BMS-654457

Factor XIa (FXIa; Kis = 0.2~0.4 nM)

在体外，BMS-654457 在人和兔子中表现出类似的针对 FXIa 的活性 (Ki=0.2 nM) (Ki=0.42 nM)，以及针对凝血相关蛋白酶（凝血酶、FXa 和 FVIIa）的选择性（超过 500 倍） ）在这些物种中。

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体外[1]
酶测定[1]
BMS-654457 是显色肽底物S-2366的竞争抑制剂，Ki值为0.4 nM（图2底部）。BMS-654457与人FXIa的关联率约为1.6 μM−1 s−1（图3上、中面板）。BMS-654457与FXIa的结合是可逆的，通过预孵育/稀释证实了这一点（图3下图）。BMS-654457的分离速率为0.001 s−1（图3），这相当于BMS-654457与FXIa分离的一半时间约为12分钟。

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凝血试验[1]
aPTT是一种对内在凝血因子如FXIa的抑制敏感的凝血时间测定。BMS-654457加入到正常人和家兔血浆中可以延长aPTT，但不能延长PT， BMS-654457在人和家兔血浆中延长aPTT的效力最高（EC2X分别为2.2和2.4µM），但在大鼠和狗血浆中很弱（EC2X =≥20µM）。

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血小板聚集[1]
兔PRP对ADP（10µM）、花生四烯酸（250µM）和胶原（10µg/ml）的体外血小板聚集反应平均分别为62±2%、70±1%和71±2%（每组n = 5）。在1µM（分别为64±2、71±1和71±2 %）和10µM（分别为56±3、67±3和73±2 %）时，BMS-654457对血小板反应无显著影响（每组n = 5）。

BMS-654457可减少出血时间，但能有效防止兔子形成动脉血栓。 BMS-654457 具有广泛的治疗窗，是一种有前途的抗血栓治疗方法 [1]。

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体内[1]
动脉血栓形成模型[1]
载药和BMS-654457对电刺激后ECAT模型颈动脉血流的影响如图4所示。载药组BMS-654457组颈动脉基底血流量平均为21±3 ml/min （n = 6 /组）。BMS-654457有效地产生剂量依赖性的损伤动脉血流保存增加。BMS-654457 （mg/kg + mg/kg/h）在0.011 + 0.008、0.037 + 0.027、0.11 + 0.08、0.37 + 0.27和1.1 + 0.8组分别使iCBF增加17±3、46±6*、48±9*、87±10*和96±5% *，而载药组为16±3% * （n = 6 /组，*P < 0.05，与载药组比较）。

BMS-654457 (mg/kg + mg/kg/h)在0.37 + 0.27和1.1 + 0.8时，离体aPTT分别显著延长1.4±0.1和1.8±0.1 (P < 0.05)，而PT和TT未延长（数据未显示）。图6显示BMS-654457在兔ECAT中的抗血栓作用与其体外aPTT活性具有良好的相关性（r2 = 0.83）。图6还显示，在该模型中，aPTT可能需要延长1.2倍才能显示50%的血流保存。

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角质层出血时间模型[1]
对照角质层BT平均为188±7、176±9、179±5和191±7 s，对照组和BMS-654457组（mg/kg + mg/kg/h）分别为0.11 + 0.08、0.37 + 0.27和1.1 + 0.8 （n = 6 /剂量）。在iCBF保存阈值~ 90%的剂量下，BMS-654457 （0.37 mg/kg + 0.27 mg/kg/h）使BT增加了1.2±0.04倍（P < 0.05）（图7）。在1.1 mg/kg + 0.8 mg/kg/h时，BMS-654457的最大BT增加（P < 0.05）为1.33±0.08倍（n = 6 /组）（图7）。

酶测定[1]
实验在0.05 M HEPES缓冲液中进行，缓冲液中含有0.145 M NaCl， 0.005 M KCl和0.1% PEG 8000，调节pH为7.4。实验在酶过量底物和抑制剂的条件下进行。为了确定抑制机制，FXIa对9个显色底物S-2366和5个BMS-654457的浓度进行了肽底物裂解实验。加入FXIa引发反应。通过使用SpectraMax 384 Plus平板阅读器和SoftMax在405 nm处连续监测吸光度60分钟，测量底物在初始弯曲时间过程区域后由于缓慢结合抑制作用的稳态水解速率。测定每个底物和抑制剂浓度对的FXIa活性。Ki值是用GraFit 将稳态底物水解速率非线性最小二乘拟合到竞争抑制方程（Eq. 1）中计算的，其中v等于反应速度（单位为OD/min）， v max等于最大反应速度，S等于底物浓度，I等于抑制剂浓度。

