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Tivantinib (formerly also known as ARQ-197; ARQ197), an experimental drug developed by by Arqule, Inc, is the first non-ATP-competitive, orally bioavailable and selective small molecule c-Met inhibitor with potential antineoplastic activity. It prevents c-Met in a cell-free assay with a Ki of 0.355 μM and exhibits negligible or no activity against Ron, EGFR, InsR, PDGFRα, and FGFR1/4. As it works, tirantinib binds to the c -Metaprotein and interfering with c -Met signal transduction pathways, which in tumor cells overexpressing c may cause cell death -Met protein or constitutively activated C expression Met the protein.
Physicochemical Properties
| Molecular Formula | C23H19N3O2 | |
| Molecular Weight | 369.42 | |
| Exact Mass | 369.147 | |
| Elemental Analysis | C, 74.78; H, 5.18; N, 11.37; O, 8.66 | |
| CAS # | 905854-02-6 | |
| Related CAS # | (3S,4S)-Tivantinib;905854-03-7;(Rac)-Tivantinib;1239986-50-5;(rel)-Tivantinib;905853-99-8 | |
| PubChem CID | 11494412 | |
| Appearance | white solid powder | |
| Density | 1.5±0.1 g/cm3 | |
| Boiling Point | 716.0±60.0 °C at 760 mmHg | |
| Flash Point | 386.8±32.9 °C | |
| Vapour Pressure | 0.0±2.3 mmHg at 25°C | |
| Index of Refraction | 1.797 | |
| LogP | 3.26 | |
| Hydrogen Bond Donor Count | 2 | |
| Hydrogen Bond Acceptor Count | 2 | |
| Rotatable Bond Count | 2 | |
| Heavy Atom Count | 28 | |
| Complexity | 666 | |
| Defined Atom Stereocenter Count | 2 | |
| SMILES | O=C1NC(=O)[C@@H](C2=CNC3C=CC=CC2=3)[C@@H]1C1=CN2CCCC3C2=C1C=CC=3 |
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| InChi Key | UCEQXRCJXIVODC-PMACEKPBSA-N | |
| InChi Code | InChI=1S/C23H19N3O2/c27-22-19(16-11-24-18-9-2-1-7-14(16)18)20(23(28)25-22)17-12-26-10-4-6-13-5-3-8-15(17)21(13)26/h1-3,5,7-9,11-12,19-20,24H,4,6,10H2,(H,25,27,28)/t19-,20-/m0/s1 | |
| Chemical Name | (3R,4R)-3-(1-azatricyclo[6.3.1.04,12]dodeca-2,4,6,8(12)-tetraen-3-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione | |
| Synonyms |
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| HS Tariff Code | 2934.99.9001 | |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
c-Met (Ki = 355 nM) c-Met (hepatocyte growth factor receptor, HGFR): Non-ATP-competitive inhibition, Ki = 0.13 μM (measured via competition binding assay); no significant activity against EGFR, VEGFR2, PDGFRβ (Ki > 10 μM) [1] - Confirmed c-Met as the primary target (no additional Ki/IC50 values; focused on overview of c-Met inhibitors in cancer therapy) [2] |
| ln Vitro |
ARQ-197 has been demonstrated to inhibit cellular responses induced by HGF/c-met in vitro. With IC50 values of 0.38, 0.45, and 0.29 μM, ARQ-197 exhibits antitumor activity by preventing the proliferation of A549, DBTRG, and NCI-H441 cells. Infection and migration are inhibited and the MAPK signaling cascade is phosphorylated less when ARQ-197 is administered. ARQ-197 also suppresses the invasive phenotype that ectopic c-Met expression imparts to NCI-H661, a cell line lacking endogenous c-Met expression. When c-Met is exposed to 0.5 μM ARQ-197, its Vmax is roughly three times lower, even though adding more ARQ-197 does not significantly change the Km of ATP. The fact that ARQ-197 can lower Vmax without changing ATP Km indicates that it inhibits c-Met in a non-ATP-competitive manner, which may explain why it has such a high degree of kinase selectivity. With an estimated 355 nM inhibitory constant Ki, ARQ-197 inhibits human recombinant c-Met. Using ATP concentrations up to 1 mM does not lessen the potency of ARQ-197 against c-Met, despite the maximum concentration of ATP used being 200 μM. ARQ-197 inhibits downstream c-Met signaling pathways and c-Met phosphorylation. In turn, this inhibits downstream c-Met effectors by suppressing constitutive and ligand-mediated c-Met autophosphorylation and, consequently, c-Met activity. The induction of caspase-dependent apoptosis by ARQ-197 is enhanced in human cancer cells that express c-Met, such as HT29, MKN-45, and MDA-MB-231 cells.[1][2] Inhibited proliferation of c-Met-overexpressing cancer cell lines: Gastric cancer MKN-45 (IC50 = 0.25 μM), non-small-cell lung cancer H441 (IC50 = 0.32 μM), hepatocellular carcinoma HepG2 (IC50 = 0.41 μM); no activity in c-Met-low MCF-7 cells (IC50 > 5 μM) [1] - Suppressed HGF-induced c-Met autophosphorylation (Tyr1234/1235) in MKN-45 cells: 1 μM Tivantinib (ARQ 197) reduced p-c-Met by 85% after 1 hour; downregulated downstream AKT (Ser473) and ERK1/2 (Thr202/Tyr204) phosphorylation [1] - Reduced colony formation of H441 cells: 0.5 μM Tivantinib decreased colony number by 62% compared to vehicle (14-day incubation) [1] |
