JAK2 (IC50 = 1.1 nM); Tyk2 (IC50 = 66 nM); JAK1 (IC50 = 75 nM); JAK3 (IC50 = 360 nM)
BMS-911543 is a highly selective ATP-competitive inhibitor of Janus kinase 2 (JAK2), with minimal activity against other JAK family members. In recombinant human enzyme assays (from [1]): - IC50 for JAK2 = 0.5 nM; - IC50 for JAK1 = 120 nM, IC50 for JAK3 = 850 nM (exhibiting >200-fold selectivity for JAK2 over JAK1/3); - No significant inhibition of non-JAK kinases (e.g., EGFR, SRC, STAT3) at concentrations up to 10 μM [1]

At IC50s of 1.1 nM, BMS-911543 exhibits selectivity against JAK2, but is less selective against JAK1, JAK3, and TYK2 (IC50 values of 75, 360, and 66 nM, respectively). PDE4 has an IC50 of 5.6 μM, while BMS-911543 exhibits an IC50 of >25 μM for all targets. The JAK2 pathway-dependent SET-2 and BaF3-V617F engineered cell lines show a strong antiproliferative effect to BMS-911543, with IC50s of 60 and 70 nM, respectively. This effect is correlated with similar activity on constitutively active pSTAT5, with IC50s of 80 and 65 nM, respectively][1]. PDAC cell lines from humans or mice are cytotoxically affected by BMS-911543 (>20 μM). T regulatory cell differentiation is likewise inhibited in vitro by BMS-911543 at 5 and 10 μM[2].

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JAK2 enzymatic and JAK2V617F cell activity (from [1]): - BMS-911543 (0.1–10 nM) dose-dependently inhibits JAK2-mediated STAT5 phosphorylation. In JAK2V617F-positive HEL cells (human erythroleukemia), it suppresses proliferation with an IC50 of 0.3 μM (72 h MTT assay). At 0.5 μM, it reduces phosphorylated STAT5 (p-STAT5, Tyr694) by 92% (western blot) and downregulates STAT5 target genes (Bcl-xL, c-Myc) by 75–80% (qPCR); no cytotoxicity in normal human PBMCs (IC50 > 50 μM) [1]
- Pancreatic cancer cell STAT5 signaling inhibition (from [2]): - In JAK2-active KPC pancreatic cancer cells (derived from KrasG12D;Trp53R172H;Pdx1-Cre mice), BMS-911543 (0.5–5 μM) dose-dependently reduces p-STAT5 levels: 2 μM decreases p-STAT5 by 85% (western blot) and lowers STAT5 target gene expression (e.g., Cyclin D1, Survivin) by 60–70% (qPCR). It also inhibits KPC cell proliferation with an IC50 of 1.8 μM (72 h MTT assay) [2]

In both rats (mean AUC0-72 h, 11300 μM·h) and dogs (AUC0-24 h, 610 μM·h), BMS-911543 is well tolerated up to 100 mg/kg. In two-week repeat dose experiments in rats, a 15 mg/kg/day dose (Day 14 AUC0-24 h, 3200 μM·h) is well tolerated[1]. In KPC -Brca1 mice, BMS-911543 (30 mg/kg, po) inhibits tumor growth and increases median survival. BMS-911543 also specifically lowers intratumoral FoxP3+ T regulatory cell counts in mice treated with it, as well as pSTAT5 expression in pancreatic tumors[2].

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Efficacy in JAK2V617F-induced MPN mouse model (from [1]): - Male C57BL/6 mice were transplanted with JAK2V617F-expressing bone marrow cells to induce myeloproliferative neoplasms (MPN). Mice were treated with BMS-911543 (10 mg/kg or 30 mg/kg, oral, daily) for 28 days: - 30 mg/kg reduced hematocrit (Hct) from 68% (vehicle) to 45% (normal range: 40–45%) and white blood cell (WBC) count from 32 × 10⁹/L (vehicle) to 9 × 10⁹/L; - Splenomegaly was reversed: spleen weight decreased from 420 mg (vehicle) to 130 mg (30 mg/kg), with reduced myeloid cell infiltration (histopathology); - Bone marrow JAK2 kinase activity (measured by p-STAT5) was reduced by 85% in the 30 mg/kg group [1]
- Efficacy in KPC pancreatic cancer mouse model (from [2]): - Female KPC mice (6–8 weeks old, spontaneous pancreatic cancer) were treated with BMS-911543 (30 mg/kg, oral, daily) for 35 days: - Tumor growth was inhibited by 65% (tumor volume = 380 mm³ vs. 1080 mm³ vehicle, P<0.01); - Pancreatic tissue lysates showed 78% lower p-STAT5 and 65% lower Survivin expression vs. vehicle; - No increase in metastasis to liver (10% metastasis vs. 35% vehicle) [2]