在S-2366存在下，用停流分光光度法测定了BMS-654457与FXIa的结合速率常数。在应用光物理SX.18MV停止流动装置中，采用循环水浴控制温度，将FXIa溶液与400µM底物S-2366和BMS-654457的预混溶液一对一快速混合，在405 nm处吸光度增加。数据是在线性时间基础上获得的，在每条曲线上创建了总共1000个数据点，在每种抑制剂浓度下使用两到三次重复测量。非线性时间过程数据符合慢结合抑制方程
式中，吸光度t为t时刻405 nm处的光密度（OD）；吸光度t=0为初始OD；初速和末速分别是初速和末速；kobs是观察到的速率常数。关联率（k on）由k ob与BMS-654457浓度的斜率计算，关系为：

为测定解离速率常数，1.25 nM FXIa与5 nM BMS-654457 BMS-654457作用60 min，形成FXIa BMS-654457复合物。稳态实验表明，在此条件下，酶被完全抑制。FXIa:BMS-654457复合物在相同的缓冲液中稀释50倍，使FXIa的最终浓度为25 pM， BMS-654457的最终浓度为100 pM，通过监测OD在405 nm处的变化来测量FXIa活性的时间依赖性增加。在这些条件下，观察到的速率常数与慢结合方程（方程2）的拟合是解离速率常数的近似值。解离的半衰期可以用2的自然对数除以解离速率常数来计算。

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凝血试验[1]
用前面描述的自动凝血分析仪测量柠檬酸血小板不良血浆（PPP）的凝血时间。从健康的狗、大鼠和家兔获得的柠檬酸血浆中，按上述方法测定使凝血时间延长两倍（EC2x）所需的BMS-654457的浓度。

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血小板聚集[1]
在水蛭素处理的兔富血小板血浆（PRP）中，根据制造商的说明，用血小板聚集计在体外测量血小板聚集。将水蛭素处理后的血液（lepirudin, 25 μg/ml）在180 g离心10 min后得到PRP， 250µl PRP分别与2.5µl载体或BMS-654457在1和10µM下混合，37℃孵育2 min。加入2.5µl激动剂（ADP为10µM，花生四烯酸为250µM，胶原为10µg/ml，终浓度）后测定血小板聚集峰。

In vivo [1]
Surgical preparation [1]
The rabbits were anesthetized and surgically prepared as described previously. Each rabbit was utilized in either an arterial thrombosis or cuticle bleeding time (BT) protocol. BMS-654457 or vehicle (10 % N-N-dimethylacetamide:90 % of 5 % dextrose) was administered as a bolus plus sustaining IV infusion begun 30 min prior to each protocol. The dosing regimen for BMS-654457 was designed based on results from pilot pharmacokinetic studies in rabbits, which determined the IV bolus and infusion doses needed to produce constant plasma levels over the duration of each experiment. Terminal plasma samples were obtained for drug exposure and ex vivo clotting times. Plasma levels of BMS-654457 were measured by a specific and sensitive liquid chromatographic mass spectrometry method.

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Arterial thrombosis model [1]
The rabbit electrolytic-mediated carotid arterial thrombosis (ECAT) model, described by Wong et al, was used in this study. Thrombosis was induced by electrical stimulation of the carotid artery. Carotid blood flow was measured by an electromagnetic flow probe over a 90-min period to monitor thrombotic occlusion. Integrated carotid blood flow (iCBF) over 90 min was measured by the area under the flow-time curve. Treatment groups included BMS-654457 (mg/kg + mg/kg/h) at 0.011 + 0.008, 0.037 + 0.027, 0.11 + 0.08, 0.37 +0.27 and 1.1 + 0.8 or vehicle (n = 6 per group). The plasma concentration that increased iCBF to 50 % of the control (EC50) was estimated for BMS-654457 as described below.

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Cuticle bleeding model [1]
The rabbit cuticle BT model, described by Himber et al., was modified and used in this study. Treatment groups included BMS-654457 (mg/kg + mg/kg/h) at 0.11 + 0.08, 0.37 + 0.27 and 1.1 + 0.8 or vehicle (n = 6 per group). BT was measured before and after treatment in opposite hind-limbs, and was expressed as a ratio of treated over the control value.

