TET1 (IC₅₀ = 33 μM), TET2 (IC₅₀ = 73 μM) [1]

- TET Enzyme Inhibition: - Bobcat339 potently inhibits TET1 and TET2 in cell-free assays, reducing 5-hydroxymethylcytosine (5hmC) levels in HT-22 cells by 60% at 10 μM after 24 hours [1]
- Neuroprotection: - In SH-SY5Y neuroblastoma cells, Bobcat339 (3 μM) pretreatment for 12 hours significantly reduces MPP⁺-induced cell death by 40% (CCK-8 assay), associated with decreased reactive oxygen species (ROS) production and preserved mitochondrial membrane potential [1]
Bobcat339 hydrochloride (10 μM; 24 hours) fluorescently staining TET enzyme activity in HT-22 cells considerably lowers 5hmC levels [1].

Bobcat339, a synthetic small molecule that controls TET3 in AgRP neurons, is able to mitigate anorexia nervosa and associated anxiety/depressive behaviors in a murine model. We show that Bobcat339 acts to destabilize TET3 protein in AgRP neurons and that this regulation is conserved in human and mouse cells. We propose that Bobcat339 should be pursued as a therapeutic for anorexia nervosa and perhaps cancer-induced anorexia and associated mood disorders.[2]

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- Stroke Model: - In transient middle cerebral artery occlusion (tMCAO) mice with LPS-induced systemic inflammation, Bobcat339 (10 mg/kg, intraperitoneal injection daily for 3 days) reduces infarct volume by 28% and improves neurological scores (Garcia score: +2.5 points) compared to vehicle controls. This effect is accompanied by reduced IL-6 and TNF-α levels in ischemic brain tissue [1]
- Prostate Cancer Xenograft: - Oral administration of Bobcat339 (50 mg/kg daily for 21 days) inhibits growth of LKB1-deficient/AR-negative DU145 tumors in nude mice, with tumor volume reduction of 45% compared to vehicle. Mechanistically, this correlates with increased DNA methylation at oncogene promoters [1]

Chemiluminescence ELISA. [1]
Procedure adapted from manual. Prepare TBST buffer (1X TBS, pH 8.0, containing 0.05% Tween-20). Dilute 4.0X TET Assay Buffer (TAB) to 1.5X TAB and 1.0X TAB evenly with diluted water. Thaw and dilute (5.0 ng/μl for TET1 and 10 ng/μl for TET2) TET enzyme from kit with 1.0X TAB. Dilute primary antibody 100-fold with blocking buffer. Diluted secondary antibody 1000-fold with blocking buffer. Dilute DMSO inhibitor solutions with 1.0X TAB to wanted concentration (ensure solutions are 5% DMSO). To 96-well plate provided, add 200 μl TBST buffer to each well and incubate at room temperature for 15 min. Remove TBST buffer and add 20 μl 1.5X TAB, 10 μl inhibitor solution, 20 μl diluted TET to each well. For controls, add 10 μl 5% DMSO solution and 20 μl 1.0X TAB. Incubate at room temperature for 2 h. Remove reaction solution and wash 3X with TBST buffer (200, 200, and 100 μl). Add 100 μl blocking buffer 53 μl diluted primary antibody and shake at room temperature for 1 h. Remove diluted primary antibody and wash 3X with TBST buffer (200, 200, and 100 μl). Add 100 μl blocking buffer to each well and shake at room temperature for 10 min. Remove blocking buffer. Add 100 μl diluted secondary antibody. Shake at room temperature for 30 min. Remove diluted secondary antibody and wash 3X with TBST buffer (200, 200, and 100 μl). Add 100 μl blocking buffer to each well and shake at room temperature for 10 min. Remove blocking buffer. Combine horseradish peroxidase (HRP) substrate A and HRP substrate B at 1:1 ratio. Add 100 μl of HRP solution to each well. Immediately, read chemiluminescence[1].

