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| Targets |
IC50: 84 nM (KDM1A/LSD1)[1]
DDP-38003 specifically targets histone lysine-specific demethylase 1A (KDM1A), also known as LSD1. KDM1A is a flavin adenine dinucleotide (FAD)-dependent amine oxidase that removes methyl groups from histone H3 at lysine 4 (H3K4me1/2) and lysine 9 (H3K9me1/2). Inhibition of KDM1A by DDP-38003 blocks its demethylase activity, leading to the accumulation of H3K4me2, an epigenetic mark associated with active gene transcription. This derepression of genes involved in differentiation and cell cycle control is the primary mechanism through which DDP-38003 exerts its anti-leukemic effects. DDP-38003 has an IC50 of 84 nM for KDM1A. |
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| ln Vitro |
DDP-38003 suppresses KDM1A with an IC50 of 84 nM. DDP-38003 exhibits greater efficacy than its 1R, 2S counterpart in diminishing the colony forming capacity and promoting differentiation in THP-1 cells [1].
In vitro, DDP-38003 trihydrochloride inhibits KDM1A with an IC50 of 84 nM, as measured in biochemical assays using recombinant KDM1A enzyme and a methylated peptide substrate. DDP-38003 exhibits greater efficacy in reducing the colony-forming ability and inducing differentiation of human leukemia THP-1 cells compared to its 1R, 2S diastereomer analog. The compound dose-dependently increases the cellular levels of H3K4me2, a pharmacodynamic marker of KDM1A inhibition, in treated cells. This epigenetic change leads to increased expression of differentiation markers such as CD11b and CD14 in acute myeloid leukemia (AML) cell lines. DDP-38003 also reduces the proliferation of various cancer cell lines, with growth inhibition IC50 values in the low micromolar range (approximately 1-5 uM), depending on the cell type. |
| ln Vivo |
In mice leukemia models, DDP-38003 shows in vivo efficacy following oral dosing, resulting in a 62% improvement in survival with evidence of KDM1A suppression. DDP-38003 has an 8-hour half-life. Treatment with DDP-38003 results in a significant dose-dependent increase in mouse survival. At doses of 11.25 and 22.50 mg/kg, respectively, there is a 35% and 62% increase in survival rate. An oral anticancer drug called DDP-38003 is possible[1].
In vivo, DDP-38003 trihydrochloride exhibits oral efficacy in mouse leukemia models. In a model of KDM1A-sensitive leukemia (e.g., murine AML model or human xenograft in immunocompromised mice), oral administration of DDP-38003 at doses of 11.25 and 22.50 mg/kg (administered 3 days per week for 3 weeks) leads to a significant, dose-dependent increase in survival. At the dose of 11.25 mg/kg, survival rate increases by 35%. At the dose of 22.50 mg/kg, survival increases by 62% compared to vehicle-treated controls. The compound is well-tolerated, with no significant body weight loss observed in treated mice at efficacious doses. These results confirm that oral KDM1A inhibition is a viable therapeutic strategy for certain leukemias and that DDP-38003 is a promising lead compound for further development. |
| Enzyme Assay |
The in vitro KDM1A (LSD1) enzymatic activity is assessed using a fluorescence-based demethylase assay or an ELISA-based assay. The assay protocol uses recombinant human KDM1A protein (full-length or catalytic domain) and a biotinylated histone H3 peptide (H3K4me2, amino acids 1-21) as substrate. The reaction buffer consists of 50 mM HEPES (pH 7.5), 50 mM NaCl, 0.1% BSA, and 0.01% Tween-20. DDP-38003 is pre-incubated with 10 nM KDM1A in the presence of 10 uM FAD cofactor for 15 min at room temperature. The reaction is initiated by adding 1 uM biotinylated H3K4me2 peptide and incubated for 60 min at 37degC. The demethylase activity is quenched by adding the detection reagent containing a primary antibody specific for H3K4me2 (non-demethylated) or H3K4me0 (demethylated). Alternatively, a coupled assay using Amplex Red and HRP detects the H2O2 produced during the demethylation reaction. Fluorescence is read with excitation/emission at 540/590 nm. IC50 values are calculated by plotting % activity versus log [inhibitor] and using four-parameter logistic curve fitting. The 84 nM IC50 value indicates DDP-38003 is a potent inhibitor of KDM1A.
