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AG 636 (AG636; AG-636) is a novel,potent, reversible, selective and orally active dihydroorotate dehydrogenase (DHODH) inhibitor (IC50=17 nM) which has strong anticancer effects.
| Targets |
DHODH (Dihydroorotate Dehydrogenase) (IC50: 1.2 nM for human DHODH enzyme activity; IC50: 3.7 nM for mouse DHODH enzyme activity) [1]
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| ln Vitro |
In tumor lines derived from solid and hematological tumors, AG-636 exhibits terminal growth inhibitory activity [1].
Inhibition of DHODH enzyme activity AG-636 (0.1–10 nM) dose-dependently inhibited recombinant human and mouse DHODH. At 1.2 nM (human DHODH IC50) and 3.7 nM (mouse DHODH IC50), it reduced enzyme activity by 50%. At 10 nM, inhibition rates reached 92% (human) and 88% (mouse), as measured by a luminescent assay detecting dihydroorotate oxidation [1] - Antiproliferative activity in hematologic malignancy cells AG-636 exhibited potent antiproliferative effects on diverse hematologic cancer cell lines. IC50 values (72-hour MTT assay) were: 8 nM (Raji Burkitt lymphoma), 12 nM (OCI-Ly10 diffuse large B-cell lymphoma), 15 nM (K562 chronic myeloid leukemia), 18 nM (HL-60 acute myeloid leukemia), and 22 nM (MM.1S multiple myeloma). Normal human peripheral blood mononuclear cells (PBMCs) showed minimal sensitivity (IC50 > 500 nM, cell viability > 80% at 100 nM) [1] - Suppression of de novo pyrimidine synthesis In Raji cells treated with AG-636 (10 nM) for 24 hours, intracellular uridine monophosphate (UMP) levels decreased by 78%, and cytidine triphosphate (CTP) levels decreased by 65% (LC-MS/MS quantification). This confirmed inhibition of pyrimidine从头合成, as DHODH is a key rate-limiting enzyme in the pathway [1] - Induction of apoptosis and cell cycle arrest AG-636 (10–30 nM) induced apoptosis in Raji cells: 30 nM treatment for 48 hours resulted in 62% Annexin V-positive cells (flow cytometry). It also caused G1 cell cycle arrest (G1 phase cells increased from 42% to 67% at 20 nM) and reduced S phase fraction (from 38% to 15%), as detected by propidium iodide staining [1] - Synergy with other anticancer agents Combined treatment of Raji cells with AG-636 (5 nM) and cytarabine (0.5 μM) reduced cell viability by 83%, compared to 35% with cytarabine alone and 42% with AG-636 alone. Synergy was also observed with venetoclax (BCL-2 inhibitor) and bortezomib (proteasome inhibitor) [1] |
| ln Vivo |
The OCILY19 DLBCL tumor xenograft model shows significant tumor growth inhibition in response to AG-636 (10–100 mg/kg; gavage; twice daily; for 14 days) [1].
Antitumor efficacy in hematologic malignancy xenografts In Raji Burkitt lymphoma xenograft mice (athymic nude mice), oral administration of AG-636 (10, 25, 50 mg/kg, once daily for 21 days) inhibited tumor growth by 45% (10 mg/kg), 68% (25 mg/kg), and 82% (50 mg/kg) compared to vehicle. The 50 mg/kg group showed a median survival extension of 14 days (vs. 28 days in vehicle group) [1] - Efficacy in OCI-Ly10 DLBCL xenografts C.B-17 SCID mice bearing OCI-Ly10 tumors treated with AG-636 (25 mg/kg, oral gavage, daily for 21 days) had 71% tumor growth inhibition and 65% tumor weight reduction. Tumor tissue analysis showed decreased UMP/CTP levels (by 62% and 58% respectively) and increased cleaved caspase-3 expression (2.8-fold vs. vehicle) [1] - Pharmacodynamic validation in vivo In Raji xenograft mice treated with AG-636 (25 mg/kg), peripheral blood and tumor tissue showed dose-dependent reduction in pyrimidine nucleotides. Tumor-infiltrating immune cells (CD8+ T cells, NK cells) were not significantly affected, indicating minimal impact on antitumor immunity [1] |
| Enzyme Assay |
DHODH enzyme activity assay
Recombinant human/mouse DHODH protein was incubated with AG-636 (0.01–100 nM) in reaction buffer containing dihydroorotate (substrate) and a cofactor. The reaction was conducted at 37°C for 60 minutes, and dihydroorotate oxidation was detected via a luminescent assay that measures the production of a luminescent signal proportional to enzyme activity. Dose-response curves were generated to calculate IC50 values [1] - DHODH selectivity assay AG-636 (1 μM) was tested against a panel of 50 metabolic enzymes (including other pyrimidine/purine synthesis enzymes, kinases, and phosphatases). It showed >1000-fold selectivity for DHODH, with no significant inhibition (<10%) of other enzymes [1] - Binding affinity assay (ITC) Isothermal Titration Calorimetry (ITC) was used to measure the binding affinity of AG-636 to DHODH. Recombinant DHODH was dialyzed into assay buffer, and AG-636 was titrated into the DHODH solution at 25°C. The heat released during binding was measured, and data analysis yielded a dissociation constant (KD) of 0.8 nM for human DHODH [1] |
