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| 25mg |
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| Targets |
Myc-Max (IC50 = 8.9 μM)
Fat mass and obesity-associated protein (FTO) demethylase. FB23 inhibits FTO-mediated m⁶A demethylation with an IC₅₀ of 0.06 μM. FB23-2 maintains inhibitory activity on FTO demethylation in vitro. [1] |
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| ln Vitro |
VPC-70063 (25 μM; 96 h) shows Myc-Max transcriptional activity inhibition of 106% and Myc-Max/UBE2C downstream pathway inhibition of 94%[1].
VPC-70063 (6.25-25 μM, 48 h) induces apoptosis in LNCaP cells, as shown by the cleavage of PARP[1]. VPC-70063 (0-500 μM; 0-600 s) disrupts the interaction of Myc-Max with DNA in a dose dependent manner[1]. FB23-2 treatment resulted in a substantial increase of m⁶A abundance in the transcriptomes of NB4 and MONOMAC6 AML cells as detected by m⁶A dot blot assay. LC-MS/MS quantitation confirmed the increase of cellular m⁶A in mRNA of AML cells after exposure to FB23-2. FB23-2 significantly suppressed the proliferation of a panel of AML cell lines (NB4, MONOMAC6, MA9.3ITD, MA9.3RAS, U937, ML-2, MV4-11) with IC₅₀ values ranging from 0.8 μM to 5.2 μM. FB23-2 minimally altered the proliferation of human normal bone marrow cells. FB23 and FB23-2 treatment significantly increased the mRNA and protein levels of ASB2 and RARA, and inhibited the expression of MYC and CEBPA in NB4 and MONOMAC6 cells. FB23-2 substantially accelerated all-trans retinoic acid (ATRA)-induced myeloid differentiation, induced apoptosis, and caused cell cycle arrest at G1 stage in AML cells. FB23-2 suppressed proliferation, induced apoptosis, decreased colony-forming unit capacity, and accelerated ATRA-mediated myeloid differentiation of primary AML cells from four patients. [1] |
| ln Vivo |
In a xeno-transplantation leukemic model using NSGS mice transplanted with MONOMAC6 AML cells, daily intraperitoneal injection of FB23-2 (2 mg/kg) for 10 days substantially delayed the onset of leukemic symptoms, significantly prolonged median survival, suppressed leukemia malignancy (reduced splenomegaly and hepatomegaly), and promoted AML cell differentiation in vivo.
In a patient-derived xeno-transplantation (PDX) AML mouse model, treatment with FB23-2 (6 mg/kg/day, i.p. for 17 days) significantly prolonged disease latency, reduced the proportion of AML blast cells in peripheral blood and bone marrow, induced differentiation of AML cells, impaired leukemia malignancy (fewer and smaller colonies), and significantly eliminated leukemia stem cells (LSCs). Secondary transplantation confirmed a reduction in functional LSCs after FB23-2 treatment. [1] |
| Enzyme Assay |
An HPLC-based assay was used to measure the inhibition of m⁶A demethylation in RNA. The reaction contained FTO protein, a single-stranded RNA substrate containing m⁶A, co-factors (2-oxoglutarate, ferrous ammonium sulfate, L-ascorbic acid), and the inhibitor at required concentrations in Tris-HCl buffer. The mixture was incubated, then the reaction was terminated by heating. The products were digested by nuclease P1 and alkaline phosphatase before HPLC analysis. The IC₅₀ values were quantitated based on the inhibitory percentages of m⁶A demethylation. [1]
A cellular thermal shift assay (CETSA) was performed to validate compound-protein interaction in cellular conditions. Cell lysates were incubated with the compound or DMSO, then subjected to heating at various temperatures to denature proteins. After centrifugation, supernatants were analyzed by western blot to detect the thermal stabilization of the target protein. [1] A drug affinity responsive target stability (DARTS) assay was conducted. Cell lysates were incubated with the compound or DMSO, then digested with Pronase. The reaction was quenched, and the lysates were subjected to western blot to determine the abundance of the target protein, which becomes protease-resistant upon ligand binding. [1] In vitro inhibitory assays on various epigenetic enzymes (HDACs, LSD1, Jumonji demethylases, BRD4, DOT1L) were performed using fluorescence, AlphaLISA, or radioactivity-based methods according to standard protocols. [1] The inhibitory effects on cyclooxygenases COX-1 and COX-2 were evaluated using a fluorescent inhibitor screening assay kit. Enzymes were incubated with test compounds, followed by addition of arachidonic acid and a detection reagent, and fluorescence was measured. [1] A broad enzymatic specificity test against 405 human kinases was conducted using a radioactivity-based filter-binding assay. Inhibition percentages were calculated and mapped. [1] |
| Cell Assay |
Cell proliferation was determined using a colorimetric cell proliferation assay. Cells were seeded and treated with compounds or DMSO for a specified duration (72 or 96 hours). The assay reagent was added, and after incubation, absorbance was measured. [1]