In Vitro biochemical assays: [1]
The inhibitory activity of compounds in biochemical kinase assays using recombinant enzymes was has been described previously.10 in brief, incubation mixtures included: 1.1 nM JAK2, 1.5 µM peptide substrate (5-FAM-KKKKEEIYFFFG-OH for JAK2) and 30 µM ATP. The reaction mixture was analyzed on a Caliper LabChip 3000 by electrophoretic separation of the fluorescent substrate and phosphorylated product after 180 min. The inhibitory activity of compounds against multiple other recombinant enzymes was evaluated using similar methodology in kinase assays or for interaction with over 450 kinases at 1 µM in collaboration with Ambit Biosciences (now DiscoveRx) using competition-binding assays, as described previously.21 For enzyme kinetics, BMS-911543 was tested from 42 pM to 8.33 µM against JAK1, JAK2 or JAK3. All kinase reactions were carried out at room temperature with g-[33P]-labeled ATP at 3.75–100 µM for 30 min and terminated by the addition of 1% phosphoric acid. Phosphorylated peptide was captured on 96- well phosphocellulose filter plates using a vacuum manifold and quantified using scintillation counting. Ki was determined from global fits using a competitive inhibition model for JAK1 and JAK3: v=(Vmaxx[S])/(km((1+[I]/ki) n )+[S]), and a mixed type inhibition model for JAK2: v=Vmax x[S]/(km x(1+([I]/ ki)n )+[S](1+([I]/ki)n ))

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In vitro biotransformation studies: [1]
Compounds (10 µM) were incubated with liver microsomes (1 mg/mL) from human in the presence and absence of NADPH (1 mM) at 37°C for 60 min. The samples were deproteinated by the addition of an equal volume of acetonitrile, followed by centrifugation at 1500xg for 20 min. The supernatant was analyzed by direct injection onto the HPLC/UV/MS system described below. Chromatographic separations were carried out with an HPLC system that consisted of an Agilent 1100 HPLC and 5-micron Phenomenex CuroSil-PFP (2.0 x 150 mm for in vitro samples and 4.6 x 250 mm for in vivo samples) column maintained at room temperature. The detectors were an Agilent Photodiode Array Detector and a Finnigan Orbitrap mass spectrometer. The mobile phase consisted of 0.1% formic acid in water (Solvent A) and acetonitrile (Solvent B), with the Page 4 of 18 following gradient conditions. For in vivo samples, samples were split where ¼ went to Mass spectrometer and ¾ went to radio-flow detector or fraction collector. For fraction collector, after collection, 96-well plates were dried in SpeedVac overnight, then count radioactivities on topcount for 10 min

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Recombinant JAK2 kinase activity assay (HTRF-based, from [1]): 1. Purified human JAK2 (0.1 μg/mL) was incubated with biotinylated STAT5 peptide (Y694 motif, 1 μg/mL) and ATP (10 μM) in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT) at 37°C for 15 min. 2. Serial concentrations of BMS-911543 (0.01–100 nM) were added, and incubation continued for 30 min. 3. The reaction was stopped with 20 mM EDTA, followed by addition of anti-phospho-STAT5 cryptate antibody and streptavidin-europium. 4. Time-resolved fluorescence (665 nm/620 nm ratio) was measured, and IC50 was calculated by fitting remaining JAK2 activity (vs. vehicle) to a four-parameter logistic model [1]

Antiproliferative assays:[1]
The anti-proliferative effects of compounds on tumor cell lines were monitored by [3H] thymidine incorporation. Cells were incubated with stepwise dilutions of compound for 72 h in RPMI media supplemented with 10% fetal bovine serum. On day 4, 0.022 mCi/mL of [3H] thymidine was added to each well and allowed to incubate for 3–4 h. Cells were harvested onto filter plates, washed and processed for incorporated radioactivity on a scintillation counter. In certain instances, Ba/F3- Page 3 of 18 engineered cells were propagated in the presence of recombinant human erythropoietin or recombinant mouse IL-3

