Cat. No.: HY-B0083

Leflunomide

(Synonyms: HWA486; RS-34821; SU101)

17 Cited Publications

Customer Review

Based on 17 publication(s) in Google Scholar

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

For research use only. We do not sell to patients.

Purity: 99.82%
CAS No.: 75706-12-6
Formula: C₁₂H₉F₃N₂O₂
Molecular Weight:270.21

Storage:

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

Leflunomide Chemical Structure

CAS No.: 75706-12-6

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Biological Activity
- Description
- Cellular Effect
- In Vitro
- Clinical Trial
Chemical Information
Publications (17)
Solvent & Solubility
- In Vitro
- In Vivo
Protocol
Purity & Documentation
References
Help & FAQs
Bioz

Biological Activity

Description

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

All Endogenous Metabolite Isoforms :

Cellular Effect

Cell Line	Type	Value	Description	References
ASPC1	IC₅₀	123 μM Compound: 58	Antiproliferative activity against human ASPC1 cells assessed as reduction in cell viability incubated for 24 hrs by CCK-8 assay Antiproliferative activity against human ASPC1 cells assessed as reduction in cell viability incubated for 24 hrs by CCK-8 assay	[PMID: 38071792]
BXPC-3	IC₅₀	32 μM Compound: 58	Antiproliferative activity against human BXPC-3 cells assessed as reduction in cell viability measured after 72 hrs by MTT assay Antiproliferative activity against human BXPC-3 cells assessed as reduction in cell viability measured after 72 hrs by MTT assay	[PMID: 38071792]
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]
Jurkat	IC₅₀	34.6 μM Compound: 1 (leflunomide)	In vitro inhibition of Jurkat cell proliferation. In vitro inhibition of Jurkat cell proliferation.	[PMID: 11384243]
Jurkat	IC₅₀	41.4 μM Compound: 1	In vitro inhibitory activity against Jurkat cell proliferation In vitro inhibitory activity against Jurkat cell proliferation	[PMID: 9719606]
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]
MIA PaCa-2	IC₅₀	32 μM Compound: 58	Antiproliferative activity against human MIA PaCa-2 cells assessed as reduction in cell viability incubated for 24 hrs by CCK8 assay Antiproliferative activity against human MIA PaCa-2 cells assessed as reduction in cell viability incubated for 24 hrs by CCK8 assay	[PMID: 38071792]
MT4	CC₅₀	178.5 μM Compound: 46	Cytotoxicity against human MT4 cells assessed as reduction in cell viability incubated for 72 hrs by celltiter glo assay Cytotoxicity against human MT4 cells assessed as reduction in cell viability incubated for 72 hrs by celltiter glo assay	[PMID: 38160620]
PANC-1	IC₅₀	47 μM Compound: 58	Antiproliferative activity against human PANC-1 cells assessed as reduction in cell viability incubated for 24 hrs by CCK-8 assay Antiproliferative activity against human PANC-1 cells assessed as reduction in cell viability incubated for 24 hrs by CCK-8 assay	[PMID: 38071792]
Sf21	IC₅₀	111.6 μM Compound: Leflunomide	Inhibition of Sprague-Dawley rat Bsep expressed in plasma membrane vesicles of Sf21 cells assessed as inhibition of ATP-dependent [3H]taurocholate uptake Inhibition of Sprague-Dawley rat Bsep expressed in plasma membrane vesicles of Sf21 cells assessed as inhibition of ATP-dependent [3H]taurocholate uptake	[PMID: 21965623]
Sf21	IC₅₀	177.4 μM Compound: Leflunomide	Inhibition of human BSEP expressed in plasma membrane vesicles of Sf21 cells assessed as inhibition of ATP-dependent [3H]taurocholate uptake Inhibition of human BSEP expressed in plasma membrane vesicles of Sf21 cells assessed as inhibition of ATP-dependent [3H]taurocholate uptake	[PMID: 21965623]

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

NCT Number	Sponsor	Condition	Start Date	Phase A549
NCT01329991	Plexxikon\|		2011-05	PHASE1

Chemical Information

CAS No. 75706-12-6
Appearance Solid
Molecular Weight 270.21
Formula C₁₂H₉F₃N₂O₂
Color White to off-white
SMILES

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

HWA486; RS-34821; SU101
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

Publications (17)

