BMS-911543   中文名称: Bms-911543

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

BMS-911543 的 IC50 值为 1.1 nM，对 JAK2 表现出选择性，但对 JAK1、JAK3 和 TYK2 的选择性较低（IC50 值分别为 75、360 和 66 nM）。 PDE4 的 IC50 为 5.6 μM，而 BMS-911543 对所有靶标的 IC50 均 >25 μM。 JAK2 途径依赖性 SET-2 和 BaF3-V617F 工程细胞系对 BMS-911543 显示出强大的抗增殖作用，IC50 分别为 60 和 70 nM。这种效应与组成型活性 pSTAT5 的类似活性相关，IC50 分别为 80 和 65 nM][1]。来自人类或小鼠的 PDAC 细胞系会受到 BMS-911543 (>20 μM) 的细胞毒性影响。 5 和 10 μM 浓度的 BMS-911543 在体外同样会抑制 T 调节细胞分化[2]。

在大鼠（平均 AUC0-72 h，11300 μM·h）和狗（AUC0-24 h，610 μM·h）中，BMS-911543 耐受性良好，最高可达 100 mg/kg。在大鼠两周重复剂量实验中，15 mg/kg/天的剂量（第 14 天 AUC0-24 h，3200 μM·h）具有良好的耐受性[1]。在 KPC -Brca1 小鼠中，BMS-911543（30 mg/kg，口服）可抑制肿瘤生长并增加中位生存期。 BMS-911543 还特异性降低接受其治疗的小鼠瘤内 FoxP3+ T 调节细胞计数，以及胰腺肿瘤中 pSTAT5 的表达 [2]。

体外生化分析：[1]
用重组酶进行生化激酶测定时，化合物的抑制活性已经被描述过。10简而言之，孵育混合物包括：1.1 nM JAK2, 1.5µM肽底物（JAK2为5-FAM-KKKKEEIYFFFG-OH）和30µM ATP。180分钟后，通过电泳分离荧光底物和磷酸化产物，在Caliper LabChip 3000上对反应混合物进行分析。使用类似的激酶测定方法评估化合物对多种其他重组酶的抑制活性，或与Ambit Biosciences（现为DiscoveRx）合作，在1µM下使用竞争结合测定方法评估化合物与450多种激酶的相互作用，如前所述酶动力学方面，BMS-911543在42 pM至8.33µM范围内对JAK1、JAK2或JAK3进行酶动力学测试。所有激酶反应均在室温下进行，g-[33P]标记的ATP在3.75-100µM下反应30 min，并以添加1%磷酸终止。磷酸化肽被捕获在96孔磷纤维素过滤板上使用真空歧管和定量使用闪烁计数。通过对JAK1和JAK3的竞争抑制模型：v=(Vmaxx[S])/(km((1+[I]/ Ki)n)+[S]) + JAK2的混合抑制模型：v=Vmax x[S]/(km x(1+([I]/ Ki)n)+[S](1+([I]/ Ki)n))从全局拟合中确定Ki。

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体外生物转化研究：[1]
化合物（10µM）与人肝微粒体（1 mg/mL）在存在和不存在NADPH （1 mM）的情况下（37°C）孵育60分钟。样品通过加入等体积的乙腈去蛋白，然后在1500xg离心20分钟。上清通过直接注射到下面描述的HPLC/UV/MS系统分析。色谱分离采用高效液相色谱系统，该系统由Agilent 1100 HPLC和5微米Phenomenex currosil - pfp（体外样品2.0 × 150 mm，体内样品4.6 × 250 mm）柱组成，保持在室温下。探测器为安捷伦光电二极管阵列探测器和芬尼根轨道阱质谱仪。流动相由0.1%甲酸水溶液（溶剂A）和乙腈（溶剂B）组成，第4页18遵循梯度条件。对于体内样品，将样品进行拆分，其中1 / 4进入质谱仪，3 / 4进入无线电流检测器或馏分收集器。对于馏分收集器，收集后，96孔板在SpeedVac中干燥过夜，然后在topcount上计数10分钟。

抗增殖实验:[1]
用[3H]胸腺嘧啶掺入法检测化合物对肿瘤细胞系的抗增殖作用。细胞在添加10%胎牛血清的RPMI培养基中，用逐步稀释的化合物孵育72小时。第4天，在每孔中加入0.022 mCi/mL的[3H]胸苷，孵育3-4小时。将细胞收集到过滤板上，清洗并在闪烁计数器上处理合并放射性。在某些情况下，18个工程细胞的Ba/F3- Page 3在重组人促红细胞生成素或重组小鼠IL-3存在下繁殖

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Western blot分析：[1]
western blotting检测BMS-911543对Ba/F3和SET2细胞株或肿瘤异种移植细胞的影响。对于细胞系，每mL培养基中约有50万个细胞以剂量-反应形式与化合物孵育2小时，随后进行western blot检测pSTAT5（酪氨酸694, 1:400稀释）、总STAT5蛋白抗体（ 1:400稀释）、p-STAT3（酪氨酸705,1:500稀释）、STAT3（1:500稀释）、ID1（1:100稀释）、PIM1（1:500稀释）、pSTAT1（酪氨酸701,1:100稀释）或STAT1（1:500稀释）。用BMS-911543处理SET2细胞24 h，分析STAT1水平。制备快速冷冻的SET2肿瘤蛋白提取物，并对pSTAT5/STAT5进行类似处理，如细胞系分析所述。

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MTT测定[2]
将人和鼠PDAC肿瘤细胞或PSC培养于96孔板中，第二天用BMS-911543或DMSO对照处理48小时。48小时后，加入MTT试剂（ATCC），在37℃下静置2小时。样品在平板阅读器上进行450 nM吸光度测试。

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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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.

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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]

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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

89.03

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.78 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (5.78 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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