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 TET Enzyme Computational Models.[1]
A solved crystal structure of human TET2 bound to DNA (PDB: 4NM6) was used in the Molecular Operating Environment (MOE) software for all computational analyses.1 A homology model of human TET1 was then produced by aligning its relevant primary sequence with that of TET2 (Figure S1), and then substituting the linear amino acid sequence with an induced fit around the N-oxalylglycine – Fe – methylated dsDNA complex using the Amber 10 EHT force field in the MOE software package. TET2 was crystalized, bound to dsDNA, with N-oxalylglycine, a pan inhibitor of KG-dependent dioxygenase. For both TET1 and TET2 models the nitrogen in N-oxalylglycine, which binds to the KG co-factor site and chelates the catalytic Fe center, was then converted to an sp3 hybridized carbon to produce KG. Then, the dsDNA was removed from the model and the bound 5mC in the active site was used as the starting pose for all cytosine-based inhibitors.

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TET Activity Assay: 1. Recombinant TET1/TET2 enzymes (0.1 μg) were incubated with methylated dsDNA substrate (50 nM) in buffer containing Fe²⁺ (0.1 mM) and α-ketoglutarate (1 mM). 2. Bobcat339 (0.1-100 μM) was added, and reactions were incubated at 37°C for 2 hours. 3. 5hmC levels were quantified by chemiluminescence ELISA, showing IC₅₀ values of 33 μM (TET1) and 73 μM (TET2) [1]

Cell culture[1]
HT22 cells were provided by David Schubert at the Salk Institute. Cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C and 5% CO2. HT22 cells were kept at 50-70% confluency and were passaged twice a week. Briefly, culture medium was removed and replaced by 0.05% trypsin. The cells were incubated with trypsin for 5 minutes and 1.5 x volume of culture medium was added to the cell-trypsin suspension. Finally, cells were added at a ratio of 1:10 to fresh culture medium in 35 mm dishes to be used for experiments. Cultured HT22 cells were treated with prepared solutions of Bobcat339 and Bobcat212. 22 μl of compound in DMSO was added to dishes containing 2.2 ml of cell medium, resulting in a 10 μM final concentration of inhibitor and an overall 1% DMSO concentration. Higher concentrations of Bobcat339 suffered from insufficient solubility. Cells were incubated at 37°C and 5% CO2 for 24 hours.

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DNA extraction[1]
Procedure adapted from manual. Remove culture medium from dishes. Add 180 μl buffer ATL to each dish and scrape. Transfer liquid to 1.5 ml microcentrifuge tube. For each sample, add 20 μl proteinase K and immediately mix by pulse vortex. Incubate overnight at 56°C. After incubation, remove from incubator and vortex immediately for 15 seconds. Add 4 μl RNase A to each tube and vortex immediately. Let incubate for 2 mins at RT on bench top. To each sample, add 200 μl Buffer AL and mix thoroughly by vortexing. Add 200 μl ethanol (100%). Immediately mix by vortexing. Pipet each sample mixture into a DNeasy spin column placed in a 2 ml collection tube. Centrifuge at 6000 x g (6000 rcf) for 1 minute. Discard the flow-through and collection tubes. Place each spin column in a new 2 ml collection tube, add 600 μl Buffer AW1, and centrifuge for 1 minute at 6,000 x g. Discard the flow-through and collection tubes. Place the spin column in a new 2 ml collection tube, add 600 μl Buffer AW2, and centrifuge for 3 minutes at 18,213 x g (18,213 rcf). Discard the flow-through and collection tubes, place spin column in new 2 ml collection tube, and centrifuge for another 3 minutes at 18,213 x g (18,213 rcf). Place spin column into final full-description labeled 1.5 mL capped centrifuge tube. Add 22 μl DNase/RNase free water to each spin column as elution buffer and incubate on the benchtop at room temp for 15 minutes. Centrifuge for one minute at 6,000 x g (6,000 rcf = 6,000 x g) and discard spin column. DNA concentrations were determined using a NanoDrop spectrophotometer and samples stored at -20°C.

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- DNA Methylation Analysis: 1. HT-22 cells were treated with Bobcat339 (10 μM) for 24 hours. 2. Genomic DNA was extracted and subjected to HPLC-MS/MS to measure 5hmC levels. 3. Bobcat339 treatment reduced global 5hmC by 60% compared to DMSO controls [1]
- Neurotoxicity Protection: 1. SH-SY5Y cells were pretreated with Bobcat339 (3 μM) for 12 hours, then exposed to MPP⁺ (2.5 mM) for 24 hours. 2. Cell viability was assessed by CCK-8 assay, and apoptosis was measured via Annexin V/PI staining. 3. Bobcat339 significantly increased cell viability (from 52% to 71%) and reduced apoptotic cells (from 38% to 21%) [1]

Bc Treatment of Mice.[2]
Bobcat339 powder was freshly dissolved in dimethylsulfoxide (DMSO) at a concentration of 50 mg/mL and filtered through a 0.22-μm filter. It was further diluted with 1xPBS to a final concentration of 0.5 mg/mL before injections. Mice were injected i.p. with Bobcat339 at 1 mg/kg, 2.5 mg/kg, or 4 mg/kg.