All Cell Imaging/StainingCell Proliferation/Viability AssayFlow CytometryWB

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Journal Impact Factor

Cancer Lett

FOXQ1/NDRG1 axis exacerbates hepatocellular carcinoma initiation via enhancing crosstalk between fibroblasts and tumor cells. [Abstract]

2018 Mar 28:417:21-34. PMID: 29248714

Leflunomide purchased from MedChemExpress. Usage Cited in: Cancer Lett. 2018 Mar 28:417:21-34. [Abstract]

Western blot indicates that the expression of pSTAT6 and CCL26 in HCC cells induced by ectopic NDRG1 expression is obviously attenuated by LEF treatment.
Haematologica

Pharmacological inhibition of dihydroorotate dehydrogenase induces apoptosis and differentiation in acute myeloid leukemia cells. [Abstract]

2018 Sep;103(9):1472-1483. PMID: 29880605

Leflunomide purchased from MedChemExpress. Usage Cited in: Haematologica. 2018 Sep;103(9):1472-1483. [Abstract]

Western blot analysis the changes of apoptosis-associated protein at day 18 of HL60 xenograft tumors treated with vehicle, Isobavachalcone (IBC) or Leflunomide (LEF). Expression levels of MYC and p21 in vehicle, IBC, or LEF treated tumors are estimated by western blot.
Neurotherapeutics

Transcriptionally distinct malignant neuroblastoma populations show selective response to adavosertib treatment. [Abstract]

2025 Apr;22(3):e00575. PMID: 40118716
Drug Des Devel Ther

Leflunomide Inhibits Proliferation and Induces Apoptosis via Suppressing Autophagy and PI3K/Akt Signaling Pathway in Human Bladder Cancer Cells. [Abstract]

2020 May 18;14:1897-1908. PMID: 32546957

Leflunomide purchased from MedChemExpress. Usage Cited in: Drug Des Devel Ther. 2020 May 18;14:1897-1908. [Abstract]

5637 and T24 cells were treated with DMSO or increasing concentrations of Leflunomide (12.5–200 μM, 48 h), the inferior cell condition and reduced cell viability were observed by microscopy.

Leflunomide purchased from MedChemExpress. Usage Cited in: Drug Des Devel Ther. 2020 May 18;14:1897-1908. [Abstract]

5637 and T24 cells were treated with DMSO or increasing concentrations of Leflunomide (12.5–200 μM, 48 h), the inferior cell condition and reduced cell viability were observed by MTS assay.

Leflunomide purchased from MedChemExpress. Usage Cited in: Drug Des Devel Ther. 2020 May 18;14:1897-1908. [Abstract]

5637 and T24 cells were treated with DMSO or increasing concentrations of Leflunomide (12.5–200 μM), the cell colonies were stained with crystal violet.

Leflunomide purchased from MedChemExpress. Usage Cited in: Drug Des Devel Ther. 2020 May 18;14:1897-1908. [Abstract]

Leflunomide induced apoptosis in bladder cancer cells. 5637 and T24 cells were treated with increasing concentrations of Leflunomide (12.5–200 μM) or DMSO for 24 or 48 hours, the apoptotic rate was determined by flow cytometry analysis with Annexin-V/PI staining.

Leflunomide purchased from MedChemExpress. Usage Cited in: Drug Des Devel Ther. 2020 May 18;14:1897-1908. [Abstract]

Western blot was performed to evaluate the expression of apoptosis-related proteins in 5637 and T24 cell lines treated with increasing concentrations of Leflunomide (12.5–200 μM) or DMSO for 48 hours.
Int J Mol Sci

In Vivo Chemical Screening in Zebrafish Embryos Identified FDA-Approved Drugs That Induce Differentiation of Acute Myeloid Leukemia Cells. [Abstract]

2024 Jul 16;25(14):7798. PMID: 39063039
Int J Antimicrob Agents

A new perspective on the antimicrobial mechanism of linezolid against Staphylococcus aureus revealed by proteomics and metabolomics analysis. [Abstract]

2025 Jun;65(6):107470. PMID: 40049374
Antiviral Res

Lapachol, a dihydroorotate dehydrogenase inhibitor, demonstrates antiviral activity against feline calicivirus in vitro and in vivo. [Abstract]