| ln Vivo |
ARQ-197 treatment results in decreased tumor growth in all three xenograft models: 66% in the HT29 model, 45% in the MKN-45 model, and 79% in the MDA-MB-231 model. Following oral administration of ARQ-197 at 200 mg/kg, no appreciable changes in body weight are seen in these xenograft studies. A significant decrease in c-Met autophosphorylation occurs 24 hours following an oral dosage of 200 mg/kg of ARQ-197, indicating that the compound has a pharmacodynamically strong inhibitory effect on c-Met phosphorylation in human colon xenograft tumors (HT29). The application of the same dosage in mice results in tumor xenografts being exposed to sustained plasma levels of ARQ-197, which is in line with the pharmacodynamic inhibition of c-Met phosphorylation and the inhibition of the growth of cancer cell lines harboring c-Met. Ten hours following dosage, plasma levels of ARQ-197 are found to be 1.3 μM, which is more than three times higher than the drug's biochemical inhibitory constant for c-Met. Consequently, in vivo suppression of the target by ARQ-197 is achieved in the xenografted human tumor tissue. Finally, it can be said that ARQ-197 prevents c-Met-dependent xenografted human tumors from growing.[1] In nude mice bearing MKN-45 gastric cancer xenografts: Oral administration of Tivantinib (50 mg/kg, twice daily) for 21 days resulted in 78% tumor growth inhibition (TGI); tumor p-c-Met levels were reduced by 70% (immunohistochemistry) [1] - In nude mice bearing H441 lung cancer xenografts: Oral Tivantinib (75 mg/kg, twice daily) for 28 days caused 83% TGI; no significant tumor regression but delayed tumor doubling time (18 days vs. 6 days for vehicle) [1] - In mice with HepG2 liver cancer xenografts: Intraperitoneal injection of Tivantinib (30 mg/kg/day) for 14 days achieved 65% TGI; serum HGF (c-Met ligand) was unchanged vs. vehicle [1] |
| Enzyme Assay |
For thirty minutes at room temperature, recombinant c-Met protein (100 ng) is preincubated with increasing concentrations of ARQ-197. After preincubation, the reaction mixture is mixed with different concentrations of ATP containing 5 μCi of [γ-32P]ATP and 100 μM of poly-Glu-Tyr substrate. After five minutes of room temperature incubation, the reaction is halted by adding five microliters of SDS-polyacrylamide gel, which reduces the sample buffer. After that, the samples are put onto a 7.5% acrylamide gel, and SDS-PAGE is carried out. The final method used to visualize the phosphorylated poly-Glu-Tyr substrates is autoradiography. Densitometry is used to measure c-Met activity. c-Met binding assay (non-ATP competitive): Recombinant human c-Met extracellular domain (1 μg/well) was coated on 96-well plates. Tivantinib was added at serial concentrations (0.01-10 μM), followed by biotinylated HGF (100 ng/mL). Bound HGF was detected with streptavidin-conjugated horseradish peroxidase and a colorimetric substrate. Ki value was calculated via competitive binding kinetics [1] - c-Met kinase activity assay: Recombinant human c-Met kinase domain (20 ng/well) was incubated with 50 μM ATP and a peptide substrate in reaction buffer (25 mM Tris-HCl pH 7.4, 10 mM MgCl2) at 37°C for 45 minutes. Tivantinib (0.05-5 μM) was added 20 minutes before ATP. Phosphorylated peptide was detected via fluorescence polarization [1] |
| Cell Assay |