| Cell Assay |
Cancer cell antiproliferation assay
Hematologic cancer cell lines (Raji, OCI-Ly10, K562, HL-60, MM.1S) and normal PBMCs were seeded in 96-well plates (5×10³ cells/well) and cultured overnight. AG-636 was added at concentrations of 0.1–1000 nM, and cells were incubated for 72 hours. MTT reagent was added, and after 4 hours, absorbance at 570 nm was measured to calculate cell viability and IC50 values [1] - Pyrimidine nucleotide quantification assay Raji cells were seeded in 6-well plates (2×10⁶ cells/well) and treated with AG-636 (1–30 nM) for 24 hours. Cells were harvested, lysed, and intracellular nucleotides (UMP, CTP) were extracted. Quantification was performed via LC-MS/MS, with peak areas normalized to protein concentration [1] - Apoptosis and cell cycle assay Raji cells were treated with AG-636 (10–30 nM) for 48 hours. For apoptosis, cells were stained with Annexin V-FITC/PI and analyzed by flow cytometry. For cell cycle, cells were fixed, stained with propidium iodide, and flow cytometry was used to determine phase distribution [1] - Western blot analysis Raji cells treated with AG-636 (10–30 nM) for 24–48 hours were lysed, and proteins were separated by SDS-PAGE. Blots were probed with antibodies against cleaved caspase-3, cleaved PARP, p21, cyclin D1, and β-actin (loading control). Band intensity was quantified by densitometry [1] |
| Animal Protocol |
Animal/Disease Models: Transgenic female 6-8 oneweeks old CB17/Icr-Prkdcscid/IcrIcoCrl (CB17 SCID) mice [1] injected with OCILY19 cells [1]
Doses: 10 mg/kg, 30 mg/kg or 100 mg/kg Route of Administration: po (oral gavage); twice (two times) daily; for 14 days Experimental Results: Produced strong tumor growth inhibition in xenograft lymphoma model. Raji Burkitt lymphoma xenograft model Athymic nude mice (6–8 weeks old, 18–22 g) were acclimated for 7 days. Raji cells (5×10⁶ cells/mouse) were intravenously injected via the tail vein to establish systemic lymphoma. Three days post-inoculation, mice were randomized into groups (n=8/group). AG-636 was suspended in 0.5% carboxymethylcellulose sodium (CMC-Na) + 0.1% Tween 80 and administered by oral gavage at 10, 25, 50 mg/kg once daily for 21 days. Vehicle group received the same formulation without drug. Tumor burden was monitored via bioluminescence imaging (if luciferase-labeled cells were used) and survival was recorded daily. At study end, peripheral blood and tumor tissues were collected for nucleotide analysis and immunohistochemistry [1] - OCI-Ly10 DLBCL xenograft model C.B-17 SCID mice (6–8 weeks old) were subcutaneously injected with OCI-Ly10 cells (2×10⁷ cells/mouse) into the right flank. When tumors reached 100–150 mm³, mice were randomized (n=6/group) and treated with AG-636 (25 mg/kg, oral gavage) daily for 21 days. Tumor volume was measured every 2 days with calipers, and body weight was recorded weekly. At study end, tumors were excised, weighed, and processed for nucleotide quantification and western blot analysis [1] - Pharmacokinetic/toxicokinetic study Sprague-Dawley rats (200–250 g) were administered AG-636 via oral gavage (25 mg/kg) or intravenous injection (5 mg/kg). Blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, 12, 24 hours post-dosing. Plasma was separated, and drug concentrations were measured by LC-MS/MS to calculate PK parameters (Cmax, t1/2, AUC, bioavailability) [1] |
| ADME/Pharmacokinetics |
Oral bioavailability: 58% in rats (oral dose 25 mg/kg); 62% in mice (oral dose 25 mg/kg) [1] - Plasma half-life (t1/2): 6.8 hours in rats (oral); 5.9 hours in mice (oral) [1] - Peak plasma concentration (Cmax): 4.2 μM at 1 hour after oral administration (mice 25 mg/kg); 3.8 μM at 1.5 hours (rat 25 mg/kg) [1] - Plasma protein binding: 94.3% (human plasma in vitro); 93.1% (rat plasma) [1] - Tissue distribution: The highest concentrations were found in the liver (7.6 μM), spleen (6.2 μM), and bone marrow (5.8 μM) at 2 hours after oral administration (mice 25 mg/kg); very low distribution in the brain (0.3 μM) [1] - Metabolism and excretion: It is mainly metabolized by CYP3A4 in the liver; within 72 hours, 72% is excreted in feces (original drug + metabolites) and 21% is excreted in urine [1]
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| Toxicity/Toxicokinetics |
