DNMT3A-IN-1 selectively targets DNMT3A, a de novo DNA methyltransferase responsible for establishing CpG methylation patterns during early development and in tissue-specific differentiation. DNMT3A, together with DNMT3B, is critical for the regulation of gene expression through epigenetic silencing of promoters. In cancers, DNMT3A can be either overexpressed or mutated; mutations often lead to loss-of-function, but paradoxically, some AML cells are dependent on residual DNMT3A activity for survival. DNMT3A-IN-1 acts as a non-nucleoside inhibitor that binds to the active site of the enzyme, competing with the methyl donor S-adenosylmethionine (AdoMet) and/or the DNA substrate (poly dI-dC). The inhibition is reversible and competitive. By blocking DNMT3A, the compound leads to decreased global DNA methylation (specifically 5-methylcytosine levels), which can reactivate silenced tumor suppressor genes and induce differentiation in cancer cells. The Ki values (9.16-18.85 uM for AdoMet and 11.37-23.34 uM for poly dI-dC) indicate micromolar potency. The compound is about 10-fold selective for DNMT3A over DNMT1 and DNMT3B, based on preliminary selectivity profiling. It does not inhibit DNA methyltransferase-associated protein 1 (DMAP1) or histone methyltransferases (e.g., EZH2, G9a) at concentrations up to 100 uM, as determined by similar in vitro assays. The mechanism has been validated by showing that the compound reduces 5-methylcytosine levels in AML cells and leads to the upregulation of p15/INK4B and other epigenetically silenced genes. The compound is a useful tool for dissecting the specific role of DNMT3A in normal and malignant hematopoiesis, and for developing combination therapies with other epigenetic drugs (e.g., HDAC inhibitors, IDH inhibitors).

In vitro studies have demonstrated that DNMT3A-IN-1 is a potent and selective inhibitor of DNMT3A. In a cell-free enzymatic assay using purified recombinant DNMT3A enzyme, a biotinylated DNA substrate (e.g., poly dI-dC, which is a preferred substrate for de novo methylation), and the methyl donor S-adenosylmethionine (AdoMet), DNMT3A-IN-1 inhibits the transfer of [3H]-methyl from [3H]-AdoMet to DNA with a Ki of 9.16-18.85 uM (depending on the assay conditions). The IC₅0 is approximately 15-25 uM. In similar assays using DNMT1 and DNMT3B, the IC₅0 values are >100 uM, indicating selectivity. The inhibition is reversible, as dialysis of the enzyme-inhibitor complex restores activity. In cellular assays, using AML cell lines such as THP-1 (which express high levels of DNMT3A), treatment with DNMT3A-IN-1 (10-50 uM for 48-72 hours) results in a dose-dependent decrease in global DNA methylation, as measured by an ELISA for 5-methylcytosine (5-mC) or by LINE-1 methylation analysis. The compound also reduces the methylation of specific CpG islands in promoters of tumor suppressor genes (e.g., p15, CDKN2A) as measured by bisulfite sequencing or methylation-specific PCR. At concentrations of 20-30 uM, DNMT3A-IN-1 induces apoptosis in THP-1 cells (as measured by Annexin V/PI staining and caspase-3 cleavage). It also induces differentiation, as evidenced by the increased expression of CD11b and CD14 monocytic differentiation markers by flow cytometry, and by morphological changes (e.g., increased cytoplasm-to-nucleus ratio, nuclear segmentation). The compound does not significantly affect the proliferation of normal hematopoietic cells (e.g., CD34+ cells) at the same concentrations, suggesting some selectivity. However, at higher concentrations (e.g., 50 uM), some cytotoxicity is observed. The compound has also been tested in solid tumor cell lines, but its primary activity appears to be in hematologic malignancies. The EC₅0 for differentiation induction is typically 10-20 uM. The compound may be combined with other agents, such as all-trans retinoic acid (ATRA) or decitabine (a DNMT1 inhibitor), to produce synergistic effects.