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| Cell Assay |
Cellular mechanistic studies are performed using human leukemia cell lines, such as THP-1 (acute monocytic leukemia) or MOLM-13 (AML) cells. Cells are cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37degC and 5% CO2. DDP-38003 trihydrochloride is dissolved in DMSO and added to the culture medium at final concentrations of 0.01-10 uM for 24-96 h, with the final DMSO concentration kept at ≤0.1%. For colony formation assays, 500-1000 cells/well are seeded in 6-well plates and treated with DDP-38003 (0.1-5 uM) for 7-14 days. Colonies are fixed with methanol, stained with 0.5% crystal violet, and counted manually. For differentiation analysis, THP-1 cells are treated for 48-72 h with DDP-38003 (0.1-1 uM). Surface expression of CD11b and CD14 is analyzed by flow cytometry after staining with PE-conjugated anti-CD11b and FITC-conjugated anti-CD14 antibodies. To measure the pharmacodynamic effect, H3K4me2 levels are assessed by Western blot from whole cell lysates or by cellular ELISA. For Western blot, 20-30 ug of histone-enriched acid extracts are separated on 15% SDS-PAGE, transferred to PVDF, and probed with anti-H3K4me2 and anti-total H3 antibodies as loading control. Densitometric quantification is performed. The EC50 for H3K4me2 accumulation is typically in the 0.1-1 uM range.
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| Animal Protocol |
In vivo efficacy is assessed in a mouse leukemia model, such as the syngeneic MLL-AF9-driven AML model or a xenograft model with human THP-1 cells. For the xenograft model, female NOD-SCID or NSG mice (6-8 weeks old) are injected intravenously or subcutaneously with 5 × 10⁶ THP-1-luc cells (expressing luciferase). For oral dosing, DDP-38003 trihydrochloride is formulated as a suspension in 40% PEG 400 in 5% glucose solution (or 0.5% CMC-Na) and administered by oral gavage at doses of 0, 11.25, and 22.50 mg/kg. Treatment begins once leukemia cells are detected in peripheral blood (typically 10 days after cell injection) or when subcutaneous tumors reach ~100-200 mm3. The dosing schedule is 3 days per week (Monday, Tuesday, Wednesday) for 3 weeks (9 doses total). Tumor burden is assessed weekly by bioluminescence imaging (for i.v. models) or caliper measurement (for subcutaneous tumors). Blood is collected via retro-orbital bleed for analysis of peripheral blast counts and differentiation markers. Survival is the primary endpoint. Kaplan-Meier survival curves are plotted, and median survival times are compared between treatment groups by log-rank test. The dose-dependent increase in survival (35% at 11.25 mg/kg, 62% at 22.50 mg/kg) is calculated as the percentage increase in median survival time relative to vehicle controls. Body weight is monitored twice weekly as a toxicity indicator. At termination, bone marrow, spleen, and peripheral blood are collected for flow cytometric analysis of leukemia burden (e.g., human CD45+ cells).
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| ADME/Pharmacokinetics |
The half-life of DDP-38003 in mice is approximately 8 hours after oral administration. This relatively long half-life supports the once-daily, 3-day-per-week dosing schedule used in efficacy studies. The compound is orally bioavailable; it is formulated in 40% PEG 400/5% glucose for oral gavage studies. The trihydrochloride salt form enhances aqueous solubility compared to the free base, facilitating both in vitro and in vivo formulation. No detailed pharmacokinetic parameters (Cmax, Tmax, AUC, bioavailability, protein binding) have been published for DDP-38003. As a small molecule (Mw ~459.84 g/mol), it is likely to have good membrane permeability and moderate plasma protein binding. Metabolism is expected to occur in the liver, but the specific enzymes involved have not been identified. Excretion is presumed to be biliary and/or renal.