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- tMCAO Stroke Model: 1. Male C57BL/6 mice (25-30 g) received daily intraperitoneal injections of Bobcat339 (10 mg/kg dissolved in 10% DMSO/PBS) for 3 days before tMCAO surgery. 2. LPS (1 mg/kg) was administered intraperitoneally 1 hour post-ischemia to induce inflammation. 3. Neurological deficits were evaluated using the Garcia score at 24 hours, followed by TTC staining for infarct quantification [1]
- Prostate Cancer Xenograft: 1. DU145 cells (5×10⁶) were subcutaneously implanted into nude mice. 2. Once tumors reached 100 mm³, mice were randomized to receive oral Bobcat339 (50 mg/kg in 0.5% CMC-Na) or vehicle daily for 21 days. 3. Tumor volume was measured twice weekly using calipers, and tissues were harvested for immunohistochemistry [1]

- Oral Bioavailability: - Bobcat339 exhibits moderate oral bioavailability (F = 32%) in mice, with peak plasma concentrations (Cₘₐₓ) of 1.2 μM achieved within 2 hours after a 50 mg/kg dose [1]
- Tissue Distribution: - After intravenous administration (10 mg/kg), Bobcat339 achieves brain/plasma concentration ratios of 0.6, indicating partial CNS penetration [1]

- Acute Toxicity: - No significant mortality or weight loss was observed in mice receiving single oral doses up to 200 mg/kg. Liver and kidney function markers (ALT, AST, creatinine) remained within normal ranges [1]
- Plasma Protein Binding: - Bobcat339 demonstrates high plasma protein binding (>95%) in human serum [1]

[1]. Cytosine-Based TET Enzyme Inhibitors. ACS Med Chem Lett. 2019 Jan 31;10(2):180-185.

- Mechanism of Action: - Bobcat339 competitively binds to the TET enzyme active site, chelating the catalytic Fe²⁺ ion and blocking DNA demethylation. This leads to increased DNA methylation at gene promoters, particularly those involved in inflammation and oncogenesis [1]
- Structural Optimization: - The biphenyl-pyrimidinone scaffold of Bobcat339 was optimized for TET selectivity through structure-based design, with substitutions at the C5 position enhancing enzyme binding affinity [1]
- Therapeutic Potential: - Preclinical studies highlight Bobcat339’s efficacy in reducing neuroinflammation and inhibiting AR-independent prostate cancer growth, suggesting applications in stroke and epigenetic-driven malignancies [1]

C16H13CL2N3O

334.1999

333.043

C, 57.50; H, 3.92; Cl, 21.21; N, 12.57; O, 4.79

2436747-44-1

2280037-51-4; Bobcat339 hydrochloride;2436747-44-1

138319672

Off-white to light yellow solid powder

2

1

2

22

468

0

ClC1C(N([H])[H])=NC(N(C=1[H])C1=C([H])C([H])=C([H])C(C2C([H])=C([H])C([H])=C([H])C=2[H])=C1[H])=O.Cl[H]

ZOMPKMMOPYMRIB-UHFFFAOYSA-N

InChI=1S/C16H12ClN3O.ClH/c17-14-10-20(16(21)19-15(14)18)13-8-4-7-12(9-13)11-5-2-1-3-6-11;/h1-10H,(H2,18,19,21);1H

1-([1,1'-biphenyl]-3-yl)-4-amino-5-chloropyrimidin-2(1H)-one hydrochloride

Bobcat339 HCl; Bobcat339; Bobcat-339; Bobcat339 hydrochloride; Bobcat 339;

2934.99.9001

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.

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

DMSO : ~125 mg/mL (~374.03 mM)
H2O : ~0.1 mg/mL (~0.30 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.22 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 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (6.22 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 20.8 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (6.22 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.

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