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 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]

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]。

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JAK2酶活性及JAK2V617F细胞活性（来自[1]）： - BMS-911543 （0.1–10 nM）剂量依赖性抑制JAK2介导的STAT5磷酸化。在JAK2V617F阳性HEL细胞（人红白血病细胞）中，其抑制增殖的IC50为0.3 μM（72小时MTT法）。0.5 μM浓度下，磷酸化STAT5（p-STAT5，Tyr694）降低92%（蛋白质印迹法），STAT5靶基因（Bcl-xL、c-Myc）表达降低75–80%（qPCR）；对人正常PBMC无细胞毒性（IC50 > 50 μM）[1]
- 胰腺癌细胞STAT5信号抑制（来自[2]）： - 在JAK2活性的KPC胰腺癌细胞（源自KrasG12D;Trp53R172H;Pdx1-Cre小鼠）中，BMS-911543 （0.5–5 μM）剂量依赖性降低p-STAT5水平：2 μM使p-STAT5降低85%（蛋白质印迹法），STAT5靶基因（如Cyclin D1、Survivin）表达降低60–70%（qPCR）。其抑制KPC细胞增殖的IC50为1.8 μM（72小时MTT法）[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]。

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JAK2V617F诱导MPN小鼠模型疗效（来自[1]）： 雄性C57BL/6小鼠移植表达JAK2V617F的骨髓细胞构建骨髓增殖性肿瘤（MPN）模型，给予BMS-911543 （10 mg/kg或30 mg/kg，口服，每日1次）处理28天： - 30 mg/kg剂量使红细胞压积（Hct）从溶剂组的68%降至45%（正常范围：40–45%），白细胞计数（WBC）从32×10⁹/L降至9×10⁹/L； - 逆转脾肿大：脾脏重量从溶剂组的420 mg降至130 mg（30 mg/kg），髓系细胞浸润减少（组织病理学）； - 30 mg/kg组骨髓JAK2激酶活性（以p-STAT5衡量）降低85%[1]
- KPC胰腺癌小鼠模型疗效（来自[2]）： 6–8周龄雌性KPC胰腺癌小鼠（自发性胰腺肿瘤）给予BMS-911543 （30 mg/kg，口服，每日1次）处理35天： - 肿瘤生长抑制率65%（肿瘤体积380 mm³ vs 溶剂组1080 mm³，P<0.01）； - 胰腺组织裂解液显示p-STAT5降低78%，Survivin表达降低65%（较溶剂组）； - 肝转移率无增加（10% vs 溶剂组35%）[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。

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重组JAK2激酶活性实验（基于HTRF，来自[1]）： 1. 将纯化人JAK2（0.1 μg/mL）与生物素化STAT5肽（Y694基序，1 μg/mL）、ATP（10 μM）在实验缓冲液（50 mM Tris-HCl pH 7.5、10 mM MgCl₂、1 mM DTT）中37°C孵育15分钟。 2. 加入系列浓度的BMS-911543 （0.01–100 nM），继续孵育30分钟。 3. 用20 mM EDTA终止反应，加入抗磷酸化STAT5穴状化合物抗体和链霉亲和素-铕。 4. 检测时间分辨荧光（665 nm/620 nm比值），将JAK2活性剩余百分比（较溶剂组）拟合四参数逻辑模型计算IC50[1]

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。

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HEL细胞增殖与p-STAT5检测（来自[1]）： 1. JAK2V617F阳性HEL细胞以5×10³细胞/孔接种于96孔板，37°C、5% CO₂过夜孵育。 2. 加入BMS-911543 （0.05–10 μM），培养72小时。加入MTT试剂（5 mg/mL，10 μL/孔），继续孵育4小时，DMSO溶解甲臜结晶，检测570 nm吸光度计算IC50。 3. 蛋白质印迹：HEL细胞用BMS-911543 （0.1–1 μM）处理2小时，用含蛋白酶/磷酸酶抑制剂的RIPA缓冲液裂解，30 μg蛋白经10% SDS-PAGE电泳后，用抗p-STAT5/STAT5抗体检测[1]
- KPC胰腺癌细胞信号实验（来自[2]）： 1. KPC胰腺癌细胞（1×10⁵细胞/孔）接种于6孔板，用BMS-911543 （0.5–5 μM）处理24小时。 2. 蛋白质印迹：裂解细胞，30 μg蛋白通过免疫印迹检测p-STAT5/STAT5。 3. qPCR：提取总RNA，逆转录为cDNA，定量STAT5靶基因（Cyclin D1、Survivin）表达（以GAPDH归一化）[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) received BMS-911543 via 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, BMS-911543 had a protein binding rate of 94% (measured by equilibrium dialysis at 37°C) [1]
- Tissue distribution in MPN mice (from [1]): Oral BMS-911543 (30 mg/kg) in MPN mice resulted in bone marrow concentrations of 5.2 μg/g and spleen concentrations of 4.8 μg/g at 2 h post-administration, ~1.3-fold higher than plasma concentrations (4.0 μg/mL) [1]

Rodent repeat-dose toxicity (from [1]): Male/female Sprague-Dawley rats (n=4/sex/group) received BMS-911543 (5, 30, 100 mg/kg, oral, daily) for 28 days: - No mortality; no-observed-adverse-effect level (NOAEL) = 30 mg/kg; - At 100 mg/kg: mild thrombocytopenia (platelet count reduced by 18% vs. control), no histopathological changes in liver/kidneys, and unchanged serum ALT/AST/creatinine [1]
- In vivo safety in pancreatic cancer mice (from [2]): KPC mice treated with BMS-911543 (30 mg/kg, oral, 35 days) showed ≤4% body weight loss, no overt toxicity (e.g., lethargy, diarrhea), and normal serum ALT (55 ± 7 U/L vs. 52 ± 6 U/L vehicle) and creatinine (0.5 ± 0.1 mg/dL vs. 0.48 ± 0.1 mg/dL vehicle) [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 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]

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Mechanism of action (from [1,2]): BMS-911543 selectively inhibits JAK2 (including the oncogenic JAK2V617F mutant) by competing with ATP for the kinase domain, blocking JAK2-mediated STAT5 phosphorylation. This suppresses downstream pro-proliferative/survival signaling in JAK2-hyperactive diseases (MPN, pancreatic cancer) [1,2]
- Therapeutic focus (from [1,2]): Preclinical data supports BMS-911543 for treating JAK2-driven myeloproliferative neoplasms (MPNs) and JAK2/STAT5-active pancreatic cancer, with high selectivity reducing off-target effects (e.g., JAK1-mediated immune suppression) [1,2]
- Drug development context (from [1]): BMS-911543 was developed to address unmet needs in MPN patients, with its high JAK2 selectivity and favorable oral bioavailability making 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

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));
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10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
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