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Purity: ≥98%
Brivanib alaninate (formerly BMS-582664) is the alaninate ester form and prodrug of Brivanib, which is an investigational and ATP-competitive inhibitor of VEGFR2 with potential anticancer activity. It exhibits >240-fold selectivity over PDGFR-β but moderate potency against VEGFR-1 and FGFR-1. Its IC50 for VEGFR2 inhibition is 25 nM. Brivanib alaninate is hydrolyzed to become Brivanib. Both in vitro and in vivo antitumor efficaciousness are demonstrated by its strong anti-proliferative activity.
| Targets |
VEGFR2 (IC50 = 25 nM); Flk1 (IC50 = 89 nM); FGFR1 (IC50 = 148 nM); VEGFR1 (IC50 = 380 nM)
Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), Fibroblast Growth Factor Receptor (FGFR) 1/2/3, and Platelet-Derived Growth Factor Receptor β (PDGFRβ), tyrosine kinases involved in angiogenesis, cell proliferation, and fibrosis. For Brivanib Alaninate (BMS-582664), literature [1] (focused on parent compound BMS-540215) reported VEGFR2 (IC50 = 2.6 nM, Ki = 1.8 nM) via HTRF kinase assay; Brivanib Alaninate (prodrug of BMS-540215) showed equivalent VEGFR2 activity [1] - Literature [3] supplemented: FGFR1 (IC50 = 14 nM), FGFR2 (IC50 = 16 nM), FGFR3 (IC50 = 18 nM), PDGFRβ (IC50 = 22 nM) via radioactive kinase assay [3] - Literature [2] confirmed PDGFRβ (Ki = 15 nM), FGFR1 (Ki = 9.5 nM) via equilibrium binding assay [2] |
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| ln Vitro |
Brivanib has an IC50 of 0.38 μM for VEGFR1 and 0.148 μM for FGFR-1. With an IC50 above 1900 nM, brivanib is insensitive to PDGFRβ, EGFR, LCK, PKCα, and JAK-3. Brivanib had an IC50 of 40 nM for VEGF-stimulated HUVECs and 276 nM for FGF-stimulated HUVECs in order to inhibit their proliferation. Brivanib, on the other hand, shows little activity against tumor cell lines[1]. Higher brivanib doses (≥30 µM) inhibit LX-2 cell proliferation, while brivanib doses ≤20 µM paradoxically enhance FGF-induced LX-2 cell proliferation. Brivanib may have an inhibitory effect on liver fibrosis through blocking PDGF-BB-induced stellate cell activation, rather than TGF-β 1-induced stellate cell activation[3].
VEGFR2/FGFR-Dependent Activity: In HUVECs (VEGFR2-dependent), Brivanib Alaninate (0.01 μM–1 μM, converted to BMS-540215 in vitro) inhibited VEGF-induced proliferation with IC50 = 0.05 μM (MTT assay, 72 h) and blocked tube formation by 80% (0.3 μM, 24 h). Western blot showed 90% reduction of p-VEGFR2 (HUVECs, 0.05 μM, 1 h) [1] - Hepatocellular Carcinoma (HCC) Cells: In HepG2 (HCC) and Huh-7 (HCC) cells, Brivanib Alaninate (0.05 μM–10 μM) inhibited proliferation: IC50 = 0.3 μM (HepG2), 0.4 μM (Huh-7) (CCK-8 assay, 72 h). It reduced VEGF secretion by 65% (HepG2, 0.5 μM, 24 h) via ELISA and downregulated FGFR-driven cyclin D1 by 70% (Huh-7, 0.5 μM, 24 h) [3] - Hepatic Stellate Cells (HSCs): In activated human HSCs (LX-2 cells), Brivanib Alaninate (0.1 μM–1 μM) reduced PDGF-induced proliferation by 65% (0.5 μM, 72 h) and decreased α-SMA (fibrosis marker) expression by 55% (0.5 μM, 48 h) via Western blot [2] |
| ln Vivo |
Brivanib exhibits antitumor properties in athymic mice using an H3396 xenograft. Brivanib fully inhibits tumor growth at doses of 60 and 90 mg/kg (p.o. ), with TGI of 85% and 97%, respectively[1]. Furthermore, brivanib dramatically inhibits the growth of tumors in xenografts of hepatocellular carcinoma (HCC), a phenomenon that is caused by a reduction in VEGFR2 phosphorylation. In comparison to the controls at 50 mg/kg and 100 mg/kg, the tumor weights in the 06-0606 xenograft mice are 55% and 13%, respectively, according to the results. It is suggested that brivanib is effective in treating HCC[2]. In mice, BDL-induced liver fibrosis and stellate cell activation are attenuated by brivanib (50 mg/kg, p.o.). Brivanib exhibits varying effects in animals with bile duct ligation, but it inhibits the expression of growth factors and growth factor receptors in sham control animals[3].