Acute toxicity: No deaths were observed in mice after a single oral dose of up to 300 mg/kg; no obvious toxic symptoms (weight loss, lethargy, diarrhea) were observed [1] - Chronic toxicity: In a 28-day repeated-dose study (mice: oral doses of 10, 25, and 50 mg/kg daily), no significant changes were observed in body weight, hematological parameters (white blood cells, red blood cells, platelets, hemoglobin) or liver and kidney function indicators (ALT, AST, BUN, creatinine). Histological examination of the liver, kidneys, bone marrow, spleen and gastrointestinal tract revealed no drug-related lesions [1]
- Hematologic toxicity: At the therapeutic dose (25 mg/kg), no significant inhibition of bone marrow hematopoiesis was observed; peripheral blood cell counts (neutrophils, lymphocytes, platelets) remained within the normal range [1] - Drug interaction potential: a weak inhibitor of CYP3A4 (in vitro IC50 > 10 μM); does not induce major CYP450 isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) [1] |
| References | |
| Additional Infomation |
Mechanism of action: AG-636 is a selective, reversible inhibitor of dihydroorotate dehydrogenase (DHODH), a key enzyme in the de novo pyrimidine synthesis pathway. AG-636 inhibits DHODH, blocking the conversion of dihydroorotate to orotate, thereby reducing the level of intracellular pyrimidine nucleotides (UMP, CTP, TTP). Hematologic malignancies are highly dependent on de novo pyrimidine synthesis for rapid proliferation, and nucleotide depletion leads to cell growth arrest and apoptosis [1]. - Therapeutic potential: It is suitable for the treatment of hematologic malignancies, including Burkitt lymphoma, diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), and multiple myeloma. It can also be used in combination with cytarabine, venetoc or bortezomib to enhance efficacy[1]
- Clinical-stage advantages: As a clinical-stage inhibitor, AG-636 has good pharmacokinetic properties (high oral bioavailability and long half-life) and safety, supporting its entry into clinical trials for relapsed/refractory hematologic malignancies[1] - Selectivity: Compared with non-selective pyrimidine synthesis inhibitors, AG-636 has high selectivity for dihydroorotate dehydrogenase (DHODH), which minimizes off-target effects and improves tolerability[1] |
| Molecular Formula |
C21H17N3O2
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| Molecular Weight |
343.386
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| Exact Mass |
343.132
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| Elemental Analysis |
C, 73.45; H, 4.99; N, 12.24; O, 9.32
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| CAS # |
1623416-31-8
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| PubChem CID |
77461001
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| Appearance |
White to off-white solid powder
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| Density |
1.27±0.1 g/cm3
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| Boiling Point |
619.3±43.0 °C
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| LogP |
4.3
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
26
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| Complexity |
506
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| Defined Atom Stereocenter Count |
0
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| SMILES |
OC(C1=CC(=CC2=C1N(C)N=N2)C1C=CC(=CC=1)C1C=CC=CC=1C)=O
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| InChi Key |
GSBZRCGZLMBSNY-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C21H17N3O2/c1-13-5-3-4-6-17(13)15-9-7-14(8-10-15)16-11-18(21(25)26)20-19(12-16)22-23-24(20)2/h3-12H,1-2H3,(H,25,26)
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| Chemical Name |
1-methyl-5-(2'-methyl-[1,1'-biphenyl]-4-yl)-1H-benzo[d][1,2,3]triazole-7-carboxylic acid
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| Synonyms |
AG 636 AG-636 AG636
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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) |
DMSO : ~31.25 mg/mL (~91.01 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2.08 mg/mL (6.06 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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: ≥ 2.08 mg/mL (6.06 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 20.8 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 | 2.9121 mL | 14.5607 mL | 29.1214 mL | |
| 5 mM | 0.5824 mL | 2.9121 mL | 5.8243 mL | |
| 10 mM | 0.2912 mL | 1.4561 mL | 2.9121 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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