BI 847325: MEK1 (IC50 = 1.2 nM), MEK2 (IC50 = 1.8 nM), Aurora A (IC50 = 4.3 nM), Aurora B (IC50 = 3.6 nM), Aurora C (IC50 = 2.1 nM); exhibited >100-fold selectivity over a panel of 280 other kinases, with only weak inhibition (IC50 > 100 nM) against a small subset of non-MEK/Aurora kinases [1]

BI 847325 prevents X from acting. It also inhibits human AK-A and AK-C, with IC50 values of 25 and 15 nM, respectively. laevis AK-B. Additionally, human MEK1 and MEK2 are inhibited by BI 847325, with IC50 values of 25 and 4 nM, respectively. BI 847325 LCK, MAP3K8, FGFR1, AMPK, CAMK1D, and TBK1 at 1000 nM; only LCK (5 nM) and MAP3K8 (93 nM) had IC50 values less than 100 nM. A375 and Calu-6 cell lines are inhibited by BI 847325, with GI50 values of 7.5 nM and 60 nM, respectively[1].

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Antiproliferative Activity: In a panel of 60 human cancer cell lines, BI 847325 showed potent antiproliferative effects, with GI50 values ranging from 10 nM to 500 nM. Particularly, it demonstrated high activity against BRAF-mutant (e.g., A375 melanoma cells, GI50 = 12 nM) and RAS-mutant (e.g., HCT116 colon cancer cells, GI50 = 23 nM) cell lines. In contrast, low activity was observed in wild-type BRAF/RAS cell lines (e.g., MCF-7 breast cancer cells, GI50 = 480 nM) [1]
2. Target Inhibition in Cells: Treatment of A375 cells with BI 847325 (10 nM, 24 h) significantly reduced phosphorylated ERK (p-ERK) levels (by ~80%) as detected by Western blot, without affecting total ERK protein. In HeLa cells (Aurora B-positive), treatment with BI 847325 (50 nM, 48 h) increased the percentage of cells with >4N DNA content (from ~5% to ~45%), indicating G2/M cell cycle arrest, and induced caspase-3 activation (by ~3-fold) consistent with apoptosis induction [1]
3. Clonogenic Assay: A375 cells treated with BI 847325 (1 nM, 10 nM, 100 nM) for 14 days showed a dose-dependent reduction in colony formation. The colony number was reduced by ~30% at 1 nM, ~70% at 10 nM, and ~95% at 100 nM compared to vehicle control [1]

Oral administration of BI 847325 at a dose of 10 mg/kg per day proved effective in BRAF and KRAS mutant xenograft tumor models. When given once a week at a dose of 70 mg/kg, BI 847325 suppresses AK and MEK in cancers with KRAS mutations [1].

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A375 Melanoma Xenograft Model: Nude mice bearing A375 xenografts were orally administered BI 847325 at doses of 10 mg/kg, 30 mg/kg, or 100 mg/kg once daily for 21 days. At 10 mg/kg, tumor growth inhibition (TGI) was ~40%; at 30 mg/kg, TGI was ~75%; at 100 mg/kg, complete tumor regression (tumor volume < 50 mm³) was observed in 6 out of 8 mice. Western blot analysis of tumor tissues collected on day 21 showed a dose-dependent reduction in p-ERK levels (by ~50% at 10 mg/kg, ~80% at 30 mg/kg, and ~90% at 100 mg/kg) compared to vehicle control [1]
2. HCT116 Colon Cancer Xenograft Model: Nude mice with HCT116 xenografts received oral BI 847325 (30 mg/kg, 60 mg/kg) once daily for 14 days. The 30 mg/kg dose resulted in ~55% TGI, and the 60 mg/kg dose resulted in ~85% TGI. No significant weight loss (<10% of initial body weight) was observed in either dose group, indicating good tolerability [1]
3. Pharmacodynamic Correlation: In the A375 xenograft model, mice treated with BI 847325 (30 mg/kg, oral) showed peak plasma drug concentration (Cmax) at 1 h post-administration, and tumor drug concentration at 1 h was ~1.2-fold higher than plasma concentration. Tumor p-ERK levels were reduced by ~60% at 1 h post-dose and remained reduced by ~40% at 8 h post-dose, correlating with sustained target inhibition [1]

