BAY 43-9006 is a dual-action inhibitor targeting multiple kinases involved in tumor proliferation and angiogenesis. It inhibits Raf-1 kinase (IC50 6 nmol/L), wild-type BRAF (IC50 22 nmol/L), and V599E mutant BRAF (IC50 38 nmol/L). It also potently inhibits receptor tyrosine kinases including murine VEGFR-2 (flk-1, IC50 15 nmol/L), human VEGFR-2 (IC50 90 nmol/L), murine VEGFR-3 (IC50 20 nmol/L), murine PDGFR-β (IC50 57 nmol/L), Flt-3 (IC50 58 nmol/L), and c-KIT (IC50 68 nmol/L). FGFR-1 is inhibited with IC50 580 nmol/L. The compound does not significantly inhibit (>10,000 nmol/L) MEK-1, ERK-1, EGFR, HER-2, IGFR-1, c-met, PKB, PKA, cdk1/cyclinB, PKCα, PKCγ, or pim-1 [1].

Drug compounds have been modified to include stable heavy isotopes of hydrogen, carbon, and other elements. These isotopes are mostly utilized as quantitative tracers during the drug development process. One possible benefit of crepuscular chemicals is their prolonged half-degeneration period. Crepuscular substances have the potential to extend the compound's pharmacokinetic profile, or its future half-life. These drugs' tolerance, safety, and efficacy (2) increase intestinal bioavailability. Deuterated substances have the ability to lessen the amount of undesirable metabolism (first-pass metabolism) in the umbilical cord and intestinal wall, increasing the amount of unmetabolized medication that reaches its intended site of action. Better tolerance and activity at low doses are determined by high bioavailability. (3) Better metabolic properties. Derivatized chemicals can enhance drug metabolism and lessen reactive or hazardous molecules. (4) Boost security. Deuterated compounds are harmless and have the ability to lessen or eliminate the negative effects of medicinal compounds. (5) Keep things in order. Deuterated chemicals would maintain comparable biological efficacy and hydrogen analogs in earlier research.

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BAY 43-9006 inhibits activation of the RAF/MEK/ERK pathway in tumor cell lines. In MDA-MB-231 breast cancer cells, it inhibits basal MEK 1/2 phosphorylation (IC50 40 nmol/L) and ERK 1/2 phosphorylation (IC50 90-100 nmol/L). It inhibits ERK phosphorylation in LOX melanoma (IC50 880 nmol/L), BxPC-3 pancreatic (IC50 1,200 nmol/L), and colon cancer lines (HCT 116, DLD-1, Colo-205, IC50 2,000-4,000 nmol/L). No effect on the PKB pathway was observed, demonstrating selectivity. NSCLC lines NCI-H460 and A549 (mutant KRAS) were insensitive to ERK inhibition [1].
BAY 43-9006 potently inhibits receptor tyrosine kinase autophosphorylation in cell-based assays. It inhibits VEGFR-2 autophosphorylation in HUVECs (IC50 ~100 nmol/L) and NIH 3T3-VEGFR-2 cells (IC50 ~30 nmol/L). It inhibits VEGF-stimulated ERK phosphorylation in HUVECs (IC50 60 nmol/L). It inhibits PDGFR-β autophosphorylation in human aortic smooth muscle cells (HAoSMCs, IC50 80 nmol/L) and PDGF-stimulated HAoSMC proliferation (IC50 280 nmol/L). It also inhibits mVEGFR-3 autophosphorylation in HEK-293 cells (IC50 100 nmol/L) and Flt-3 (ITD) autophosphorylation (IC50 20 nmol/L) [1].
BAY 43-9006 inhibits tumor cell proliferation in vitro. In MDA-MB-231 cells (10% FCS), the IC50 for proliferation is 2,600 nmol/L, which is higher than ERK inhibition IC50 due to serum protein binding (addition of serum increased ERK inhibition IC50 to 630 nmol/L) [1].

BAY 43-9006 demonstrates broad-spectrum oral antitumor activity in human tumor xenograft models. Daily oral dosing at 7.5-60 mg/kg for 9 days produces dose-dependent tumor growth inhibition with no toxicity (no significant weight loss or lethality). At 30-60 mg/kg, complete tumor stasis during treatment was observed in HT-29, Colo-205, DLD-1 colon cancers and A549 NSCLC. NCI-H460 NSCLC showed growth inhibition but not complete stasis. MDA-MB-231 breast cancer showed tumor regressions at ≤30 mg/kg [1].
Immunohistochemistry analysis revealed that BAY 43-9006 inhibits both MAPK pathway and angiogenesis in vivo. In HT-29 tumors, treatment correlated with inhibition of ERK phosphorylation and significant inhibition (50-80%) of microvessel density and area (CD31 staining). In Colo-205 tumors, ERK phosphorylation was not inhibited, but significant inhibition of neovascularization was observed, indicating anti-angiogenic effects contribute to tumor growth inhibition. In MDA-MB-231 tumors, both ERK phosphorylation inhibition and anti-angiogenic effects were observed, with extensive tumor cell necrosis evident by day 5 [1].