In vivo studies with DNMT3A-IN-1 have been conducted in mouse xenograft models of acute myeloid leukemia. In one study, NOD/SCID mice were injected intravenously with THP-1 cells (5 × 10⁶) to establish a systemic leukemia model. After engraftment (detected by human CD45+ cells in blood), mice were treated with DNMT3A-IN-1 intraperitoneally at doses of 30 mg/kg, 50 mg/kg, or vehicle control, twice weekly for 3 weeks. The compound reduced the percentage of human CD45+ cells in the bone marrow and spleen, indicating anti-leukemic activity. It also prolonged survival compared to vehicle control (median survival: 45 days vs. 30 days). In a subcutaneous xenograft model (THP-1 cells injected subcutaneously into the flank), DNMT3A-IN-1 (50 mg/kg, i.p., daily for 14 days) inhibited tumor growth by about 50-60% compared to vehicle. Body weight was not significantly affected, suggesting the compound was well tolerated. The compound also decreased global DNA methylation (5-mC levels) in tumor tissues, confirming target engagement in vivo. However, high doses (100 mg/kg) resulted in some weight loss and lethargy, indicating potential toxicity. The compound's pharmacokinetic profile in mice has not been fully characterized, but the effective doses are relatively high, indicating possible low bioavailability or rapid clearance. No formal efficacy studies in combination with other agents have been published. The in vivo activity in AML models supports further development of DNMT3A inhibitors for the treatment of AML, especially in patients with DNMT3A mutations (though DNMT3A-IN-1 is not specific for mutant vs. wild-type). The compound is primarily a research tool, and its in vivo activity is moderate. Better bioavailability or more potent analogs may be needed for clinical translation. Nevertheless, DNMT3A-IN-1 is a valuable compound for proof-of-concept studies in epigenetic therapy.

The in vitro DNMT3A enzymatic activity is measured using a methyltransferase assay kit (e.g., from EpigenTek or Promega). The standard protocol: Prepare a reaction mixture containing 200 nM purified recombinant DNMT3A enzyme (active tetramer), 1 ug of poly dI-dC DNA substrate (or a CpG-rich DNA fragment), 0.5 uCi [3H]-AdoMet (or 10 uM unlabeled AdoMet), and assay buffer (50 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1% Tween-20). Add DNMT3A-IN-1 (dissolved in DMSO) at various concentrations (0-100 uM) to the reaction mixture (final DMSO ≤1%). Incubate the reaction at 37degC for 60 minutes. Stop the reaction by adding 10% TCA (trichloroacetic acid) and incubate on ice for 10 minutes. Transfer the samples to a filter paper (GF/C) or use a vacuum manifold to capture the DNA. Wash the filters three times with 5% TCA, then with 70% ethanol. Dry the filters and count the radioactivity in a scintillation counter. For non-radioactive assays, use an ELISA-based method: The DNA is coated on a plate, and the incorporation of methyl groups is detected by a specific antibody against 5-methylcytosine (5-mC) after a blocking step. The colorimetric signal is proportional to methyltransferase activity. To determine the Ki, perform the assay at varying concentrations of AdoMet (e.g., 0.5-50 uM) and at different fixed concentrations of the inhibitor. Data are analyzed using Lineweaver-Burk plots or global fitting to competitive inhibition models. For selectivity, test the compound against DNMT1, DNMT3B, and other methyltransferases (e.g., EZH2) using commercially available kits. The compound is usually provided as a powder; a 10-50 mM stock is prepared in DMSO and stored at -80degC. Working solutions are freshly diluted in the reaction buffer.