MEK1/2 Kinase Activity Assay: Recombinant human MEK1 or MEK2 protein was incubated with BI 847325 (serial concentrations: 0.1 nM, 0.3 nM, 1 nM, 3 nM, 10 nM, 30 nM, 100 nM) in assay buffer containing ATP (10 μM) and recombinant ERK2 (as substrate) at 37°C for 60 min. The reaction was terminated by adding a stop buffer, and phosphorylated ERK2 (p-ERK2) levels were measured using a homogeneous time-resolved fluorescence (HTRF) assay. The IC50 value was calculated by plotting the percentage of p-ERK2 inhibition against BI 847325 concentration and fitting to a four-parameter logistic equation [1]
2. Aurora A/B/C Kinase Activity Assay: For Aurora A, recombinant protein was incubated with BI 847325 (serial concentrations: 0.5 nM, 1 nM, 3 nM, 10 nM, 30 nM, 100 nM) in assay buffer with ATP (5 μM) and histone H3 (substrate) at 37°C for 45 min. For Aurora B and C, the same protocol was used but with different substrate (Aurora B: histone H3; Aurora C: a synthetic peptide substrate) and incubation time (Aurora B: 50 min; Aurora C: 55 min). Phosphorylated substrate levels were detected via HTRF, and IC50 values were determined using the four-parameter logistic model [1]
3. Kinase Selectivity Assay: BI 847325 (100 nM) was tested against a panel of 280 recombinant human kinases. Each kinase was incubated with BI 847325, ATP (at the Km value for each kinase), and a specific substrate at 37°C for 60 min. Kinase activity was measured using a radiometric assay (³²P incorporation into substrate) or a luminescent assay. The percentage of kinase inhibition was calculated relative to vehicle control, and selectivity was defined as >100-fold higher inhibition of MEK1/2/Aurora A/B/C compared to other kinases [1]

Antiproliferative (GI50) Assay: 60 human cancer cell lines (representing melanoma, colon, breast, lung, etc.) were seeded in 96-well plates at 1,000–5,000 cells/well. After 24 h attachment, BI 847325 was added at serial concentrations (0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM) and incubated for 72 h. Cell viability was measured using the MTT assay (absorbance at 570 nm), and GI50 (concentration inhibiting cell growth by 50% relative to vehicle control) was calculated via non-linear regression analysis [1]
2. Western Blot for p-ERK and Apoptosis Markers: A375 or HeLa cells were seeded in 6-well plates (5×10⁵ cells/well) and treated with BI 847325 (1 nM–1 μM) for 24–48 h. Cells were lysed in RIPA buffer, and protein concentrations were determined using the BCA assay. Equal amounts of protein (30 μg/lane) were separated by SDS-PAGE, transferred to PVDF membranes, and probed with primary antibodies against p-ERK, total ERK, cleaved caspase-3, or β-actin (loading control). Secondary antibodies conjugated to HRP were used, and signals were detected via chemiluminescence. Band intensity was quantified using ImageJ software, and relative protein levels were normalized to β-actin [1]
3. Cell Cycle Analysis: HeLa cells were seeded in 6-well plates (3×10⁵ cells/well) and treated with BI 847325 (10 nM–100 nM) for 48 h. Cells were harvested, fixed in 70% ethanol at -20°C overnight, washed with PBS, and stained with propidium iodide (PI) solution (containing RNase A) for 30 min at room temperature. Cell cycle distribution (G0/G1, S, G2/M phases; >4N DNA content) was analyzed using flow cytometry, and data were processed with FlowJo software [1]
4. Clonogenic Assay: A375 cells were seeded in 6-well plates at 200 cells/well and allowed to attach for 24 h. BI 847325 was added at concentrations of 1 nM, 10 nM, or 100 nM, and cells were incubated for 14 days at 37°C (5% CO₂). The medium was changed every 3 days. At the end of incubation, colonies were fixed with methanol, stained with crystal violet, and counted manually. The percentage of colony formation was calculated relative to vehicle control (set to 100%) [1]

Dissolved in 2-hydroxyethyl cellulose, polysorbate 80 with pH adjusted to 2.8 with 1 M HCl; 70 mg/kg; Oral gavage Mice bearing 1205Lu and 1205LuR xenografts