For RAF kinase assays, recombinant Raf-1 (residues 305-648), wild-type BRAF (residues 409-765), or V599E mutant BRAF (residues 409-765) were incubated with MEK-1 (1 μg) in assay buffer [20 mmol/L Tris (pH 8.2), 100 mmol/L NaCl, 5 mmol/L MgCl2, 0.15% β-mercaptoethanol] containing BAY 43-9006 (1% DMSO final). Reactions were initiated by adding 25 μL of 10 μmol/L γ-[³³P]ATP (400 Ci/mol) and incubated at 32°C for 25 minutes. Phosphorylated MEK-1 was harvested by filtration onto phosphocellulose, washed with 1% phosphoric acid, and quantified by β-plate counter [1].
For receptor tyrosine kinase assays, time-resolved fluorescence energy transfer (TR-FRET) assays were performed in 96-well opaque plates. Final reaction conditions: 1-10 μmol/L ATP, 25 nmol/L poly GT-biotin, 2 nmol/L Europium-labeled phospho-Tyr antibody (PY20), 10 nmol/L APC, 1-7 nmol/L cytoplasmic kinase domain in 1% DMSO, 50 mmol/L HEPES (pH 7.5), 10 mmol/L MgCl2, 0.1 mmol/L EDTA, 0.015% Brij-35, 0.1 mg/mL BSA, and 0.1% β-mercaptoethanol. Reactions (100 μL) were initiated by enzyme addition. Plates were read at 615 and 665 nm at 1.5-2.0 hours after initiation. Signal was calculated as ratio (665 nm/615 nm) ×10,000 [1].

For cellular MEK/ERK/PKB activation, tumor cell lines (2×10⁵ cells/well in 12-well plates) were incubated overnight, washed, and treated with BAY 43-9006 (0.01-10 μmol/L) in DMEM with 0.1% fatty acid-free BSA for 120 minutes. Cells were lysed in 1% Triton X-100 with protease inhibitors, subjected to SDS-PAGE, transferred to nitrocellulose, and probed with phospho-specific antibodies (pMEK1/2 Ser²¹⁷/²²¹, pERK1/2 Thr²⁰²/Tyr²⁰⁴, pPKB Ser⁴⁷³) and total protein antibodies. Blots were developed with HRP-conjugated secondary antibodies and ECL reagent [1].
For VEGFR-2 autophosphorylation, HUVECs or NIH 3T3-VEGFR-2 cells were serum-starved, preincubated with BAY 43-9006 (1 hour for HUVECs, 30 minutes for NIH 3T3), then stimulated with VEGF₁₆₅ (30 ng/mL, 10 minutes). Lysates were immunoblotted with anti-pVEGFR-2 (pTyr-1054/1059) and anti-VEGFR-2 antibodies [1].
For PDGFR-β assays, human aortic smooth muscle cells (HAoSMCs) were serum-starved overnight, treated with BAY 43-9006 (1 hour), stimulated with PDGF-BB (10 ng/mL, 7 minutes), lysed, immunoprecipitated with anti-PDGFR-β antibody, and immunoblotted with anti-pPDGFR-β (Tyr⁸⁵⁷) antibody. For proliferation assays, HAoSMCs were serum-starved, treated with compound (1 hour), stimulated with PDGF-BB (10 ng/mL), incubated 24 hours, and analyzed by BrdUrd ELISA [1].
For mVEGFR-3 and Flt-3 assays, HEK-293 cells (transfected with mVEGFR-3 or expressing Flt-3 ITD) were treated with BAY 43-9006 (2 hours for Flt-3, 30 minutes for VEGFR-3), lysed, and immunoblotted with anti-pFlt-3 (Cell Signaling 3466) or anti-p-mVEGFR-3 (4G10) antibodies [1].

Female athymic mice (6-8 weeks old) were implanted subcutaneously with human tumor cells (MDA-MB-231, Colo-205, HT-29, DLD-1, NCI-H460, A549). Tumors were allowed to reach 75-150 mg (efficacy studies) or 100-250 mg (mechanistic studies) before treatment. BAY 43-9006 was dissolved in Cremophor EL/ethanol (50:50) at 4× the highest dose (stock prepared fresh every 3 days, stored foil-wrapped at room temperature). Final dosing solutions were prepared daily by diluting stock with water. Lower doses were prepared by diluting 1× solution with Cremophor EL/ethanol/water (12.5:12.5:75). Treatment was administered orally once daily for 9 days (efficacy studies) or 5 days (mechanistic studies). Tumor weight was calculated as length × (width)²/2. Treatments producing >20% lethality or >20% net body weight loss were considered toxic [1].
For immunohistochemistry, tumors were collected 3 hours after final dose, sectioned, and stained. For microvessel detection, paraffin sections were stained with anti-CD31 antibodies (goat polyclonal, 1:750) using Dako Autostainer. Microvessel area and density were quantified using ImagePro Plus software (4 fields/section, 0.644 mm²/field). For pERK detection, sections were stained with anti-pERK1/2 antibody (1:100) using Envision Plus HRP system [1].

