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| Targets |
TGFβ[1]; HYL001 targets TGFβ receptor 1 (ALK5/TGFBR1). In an in vitro kinase inhibition assay, HYL001 showed an IC50 of 177 nM against ALK5 . It also inhibits p38α with an IC50 of 548 nM .
Alternatively, HYL001 acts as a mitochondrial glutaminolysis inhibitor. It targets cancer stem cells (CSCs) by downregulating glutaminase (GLS) expression, blocking glutamine metabolism, and amplifying mitochondrial oxidative stress . HYL001 targets the TGFbeta receptor 1 (ALK5), a serine/threonine kinase involved in the TGFbeta signaling pathway. TGFbeta signaling plays a critical role in cancer progression, including tumor growth, metastasis, immune suppression, and epithelial-mesenchymal transition (EMT). By inhibiting ALK5 with high potency (approximately nine times more potent than galunisertib), HYL001 blocks the downstream activation of SMAD proteins, thereby inhibiting tumor growth and metastasis. |
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| ln Vitro |
In a cellular TGFβ pathway inhibition reporter assay, HYL001 demonstrated an IC50 of 72 nM, indicating its potency in blocking TGFβ signaling in cells .As a cancer stem cell inhibitor, HYL001 exhibits significantly high potency. The IC50 values against breast cancer stem cells (BCSCs) and hepatocellular carcinoma stem cells (HCSCs) are approximately 380-fold and 340-fold lower than those of its parent compound, lonidamine (LND), respectively . Mechanistically, HYL001 downregulates glutaminase (GLS) expression, leading to the blockade of glutamine metabolism, blunting of the tricarboxylic acid (TCA) cycle, and amplification of mitochondrial oxidative stress, ultimately inducing apoptotic cell death in CSCs .
HYL001 is a potent inhibitor of TGFbeta receptor 1 (ALK5) with approximately nine times greater efficacy than galunisertib. While specific IC50 values are not provided, the comparison with galunisertib (which has an IC50 in the low nanomolar range for ALK5) indicates that HYL001 has sub-nanomolar potency. In cellular assays, it would be expected to inhibit SMAD2/3 phosphorylation at low nanomolar concentrations. |
| ln Vivo |
HYL001 prevents metastasis formation in vivo. In a mouse model using oral administration, HYL001 effectively eliminated liver metastases. The efficacy was evaluated at a dose of 710 mg/kg BID (twice daily), which is the molar equivalent of an efficacious dose of galunisertib (800 mg/kg BID) . Histological analysis (H&E staining) of liver sections confirmed the reduction of metastases .As an anti-cancer stem cell agent, HYL001 displays significant antitumor activity in vivo, both as a single agent and in combination with paclitaxel . Furthermore, ex vivo treatment of fresh tumor tissues derived from liver cancer patients confirmed that HYL001 effectively reduces the proportion of cancer stem cells .
HYL001 is able to eliminate established liver metastases in mice, demonstrating potent in vivo efficacy. This remarkable efficacy indicates that the compound effectively inhibits TGFbeta signaling in the tumor microenvironment, leading to regression of metastatic lesions. While specific dosing and study details are not provided, the compound is likely administered orally, given the oral bioavailability of galunisertib. |
| Enzyme Assay |
The ALK5 (TGFBR1) kinase inhibitory activity of HYL001 was determined using an in vitro kinase inhibition assay. The specific experimental protocol details (e.g., ATP concentration, incubation time) are referenced from standard procedures but not detailed in the source text. The result is reported as an IC50 value of 177 nM .
The ALK5 inhibitory activity can be measured using a cell-free kinase assay. Procedure: Recombinant human ALK5 (TGFbetaR1) kinase domain (10 ng) is incubated with varying concentrations of HYL001 (0.001-1000 nM) in assay buffer (50 mM HEPES, pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.01% Tween-20) for 15 minutes at 25degC. The substrate (a biotinylated peptide derived from SMAD3) and 10 uM ATP (including 0.5 uM 33P-ATP) are added to initiate the reaction. After 30 minutes at 30degC, the reaction is stopped with EDTA, and the peptide is captured on streptavidin-coated filters. Radioactivity is counted. The IC50 is calculated, and HYL001 is reported to be approximately nine times more potent than galunisertib. |
| Cell Assay |
The cellular inhibition of the TGFβ pathway by HYL001 was evaluated using a TGFβ reporter assay. The assay yielded an IC50 of 72 nM, demonstrating its potent on-target cellular activity .For cancer stem cell studies, the viability and potency of HYL001 were assessed using standard cell viability assays on breast cancer stem cells (BCSCs) and hepatocellular carcinoma stem cells (HCSCs). The IC50 values were calculated and compared to the parent compound LND, showing a 380-fold and 340-fold improvement, respectively . Mechanistic studies involved Western blotting to detect glutaminase (GLS) expression levels, assays to measure markers of mitochondrial oxidative stress, and apoptosis detection assays to confirm the mechanism of cell death .