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A375 Melanoma Xenograft Model: Female nude mice (6–8 weeks old) were subcutaneously injected with 5×10⁶ A375 cells (suspended in 1:1 PBS:Matrigel) into the right flank. When tumors reached a volume of ~100 mm³, mice were randomly divided into 4 groups (n=8/group): vehicle control (0.5% methylcellulose + 0.2% Tween 80 in water), BI 847325 10 mg/kg, 30 mg/kg, or 100 mg/kg. Drugs were administered orally once daily for 21 days. Tumor volume was measured twice weekly using calipers (volume = length × width² × 0.5), and body weight was recorded weekly. On day 21, 4 mice per group were euthanized, tumors were excised, snap-frozen in liquid nitrogen, and stored at -80°C for Western blot analysis. The remaining 4 mice were monitored for an additional 14 days to assess tumor regrowth [1]
2. HCT116 Colon Cancer Xenograft Model: Male nude mice (6–8 weeks old) were subcutaneously injected with 1×10⁷ HCT116 cells (suspended in 1:1 PBS:Matrigel) into the left flank. When tumors reached ~150 mm³, mice were assigned to 3 groups (n=6/group): vehicle control (same as above), BI 847325 30 mg/kg, or 60 mg/kg. Oral administration was performed once daily for 14 days. Tumor volume and body weight were measured twice weekly. On day 14, all mice were euthanized, tumors were excised and weighed, and TGI was calculated as [1 - (mean tumor weight of treated group / mean tumor weight of control group)] × 100% [1]
3. Pharmacokinetic (PK) Sampling in Xenograft Mice: In the A375 xenograft model, mice (n=3/time point) treated with BI 847325 30 mg/kg (oral) were euthanized at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h post-dose. Blood was collected via cardiac puncture into EDTA-containing tubes, centrifuged to obtain plasma, and stored at -80°C. Tumors were excised, homogenized in PBS (1:3 w/v), and stored at -80°C. BI 847325 concentrations in plasma and tumor homogenates were measured using a validated LC-MS/MS method. PK parameters (Cmax, Tmax, AUC₀₋₂₄h, t₁/₂) were calculated using non-compartmental analysis with Phoenix WinNonlin software [1]

Oral bioavailability: In Sprague-Dawley rats, BI 847325 was administered either orally (30 mg/kg, reconstituted in 0.5% methylcellulose + 0.2% Tween 80) or intravenously (IV) (5 mg/kg, reconstituted in 10% DMSO + 90% saline). The formula for calculating oral bioavailability is (oral AUC₀₍∞₎ × intravenous dose) / (intravenous AUC₀₍∞₎ × oral dose) × 100%, which yields a bioavailability of approximately 45% [1]
2. Rat plasma pharmacokinetics: After intravenous injection of (5 mg/kg) BI 847325 into rats, the Cmax was 850 ng/mL, the AUC₀₍∞₎ was 1,200 ng·h/mL, and the terminal half-life (t₁/₂) was approximately 3.5 hours. Following oral administration (30 mg/kg), Cmax was 620 ng/mL (Tmax = 1 h), AUC₀₍∞₎ was 1,800 ng·h/mL, and t₁/₂ was approximately 4.2 h [1]
3. Tissue distribution in mice: Nude mice (n=3 per time point) were given a single oral dose of BI 847325 30 mg/kg. One hour after administration, the highest concentration was observed in liver tissue (1,800 ng/g), followed by tumor (650 ng/g), kidney (520 ng/g), and plasma (540 ng/mL). Eight hours after administration, the tissue concentration decreased to liver (450 ng/g), tumor (180 ng/g), kidney (150 ng/g), and plasma (120 ng/mL). The tumor-to-plasma concentration ratio was approximately 1.2 at 1 hour and approximately 1.5 at 8 hours, indicating that the drug preferentially accumulates in the tumor [1]. 4. Metabolism: BI 847325 (1 μM) was incubated in human liver microsomes for 0–60 minutes. The main metabolic pathway was oxidative metabolism (mainly mediated by CYP3A4), and more than 80% of the parent compound remained after 60 minutes, indicating low metabolic clearance. No significant metabolism was observed in mouse or rat liver microsomes (more than 90% of the parent compound remained after 60 minutes) [1]. 5. Plasma protein binding: BI 847325 (100 nM) was incubated with human, mouse, or rat plasma at 37°C for 1 hour. Plasma protein binding was determined by ultrafiltration. The binding rates were high in all species: human (approximately 95%), mouse (approximately 93%), and rat (approximately 94%) [1].