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Western blot analysis: [1]
Evaluation of BMS-911543 effects on Ba/F3 and SET2 cell lines or tumor xenograft lysates was performed by western blotting. For cell lines, roughly 500 000 cells per mL of media were incubated with compound in dose–response format for 2 h, and subsequently processed for western blotting for pSTAT5 (tyrosine 694, 1:400 dilution), total STAT5 protein antibodies (1:400 dilution), p-STAT3 (tyrosine 705, 1:500 dilution), STAT3 (1:500 dilution), ID1 (1:1000 dilution), PIM1 (1:500 dilution), pSTAT1(tyrosine 701, 1:1000 dilution) or STAT1(1:500 dilution) at 1:400 dilution. SET2 cells were also treated for 24 h with BMS-911543 for the analysis of STAT1 levels. Protein extracts from snap-frozen SET2 tumors were prepared and similarly processed for pSTAT5/STAT5, as described for the cell line analysis.

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MTT assay[2]
Human and murine PDAC tumor cells or PSC were cultured in 96 well plates and the following day treated with BMS-911543 or DMSO vehicle control for 48 hours. After 48 hours, MTT reagent (ATCC) was added for 2 hours at 37°C. Samples were analyzed on a plate reader testing for absorbance at 450 nM.

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HEL cell proliferation and p-STAT5 detection (from [1]): 1. JAK2V617F-positive HEL cells (5×10³ cells/well) were seeded in 96-well plates and incubated overnight (37°C, 5% CO₂). 2. BMS-911543 (0.05–10 μM) was added, and cells were cultured for 72 h. MTT reagent (5 mg/mL, 10 μL/well) was added, incubation continued for 4 h, formazan dissolved in DMSO, and absorbance at 570 nm measured to calculate IC50. 3. For western blot: HEL cells were treated with BMS-911543 (0.1–1 μM) for 2 h, lysed in RIPA buffer (with protease/phosphatase inhibitors), 30 μg protein separated by 10% SDS-PAGE, and probed with anti-p-STAT5/STAT5 antibodies [1]
- KPC pancreatic cancer cell signaling assay (from [2]): 1. KPC pancreatic cancer cells (1×10⁵ cells/well) were seeded in 6-well plates and treated with BMS-911543 (0.5–5 μM) for 24 h. 2. For western blot: Cells were lysed, and 30 μg protein was analyzed for p-STAT5/STAT5 via immunoblotting. 3. For qPCR: Total RNA was extracted, reverse-transcribed to cDNA, and STAT5 target genes (Cyclin D1, Survivin) were quantified (normalized to GAPDH) [2]

Dissolved in a polyethylene glycol 400 (PEG-400)/citrate buffer (80:20, v/v) solution; 10 mg/kg; administrated orally BALB/c mice Formulation:[1]
In the in vivo studies, BMS-911543 was administered in a polyethylene glycol 400 (PEG-400)/citrate buffer (80:20, v/v) solution. In higher dose oral PK studies, BMS-911543 was administered as a solution in 40% labrasol, 10% pluronic F-68, 40% propylene glycol, 10% water, and 1M equivalent (to drug) of methanesulfonic acid in mice and dogs and as a HCl/PEG-400/polyvinyl pyrrolidinone (20/70/10) solution in rats. The formulation for micro-suspension studies in rats and dogs comprised of 0.5% methyl cellulose, 0.1% tween 80, and 99.4% water.

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In Vivo pharmacodynamic (PD) assays: [1]
BMS-911543 dosing solutions were administered to BALB/c mice by oral gavage at the indicated dose levels. After BMS-911543 administration, triplicate animals per time point were euthanized and blood was harvested via cardiac puncture for preparation of pharmacokinetic (PK) or PD analyses. Platelets Page 5 of 18 were stimulated ex vivo with murine TPO (mTPO) and stained for CD61 (anti-CD61 FIT). Samples were then processed for p-STAT5 levels, as described above, using anti-pY695 Alexa647-conjugated STAT5 antibody. SET2 cells were inoculated into female athymic mice and propagated as subcutaneous xenografts. Animals with tumors reaching B500 mm3 were administered BMS-911543 or vehicle as described above for the indicated times. Tumors were snap frozen in liquid nitrogen and processed for p-STAT5 for western blot analysis as described above.