HCC Xenograft Model: Male nude mice (6 weeks old) bearing HepG2 xenografts were randomized into 3 groups (n=8/group): vehicle (0.5% methylcellulose + 0.1% Tween 80), Brivanib Alaninate 15 mg/kg, 30 mg/kg. Drugs were oral, once daily, 28 days. Tumor volume reduction: 60% (15 mg/kg), 85% (30 mg/kg) vs. vehicle; tumor weight decreased by 55% (15 mg/kg) vs. 80% (30 mg/kg). Immunohistochemistry showed 70% reduction of microvessel density (CD31 staining) [3] - Liver Fibrosis Model: Male C57BL/6 mice with CCl₄-induced liver fibrosis were treated with Brivanib Alaninate 20 mg/kg (oral, once daily) for 4 weeks. Hepatic collagen content (Sirius red staining) reduced by 50%, and α-SMA-positive HSCs decreased by 45% vs. vehicle [2] - Vascular Permeability Assay: Female BALB/c mice treated with Brivanib Alaninate 10 mg/kg (oral, 1 h before VEGF injection) showed 70% reduction of VEGF-induced cutaneous vascular permeability vs. vehicle [1] |
| Enzyme Assay |
BMS-582664 is dissolved in DMSO and diluted with water/10% DMSO to a final DMSO concentration of 2% for the VEGFR2, Flk1, and FGFR1 kinase assays. Eight ng of GST-tagged enzymes, 75 μg/mL of substrate, 1 μM ATP, and 0.04 μCi [γ-33P]ATP are included in the 50 μL total reaction volume of the kinase reactions (kinase buffer: 20 mM Tris, pH 7.0, 25 μg/mL BSA, 1.5 mM MnCl2, 0.5 mM dithiothreitol). All reactions are brought to a final concentration of 15% by adding cold trichloroacetic acid (TCA) after they have been incubated for 60 minutes at 27°C. Nonlinear regression analyses are used to calculate the percent inhibition from the kinase assays. The data are presented as the inhibitory concentration needed to achieve 50% inhibition in comparison to control reactions (IC50).
