| Size | Price | Stock | Qty |
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| 1mg |
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| 5mg |
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| Other Sizes |
| Targets |
WDR5 (WD repeat domain 5 protein) - binds to the WIN site of WDR5. IC50 (FP assay) = 8.6 ± 1.3 nM. Kd (ITC assay) = 11.6 ± 4.0 nM. [1]
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| ln Vitro |
The expression of MLL-fusion protein-dependent genes (HOXA9 and Meis1) is inhibited by DDO-2093 (5 μM; pretreated 7 days) dihydrochloride[1].
- DDO-2093 showed high binding affinity to WDR5 protein in a fluorescence polarization (FP) assay with an IC50 of 8.6 ± 1.3 nM. [1] - Isothermal titration calorimetry (ITC) confirmed direct binding of DDO-2093 to WDR5 with a Kd value of 11.6 ± 4.0 nM, a 1:1 binding stoichiometry, and thermodynamic parameters: ΔH = -10.2 ± 0.2 kcal·mol⁻¹, -TΔS = -0.629 kcal·mol⁻¹, indicating a synergistic enthalpy/entropy-driven process. [1] - DDO-2093 selectively inhibited MLL1 HMT activity in vitro and did not affect other methyltransferases including G9a (H3K9), DOT1L (H3K79), EZH2 (H3K27), SET8 (H4K20), and PRMT5 within the tested concentration range. [1] - DDO-2093 inhibited the proliferation of MV4-11 human acute leukemia cells (harboring MLL-AF4 fusion) with an IC50 of 8.0 ± 1.2 μM, and MOLM-13 cells (harboring MLL-AF9 fusion) with an IC50 of 9.9 ± 1.9 μM. It showed no significant toxicity against HUVEC non-cancerous human cells (IC50 > 100 μM). [1] - In MV4-11 cells, DDO-2093 (1, 2.5, 5, 10 μM for 7 days) dose-dependently reduced the mono- (H3K4me1), di- (H3K4me2), and trimethylation (H3K4me3) of histone H3 lysine 4. [1] - DDO-2093 (1, 2.5, 5 μM for 7 days) significantly decreased the expression of MLL1 target genes HOXA9 and Meis1 in MV4-11 cells. Approximately 60% reduction in HOXA9 and 70% reduction in Meis1 gene expression were observed at 5 μM. [1] - Docking studies showed that DDO-2093 occupies the WIN site of WDR5 (PDB: 4IA9). The phenyltriazole moiety forms a π-π stacking interaction with Phe133. The amide proton and carbonyl of the benzamide core form a direct H-bond with Ser91 and a water-bridged interaction with Cys261, respectively. The N-methylpiperazine moiety engages a water-mediated hydrogen bond with the backbone carbonyl of Cys261. The 2-chloro-3-methyl-4-fluoro-5-aminophenyl occupies the hydrophobic P4 pocket and forms an additional H-bond with Asp107. [1] |
| ln Vivo |
DDO-2093 dihydrochloride (20–80 mg/kg; ip; every other day for 21 days) substantially and dose-dependently decreases the size and weight of tumors[1].
- MV4-11 xenograft model: In female nude mice bearing MV4-11 xenografts, DDO-2093 administered intraperitoneally (i.p.) at doses of 20, 40, and 80 mg/kg for 21 consecutive days significantly suppressed tumor growth in a dose-dependent manner. Tumor volume growth inhibition (GI) values were 13.7%, 37.6%, and 63.9% at 20, 40, and 80 mg/kg, respectively. Tumor weight growth inhibition (TGI) was 60.0% at 80 mg/kg, comparable to the reference drug cytarabine (ARAC, 100 mg/kg). No obvious changes in mouse behavior or body weight were observed during treatment. [1] - In the same xenograft model, DDO-2093 dose-dependently reduced H3K4me1, H3K4me2, and H3K4me3 levels in tumor tissues and decreased the expression of HOXA9 and Meis1 genes, confirming the pharmacodynamic biomarker changes. [1] |
| Enzyme Assay |
- Fluorescence Polarization (FP) Assay: The binding affinities of compounds to WDR5 were evaluated using a fluorescence polarization assay. This assay measures the displacement of a fluorescently labeled probe from the WDR5 binding site. IC50 values were calculated using nonlinear regression with normalized dose-response fit using GraphPad Prism 6.0. [1]
- Isothermal Titration Calorimetry (ITC): ITC was carried out at 25°C using an ITC200 system. Compound was injected into the sample cell containing 300 μL of 20 μM WDR5 using 2 μL injections with a total of 19 injections at 2.5 min intervals from a stirring syringe (750 rpm) into the sample cell. Data were analyzed with Origin version 7.0. The binding parameters (Kd, ΔH, -TΔS, stoichiometry) were determined. [1] - In vitro MLL HMT Functional Assay: MLL1 enzymatic reactions were conducted at room temperature for 60 min in a 50 μL mixture containing methyltransferase assay buffer (50 mM HEPES, 100 mM NaCl, 1.0 mM EDTA, 5% glycerol, pH 7.8), 1 μM SAM, MLL protein complex, and test compound. Reactions were carried