For m⁶A dot blot assay, total RNA was isolated from treated cells, and poly(A)+ mRNA was enriched. RNA samples were denatured, dotted onto a membrane, and cross-linked. The membrane was blocked, incubated with an m⁶A antibody, followed by an HRP-conjugated secondary antibody, and developed. [1] LC-MS/MS quantitation of m⁶A and m⁶Aₘ in AML cells involved mRNA isolation, decapping, digestion by nuclease P1 and alkaline phosphatase, and analysis using liquid chromatography coupled with tandem mass spectrometry. [1] For apoptosis analysis, cells were treated with compounds, then collected, washed, and stained with Annexin V and 7-AAD or PI. The stained cells were analyzed by flow cytometry. [1] Myeloid differentiation was analyzed by flow cytometry. Cells were induced with ATRA in the presence of compounds, then stained with fluorescently conjugated antibodies against differentiation markers (e.g., CD11b, CD14, CD15) and analyzed. [1] Cell cycle analysis was performed by flow cytometry. Treated cells were fixed/permeabilized and stained with propidium iodide (PI) or a combination of Hoechst 33342 and Pyronin Y, then analyzed. [1] Colony formation assay for primary AML cells was performed by suspending cells in methylcellulose-based media with or without compounds, seeding in culture dishes, and incubating for 12 days before counting colonies. [1] |
| Animal Protocol |
For the toxicity study in BALB/c mice, FB23-2 was formulated in a vehicle (composition not specified) and administered intraperitoneally daily at doses of 10, 20, 40, and 80 mg/kg for 14 days. Body weight and organ conditions were monitored. [1]
For pharmacokinetic profiling in Sprague Dawley rats, a single dose of 3 mg/kg FB23-2 (formulated in DMSO) was administered intraperitoneally. Blood samples were collected at multiple time points via retro-orbital bleeding for LC-MS/MS analysis. [1] For the MONOMAC6 xeno-transplantation efficacy study, NSGS mice were transplanted with AML cells via tail vein. After 10 days, mice received daily intraperitoneal injections of FB23-2 (2 mg/kg) or vehicle control for 10 days. Mice were monitored for survival and symptoms. [1] For the PDX model efficacy study, NSGS mice were sublethally irradiated and transplanted with primary human AML cells via tail vein. When engraftment reached 3–5% in peripheral blood, mice received daily intraperitoneal injections of FB23-2 (6 mg/kg) or vehicle control (DMSO) for 17 days. Mice were monitored for survival and engraftment levels. [1] |
| ADME/Pharmacokinetics |
Following a single intraperitoneal injection of 3 mg/kg FB23-2 in rats, the Cₘₐₓ was 2421.3 ± 90.9 ng/ml, the Tₘₐₓ was 0.08 h, the elimination half-life (T₁/₂) was 6.7 ± 1.3 h, and the AUC₀–₂₄ was 2184 ± 152 h × ng/ml. FB23 (a hydrolytic metabolite of FB23-2) was also detected, with a Cₘₐₓ of 142.5 ± 26.1 ng/ml and a Tₘₐₓ of 0.4 ± 0.1 h.
The metabolic stability of FB23-2 in rat liver microsomes showed that its half-life (T₁/₂) was estimated to be 128 min, and its intrinsic clearance was 19.7 ml/min/kg. The plasma protein binding of FB23-2 was close to 100%. [1] |
| Toxicity/Toxicokinetics |
In a 14-day repeated-dose toxicity study in BALB/c mice, no weight loss or physical damage to vital organs (heart, kidneys, lungs, liver, spleen) was observed after daily intraperitoneal injection of 20 mg/kg FB23-2. Hematological and plasma biochemical analyses showed no significant differences between the carrier control group and the 20 mg/kg FB23-2 treatment group. [1]
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| References | |
| Additional Infomation |
FB23 and FB23-2 are small molecule inhibitors designed based on a well-structured approach that target the oncogenic mRNA m⁶A demethylase FTO, which is overexpressed in some acute myeloid leukemia (AML) cases. FB23-2 is a benzo[a]hydroxyxamic acid derivative of FB23 designed to enhance cell permeability. These inhibitors are highly selective for FTO, but less selective for the associated demethylase ALKBH5 and a range of other epigenetic targets and kinases. Their antileukemic effect is mediated by FTO inhibition, which leads to increased m⁶A RNA methylation, thereby regulating key genes and signaling pathways (e.g., downregulation of MYC and CEBPA and upregulation of RARA and ASB2), ultimately inhibiting the proliferation of AML cells and leukemia stem cells (LSCs) and inducing their differentiation and apoptosis. [1]
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| Molecular Formula |
C16H12N2F6S
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|---|---|
| Molecular Weight |
378.335
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| Exact Mass |
378.06
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| Elemental Analysis |
C, 50.80; H, 3.20; F, 30.13; N, 7.40; S, 8.47
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| CAS # |
13571-44-3
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| Related CAS # |
13571-44-3
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| PubChem CID |
4142933
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| Appearance |
White to off-white solid powder
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| LogP |
4.8
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
25
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| Complexity |
423
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
PIQMVCPITQIXGJ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C16H12F6N2S/c17-15(18,19)11-6-12(16(20,21)22)8-13(7-11)24-14(25)23-9-10-4-2-1-3-5-10/h1-8H,9H2,(H2,23,24,25)
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| Chemical Name |
1-benzyl-3-[3,5-bis(trifluoromethyl)phenyl]thiourea
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| Synonyms |
VPC70063; VPC-70063; VPC 70063
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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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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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 | 2.6431 mL | 13.2156 mL | 26.4313 mL | |
| 5 mM | 0.5286 mL | 2.6431 mL | 5.2863 mL | |
| 10 mM | 0.2643 mL | 1.3216 mL | 2.6431 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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