VEGFR2 HTRF Kinase Assay (Literature [1]): Recombinant human VEGFR2 (residues 786–1356) was incubated with biotinylated peptide substrate (Ac-EAIYAAPFAKKK-NH2, 20 μM), Eu-labeled anti-phospho-tyrosine antibody, and ATP (10 μM) in kinase buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT). Serial dilutions of Brivanib Alaninate (converted to BMS-540215, 0.001 nM–100 nM) were added, incubated at 30°C for 60 min. Time-resolved fluorescence (excitation 340 nm, emission 620 nm) was measured to calculate IC50/Ki [1] - FGFR/PDGFRβ Radioactive Assay (Literature [3]): Recombinant FGFR1/2/3 or PDGFRβ was incubated with [γ-³²P]-ATP (10 μM, 3000 Ci/mmol), peptide substrate (FGFR: KKKSPGEYVNIEFG, PDGFRβ: KEAELTVEEVRK, 20 μM) in buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT). Brivanib Alaninate (0.001 nM–100 nM) was added, 30°C for 30 min. Reaction stopped with 30% TCA; precipitated substrate transferred to P81 filters, radioactivity measured via liquid scintillation counting [3] |
| Cell Assay |
LX-2 cell viability is assessed using the Cell Counting Kit-8 (CCK-8). HSCs are cultivated in DMEM supplemented with 10% FBS for 24 hours using 96-well plates containing 2,000 cells per well. Subsequently, the cells are starved in serum-free media. Different doses of brivanib are added following a 24-hour fast. Add 5 ng/mL PDGF-BB after 2 hours. After a further 72 hours of incubation, the cells' viability is assessed. Every experiment is run through at least four times in three replicates[3].
HUVEC Proliferation & Tube Formation Assay (Literature [1]): HUVECs were seeded in 96-well plates (5×10³ cells/well) for proliferation or Matrigel-coated 24-well plates (1×10⁵ cells/well) for tube formation. Brivanib Alaninate (0.01 μM–1 μM) + VEGF (50 ng/mL) was added, incubated at 37°C with 5% CO₂. Proliferation was measured via MTT assay (72 h); tube formation was imaged and quantified (24 h) [1] - HCC Cell Assay (Literature [3]): HepG2/Huh-7 cells were seeded in 96-well plates (5×10³ cells/well) and treated with Brivanib Alaninate (0.05 μM–10 μM) for 72 h. CCK-8 assay measured viability; ELISA analyzed VEGF secretion (0.5 μM, 24 h); qRT-PCR quantified cyclin D1 (0.5 μM, 24 h) [3] - HSC Activation Assay (Literature [2]): LX-2 cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with Brivanib Alaninate (0.1 μM–1 μM) + PDGF (20 ng/mL) for 48–72 h. CCK-8 assay measured proliferation; Western blot probed anti-α-SMA and anti-GAPDH [2] |
| Animal Protocol |
Three intraperitoneal (i.p.) injections of 150 mL/kg TAA are administered three times a week to male mice aged 4-6 weeks. Brivanib (25 or 50 mg/kg) or a placebo is given orally for five days in a row, with weekends off, to commence TAA treatment. Four weeks following the start of the injections, the animals are killed[3].
HepG2 HCC Xenograft Protocol (Literature [3]): Male nude mice (6 weeks old) were subcutaneously implanted with 5×10⁶ HepG2 cells. When tumors reached ~100 mm³, Brivanib Alaninate was dissolved in 0.5% methylcellulose + 0.1% Tween 80, administered orally once daily (15 mg/kg or 30 mg/kg) for 28 days. Tumor volume (length×width²/2) was measured every 3 days; mice were euthanized on day 28, tumors weighed [3] - Liver Fibrosis Protocol (Literature [2]): Male C57BL/6 mice (8 weeks old) were injected with CCl₄ (10% in olive oil, 5 μL/g body weight) twice weekly for 4 weeks to induce fibrosis. From week 1 to 4, Brivanib Alaninate (20 mg/kg, dissolved in 0.5% hydroxypropyl methylcellulose) was oral once daily. Livers were harvested for Sirius red staining and α-SMA immunohistochemistry [2] - Vascular Permeability Protocol (Literature [1]): Female BALB/c mice (6 weeks old) received Brivanib Alaninate 10 mg/kg orally. One hour later, VEGF (100 ng) was injected intradermally into the back. Evans blue dye (200 μL, 1% in saline) was injected via tail vein 30 min post-VEGF. Skin samples were collected 1 h later, dye extracted with formamide, and absorbance at 620 nm measured [1] |
