JAK2 (IC50 = 1.1 nM); Tyk2 (IC50 = 66 nM); JAK1 (IC50 = 75 nM); JAK3 (IC50 = 360 nM)

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].

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].

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 ))

---

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

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

---

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.

---

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.

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.

---

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.

---

Dissolved in a polyethylene glycol 400 (PEG-400)/citrate buffer (80:20, v/v) solution; 10 mg/kg; administrated orally

BALB/c mice

[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 has been used in trials studying the treatment of Cancer.
JAK2 Inhibitor BMS-911543 is an orally available small molecule targeting a subset of Janus-associated kinase (JAK) with potential antineoplastic activity. JAK2 inhibitor BMS-911543 selectively inhibits JAK2, thereby preventing the JAK/STAT (signal transducer and activator of transcription) signaling cascade, including activation of STAT3. This may lead to an induction of tumor cell apoptosis and a decrease in cellular proliferation. JAK2, often upregulated or mutated in a variety of cancer cells, mediates STAT3 activation and plays a key role in tumor cell proliferation and survival.

---

JAK2 kinase inhibitors are a promising new class of agents for the treatment of myeloproliferative neoplasms and have potential for the treatment of other diseases possessing a deregulated JAK2-STAT pathway. X-ray structure and ADME guided refinement of C-4 heterocycles to address metabolic liability present in dialkylthiazole 1 led to the discovery of a clinical candidate, BMS-911543 (11), with excellent kinome selectivity, in vivo PD activity, and safety profile.[1]

---

The Jak/STAT pathway is activated in human pancreatic ductal adenocarcinoma (PDAC) and cooperates with mutant Kras to drive initiation and progression of PDAC in murine models. We hypothesized that the small-molecule Jak2 inhibitor (BMS-911543) would elicit anti-tumor activity against PDAC and decrease immune suppressive features of the disease. We used an aggressive genetically engineered PDAC model with mutant KrasG12D, tp53R270H, and Brca1 alleles (KPC-Brca1 mice). Mice with confirmed tumor burden were treated orally with vehicle or 30 mg/kg BMS-911543 daily for 14 days. Histologic analysis of pancreata from treated mice revealed fewer foci of adenocarcinoma and significantly decreased Ki67+ cells versus controls. In vivo administration of BMS-911543 significantly reduced pSTAT5 and FoxP3 positive cells within the pancreas, but did not alter STAT3 phosphorylation. Continuous dosing of KPC-Brca1 mice with BMS-911543 resulted in a median survival of 108 days, as compared to a median survival of 87 days in vehicle treated animals, a 23% increase (p = 0.055). In vitro experiments demonstrated that PDAC cell lines were poorly sensitive to BMS-911543, requiring high micromolar concentrations to achieve targeted inhibition of Jak/STAT signaling. Similarly, BMS-911543 had little in vitro effect on the viability of both murine and human PDAC-derived stellate cell lines. However, BMS-911543 potently inhibited phosphorylation of pSTAT3 and pSTAT5 at low micromolar doses in human PBMC and reduced in vitro differentiation of Foxp3+ T regulatory cells. These results indicate that single agent Jak2i deserves further study in preclinical models of PDAC and has distinct inhibitory effects on STAT5 mediated signaling.[2]

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.

---

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.

---

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