out in wells of a histone substrate pre-coated plate. After stopping the methylation reactions with Blocking Buffer, primary and secondary antibodies were added. HRP chemiluminescent substrates were added and luminescence was measured in a microplate reader. IC50 values were calculated using GraphPad Prism 6.0. [1] - AlphaScreen Assay for Selectivity: The AlphaScreen assay was used to evaluate compound activity on other methyltransferases (G9a, Dot1L, EZH2, PRMT5, SET8). The assay was performed in a 384-well white plate. Buffer contained 10 mM PBS, compound, SAM, and proteins mixed at room temperature for 30 min. Receptor beads and substrate antibodies were then added and incubated for 30 min, followed by addition of AlphaScreen Streptavidin coupled donor beads. The plate was read in an AlphaScreen microplate reader. [1] |
| Cell Assay |
Western Blot Analysis[1]
Cell Types: MV4-11 cells Tested Concentrations: 1, 2.5, 5, and 10 μM Incubation Duration: 7 days Experimental Results: Dose-dependently decreased the mono-, di-, and trimethylation of H3K4. - Cell Proliferation Assay (CCK8): MV4-11, MOLM-13, and K562 cells were seeded at 8,000 cells/well in 96-well plates and treated with compounds for 72 h at different concentrations in culture media containing 0.2% DMSO. Cell viability was assessed by CCK8 (WST-8) and absorbance was measured at 450 nm. For HUVEC cells, an MTT assay was used after 24 h treatment. IC50 values were calculated using GraphPad Prism 6.0. [1] - Western Blot Analysis: MV4-11 cells were pretreated with various concentrations of DDO-2093 or DMSO for 7 days. Cells were lysed in lysis buffer (50 mM Tris-Cl pH 7.6, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.2 mM PMSF, 0.1 mM NaF, 1.0 mM DTT) for 30 min. After centrifugation, protein concentration was determined by BCA assay. Equal amounts of protein were separated by SDS-PAGE, electroblotted onto PVDF membranes, blocked with 5% defatted milk, and incubated with primary antibodies (anti-H3K4me1, anti-H3K4me2, anti-H3K4me3, anti-β-actin) overnight at 4°C, followed by DyLight 800 labeled secondary antibody. Membranes were scanned using an Odyssey infrared imaging system. [1] - RNA Extraction and RT-PCR: MV4-11 cells were treated with different concentrations of DDO-2093 or DMSO for 7 days. Total RNA was isolated using TRIzol. RNA concentration was assessed, and RNA was reverse transcribed using PrimeScript RT reagent kit. Real-time PCR analysis of HOXA9 and Meis1 was performed using a STEPONE SYSTEM Fast Real Time PCR system. GAPDH was used for normalization. Primers: HOXA9 (sense: TACGTGGACTCGTTCTCGTT, antisense: CGTCGCTCGTTGAGCTAGGAAG); Meis1 (sense: GGCGATGGATGGAGTAGGC, antisense: GGGTACTGATGCCGAGTGCAG). [1] |
| Animal Protocol |
Animal/Disease Models: Female nude mice (MV4-11 human leukemia cancer xenografts)[1]
Doses: 20, 40, and 80 mg/kg Route of Administration: intraperitoneal (ip)injection; every other day for 21 days Experimental Results: Had the tumor volume growth inhibition (GI) values were calculated to be 13.7%, 37.6% and 63.9% with doses of 20 mg/kg, 40 mg/kg and 80 mg/kg, respectively. - In vivo xenograft study: Female nude mice were injected subcutaneously with MV4-11 cells (5 × 10⁶) in the right flank. When average tumor volume reached approximately 100 mm³, mice were randomized into different cohorts (n = 6 per group). DDO-2093 was administered intraperitoneally (i.p.) every other day for 21 days at doses of 20, 40, and 80 mg/kg. Cytarabine (ARAC, 100 mg/kg) was used as a reference drug. Tumor growth and body weight were monitored every 2 days. Tumor volume (TV) was calculated using the formula: (smaller diameter)² × (larger diameter)/2. GI% (growth inhibition) = [1 - (TVt - TV₀)/(CVt - CV₀)] × 100%. TGI% (tumor weight growth inhibition) = (mean W_control - mean W_tumor)/mean W_control × 100%. After treatment, tumors were harvested for Western blot and RT-PCR analysis. [1] - Initial short-time toxicity: Balb/c female mice were randomized into different groups and intraperitoneally treated with compounds for 10 days at a dose of 80 mg/kg/day. Body weight and death rate were monitored to evaluate toxicity. [1] - Subacute toxicity evaluation: Female ICR mice (6-8 weeks) were divided into 4 groups (n = 6): control group, DDO-2093 efficacious dose group (80 mg/kg), middle-dose group (200 mg/kg), and high-dose group (300 mg/kg). All mice were administered i.p. with the corresponding dose every day for 14 days. Body weights were measured and recorded. On day 15, mice were dissected, and heart, liver, spleen, lung, and kidney were extracted. Organ weights were measured, and organs were examined by hematoxylin-eosin (HE) staining. [1] |