2026 Mar:247:106354. PMID: 41577199
Sci Rep

Butin regulates the NLRP3/CASP-1/GSDMD pyroptosis axis to inhibit proliferation and pyroptosis in human fibroblast-like synoviocytes of rheumatoid arthritis. [Abstract]

2025 Sep 26;15(1):33090. PMID: 41006493
Sci Rep

Transcriptomic analysis of the anti-tumor effects of leflunomide in prolactinoma. [Abstract]

2025 Apr 5;15(1):11703. PMID: 40188147
Sci Rep

Biocompatible ionized air alleviates rat osteoarthritis by modulating polarization from M1 to M2 macrophages. [Abstract]

2024 Dec 30;14(1):31901. PMID: 39738316
Biotechnol Bioeng

An integrated biomimetic array chip for establishment of collagen-based 3D primary human hepatocyte model for prediction of clinical drug-induced liver injury. [Abstract]

2021 Dec;118(12):4687-4698. PMID: 34478150
Chin J Integr Med

Arsenic Trioxide Combining Leflunomide Activates Nrf2-ARE-HO-1 Signaling Pathway and Protects Heart Xenografts. [Abstract]

2021 Oct;27(10):760-766. PMID: 34319507
Inhal Toxicol

GRP78 mediates mitochondrial fusion and fission in cigarette smoke-induced inflammatory responses in airway epithelial cells. [Abstract]

2024 Nov 20:1-10. PMID: 39565149
Res Sq

2025 Sep 14.
bioRxiv

Closing the loop: Teaching single-cell foundation models to learn from perturbations. [Abstract]

2025 Jul 12:2025.07.08.663754. PMID: 40672312
bioRxiv

2025 June 01.
SSRN

2024 Jan 26.

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.

View the Complete Stock Solution Preparation Table

Preparing Stock Solutions

View the Complete Stock Solution Preparation Table

Concentration Solvent Mass

1 mg

5 mg

10 mg

Concentration

Solvent

Mass

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

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

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

Mass Mass

Concentration Concentration

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Molecular Weight Molecular Weight

Dilution Calculator

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

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In Vivo:

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

Stock Solution Working Solution

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.

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.

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 Dosage
mg/kg
Animal Weight (Per Animal) Animal Weight (Per Animal)
g
Dosing Volume (Per Animal) Dosing Volume (Per Animal)
μL
Number of Animals Number of Animals

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

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

Please enter your animal formula composition:

%
DMSO +

Recommended: Keep the proportion of DMSO in working solution below 2% if your animal is weak.
%
+
+
%
Tween-80 + +
%
Saline +

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).

Working solution concentration: 0.22 mg/mL

Protocol

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.

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

Purity & Documentation

Select Batch:

Purity: 99.98%

Assay: 99.98%

ee.: 99.98%

Data Sheet (280 KB)
SDS (480 KB)
- English - EN (480 KB)
- Français - FR (480 KB)
- Deutsch - DE (480 KB)
- Norwegian - NO (480 KB)
- Español - ES (480 KB)
- Swedish - SV (480 KB)
- Italian - IT (480 KB)
- Portuguese - PT (480 KB)
COA (2659 KB)
Elemental Analysis Report (2659 KB)
HNMR (2659 KB)
CNMR (2659 KB)
RP-HPLC (2659 KB)
NP-HPLC (2659 KB)
MS (2659 KB)
LCMS (2659 KB)
IR (2659 KB)
GC (2659 KB)
OR (2659 KB)
SDS-PAGE (2659 KB)
SEC-HPLC (2659 KB)
Other Report (2659 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]

Help & FAQs

How should lyophilized recombinant proteins be reconstituted and stored?

1. Before opening the cap, centrifuge the vial at 13000 rpm for 20-30 seconds. This step will ensure that any lyophilized powder that may have adhered to the cap or walls is collected at the bottom of the vial, minimizing the risk of product loss.

Complete Stock Solution Preparation Table

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.

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

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Leflunomide

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Publications Citing Use of MedChemExpress (MCE) Leflunomide

All Endogenous Metabolite Isoforms

Biological Activity

Chemical Information

Publications (17)

Solvent & Solubility

Protocol

Purity & Documentation

References

Help & FAQs

Complete Stock Solution Preparation Table

Leflunomide Related Classifications

Cat. No.: HY-B0083