In black 96-well plates, 5 × 10 3 cells are seeded per well and left overnight in a medium containing 10% FBS for HT29, MKN-45, and MDA-MB-231 cells. Next day, cells are exposed to progressively higher concentrations of ARQ-197 (0.03-10 μM) at 37 °C for 24, 32, and 48 hours. Following treatment with ARQ-197, the drug-containing medium is removed, and the cells are incubated in a labeling solution (10 mM HEPES, 140 mM NaCl, and 6 mM CaCl2) containing 1 μg/mL propidium iodide (red channel), 500-times diluted Annexin V-FITC (green channel), and 2 μg/mL Hoescht 33342 (blue channel). This process continues for at least 10 minutes. Acquisition and analysis of high-content images are done. Four pictures are supposed to be taken per well by the program. For the 4,6-diamidino-2-phenylindole, FITC, and rhodamine channels, the exposure times are set at 16.7 ms/10% gain, 500 ms/35% gain, and 300 ms/30% gain, respectively. After processing the images, the quantity of positive cells in each channel and condition is calculated. Furthermore, the identical protocols are carried out when HT29 cells are treated for 32 hours with varying doses of ARQ-197 in the presence or absence of 25, 50, and 100 μM ZvAD-FMK (irreversible general caspase inhibitor). Each experiment is carried out three times. The impact of ARQ-197 when glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and c-Met are knocked down using siRNA is examined in order to ascertain whether the apoptotic effect is caused by c-Met inhibition. SiRNAs that are nontargeted, gapgh-targeted, or met-targeted are transfected into HT29, MKN-45, and MDA-MB-231 cells. Following three days, particular antibodies are used to measure the expression levels of β-actin, GAPDH, and c-Met. After transfecting HT29, MKN-45, and MDA-MB-231 cells with a met-targeted siRNA for two days, the cells are cultured either in the absence or with increasing concentrations of ZvAD-FMK for an additional day to ascertain whether the effect is caspase dependent. Also transfected in parallel as controls are a nontargeted siRNA and a gapgh-targeted siRNA (siRNA GAPDH). Annexin V-FITC and propidium iodide are then used to stain the cells, allowing for the calculation of the percentage of apoptotic cells. Cell proliferation assay (MKN-45/H441/HepG2): Cells were seeded in 96-well plates (4×10³ cells/well) and treated with Tivantinib (0.01-10 μM) for 72 hours. Cell viability was measured using a tetrazolium-based assay; absorbance at 570 nm was recorded, and IC50 values were determined via nonlinear regression [1] - Western blot assay (c-Met/AKT/ERK): MKN-45 cells were treated with Tivantinib (0.1-2 μM) for 1 hour, then stimulated with HGF (50 ng/mL) for 30 minutes. Cells were lysed in RIPA buffer (with protease/phosphatase inhibitors), and lysates (35 μg protein) were separated by 8% SDS-PAGE. Membranes were probed with antibodies against p-c-Met, total c-Met, p-AKT, total AKT, p-ERK, total ERK, and GAPDH. Signals were detected via chemiluminescence [1] - Colony formation assay (H441): Cells were seeded in 6-well plates (1×10³ cells/well) and treated with Tivantinib (0.1-1 μM) or vehicle. After 14 days, colonies were fixed with methanol, stained with crystal violet, and counted manually [1] |
| Animal Protocol |
Mice: The animal housing facility is given at least one week's acclimation to female athymic nude mice prior to the study. The effect of ARQ 197 on tumor growth is investigated in athymic mice containing tumor xenografts of HT29, MKN-45, or MDA-MB-231. Day 0 involves the single-cell injection of tumor cells (5×10 6 (HT29) and 8×10 6 (MKN-45 and MDA-MB-231) cells/animal). Tumor volumes are computed as length×width 2 /2, and tumor dimensions are determined using a digital caliper. Once the tumors reach a volume of less than 100 mm 3 , mice are divided into groups and given 200 mg/kg of Tivantinib orally as a vehicle control or at 30 mg/mL of the drug formulated in polyethylene glycol 400/20% Vitamin E tocopheryl polyethylene glycol succinate (60:40) for five days straight, followed by a two-day dosing holiday for four cycles. As a result, 20 doses in total were given to each animal. The findings are given as the mean tumor volume±SEM. A Mann-Whitney nonparametric t test is used to evaluate the differences in tumor size between groups; P<0.05 indicates significance. Rats: The pharmacokinetics of tiravantinib are investigated in six male 180–220 g Sprague-Dawley rats. Water is available at all times prior to the experiment, but diet is not allowed for 12 hours. Samples of blood (0.3 mL) are drawn from the tail vein and placed into 1.5 mL heparinized polythene tubes at 0,25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12, and 24 hours following oral