HY-B0083-100mg | Leflunomide [75706-12-6] Biotrend

Description

Leflunomide is a pyrimidine synthesis inhibitor, inhibiting dihydroorotate dehydrogenase (DHODH), and acts as a disease-modifying antirheumatic agent.

Cellular Effect

Cell Line	Type	Value	Description	References
HepG2	IC₅₀	109.5 μM Compound: Leflunomide	Cytotoxicity against human HepG2 cells assessed as reduction in glucose induced mitochondrial toxicity by measuring ATP depletion incubated for 24 hrs by CellTiter-Glo luminescent cell viability assay Cytotoxicity against human HepG2 cells assessed as reduction in glucose induced mitochondrial toxicity by measuring ATP depletion incubated for 24 hrs by CellTiter-Glo luminescent cell viability assay	[PMID: 32942072]
HT-1080	IC₅₀	30 μM Compound: Leflunomide	Antiproliferative activity against human HT-1080 cells assessed as cell growth inhibition measured after 48 hrs by SRB assay Antiproliferative activity against human HT-1080 cells assessed as cell growth inhibition measured after 48 hrs by SRB assay	[PMID: 33007554]
HT-1080	IC₅₀	38 μM Compound: Leflunomide	Antiproliferative activity against human HT-1080 cells assessed as cell growth inhibition measured after 48 hrs in presence of uridine by SRB assay Antiproliferative activity against human HT-1080 cells assessed as cell growth inhibition measured after 48 hrs in presence of uridine by SRB assay	[PMID: 33007554]
HT-29	IC₅₀	75 μM Compound: Leflunomide	Antiproliferative activity against human HT-29 cells assessed as cell growth inhibition measured after 48 hrs by SRB assay Antiproliferative activity against human HT-29 cells assessed as cell growth inhibition measured after 48 hrs by SRB assay	[PMID: 33007554]
HT-29	IC₅₀	75 μM Compound: Leflunomide	Antiproliferative activity against human HT-29 cells assessed as cell growth inhibition measured after 48 hrs in presence of uridine by SRB assay Antiproliferative activity against human HT-29 cells assessed as cell growth inhibition measured after 48 hrs in presence of uridine by SRB assay	[PMID: 33007554]
M21	IC₅₀	83 μM Compound: Leflunomide	Antiproliferative activity against human M21 cells assessed as cell growth inhibition measured after 48 hrs by SRB assay Antiproliferative activity against human M21 cells assessed as cell growth inhibition measured after 48 hrs by SRB assay	[PMID: 33007554]
M21	IC₅₀	83 μM Compound: Leflunomide	Antiproliferative activity against human M21 cells assessed as cell growth inhibition measured after 48 hrs in presence of uridine by SRB assay Antiproliferative activity against human M21 cells assessed as cell growth inhibition measured after 48 hrs in presence of uridine by SRB assay	[PMID: 33007554]
MCF7	IC₅₀	55 μM Compound: Leflunomide	Antiproliferative activity against human MCF7 cells assessed as cell growth inhibition measured after 48 hrs by SRB assay Antiproliferative activity against human MCF7 cells assessed as cell growth inhibition measured after 48 hrs by SRB assay	[PMID: 33007554]
MCF7	IC₅₀	57 μM Compound: Leflunomide	Antiproliferative activity against human MCF7 cells assessed as cell growth inhibition measured after 48 hrs in presence of uridine by SRB assay Antiproliferative activity against human MCF7 cells assessed as cell growth inhibition measured after 48 hrs in presence of uridine by SRB assay	[PMID: 33007554]

In Vitro

Leflunomide is actually a prodrug that has been shown to inhibit proliferation of mononuclear and T-cells. Leflunomide is an inhibitor of several protein tyrosine kinases, with IC₅₀ values between 30 mM and 100 mM in vitro cellular and enzymatic assays^[1].
Leflunomide is capable of inhibiting anti-CD3- and interleukin-2 (IL-2)-stimulated T cell proliferation. Leflunomide is able to inhibit p59fyn and p56lck activity in in vitro tyrosine kinase assays. Leflunomide also inhibits Ca²⁺ mobilization in Jurkat cells stimulated by anti-CD3 antibody but not in those stimulated by ionomycin. Leflunomide also inhibits distal events of anti-CD3 monoclonal antibody stimulation, namely, IL-2 production and IL-2 receptor expression on human T lymphocytes. Leflunomide also inhibits tyrosine phosphorylation in CTLL-4 cells stimulated by IL-2^[2].
Leflunomide is an immunomodulatory drug that may exert its effects by inhibiting the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH), which plays a key role in the de novo synthesis of the pyrimidine ribonucleotide uridine monophosphate (rUMP). Leflunomide prevents the expansion of activated and autoimmune lymphocytes by interfering with the cell cycle progression due to inadequate production of rUMP and utilizing mechanisms involving p53^[3].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

Clinical Trial

Molecular Weight

270.21

Formula

C₁₂H₉F₃N₂O₂

CAS No.