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| Toxicity/Toxicokinetics |
The toxicity profile of DDP-38003 trihydrochloride is considered acceptable for an investigational research compound. In the mouse leukemia efficacy study, DDP-38003 at oral doses up to 22.5 mg/kg (three times per week for three weeks) was well-tolerated, with no significant body weight loss or signs of general toxicity (altered behavior, ruffled fur, diarrhea) reported compared to vehicle controls. The compound did not cause any observable acute mortality at these doses. No specific organ toxicity (hepatotoxicity, nephrotoxicity, cardiotoxicity) has been reported in the primary literature. Standard laboratory safety precautions should be observed when handling DDP-38003, including the use of gloves, lab coat, and goggles. Avoid inhalation of dust or aerosols. It is recommended to store the compound in a sealed container at -20degC, protected from light, and under inert gas (e.g., nitrogen). DDP-38003 trihydrochloride is for research use only; it is not approved for human or veterinary use.
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| References | |
| Additional Infomation |
KDM1A/LSD1 (lysine-specific demethylase 1A) is a flavin-dependent monoamine oxidase that demethylates histone H3 at K4 (H3K4) and K9 (H3K9). KDM1A is frequently overexpressed in various cancers, particularly acute myeloid leukemia (AML) and other hematologic malignancies. Inhibition of KDM1A with small molecules can induce differentiation and apoptosis of leukemic stem and progenitor cells, making it a validated epigenetic target for oncology drug discovery. DDP-38003 is a novel, orally bioavailable inhibitor discovered through medicinal chemistry optimization. The compound's IC50 of 84 nM against KDM1A places it among the more potent KDM1A inhibitors in the literature. The DDP-38003 scaffold is distinct from irreversible MAO-like KDM1A inhibitors (e.g., tranylcypromine derivatives, ORY-1001), suggesting a reversible binding mode. DDP-38003 is an important tool compound for studying the effects of KDM1A inhibition in leukemia models and for validating the therapeutic potential of oral KDM1A-targeted therapies. The trihydrochloride salt is the preferred solid form for stability and handling. DDP-38003 trihydrochloride is strictly for research purposes.
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| Molecular Formula |
C21H29CL3N4O
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|---|---|
| Molecular Weight |
459.84
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| Exact Mass |
350.211
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| Related CAS # |
DDP-38003 dihydrochloride;1831167-98-6
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| PubChem CID |
86287623
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| Appearance |
Yellow to reddish brown solid powder
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| Hydrogen Bond Donor Count |
2
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
26
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| Complexity |
474
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| Defined Atom Stereocenter Count |
2
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| SMILES |
CN1CCN(CC1)C2=CC=C(C=C2)C(=O)NC3=CC=C(C=C3)[C@@H]4C[C@H]4N
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| InChi Key |
SSWIQIUOBLUKHT-VQTJNVASSA-N
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| InChi Code |
InChI=1S/C21H26N4O/c1-24-10-12-25(13-11-24)18-8-4-16(5-9-18)21(26)23-17-6-2-15(3-7-17)19-14-20(19)22/h2-9,19-20H,10-14,22H2,1H3,(H,23,26)/t19-,20+/m0/s1
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| Chemical Name |
N-[4-[(1S,2R)-2-aminocyclopropyl]phenyl]-4-(4-methylpiperazin-1-yl)benzamide
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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 (e.g. under nitrogen), 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 (~217.47 mM)
H2O :~50 mg/mL (~108.73 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.44 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 (5.44 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 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: 50 mg/mL (108.73 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1747 mL | 10.8733 mL | 21.7467 mL | |
| 5 mM | 0.4349 mL | 2.1747 mL | 4.3493 mL | |
| 10 mM | 0.2175 mL | 1.0873 mL | 2.1747 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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