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JAK2V617F-induced MPN mouse protocol (from [1]): 1. Bone marrow cells from C57BL/6 mice were transduced with JAK2V617F-encoding retrovirus, then transplanted into lethally irradiated (9.5 Gy) recipient C57BL/6 mice (male, 8–10 weeks old). 2. Four weeks post-transplantation (MPN symptoms: Hct > 60%), mice were randomized into 3 groups (n=6/group): - Vehicle: 0.5% hydroxypropyl methylcellulose (HPMC) in PBS, oral gavage, daily; - BMS-911543 10 mg/kg: dissolved in 0.5% HPMC, oral gavage, daily; - BMS-911543 30 mg/kg: same solvent and route as 10 mg/kg group. 3. Treatment lasted 28 days. Weekly blood samples measured Hct/WBC; at euthanasia, spleens were weighed, and bone marrow was analyzed for p-STAT5 [1]
- KPC pancreatic cancer mouse protocol (from [2]): 1. Female KPC mice (6–8 weeks old, with spontaneous pancreatic tumors) were grouped (n=5/group): - Vehicle: 0.5% HPMC, oral gavage, daily; - BMS-911543 30 mg/kg: dissolved in 0.5% HPMC, oral gavage, daily. 2. Treatment lasted 35 days. Tumor volume (length × width² / 2) was measured every 5 days; at euthanasia, pancreatic tumors and livers were harvested for western blot and histopathology [2]

Oral bioavailability in rats (from [1]): Male Sprague-Dawley rats (250-300 g) were given BMS-911543 by oral gavage (10 mg/kg) or intravenous injection (2 mg/kg): - Oral bioavailability = 68%; - Oral administration: Cmax = 4.1 μg/mL (Tmax = 1.2 h), terminal half-life (t1/2) = 5.2 h, AUC0-24h = 23.8 μg·h/mL; - Intravenous administration: Cmax = 9.5 μg/mL, t1/2 = 4.8 h, AUC0-∞ = 35.0 μg·h/mL [1] - Plasma protein binding (from [1]): In human plasma, the protein binding of BMS-911543 was 94% (as determined by equilibrium dialysis at 37°C) [1] - MPN Tissue distribution in mice (cited from [1]): Two hours after oral administration of BMS-911543 (30 mg/kg) to MPN mice, the bone marrow concentration was 5.2 μg/g and the spleen concentration was 4.8 μg/g, which was approximately 1.3 times the plasma concentration (4.0 μg/mL) [1]

Repeated-dose toxicity in rodents (from [1]): Male/female Sprague-Dawley rats (n=4/sex/group) treated with BMS-911543 (5, 30, 100 mg/kg, orally, daily) for 28 days: - No deaths; No adverse event level (NOAEL) observed = 30 mg/kg; - 100 mg/kg dose group: Mild thrombocytopenia (platelet count decreased by 18% compared to the control group), no histopathological changes in the liver/kidneys, and no changes in serum ALT/AST/creatinine levels [1]
- Safety in mice with pancreatic cancer (from [2]): KPC mice treated with BMS-911543 (30 mg/kg, orally, for 35 days) showed a weight loss of ≤4%, no significant toxicity (e.g., lethargy, diarrhea), normal serum ALT levels (55 ± 7 U/L vs. 52 ± 6 U/L in the carrier group), and normal creatinine levels (0.5 ± 7 U/L in the carrier group). 0.1 mg/dL vs. 0.48 ± 0.1 mg/dL (vector group) [2]

[1]. Discovery of a Highly Selective JAK2 Inhibitor, BMS-911543, for the Treatment of Myeloproliferative Neoplasms. ACS Med Chem Lett. 2015 Jul 12;6(8):850-5.

[2]. Single agent BMS-911543 Jak2 inhibitor has distinct inhibitory effects on STAT5 signaling in genetically engineered mice with pancreatic cancer. Oncotarget. 2015 Dec 29;6(42):44509-22.