75706-12-6

Appearance

Solid

Color

White to off-white

SMILES

O=C(C1=C(C)ON=C1)NC2=CC=C(C(F)(F)F)C=C2

Structure Classification

Others

Initial Source

Endogenous metabolite

Shipping

Room temperature in continental US; may vary elsewhere.

Storage

Powder	-20°C	3 years
	4°C	2 years
In solvent	-80°C	1 year
	-20°C	6 months

Solvent & Solubility

In Vitro:

DMSO : ≥ 50 mg/mL (185.04 mM; Hygroscopic DMSO has a significant impact on the solubility of product, please use newly opened DMSO)

*"≥" means soluble, but saturation unknown.

Preparing
Stock Solutions

Concentration Solvent Mass	1 mg	5 mg	10 mg

1 mM	3.7008 mL	18.5041 mL	37.0083 mL
5 mM	0.7402 mL	3.7008 mL	7.4017 mL
10 mM	0.3701 mL	1.8504 mL	3.7008 mL

View the Complete Stock Solution Preparation Table

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 1 year; -20°C, 6 months. When stored at -80°C, please use it within 1 year. When stored at -20°C, please use it within 6 months.

Molarity Calculator
Dilution Calculator

Mass (g) = Concentration (mol/L) × Volume (L) × Molecular Weight (g/mol)

Concentration _(start) × Volume _(start) = Concentration _(final) × Volume _(final)

This equation is commonly abbreviated as: C₁V₁ = C₂V₂

Concentration _(start)

Volume _(start)

Concentration _(final)

Volume _(final)

In Vivo:

Select the appropriate dissolution method based on your experimental animal and administration route.

For the following dissolution methods, please ensure to first prepare a clear stock solution using an In Vitro approach and then sequentially add co-solvents:
To ensure reliable experimental results, the clarified stock solution can be appropriately stored based on storage conditions. As for the working solution for in vivo experiments, it is recommended to prepare freshly and use it on the same day.
The percentages shown for the solvents indicate their volumetric ratio in the final prepared solution. If precipitation or phase separation occurs during preparation, heat and/or sonication can be used to aid dissolution.

Protocol 1

Add each solvent one by one: 10% DMSO 40% PEG300 5% Tween-80 45% Saline
Solubility: 2.5 mg/mL (9.25 mM); Suspended solution; Need ultrasonic and warming

This protocol yields a suspended solution of 2.5 mg/mL. Suspended solution can be used for oral and intraperitoneal injection.
Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (25.0 mg/mL) to 400 μL PEG300, and mix evenly; then add 50 μL Tween-80 and mix evenly; then add 450 μL Saline to adjust the volume to 1 mL.
Preparation of Saline: Dissolve 0.9 g sodium chloride in ddH₂O and dilute to 100 mL to obtain a clear Saline solution.
Protocol 2

Add each solvent one by one: 10% DMSO 90% (20% SBE-β-CD in Saline)
Solubility: ≥ 2.5 mg/mL (9.25 mM); Clear solution

This protocol yields a clear solution of ≥ 2.5 mg/mL (saturation unknown).
Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (25.0 mg/mL) to 900 μL 20% SBE-β-CD in Saline, and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C, storage for one week): 2 g SBE-β-CD powder is dissolved in 10 mL Saline, completely dissolve until clear.
Protocol 3

Add each solvent one by one: 10% DMSO 90% Corn Oil
Solubility: ≥ 2.5 mg/mL (9.25 mM); Clear solution

This protocol yields a clear solution of ≥ 2.5 mg/mL (saturation unknown). If the continuous dosing period exceeds half a month, please choose this protocol carefully.
Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (25.0 mg/mL) to 900 μL Corn oil, and mix evenly.

In Vivo Dissolution Calculator

Please enter the basic information of animal experiments:

Dosage

mg/kg

Animal weight
(per animal)

Dosing volume
(per animal)

μL

Number of animals

Recommended: Prepare an additional quantity of animals to account for potential losses during experiments.