| ADME/Pharmacokinetics |
- Permeability (PAMPA): DDO-2093 exhibited a permeability (Pe) value of 17.2 ± 4.8 × 10⁻⁶ cm/s at pH 7.4, as determined by parallel artificial membrane permeability assay (PAMPA). This represents significantly improved permeability compared to the biphenyl compound 6 (1.6 × 10⁻⁶ cm/s). [1]
- Solubility: DDO-2093 showed an aqueous solubility of 245.9 μmol/L at pH 7.4, as determined by potentiometric titration on a Gemini Profiler instrument. This represents significantly improved solubility compared to the biphenyl compound 6 (199.1 μmol/L). [1] |
| Toxicity/Toxicokinetics |
- In the initial short-time toxicity study in Balb/c mice (80 mg/kg/day i.p. for 10 days), no significant toxicity was observed based on body weight and death rate monitoring. [1]
- In the subacute toxicity evaluation in ICR mice (80, 200, 300 mg/kg/day i.p. for 14 days), no apparent signs of animal toxicity were observed during the treatment period. Body weights of treatment groups increased gradually compared to the control group. No behavioral abnormalities were observed. Organ/body weight ratios of heart, liver, spleen, lungs, and kidneys did not change significantly after administration of DDO-2093. Hematoxylin-eosin (HE) staining of organs showed no obvious tissue damage in normal mice even at the high dose (300 mg/kg). [1] |
| References | |
| Additional Infomation |
- DDO-2093 (compound 24) is a phenyltriazole derivative synthesized via click chemistry. It was optimized from the biphenyl scaffold compound DDO-2117 (compound 6) to improve physicochemical properties while maintaining high binding affinity to WDR5. [1]
- The compound binds to the WIN site of WDR5, blocking the MLL1-WDR5 protein-protein interaction, which is essential for MLL complex H3K4 methyltransferase activity. Disrupting this interaction inhibits MLL1-dependent gene expression (e.g., HOXA9 and Meis1) and suppresses leukemia cell growth. [1] - DDO-2093 showed selectivity for MLL-rearranged leukemia cells (MV4-11, MOLM-13) over K562 cells (no MLL fusion) and non-cancerous HUVEC cells. [1] - The compound demonstrated a favorable safety profile with a large therapeutic window (no significant subacute toxicity at 300 mg/kg, which is 3.75 times the efficacious dose of 80 mg/kg). [1] |
| Molecular Formula |
C29H39CL3FN9O3
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|---|---|
| Molecular Weight |
687.04
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| Exact Mass |
685.222547
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| Related CAS # |
DDO-2093;2250024-74-7
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| Appearance |
Off-white to light yellow solid powder
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| SMILES |
CC1=C(C(=CC(=C1F)N)C(=O)NC2=C(C=CC(=C2)N3C=C(N=N3)C(=O)NCCCN4CCOCC4)N5CCN(CC5)C)Cl.Cl.Cl
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| InChi Key |
RIMZKQNHTPMNER-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C29H37ClFN9O3.2ClH/c1-19-26(30)21(17-22(32)27(19)31)28(41)34-23-16-20(4-5-25(23)39-10-8-37(2)9-11-39)40-18-24(35-36-40)29(42)33-6-3-7-38-12-14-43-15-13-38;;/h4-5,16-18H,3,6-15,32H2,1-2H3,(H,33,42)(H,34,41);2*1H
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| Chemical Name |
1-[3-[(5-amino-2-chloro-4-fluoro-3-methylbenzoyl)amino]-4-(4-methylpiperazin-1-yl)phenyl]-N-(3-morpholin-4-ylpropyl)triazole-4-carboxamide;dihydrochloride
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| Synonyms |
DDO-2093 (dihydrochloride); DDO-2093 dihydrochloride; orb1745033; DDO-2093 diHCl; CHEMBL5723357; DDO-2093 DIHYDROCHLORIDE;
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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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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 :~100 mg/mL (~145.55 mM)
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| 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
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] 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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.4555 mL | 7.2776 mL | 14.5552 mL | |
| 5 mM | 0.2911 mL | 1.4555 mL | 2.9110 mL | |
| 10 mM | 0.1456 mL | 0.7278 mL | 1.4555 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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