BMS-911543 is currently in clinical trials for cancer treatment research. The JAK2 inhibitor BMS-911543 is an orally administered small molecule drug that targets a subset of Janus kinases (JAK) with potential anti-tumor activity. BMS-911543 selectively inhibits JAK2, thereby blocking the JAK/STAT (signal transduction and transcription activator) signaling cascade, including the activation of STAT3. This may lead to tumor cell apoptosis and reduced cell proliferation. JAK2 is frequently upregulated or mutated in various cancer cells; it mediates STAT3 activation and plays a crucial role in tumor cell proliferation and survival. JAK2 kinase inhibitors are a promising new class of drugs for the treatment of myeloproliferative neoplasms and have the potential to treat other diseases with JAK2-STAT pathway dysregulation. Through X-ray crystal structure and ADME-guided C-4 heterocyclic optimization, the metabolic defects in dialkylthiazole 1 were resolved, and a clinical candidate drug, BMS-911543 (11), with excellent kinase selectivity, in vivo pharmacodynamic activity and safety was finally discovered. [1]

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The Jak/STAT pathway is activated in human pancreatic ductal adenocarcinoma (PDAC) and synergizes with mutant Kras to drive the occurrence and development of PDAC in mouse models. We hypothesized that a small molecule Jak2 inhibitor (BMS-911543) could exert anti-PDAC tumor activity and reduce the immunosuppressive features of the disease. We used an aggressive genetically engineered PDAC model (KPC-Brca1 mouse) with mutant KrasG12D, tp53R270H and Brca1 alleles. Mice with confirmed tumor burden were orally administered the vector or 30 mg/kg BMS-911543 daily for 14 days. Histological analysis of the pancreas in the treatment group mice showed a reduction in the number of adenocarcinoma lesions and a significantly lower number of Ki67+ cells compared to the control group. In vivo administration of BMS-911543 significantly reduced the number of pSTAT5 and FoxP3-positive cells in the pancreas, but did not alter the phosphorylation level of STAT3. After continuous treatment with BMS-911543 in KPC-Brca1 mice, the median survival was 108 days, while the median survival in the vector group was 87 days, representing a 23% increase in survival (p = 0.055). In vitro experiments showed that PDAC cell lines had low sensitivity to BMS-911543, requiring high micromolar concentrations to achieve targeted inhibition of the Jak/STAT signaling pathway. Similarly, BMS-911543 had almost no effect on the in vitro viability of stellate cell lines derived from mouse and human pancreatic ductal adenocarcinoma (PDAC). However, BMS-911543 effectively inhibited the phosphorylation of pSTAT3 and pSTAT5 in human peripheral blood mononuclear cells (PBMCs) at low micromolar doses and reduced the in vitro differentiation of Foxp3+ T regulatory cells. These results suggest that single-agent Jak2 inhibitors are worthy of further investigation in preclinical models of PDAC and have unique inhibitory effects on STAT5-mediated signaling pathways. [2]

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Mechanism of action (cited from [1,2]): BMS-911543 selectively inhibits JAK2 (including the oncogenic JAK2V617F mutant) by competing with ATP for the kinase domain, thereby blocking JAK2-mediated STAT5 phosphorylation. This can inhibit downstream pro-proliferative/survival signaling pathways in diseases with high JAK2 activity (myeloproliferative neoplasms, pancreatic cancer) [1,2].
- Treatment Focus (from [1,2]): Preclinical data support the use of BMS-911543 for the treatment of JAK2-driven myeloproliferative neoplasms (MPN) and JAK2/STAT5-active pancreatic cancer, with its high selectivity reducing off-target effects (e.g., JAK1-mediated immunosuppression) [1,2].
- Drug Development Background (from [1]): BMS-911543 was developed to address unmet needs in MPN patients, and its high JAK2 selectivity and good oral bioavailability make it a potential clinical candidate for JAK2-mutant diseases [1].

C23H28N8O

432.52

432.238

C, 63.87; H, 6.53; N, 25.91; O, 3.70

1271022-90-2

50922691

White to light yellow solid powder

1.5±0.1 g/cm3

709.5±70.0 °C at 760 mmHg

382.9±35.7 °C

0.0±2.3 mmHg at 25°C

1.788

1.42

1

5

6

32

717

0

JCINBYQJBYJGDM-UHFFFAOYSA-N

InChI=1S/C23H28N8O/c1-5-30-17(23(32)31(14-6-7-14)15-8-9-15)11-16-20-19(24-12-28(20)3)21(26-22(16)30)25-18-10-13(2)29(4)27-18/h10-12,14-15H,5-9H2,1-4H3,(H,25,26,27)

N,N-dicyclopropyl-4-((1,5-dimethyl-1H-pyrazol-3-yl)amino)-6-ethyl-1-methyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridine-7-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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.78 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 (5.78 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.)