Please enter your animal formula composition:

DMSO +

Tween-80 +

Saline

Recommended: Keep the proportion of DMSO in working solution below 2% if your animal is weak.

The co-solvents required include: DMSO, . All of co-solvents are available by MedChemExpress (MCE). , Tween 80. All of co-solvents are available by MedChemExpress (MCE).

Calculation results:

Working solution concentration: mg/mL

Method for preparing stock solution: mg drug dissolved in μL DMSO (Stock solution concentration: mg/mL).

The concentration of the stock solution you require exceeds the measured solubility. The following solution is for reference only. If necessary, please contact MedChemExpress (MCE).

Method for preparing in vivo working solution for animal experiments: Take μL DMSO stock solution, add μL . μL , mix evenly, next add μL Tween 80, mix evenly, then add μL Saline.

If the continuous dosing period exceeds half a month, please choose this protocol carefully.

Please ensure that the stock solution in the first step is dissolved to a clear state, and add co-solvents in sequence. You can use ultrasonic heating (ultrasonic cleaner, recommended frequency 20-40 kHz), vortexing, etc. to assist dissolution.

Purity & Documentation

Purity: 99.82%

Select Batch:

SDS (480 KB)

COA (250 KB) LCMS (93 KB)

Handling Instructions (2659 KB)

References

[1]. Davis JP, et al. The immunosuppressive metabolite of leflunomide is a potent inhibitor of human dihydroorotate dehydrogenase. Biochemistry. 1996 Jan 30;35(4):1270-3. [Content Brief]

[2]. Xu X, et al. Inhibition of protein tyrosine phosphorylation in T cells by a novel immunosuppressive agent, leflunomide. J Biol Chem. 1995 May 26;270(21):12398-403. [Content Brief]

[3]. Fox RI, et al. Mechanism of action for leflunomide in rheumatoid arthritis. Clin Immunol. 1999 Dec;93(3):198-208. [Content Brief]

Kinase Assay ^[1]	DHODase activity is measured by the DCIP colorimetric assay. This is a coupled assay in which oxidation of DHO and subsequent reduction of ubiquinone are stoichiometrically equivalent to the reduction of DCIP. Reduction of DCIP is accompanied by a loss of absorbance at 610 nm (ε=21500 M/cm). The assay is performed in a 96-well microtiter plate at ambient temperature (ca. 25°C). Stock solutions of 10 mM leflunomide and A771726 are prepared in dimethyl sulfoxide (DMSO) and these are diluted with reaction buffer (100 mM Tris and 0.1 % Triton X-100, pH 8.0) to prepare working stocks of the inhibitors at varying concentrations. For each reaction, the well contained 10 nM DHODase, 68 μM DCIP, 0.16 mg/mL gelatin, the stated concentration of ubiquinone, 10 μL of an inhibitor working stock to give the stated final concentration, and reaction buffer. After a 5-min equilibration period, the reaction is initiated by addition of DHO to the stated final concentrations. The total volume of reaction mixture for each assay is 150 μL, and the final DMSO concentration is ≤ 0.01% (v/v). The reaction progress is followed by recording the loss of absorbance at 610 nm over a 10-min period (during which the velocity remained linear). Velocities are reported as the change in absorbance at 610 nm per minute, and each reported value is the average of three replicates. In experiments where the DHO or ubiquinone concentration is varied, the other substrate is held constant at 200 μM. To determine the inhibitor potency of leflunomide and A771726, the effects of varying concentrations of the two compounds on the initial velocity of the DHODase reaction is measured over a concentration range of 0.01−1.0 μM. In these experiments the DHO and ubiquinone concentrations are held constant at 200 and 100 μM, respectively. MCE has not independently confirmed the accuracy of these methods. They are for reference only.
References	[1]. Davis JP, et al. The immunosuppressive metabolite of leflunomide is a potent inhibitor of human dihydroorotate dehydrogenase. Biochemistry. 1996 Jan 30;35(4):1270-3. [Content Brief] [2]. Xu X, et al. Inhibition of protein tyrosine phosphorylation in T cells by a novel immunosuppressive agent, leflunomide. J Biol Chem. 1995 May 26;270(21):12398-403. [Content Brief] [3]. Fox RI, et al. Mechanism of action for leflunomide in rheumatoid arthritis. Clin Immunol. 1999 Dec;93(3):198-208. [Content Brief]

Kinase Assay
^[1]