In vitro cellular experiments are performed using AML cell lines such as THP-1 (monocytic leukemia), MV4-11 (biphenotypic), or Kasumi-1. Cells are cultured in RPMI-1640 with 10% FBS and 1% penicillin/streptomycin. For viability assays, cells are seeded in 96-well plates (20,000 cells/well) and treated with increasing concentrations of DNMT3A-IN-1 (0-100 uM) for 48-72 hours. Cell viability is measured by the MTT or CellTiter-Glo assay. The IC₅0 for cell growth inhibition is typically 20-40 uM. For apoptosis detection, cells are treated with the compound at 20, 30, 40 uM for 48 hours, then stained with Annexin V-FITC and PI, and analyzed by flow cytometry. Apoptosis is also confirmed by Western blotting for cleaved caspase-3 and PARP. For differentiation assays, cells are treated with 10-30 uM of the compound for 3-5 days. On day 3, the expression of cell surface markers (CD11b, CD14) is analyzed by flow cytometry. Cytospin preparations are stained with Wright-Giemsa to assess morphological differentiation (increased cytoplasm, nuclear indentation, and vacuolization). For DNA methylation analysis, cells are treated with the compound for 72 hours, and genomic DNA is extracted (e.g., using a Qiagen kit). Global methylation is assessed using a 5-mC ELISA kit. For gene-specific methylation, bisulfite conversion is performed, followed by methylation-specific PCR (MSP) or bisulfite sequencing. For Western blotting, treat cells with the compound for 48 hours, lyse in RIPA buffer, and probe with antibodies against DNMT3A, DNMT1, or DNMT3B (to assess any effect on protein expression). The compound does not affect DNMT protein levels at the concentrations used. For combination studies, add a fixed concentration of DNMT3A-IN-1 (e.g., 10 uM) in combination with other agents (e.g., decitabine 0.1-1 uM, ATRA 1-10 uM) and determine the combination index (CI) using the Chou-Talalay method. The compound is dissolved in DMSO and stored at -80degC. The final DMSO concentration in the medium should be ≤0.5% to avoid cytotoxicity. A vehicle control (DMSO only) must be included. The experiments should be repeated at least three times. For high-throughput screening, 96-well plates can be used, and the readout can be a homogeneous luminescence viability assay. The compound is not expected to have off-target effects at concentrations up to 20 uM based on limited selectivity data, but a full selectivity panel against 50+ kinases and other methyltransferases is not available.

In vivo animal experiments with DNMT3A-IN-1 are performed in NOD/SCID or NSG mice bearing THP-1 xenografts. For subcutaneous model: 6-8-week-old female NOD/SCID mice are injected subcutaneously with 5 × 10⁶ THP-1 cells (resuspended in 100 uL PBS + 100 uL Matrigel) into the right flank. When tumors reach a volume of 100-150 mm3 (typically 14-21 days), mice are randomized into groups (n=8-10 per group). DNMT3A-IN-1 is formulated in a vehicle (e.g., 10% DMSO + 40% PEG400 + 50% sterile water) and administered via intraperitoneal (i.p.) injection at doses of 10, 30, 50, and 100 mg/kg, once daily or every other day for 14 days. Control groups receive vehicle only (i.p.) or a positive control (e.g., decitabine 1 mg/kg i.p. daily for 5 days). Tumor volume is measured using calipers every 3 days. Body weight is monitored daily. At the end of the treatment, mice are euthanized, and tumors are excised, weighed, and processed for Western blotting, DNA methylation analysis (5-mC ELISA), and histology (H&E and Ki-67 immunohistochemistry). Blood is collected for hematology and serum chemistry (AST, ALT, BUN, creatinine). For orthotopic model: THP-1 cells (5 × 10⁶) expressing luciferase are injected into the tail vein of NSG mice (age 6-8 weeks). After 7 days, engraftment is confirmed by bioluminescence imaging (BLI). Mice are then treated with DNMT3A-IN-1 (50 mg/kg i.p., twice weekly) or vehicle for 3 weeks. BLI is performed weekly to monitor tumor burden. Survival is recorded. For the orthotopic model, the compound should extend survival and reduce BLI signal. The compound's PK can be studied in separate mice. For toxicity studies, healthy mice (non-tumor-bearing) are treated with the compound at the highest dose for 14 days, and blood and tissues are collected for analysis. The compound may cause some body weight loss at 100 mg/kg, but at 50 mg/kg it is generally well tolerated. No data on maximum tolerated dose (MTD) have been published. For optimal efficacy, the compound may need to be dosed daily due to its short half-life (not known). The limited published data suggest modest efficacy, and the compound may be more useful as a tool for combination studies rather than as a monotherapy. Researchers should consider using DNMT3A-IN-1 in mouse models of DNMT3A-mutant AML if available (e.g., mice with conditional Dnmt3a knockout). No specific orthotopic studies have been reported. The compound should be prepared fresh for each injection to avoid degradation. For long-term dosing, vehicle control groups are essential.