[1]. In vitro, antithrombotic and bleeding time studies of BMS-654457, a small-molecule, reversible and direct inhibitor of factor XIa. J Thromb Thrombolysis. 2015 Nov;40(4):416-23.

BMS-654457 ((+) 3′-(6-carbamimidoyl-4-methyl-4-phenyl-1,2,3,4-tetrahydro-quinolin-2-yl)-4-carbamoyl-5′-(3-methyl-butyrylamino)-biphenyl-2-carboxylic acid) is a small-molecule factor XIa (FXIa) inhibitor. We evaluated the in vitro properties of BMS-654457 and its in vivo activities in rabbit models of electrolytic-induced carotid arterial thrombosis and cuticle bleeding time (BT). Kinetic studies conducted in vitro with a chromogenic substrate demonstrated that BMS-654457 is a reversible and competitive inhibitor for FXIa. BMS-654457 increased activated partial thromboplastin time (aPTT) without changing prothrombin time. It was equipotent in prolonging the plasma aPTT in human and rabbit, and less potent in rat and dog. It did not alter platelet aggregation to ADP, arachidonic acid and collagen. In vivo, BMS-654457 or vehicle was given IV prior to initiation of thrombosis or cuticle transection. Preservation of integrated carotid blood flow over 90 min (iCBF, % control) was used as a marker of antithrombotic efficacy. BMS-654457 at 0.37 mg/kg + 0.27 mg/kg/h produced almost 90 % preservation of iCBF compared to its vehicle (87 ± 10 and 16 ± 3 %, respectively, n = 6 per group) and increased BT by 1.2 ± 0.04-fold (P < 0.05). At a higher dose (1.1 mg/kg + 0.8 mg/kg/h), BMS-654457 increased BT by 1.33 ± 0.08-fold. This compares favorably to equivalent antithrombotic doses of reference anticoagulants (warfarin and dabigatran) and antiplatelet agents (clopidogrel and prasugrel) which produced four- to six-fold BT increases in the same model. In summary, BMS-654457 was effective in the prevention of arterial thrombosis in rabbits with limited effects on BT. This study supports inhibition of FXIa, with a small-molecule, reversible and direct inhibitor as a promising antithrombotic therapy with a wide therapeutic window. [1]

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In summary, BMS-654457 is a small-molecule, reversible and direct inhibitor of FXIa. BMS-654457 pretreatment was able to prevent the rapid formation of thrombotic occlusion in the rabbit ECAT model at doses that had a minimal impact on primary hemostasis. The ex vivo aPTT appeared to be a reasonable biomarker for monitoring the antithrombotic activity in rabbits. Overall this study supports FXIa as an attractive target in the development of anticoagulants possessing a wide therapeutic window. [1]

C36H37N5O4

603.71

603.284

C, 71.62; H, 6.18; N, 11.60; O, 10.60

1004551-41-0

60154989

Typically exists as solid at room temperature

5.3

171

6

6

9

45

1090

2

CC(C)CC(=O)NC1=CC(=CC(=C1)[C@@H]2C[C@](C3=C(N2)C=CC(=C3)C(=N)N)(C)C4=CC=CC=C4)C5=C(C=C(C=C5)C(=O)N)C(=O)O

PDUMJXCNOKHQKH-SVXHESJVSA-N

InChI=1S/C36H37N5O4/c1-20(2)13-32(42)40-26-15-23(27-11-9-22(34(39)43)17-28(27)35(44)45)14-24(16-26)31-19-36(3,25-7-5-4-6-8-25)29-18-21(33(37)38)10-12-30(29)41-31/h4-12,14-18,20,31,41H,13,19H2,1-3H3,(H3,37,38)(H2,39,43)(H,40,42)(H,44,45)/t31-,36+/m0/s1

2-[3-[(2S,4R)-6-carbamimidoyl-4-methyl-4-phenyl-2,3-dihydro-1H-quinolin-2-yl]-5-(3-methylbutanoylamino)phenyl]-5-carbamoylbenzoic acid

BMS-654457; CHEMBL3127491; 1004551-41-0; 3'-[(2s,4r)-6-Carbamimidoyl-4-Methyl-4-Phenyl-1,2,3,4-Tetrahydroquinolin-2-Yl]-4-Carbamoyl-5'-[(3-Methylbutanoyl)amino]biphenyl-2-Carboxylic Acid; SCHEMBL4936907; BDBM50448583;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。