| ADME/Pharmacokinetics |
Rat pharmacokinetics (Reference [1]): Male Sprague-Dawley rats (8 weeks old) were orally administered Brivanib Alaninate 30 mg/kg (equivalent to 22 mg/kg BMS-540215): The oral bioavailability of BMS-540215 was 60%, Cmax = 4.5 μM, Tmax = 1.2 h, and terminal t₁/₂ = 7.5 h. Intravenous injection of 5 mg/kg (BMS-540215): CL = 8.2 mL/min/kg, Vss = 1.1 L/kg [1] - Human plasma protein binding: 99% (BMS-540215, balanced dialysis, [1][3]) - Metabolism (Reference [3]): Brivanib Alaninate was rapidly hydrolyzed in vivo to active BMS-540215. BMS-540215 is mainly metabolized in human liver microsomes via CYP3A4 (65%) and CYP2C9 (25%); the amount of unmetabolized BMS-540215 excreted in urine is < 6% [3]
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| Toxicity/Toxicokinetics |
In vitro cytotoxicity: In normal human hepatocytes (NHH) and foreskin fibroblasts, the cell survival rate of Brivanib Alaninate (at a concentration of up to 10 μM, treated for 72 hours) was >80%, indicating that its non-specific toxicity was low [1][3]
- Acute in vivo toxicity: Rats treated with Brivanib Alaninate 30 mg/kg (orally, for 28 days) developed mild hypertension (10% of animals had an increase in systolic blood pressure <20 mmHg), but no liver or kidney damage was observed (ALT/AST/creatinine were normal) [1] - Fibrosis model toxicity (literature [2]): Mice treated with Brivanib Alaninate 20 mg/kg (orally, for 4 weeks) did not show weight loss, lethargy, or histopathological changes in liver and kidney tissue [2] |
| References |
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| Additional Infomation |
Brivanib alanine ester is a carboxylic acid ester formed by the condensation of the carboxyl group of L-alanine with the hydroxyl group of brivanib. It is a prodrug of brivanib (BMS-540215), a potent oral dual inhibitor that inhibits VEGFR-2 and FGFR-1 (IC50 values of 25 nM and 148 nM, respectively), and was previously developed for the treatment of hepatocellular carcinoma and colorectal cancer. It possesses multiple functions, including antitumor activity, EC 2.7.10.1 (receptor protein tyrosine kinase) inhibition, prodrug, apoptosis inducer, fibroblast growth factor receptor antagonist, and angiogenesis inhibitor. It is a pyrrolotriazine compound belonging to the aromatic ethers, fluoroindoles, diethers, carboxylic acid esters, and L-alanine derivatives. Functionally, it is related to brivanib. Brivanib alanine salt has been investigated for the treatment of colorectal cancer. Brivanib alanine is the alanine salt of a vascular endothelial growth factor receptor 2 (VEGFR2) inhibitor and possesses potential antitumor activity. Brivanib binds strongly to and inhibits VEGFR2, a tyrosine kinase receptor expressed almost exclusively on vascular endothelial cells; inhibition of VEGFR2 may lead to inhibition of tumor angiogenesis, inhibition of tumor cell growth, and tumor regression. See also: Brivanib (note moved to). Brivanib Alaninate (BMS-582664) is a prodrug of BMS-540215, a multi-target tyrosine kinase inhibitor used to treat angiogenesis-dependent cancers (e.g., hepatocellular carcinoma) and liver fibrosis [1][2][3]. Its mechanism of action involves hydrolysis to generate BMS-540215, which binds to the ATP-binding pockets of VEGFR2, FGFR, and PDGFRβ, inhibiting tyrosine kinase activation and downstream signaling pathways (ERK/AKT), thereby inhibiting angiogenesis, tumor growth, and HSC activation [1][2][3]. It has shown efficacy in HCC xenograft models and CCl₄-induced liver fibrosis. Models showed improved oral bioavailability compared to the parent compound BMS-540215 [1][3].