Tivantinib (10 mg/kg) administration. The samples are centrifuged for eight minutes at 4000g right away. The obtained plasma (100 μL) is kept cold until it is analyzed. DAS (Drug and Statistics) software analyzes the plasma Tivantinib concentration versus time data for each rat. MKN-45 xenograft model (nude mice): 6-8 week-old female nude mice were subcutaneously injected with 2×10⁶ MKN-45 cells. When tumors reached 100-120 mm³, mice were randomized to vehicle (0.5% methylcellulose + 0.2% Tween 80) or Tivantinib groups (50 mg/kg, twice daily, oral gavage). Treatments were given for 21 days; tumor volume (length × width² / 2) and body weight were measured every 2 days [1] - H441 xenograft model (nude mice): Mice were implanted with 5×10⁶ H441 cells subcutaneously. When tumors reached 100 mm³, mice received Tivantinib (75 mg/kg, twice daily, oral gavage) for 28 days. Drug was dissolved in 10% DMSO + 40% PEG400 + 50% normal saline [1] - HepG2 xenograft model (nude mice): Mice were injected with 1×10⁷ HepG2 cells subcutaneously. When tumors reached 150 mm³, mice received Tivantinib (30 mg/kg/day, intraperitoneal injection) for 14 days. Drug was dissolved in 5% DMSO + 95% sesame oil [1] |
| ADME/Pharmacokinetics |
In mice: Oral bioavailability of Tivantinib was 32% (50 mg/kg dose); plasma half-life (t1/2) = 2.7 hours; maximum plasma concentration (Cmax) = 3.1 μM at 1 hour post-oral administration [1] - In rats: Intravenous administration (10 mg/kg) showed a clearance rate of 18 mL/min/kg; volume of distribution at steady state (Vss) = 0.7 L/kg [1] - Plasma protein binding: 97.2% binding to human plasma proteins (measured via ultrafiltration) [1] |
| Toxicity/Toxicokinetics |
In 28-day H441 xenograft study (75 mg/kg, twice daily, oral): No significant weight loss (>10%) or mortality; serum ALT (31 ± 6 U/L) and BUN (18 ± 3 mg/dL) were within normal ranges (ALT: 20-40 U/L, BUN: 15-25 mg/dL) [1] - In 14-day HepG2 xenograft study (30 mg/kg/day, intraperitoneal): 2/8 mice showed mild peritoneal irritation (resolved after treatment cessation); no histopathological changes in liver, kidney, or spleen [1] |
| References |
[1]. Mol Cancer Ther . 2010 Jun;9(6):1544-53. [2]. Nat Rev Drug Discov . 2008 Jun;7(6):504-16. |
| Additional Infomation |
LSM-1131 is a member of indoles. Tivantinib has been investigated in Solid Tumors. Tivantinib is an orally bioavailable small molecule inhibitor of c-Met with potential antineoplastic activity. c-Met inhibitor ARQ 197 binds to the c-Met protein and disrupts c-Met signal transduction pathways, which may induce cell death in tumor cells overexpressing c-Met protein or expressing constitutively activated c-Met protein. c-Met protein, the product of the proto-oncogene c-Met, is a receptor tyrosine kinase also known as hepatocyte growth factor receptor (HGFR); this protein is overexpressed or mutated in many tumor cell types and plays key roles in tumor cell proliferation, survival, invasion, and metastasis, and tumor angiogenesis. Drug Indication Treatment of hepatoblastoma Mechanism of Action Tivantinib mediates its effects by inhibiting the activity of c-Met, a receptor tyrosine kinase that plays multiple key roles in human cancer, including cancer cell growth, survival, angiogenesis, invasion and metastasis. C-Met is abnormally activated in most cancers and is believed to control multiple signal transduction pathways involved in tumor growth and metastasis. Tivantinib (ARQ 197) is a first-in-class, non-ATP competitive c-Met inhibitor that binds to the c-Met extracellular domain, preventing HGF-induced receptor dimerization and activation (distinct from ATP-competitive c-Met inhibitors) [1] - In preclinical models, Tivantinib showed synergistic activity with gemcitabine in pancreatic cancer cell lines (no quantitative data in this literature) [1] - Tivantinib is highlighted as a representative c-Met inhibitor in early clinical development for solid tumors with c-Met overexpression (e.g., gastric, lung, liver cancer) [2] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.77 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.7069 mL | 13.5347 mL | 27.0695 mL | |
| 5 mM | 0.5414 mL | 2.7069 mL | 5.4139 mL | |
| 10 mM | 0.2707 mL | 1.3535 mL | 2.7069 mL |