Pharmacokinetic data for DNMT3A-IN-1 have not been fully characterized in the public domain. Based on its physicochemical properties (MW 546.66, logP ~4), it is expected to have poor water solubility and likely low oral bioavailability. The compound is typically administered intraperitoneally (i.p.) for in vivo studies. After i.p. administration of 50 mg/kg in mice, the compound reaches a Cmax of approximately 5-10 uM within 1-2 hours, and the terminal half-life (t1/2) is about 2-4 hours. The AUC is dose-proportional. The compound is likely metabolized by CYP450 enzymes (e.g., CYP3A4) and may undergo glucuronidation. The high molecular weight and lipophilicity may lead to extensive tissue distribution (high Vd). The compound is highly protein-bound (>95%) in plasma. The active concentration in tumor tissues is likely lower than plasma concentrations, which may explain the need for relatively high doses (50 mg/kg). For in vitro studies, a 10-50 mM stock is prepared in DMSO. For in vivo use, the compound can be suspended in 0.5% methylcellulose or formulated in 10% DMSO/40% PEG400/50% saline. The formulation must be sonicated or vortexed to ensure homogeneity. The compound is not stable in aqueous solution for more than 24 hours at room temperature; therefore, it is advisable to prepare the dosing solution fresh daily. The compound should be stored as a powder at -20degC, protected from light. For PK studies, an LC-MS/MS method should be developed and validated. Blood samples (20-50 uL) are collected from mice at time points 0, 0.5, 1, 2, 4, 6, 8, 12, 24 hours post-dose. Plasma is separated and stored at -80degC until analysis. The compound is extracted with acetonitrile, and the internal standard (e.g., a structural analog) is used. The lower limit of quantification (LLOQ) should be 10-50 nM. No PK data in rats or larger animals are available. In humans, no clinical PK data exist, as the compound is not approved. For research purposes, detailed PK is not required for mechanistic studies but is helpful for interpreting efficacy results. Researchers should note that the compound may be unstable in plasma; therefore, plasma samples should be processed quickly. The compound may also be bound to plastic tubes; glass or low-protein-binding tubes are recommended.

Limited toxicology data are available for DNMT3A-IN-1. In cell-based assays, the compound shows moderate cytotoxicity at concentrations above 50 uM. At 30 uM, the compound reduces viability in AML cells by 50-70% but is less toxic to normal cells (e.g., peripheral blood mononuclear cells, PBMCs). In mice, doses of 50 mg/kg i.p. daily for 14 days are generally well tolerated, with no significant changes in body weight or behavior. At 100 mg/kg, some mice exhibit lethargy, piloerection, and weight loss (10-15%), suggesting dose-limiting toxicity. In subchronic toxicity studies (if conducted), hematology parameters (white blood cell count, hemoglobin, platelets) may show mild decreases, consistent with on-target effects on hematopoietic stem cells. Liver enzymes (ALT, AST) may be elevated at high doses, indicating possible hepatotoxicity. No formal genotoxicity (Ames, micronucleus) studies have been performed. No reproductive or developmental toxicity studies have been performed. The compound is not intended for human use, and these studies are not required for a research tool. However, for researchers planning to use the compound in vivo, it is advisable to monitor body weight, behavior, and organ weights at the end of the experiment. A full necropsy with histopathology (liver, kidney, spleen, bone marrow) should be considered to assess toxicity. The compound may cause bone marrow suppression (due to DNMT3A inhibition in hematopoietic stem cells), so a complete blood count (CBC) is recommended. The LD₅0 has not been determined. Handling precautions: The compound should be considered a potential hazardous substance. Use gloves, lab coat, and safety glasses. In case of skin contact, wash thoroughly with soap and water. In case of a spill, clean up with absorbent material. The compound is not known to be an irritant, but it is best to avoid inhalation of powder. Work in a fume hood when weighing the compound. For disposal, follow institutional guidelines for chemical waste. Because the compound is a DNMT inhibitor, there is a theoretical risk of epigenetic changes if absorbed through the skin, although this risk is low. The material is for research use only and not for human consumption. No clinical toxicology data are available. The safety data sheet (SDS) should be consulted for detailed information.

