 ml) appears in the Volume (Start) box

Molecular formula

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Molecular Weight Calculator allows you to calculate the molar mass and elemental composition of a compound, as detailed below:

Note: Chemical formula is case sensitive: C12H18N3O4 c12h18n3o4
Instructions to calculate molar mass (molecular weight) of a chemical compound:

To calculate molar mass of a chemical compound, please enter the chemical/molecular formula and click the “Calculate’ button.

Definitions of molecular mass, molecular weight, molar mass and molar weight:

Molecular mass (or molecular weight) is the mass of one molecule of a substance and is expressed in the unified atomic mass units (u). (1 u is equal to 1/12 the mass of one atom of carbon-12)
Molar mass (molar weight) is the mass of one mole of a substance and is expressed in g/mol.

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Reconstitution Calculator allows you to calculate the volume of solvent required to reconstitute your vial.

Enter the mass of the reagent and the desired reconstitution concentration as well as the correct units
Click the “Calculate” button
The answer appears in the Volume (to add to vial) box

In vivo Formulation Calculator (Clear solution)

Step 1: Enter information below (Recommended: An additional animal to make allowance for loss during the experiment)

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Step 2: Enter in vivo formulation (This is only a calculator, not the exact formulation for a specific product. Please contact us first if there is no in vivo formulation in the solubility section.)

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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.

Note： (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.

Biological Data

The anti-apoptotic function of BTK.Wild-type and BTK-deficient (BTK−) DT40 lymphoma B cells (A) as well as BTK− DT40 cells reconstituted with wild-type or mutant human BTK (B) were treated with C2-ceramide (C2-CER), vincristine (VCR), or anti-Fas antibody, as described under “Experimental Procedures.” BTK-deficient DT40 (BTK −) cells expressing wild-type BTK, BTK(Arg525 → Gln), BTK(Arg28 → Cys), and BTK(Arg307 → Ala) were designated as BTK−,rBTK(WT), BTK−,rBTK(K−), BTK−,rBTK(mPH) and BTK−,rBTK(mSH2), respectively. Vehicle (0.1% Me2SO in phosphate-buffered saline) treated as well as drug-treated cells were maintained in culture medium for 24 h at 37 °C and 5% CO2 before harvesting. DNA from Triton X-100 lysates was analyzed for fragmentation, as described (32). bp, base pairs;WT, wild type; M, size markers. J Biol Chem . 1999 Apr 2;274(14):9587-99.

A, ribbon representation of the homology model of the BTK kinase domain. The LFM-A13 molecule is shown as a space filling model in the catalytic site of BTK. Prepared using Molscript and Raster3D programs (38, 39, 40). B, space filling representation of the backbone of the catalytic site residues of the BTK kinase domain. The C-α chain of BTK is represented as ablue ribbon. Shown in yellow, green, pink, andblue are the residues at the four corners of therectangular-shaped binding pocket (other residues in the cavity are shown in gray). A ball and stick model of the BTK inhibitor LFM-A13 is shown inmulticolor and represents the favorable orientation of this molecule in the kinase active site of BTK. J Biol Chem . 1999 Apr 2;274(14):9587-99.

Docked position of the LFM-A13 molecule (multicolor) at the catalytic site (blueribbon) of the kinase domain of BTK. Dashed linesrepresent hydrogen bonds between LFM-A13 and the kinase domain residues of BTK. J Biol Chem . 1999 Apr 2;274(14):9587-99.