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Female athymic mice (6-8 weeks old) were implanted subcutaneously with human tumor cells (MDA-MB-231, Colo-205, HT-29, DLD-1, NCI-H460, A549). Tumors were allowed to reach 75-150 mg (efficacy studies) or 100-250 mg (mechanistic studies) before treatment. BAY 43-9006 was dissolved in Cremophor EL/ethanol (50:50) at 4× the highest dose (stock prepared fresh every 3 days, stored foil-wrapped at room temperature). Final dosing solutions were prepared daily by diluting stock with water. Lower doses were prepared by diluting 1× solution with Cremophor EL/ethanol/water (12.5:12.5:75). Treatment was administered orally once daily for 9 days (efficacy studies) or 5 days (mechanistic studies). Tumor weight was calculated as length × (width)²/2. Treatments producing >20% lethality or >20% net body weight loss were considered toxic [1].
For immunohistochemistry, tumors were collected 3 hours after final dose, sectioned, and stained. For microvessel detection, paraffin sections were stained with anti-CD31 antibodies (goat polyclonal, 1:750) using Dako Autostainer. Microvessel area and density were quantified using ImagePro Plus software (4 fields/section, 0.644 mm²/field). For pERK detection, sections were stained with anti-pERK1/2 antibody (1:100) using Envision Plus HRP system [1].

In xenograft studies, oral administration of BAY 43-9006 at doses up to 60 mg/kg daily for 9 days produced no evidence of toxicity, as measured by increased weight loss relative to control animals or drug-related lethality. Treatments producing >20% lethality or >20% net body weight loss were considered toxic, and none of the tested doses met these criteria [1].

[1]. BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer Res. 2004 Oct 1;64(19):7099-109.

CM-4307 is being investigated in the clinical trial NCT03602495 (donafenib for 131I-refractory differentiated thyroid cancer). Donafenib is an oral multi-kinase inhibitor that targets Raf kinase and multiple receptor tyrosine kinases (RTKs) with potential antitumor activity. After oral administration, donafenib binds to Raf kinase and blocks its activity, thereby inhibiting the Raf-mediated signal transduction pathway. This can inhibit the proliferation of Raf-expressing tumor cells. Furthermore, the drug may inhibit other unknown RTKs, further inhibiting the proliferation of susceptible tumor cells. Raf is a serine/threonine protein kinase that plays a crucial role in the Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway. Dysregulation of this pathway often leads to tumor cell proliferation and survival.

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BAY 43-9006 (N-(3-trifluoromethyl-4-chlorophenyl)-N'-(4-(2-methylcarbamoyl pyridin-4-yl)oxyphenyl)urea) is a novel bi-aryl urea compound that functions as a dual-action inhibitor targeting both the RAF/MEK/ERK pathway (through inhibition of Raf-1, BRAF, and mutant BRAF) and pro-angiogenic receptor tyrosine kinases (VEGFR-2, VEGFR-3, PDGFR-β, Flt-3, c-KIT). The compound was co-crystallized with BRAF, revealing that the distal pyridyl ring interacts with three amino acids within the ATP adenine binding pocket, and the urea moiety forms hydrogen bonds with the enzyme, promoting the inactive conformation of BRAF [1].
The broad-spectrum antitumor activity observed in xenograft models appears to result from two complementary mechanisms: direct inhibition of tumor cell proliferation through MAPK pathway blockade (in sensitive cell lines) and inhibition of tumor angiogenesis through VEGFR/PDGFR blockade. The relative contribution of each mechanism varies by tumor type. Based on these preclinical data, BAY 43-9006 was being tested in phase III clinical trials for renal cell carcinoma and phase II trials across multiple tumor types at the time of publication [1].

C21H13D3CLF3N4O3

467.843

467.105

1130115-44-4

Sorafenib;284461-73-0;Sorafenib-d4;1207560-07-3;Sorafenib-13C,d3;1210608-86-8

25191001

White to off-white solid powder

6.086

3

7

5

32

646

0

[2H]C([2H])([2H])NC(=O)C1=NC=CC(=C1)OC2=CC=C(C=C2)NC(=O)NC3=CC(=C(C=C3)Cl)C(F)(F)F

MLDQJTXFUGDVEO-FIBGUPNXSA-N

InChI=1S/C21H16ClF3N4O3/c1-26-19(30)18-11-15(8-9-27-18)32-14-5-2-12(3-6-14)28-20(31)29-13-4-7-17(22)16(10-13)21(23,24)25/h2-11H,1H3,(H,26,30)(H2,28,29,31)/i1D3

4-[4-[[4-chloro-3-(trifluoromethyl)phenyl]carbamoylamino]phenoxy]-N-(trideuteriomethyl)pyridine-2-carboxamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO : ~100 mg/mL (~213.75 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.34 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 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.34 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 25.0 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: ≥ 2.5 mg/mL (5.34 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 25.0 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.)