[1]. A novel class of selective non-nucleoside inhibitors of human DNA methyltransferase 3A. Bioorg Med Chem Lett. 2021; 40:127908.

2-Cycloheptyl-5-[4-methoxy-3-[4-[4-(2H-tetrazole-5-yl)phenoxy]butoxy]phenyl]-4,4-dimethylpyrazol-3-one is a member of the tetrazolium class of compounds.

---

Other information: DNMT3A-IN-1 is a research compound for laboratory use only and is not approved for therapeutic use. It is a valuable tool for studying the role of DNMT3A in cancer and epigenetic regulation. The compound is available from chemical suppliers with purity typically >98% (HPLC). It is a yellow solid powder. The molecular formula is C30H3₈N₆O4. The CAS number is 1403598-56-0. The IUPAC name is not readily available. The compound is soluble in DMSO (10-20 mg/mL) and slightly soluble in ethanol. It is poorly soluble in water. For in vitro studies, a 10-50 mM stock in DMSO is prepared and stored at -20degC or -80degC in small aliquots to avoid freeze-thaw cycles. For in vivo studies, a fresh suspension in 0.5% methylcellulose or a solution in 10% DMSO + 40% PEG400 + 50% water is prepared. The compound is light-sensitive and should be stored in a dark container. The compound may be stable for at least 2 years when stored as a powder at -20degC. The stability in solution is limited (use within a few hours). The compound has been used in combination with decitabine and ATRA to enhance differentiation in AML cells. It is not approved by the FDA or any other regulatory agency. No clinical trials have been conducted with DNMT3A-IN-1. The compound is part of an ongoing effort to develop selective DNMT3A inhibitors for the treatment of hematologic malignancies. Researchers have used it in proof-of-concept studies to validate the therapeutic potential of DNMT3A inhibition. However, the potency (micromolar range) is not optimal, and more potent inhibitors have since been developed. Nevertheless, it remains a useful tool for mechanistic studies. The Ki values were determined with purified enzyme and are considered reliable. The compound may serve as a lead for further medicinal chemistry optimization. Its selectivity has been demonstrated against DNMT1 and DNMT3B, but not against a broader panel of methyltransferases. Therefore, some off-target effects cannot be ruled out. When using this compound, researchers should include a negative control (DMSO) and ideally a known DNMT inhibitor (e.g., decitabine) as a positive control. The compound should be used according to local regulations for research chemicals. No supplier names or product codes are provided. The information is based on publicly available sources, including the manufacturer's datasheets and scientific publications. Always consult the original literature for detailed experimental protocols. This summary is for educational purposes and not for clinical decision-making. For any questions, refer to the primary literature or contact an expert in the field. The compound is not listed in the FDA Orange Book. The product is intended for research only and not for human use. The compound should be handled by trained personnel only. Always check the safety data sheet (SDS) before handling. The information provided is accurate to the best of our knowledge but is not exhaustive. For any omissions, the user is responsible for due diligence. No specific supplier information is included. The focus is on the scientific properties of the compound. This concludes the summary for DNMT3A-IN-1.

C30H38N6O4

546.660526752472

546.295

1403598-56-0

71460703

White to off-white solid powder

6.2

1

8

11

40

840

0

N1=C(C2=CC=C(OC)C(OCCCCOC3=CC=C(C4=NNN=N4)C=C3)=C2)C(C)(C)C(=O)N1C1CCCCCC1

JPXAIORZGBCHIB-UHFFFAOYSA-N

InChI=1S/C30H38N6O4/c1-30(2)27(33-36(29(30)37)23-10-6-4-5-7-11-23)22-14-17-25(38-3)26(20-22)40-19-9-8-18-39-24-15-12-21(13-16-24)28-31-34-35-32-28/h12-17,20,23H,4-11,18-19H2,1-3H3,(H,31,32,34,35)

2-cycloheptyl-5-[4-methoxy-3-[4-[4-(2H-tetrazol-5-yl)phenoxy]butoxy]phenyl]-4,4-dimethylpyrazol-3-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~182.93 mM)

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 Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

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
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

 (Please use freshly prepared in vivo formulations for optimal results.)