DHODase activity is measured by the DCIP colorimetric assay. This is a coupled assay in which oxidation of DHO and subsequent reduction of ubiquinone are stoichiometrically equivalent to the reduction of DCIP. Reduction of DCIP is accompanied by a loss of absorbance at 610 nm (ε=21500 M/cm). The assay is performed in a 96-well microtiter plate at ambient temperature (ca. 25°C). Stock solutions of 10 mM leflunomide and A771726 are prepared in dimethyl sulfoxide (DMSO) and these are diluted with reaction buffer (100 mM Tris and 0.1 % Triton X-100, pH 8.0) to prepare working stocks of the inhibitors at varying concentrations. For each reaction, the well contained 10 nM DHODase, 68 μM DCIP, 0.16 mg/mL gelatin, the stated concentration of ubiquinone, 10 μL of an inhibitor working stock to give the stated final concentration, and reaction buffer. After a 5-min equilibration period, the reaction is initiated by addition of DHO to the stated final concentrations. The total volume of reaction mixture for each assay is 150 μL, and the final DMSO concentration is ≤ 0.01% (v/v). The reaction progress is followed by recording the loss of absorbance at 610 nm over a 10-min period (during which the velocity remained linear). Velocities are reported as the change in absorbance at 610 nm per minute, and each reported value is the average of three replicates. In experiments where the DHO or ubiquinone concentration is varied, the other substrate is held constant at 200 μM. To determine the inhibitor potency of leflunomide and A771726, the effects of varying concentrations of the two compounds on the initial velocity of the DHODase reaction is measured over a concentration range of 0.01−1.0 μM. In these experiments the DHO and ubiquinone concentrations are held constant at 200 and 100 μM, respectively.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

References

[1]. Davis JP, et al. The immunosuppressive metabolite of leflunomide is a potent inhibitor of human dihydroorotate dehydrogenase. Biochemistry. 1996 Jan 30;35(4):1270-3. [Content Brief]

[2]. Xu X, et al. Inhibition of protein tyrosine phosphorylation in T cells by a novel immunosuppressive agent, leflunomide. J Biol Chem. 1995 May 26;270(21):12398-403. [Content Brief]

[3]. Fox RI, et al. Mechanism of action for leflunomide in rheumatoid arthritis. Clin Immunol. 1999 Dec;93(3):198-208. [Content Brief]

Complete Stock Solution Preparation Table

Optional Solvent	Concentration Solvent Mass	1 mg	5 mg	10 mg	25 mg

DMSO	1 mM	3.7008 mL	18.5041 mL	37.0083 mL	92.5206 mL
	5 mM	0.7402 mL	3.7008 mL	7.4017 mL	18.5041 mL
	10 mM	0.3701 mL	1.8504 mL	3.7008 mL	9.2521 mL
	15 mM	0.2467 mL	1.2336 mL	2.4672 mL	6.1680 mL
	20 mM	0.1850 mL	0.9252 mL	1.8504 mL	4.6260 mL
	25 mM	0.1480 mL	0.7402 mL	1.4803 mL	3.7008 mL
	30 mM	0.1234 mL	0.6168 mL	1.2336 mL	3.0840 mL
	40 mM	0.0925 mL	0.4626 mL	0.9252 mL	2.3130 mL
	50 mM	0.0740 mL	0.3701 mL	0.7402 mL	1.8504 mL
	60 mM	0.0617 mL	0.3084 mL	0.6168 mL	1.5420 mL
	80 mM	0.0463 mL	0.2313 mL	0.4626 mL	1.1565 mL
	100 mM	0.0370 mL	0.1850 mL	0.3701 mL	0.9252 mL

Leflunomide Related Classifications

Cancer
Cancer Targeted Therapy Cancer Immunotherapy Cancer Metabolism and Metastasis

Metabolic Enzyme/Protease Anti-infection
Dihydroorotate Dehydrogenase Endogenous Metabolite Bacterial

Leflunomide [75706-12-6]

Cat# HY-B0083-100mg

Contact local distributor :

Brand : MedChemExpress

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Complete Stock Solution Preparation Table

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Leflunomide [75706-12-6]

Cat# HY-B0083-100mg

Contact local distributor :

Brand : MedChemExpress

Contact local distributor :

Other Forms of Leflunomide:

Leflunomide Related Antibodies

Molarity Calculator

Dilution Calculator

Complete Stock Solution Preparation Table

Leflunomide Related Classifications

Keywords:

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