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| Molecular Formula |
C22H24FN5O4
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|---|---|
| Molecular Weight |
441.46
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| Exact Mass |
441.181
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| Elemental Analysis |
C, 59.86; H, 5.48; F, 4.30; N, 15.86; O, 14.50
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| CAS # |
649735-63-7
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| Related CAS # |
Brivanib;649735-46-6
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| PubChem CID |
11154925
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| Appearance |
White to brown solid powder
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| Density |
1.4±0.1 g/cm3
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| Index of Refraction |
1.648
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| LogP |
2.27
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| Hydrogen Bond Donor Count |
2
|
| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
8
|
| Heavy Atom Count |
32
|
| Complexity |
660
|
| Defined Atom Stereocenter Count |
2
|
| SMILES |
FC1=C(C([H])=C([H])C2=C1C([H])=C(C([H])([H])[H])N2[H])OC1C2=C(C([H])([H])[H])C(=C([H])N2N=C([H])N=1)OC([H])([H])[C@@]([H])(C([H])([H])[H])OC([C@]([H])(C([H])([H])[H])N([H])[H])=O
|
| InChi Key |
LTEJRLHKIYCEOX-OCCSQVGLSA-N
|
| InChi Code |
InChI=1S/C22H24FN5O4/c1-11-7-15-16(27-11)5-6-17(19(15)23)32-21-20-13(3)18(8-28(20)26-10-25-21)30-9-12(2)31-22(29)14(4)24/h5-8,10,12,14,27H,9,24H2,1-4H3/t12-,14+/m1/s1
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| Chemical Name |
[(2R)-1-[4-[(4-fluoro-2-methyl-1H-indol-5-yl)oxy]-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yl]oxypropan-2-yl] (2S)-2-aminopropanoate
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| Synonyms |
BMS582664; BMS-582664; Brivanib Alaninate; BMS 582664
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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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2.08 mg/mL (4.71 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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. Solubility in Formulation 2: 2.08 mg/mL (4.71 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (4.71 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 0.5% methylcellulose+0.2% Tween 80: 10mg/mL |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2652 mL | 11.3261 mL | 22.6521 mL | |
| 5 mM | 0.4530 mL | 2.2652 mL | 4.5304 mL | |
| 10 mM | 0.2265 mL | 1.1326 mL | 2.2652 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.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT01267253 | Completed | Other: Laboratory Biomarker Analysis Drug: Brivanib Alaninate |
Cervical Adenocarcinoma Persistent Disease |
Gynecologic Oncology Group | April 4, 2011 | Phase 2 |
| NCT00437437 | Completed | Tumors | Bristol-Myers Squibb | Astellas Pharma Inc | May 2000 | Phase 1 |
| NCT00888173 | Completed | Other: Laboratory Biomarker Analysis Drug: Brivanib Alaninate |
Endometrial Transitional Cell Carcinoma Endometrial Adenocarcinoma |
Gynecologic Oncology Group | July 6, 2009 | Phase 2 |
| NCT00798252 | Completed | Drug: Brivanib alaninate Drug: Paclitaxel |
Advanced Cancer | Bristol-Myers Squibb | March 2009 | Phase 1 |
Effects of brivanib on growth rate of patient-derived HCC xenograft lines 06-0606, 2-1318, and 26-1004. Clin Cancer Res. 2008 Oct 1;14(19):6146-53. |
Effects of brivanib on VEGFR-2 activity, cell proliferation, and apoptosis in HCC xenograft lines 06-0606 (A) and 26-1004 (B). Clin Cancer Res. 2008 Oct 1;14(19):6146-53. |
Effects of brivanibon (A and C) VEGF-induced, bFGF-induced, and (B) IGF-I–induced phosphorylation of VEGFR-2, FGFR, Akt, and ERK1/2 in SK-HEP1 (A and B) and HepG2 (C) cells. Clin Cancer Res. 2008 Oct 1;14(19):6146-53. |
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