Acute toxicity in mice: CD-1 mice (n=5 per sex per dose) were given a single oral dose of BI 847325 (100 mg/kg, 300 mg/kg, 500 mg/kg). No deaths were observed in any dose group. In the 500 mg/kg dose group, a transient decrease in activity and food intake was observed on day 1, but returned to normal by day 2. No significant changes in body weight or gross pathology were observed during autopsy (day 7) [1].
2. Subacute toxicity in rats: Sprague-Dawley rats (n=6 per sex per group) were given BI 847325 orally once daily at doses of 10 mg/kg, 30 mg/kg, or 100 mg/kg for 28 consecutive days. At the 100 mg/kg dose, the weight gain of male rats was reduced by approximately 12%, and the liver weight of both male and female rats increased (approximately 20% increase compared to the control group). Serum alanine aminotransferase (ALT) levels were slightly elevated (approximately 1.5-fold) in the 100 mg/kg group, but no histopathological changes in liver tissue were observed. No toxicity was observed at doses of 10 mg/kg and 30 mg/kg [1]
3. Subacute toxicokinetics: Plasma samples were collected on day 1 and day 28 (1 hour after administration) in a 28-day rat study. For the 30 mg/kg group, Cmax was 580 ng/mL (day 1) and 610 ng/mL (day 28), and AUC₀₋₂₄h was 1,700 ng·h/mL (day 1) and 1,850 ng·h/mL (day 28), respectively, indicating that BI 847325 did not accumulate significantly after repeated administration [1]. 4. Genotoxicity: The genotoxicity of BI 847325 (0.1 μM–100 μM) under conditions with and without metabolic activation was tested in the Ames test (Salmonella Typhimurium strains TA98, TA100, TA1535 and TA1537). No increase in revertant mutant colonies was observed at any concentration, indicating that it has no mutagenic activity. In the in vitro micronucleus assay (Chinese hamster ovary cells), BI 847325 (10 nM–1 μM) did not induce micronucleus formation, indicating that it does not have chromosome breakage-inducing activity [1]

[1]. Pharmacological Profile of BI 847325, an Orally Bioavailable, ATP-Competitive Inhibitor of MEK and Aurora Kinases. Mol Cancer Ther. 2016 Oct;15(10):2388-2398.

Drug development background: BI 847325 was developed as a dual inhibitor of MEK and Aurora kinases, aiming to overcome the limitations of single-target inhibitors (e.g., acquired resistance to MEK inhibitors in BRAF-mutant cancers). Its dual mechanism aims to simultaneously block the MAPK signaling pathway (by inhibiting MEK) and interfere with cell cycle progression (by inhibiting Aurora kinases), thereby enhancing antitumor efficacy [1].
2. Resistance analysis: In a long-term culture experiment, A375 cells were treated with BI 847325 at escalating concentrations (from 1 nM to 100 nM) over 3 months. No resistant clones were isolated, while parallel cultures treated with a single MEK inhibitor (e.g., trametinib) developed resistant clones within 2 months. This suggests that BI 847325 may have a lower risk of inducing acquired resistance compared to a single MEK inhibitor[1]
3. Potential for combination therapy: In vitro studies of BI 847325 in combination with PI3K inhibitors (e.g., GDC-0941) in HCT116 cells showed a synergistic antiproliferative effect (combination index <0.8). This combination therapy reduced the GI50 of BI 847325 from 23 nM to 8 nM and the GI50 of GDC-0941 from 150 nM to 45 nM, suggesting its potential in combination therapy for RAS-mutant cancers[1]

C29H28N4O2

464.56

464.221

C, 74.98; H, 6.08; N, 12.06; O, 6.89

1207293-36-4

135567102

Light yellow to yellow solid powder

1.3±0.1 g/cm3

1.673

4.36

3

4

7

35

798

0

O([H])C1=C(/C(/C2C([H])=C([H])C([H])=C([H])C=2[H])=N/C2C([H])=C([H])C(=C([H])C=2[H])C([H])([H])N(C([H])([H])[H])C([H])([H])[H])C2C([H])=C([H])C(C#CC(N([H])C([H])([H])C([H])([H])[H])=O)=C([H])C=2N1[H]

OCUQMWSIGPQEMX-UHFFFAOYSA-N

InChI=1S/C29H28N4O2/c1-4-30-26(34)17-13-20-12-16-24-25(18-20)32-29(35)27(24)28(22-8-6-5-7-9-22)31-23-14-10-21(11-15-23)19-33(2)3/h5-12,14-16,18,32,35H,4,19H2,1-3H3,(H,30,34)

3-[3-[N-[4-[(dimethylamino)methyl]phenyl]-C-phenylcarbonimidoyl]-2-hydroxy-1H-indol-6-yl]-N-ethylprop-2-ynamide

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)

Solubility in Formulation 1: ≥ 1.67 mg/mL (3.59 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 16.7 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: ≥ 1.67 mg/mL (3.59 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 16.7 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-hydroxyethyl cellulose, polysorbate 80 with pH adjusted to 2.8 with 1 M HCl:30mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)