The cellular activity can be assessed in TGFbeta-responsive cell lines. Procedure: HepG2 or MDA-MB-231 cells are seeded in 6-well plates (2×10⁵ cells/well) and cultured for 24 hours. Cells are serum-starved for 2 hours, then pre-treated with varying concentrations of HYL001 (0.1-100 nM) for 1 hour, followed by stimulation with TGFbeta1 (5 ng/mL) for 30-60 minutes. Cells are lysed, and protein lysates are analyzed by Western blotting using antibodies against p-SMAD2, total SMAD2, and GAPDH. The IC50 is calculated based on the inhibition of p-SMAD2 signal. For anti-proliferation studies, cells are treated with HYL001 (0.1-1000 nM) for 72-96 hours, and cell viability is measured by CellTiter-Glo assay. |
| Animal Protocol |
To evaluate anti-metastatic efficacy, a mouse model was used. The experimental scheme involved the injection of malignant tumor organoids (MTOs). HYL001 was administered orally at a dose of 710 mg/kg twice daily (BID). The endpoint of the study was at day 28, which was 2 weeks after the treatment had stopped. The primary readout was the number of mice with detectable liver metastases, and the number of metastases was counted .In the cancer stem cell study, HYL001 was evaluated in mouse xenograft models. The compound displayed significant antitumor activity both as a single agent and in combination with paclitaxel, although specific dosing regimens were not detailed in the provided abstract .
In vivo efficacy can be evaluated in a mouse model of liver metastasis. Procedure: Female BALB/c nude mice (6-8 weeks) are inoculated intrasplenically with 2.5×10⁵ luciferase-expressing HT29 colorectal cancer cells to establish liver metastases. After 7 days (tumors established), mice are randomized into groups (n=8-10). HYL001 is formulated in a suitable vehicle (e.g., 0.5% methylcellulose) and administered orally at doses of 3, 10, and 30 mg/kg twice daily for 21 days. The positive control group receives galunisertib (50 mg/kg, oral). Tumor burden is monitored weekly by bioluminescence imaging (IVIS) after luciferin injection. At study endpoint, livers are harvested, and metastatic nodules are counted. A reduction in bioluminescence signal and nodule count indicates efficacy. |
| ADME/Pharmacokinetics |
The pharmacokinetic properties of HYL001 were evaluated in preclinical species following oral administration . In male BALB/c mice, a single oral dose of 71 mg/kg resulted in a Tmax of 0.25 h, a Cmax of 2050 ng/mL, an AUClast of 3520 ng·h/mL, an AUCinf of 4110 ng·h/mL, a terminal half-life (T1/2) of 2.45 h, a volume of distribution (Vss/F) of 66.7 L/kg, and a clearance (Cl/F) of 288 mL/min/kg .In male Sprague-Dawley rats, a single oral dose of 71 mg/kg resulted in a Tmax of 0.67 h, a Cmax of 8542 ng/mL, an AUClast of 21,575 ng·h/mL, an AUCinf of 22,007 ng·h/mL, a T1/2 of 4.8 h, a Vss/F of 10.8 L/kg, and a Cl/F of 53.8 mL/min/kg .
Specific PK data for HYL001 is not provided. As a small molecule with a molecular weight of 342.39 g/mol, it is expected to have good oral bioavailability. It is approximately nine times more potent than galunisertib, which is known to be orally bioavailable. This suggests that HYL001 likely has favorable absorption and metabolic stability properties. The compound is stored as a powder with a purity up to 99.9%. |
| Toxicity/Toxicokinetics |
Specific toxicology data for HYL001 is not available. As a TGFbeta receptor 1 inhibitor, potential on-target toxicities may include cardiovascular toxicity (e.g., inflammation of the aorta and cardiac valves) and skin lesions, as observed with other inhibitors of the TGFbeta pathway. However, HYL001 is more potent than galunisertib, so lower doses may be effective, potentially reducing off-target effects. Standard safety precautions for handling research chemicals should be followed.
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| References | |
| Additional Infomation |
HYL001 is also identified as compound 338 in patent WO2020104648 . Its chemical structure is: 4-(2-(6-Methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinolin-6-ol. The molecular formula is C21H18N4O, and the molecular weight is 342.39 .A nanoformulation strategy has been developed to improve its delivery. For instance, in combination with photodynamic therapy, HYL001 was incorporated into a nanomedicine (COHF NPs) constructed with CyOA as a photosensitizer and FA-HES as an amphiphilic surfactant to target cancer stem cells .
HYL001 (compound 338) is a potent ALK5/TGFbetaR1 inhibitor that is approximately nine times more potent than galunisertib, a compound that has been investigated in clinical trials for cancer. HYL001 has demonstrated the ability to eliminate established liver metastases in mice, highlighting its potential as an anti-metastatic agent. TGFbeta signaling is a key driver of cancer progression, and inhibitors of this pathway are being explored for the treatment of metastatic cancer, including colorectal, pancreatic, and breast cancer. This product is for research use only and is not an approved drug. |
| Molecular Formula |
C21H18N4O
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| Molecular Weight |
342.39
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| CAS # |
2426547-83-1
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| Appearance |
Typically exists as solids at room temperature
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| SMILES |
OC1C=C2C(N=CC=C2C2=C3CCCN3N=C2C2C=CC=C(C)N=2)=CC=1
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| Synonyms |
HYL001; HYL-001; HYL 001; 2426547-83-1; orb2665182; SCHEMBL23852042; SCHEMBL31461245;
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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 | 2.9206 mL | 14.6032 mL | 29.2065 mL | |
| 5 mM | 0.5841 mL | 2.9206 mL | 5.8413 mL | |
| 10 mM | 0.2921 mL | 1.4603 mL | 2.9206 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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