Nemtabrutinib (ARQ-531)

Wild type BTK (IC50 = 0.85 nM), C481S-BTK (IC50 = 0.39 nM)[1].

在 TMD8 细胞系中，ARQ 531 表现出显着的靶标抑制作用。 WT-BTK和C481S-BTK在生化实验中的IC50值分别为0.85 nM和0.39 nM。此外，ARQ 531 对 TEK 激酶也具有显着的抑制作用，IC50 值为 3.6.4 nM (TXK)、5.80 nM (TEC) 和 5.23 nM (BMX)。 3.86 nM (LCK)、4.22 nM (YES)、9.71 nM (BLK)、18.3 nM (HCK)、18.8 nM (LYNa)、25.9 nM (FGR)、32.2 nM (FYN)、48.0 nM (FRK) 和 TRK 激酶ARQ 531 针对 SRC 激酶的 IC50 值为 11.7 nM (TrkB)、13.1 nM (TrkA) 和 19.1 nM (TrkC)。 ARQ 531 可有效抑制多种细胞系的生长（TMD8：GI50=0.13 μM，REC1：GI50=0.18 nM），并且可有效对抗依赖 PI3K/AKT 途径、Src 家族激酶和 BCR 的细胞系。

ARQ 531 在肿瘤异种移植模型（例如 TMD-8）中效果良好。 14 天后，ARQ 531 疗法完全缩小了肿瘤。在胶原蛋白引起的关节炎模型中，ARQ 531 也有益。在动物研究中，ARQ 531 显示出强大的抗关节炎功效。 ARQ 531 在 BTK 驱动的 TMD8 异种移植小鼠模型中表现出显着的抗癌功效和持久效果。在胶原诱导关节炎 (CIA) 的小鼠模型中，ARQ 531 显示出体内有效性 [1]。

BTK生化和激酶选择性试验[1]
采用野生型和C481S突变体全长BTK构建体进行生化抑制实验。进行了激酶谱分析。对36个激酶的分析发现，在200 nM浓度的arq531下，45个激酶的抑制作用>50%。随后，在生理的1mM ATP浓度下，测定这种ATP竞争抑制剂在这些激酶上的效价。

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体外PD测定[1]
细胞用浓度增加的SUDHL-4抑制剂处理2小时，在抗igm或生长因子刺激后，细胞裂解进行Western blot分析。

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BTK人PBMC功能测定[1]
采集献血者新鲜血液，采用淋巴细胞密度梯度法分离PBMC。CD69在CD20+ b细胞上的表达通过增加arq531浓度和IgM刺激来检测。通过流式细胞术检测CD69在CD20+ b细胞上的表达水平百分比，以估计arq531的效价。

细胞生长试验[1]
细胞接种于96孔组织培养板中，用化合物或载体处理72小时。加入CellTiter-Glo试剂，轻摇培养2分钟。反应再进行10分钟，然后在Victor®微孔板阅读器上读板。采用四参数logistic曲线拟合确定GI50值。

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逆相蛋白测定法(RPPA) [1]
在6孔板中培养TMD8细胞，用0.3 μM或3 μM ARQ 531或DMSO处理2小时。然后按照Carna Bio Science提供的RPPA程序裂解细胞，并由Bradford测定蛋白质浓度。样品被运往Carna进行磷酸化蛋白表达分析。将数据归一化为对照样本，并将单个目标的相对值按其指定的途径分组。

Animal care [1]
Six week old female CB-17 SCID mice were purchased commercially and allowed to acclimate 1-2 weeks. Mice were housed in sterile micro isolator cages, five mice per cage and receive food and water ad libitum. All experimental procedures were approved in accordance with ArQule’s Institutional Animal Care and Use Committee (IACUC).

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Tumor model [1]
Female SCID mice were implanted subcutaneously with 8x106 TMD8 cells in 0.2ml sterile Hanks Balanced Salt Solution (HBSS) with 50% standard concentration BD matrigel in the upper right flank area. Mice were monitored and staged on day 14 (post injection of tumor cells) when size reached approximately 400mg. Oral daily dosing with ARQ 531 at 100 mg/kg, vinblastine or vehicle began on stage day. Tumor measurements and body weights were collected three times a week with electronic calipers and balance. Tumor weight (mg) was calculated from the equation, length x (width)2 /2 and the tumor volume was calculated by assuming unit density 1mg equals to 1mm3.

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In vivo PD analysis [1]
In vivo Target and pathway inhibition was studied in mouse TMD8 xenograft model. Following oral dosing of BTK inhibitors tumor tissues and plasma were collected for Western blot analysis of phosphorylated proteins and ARQ 531 levels in plasma. Percent inhibition relative to the vehicle control was determined using densitometry analysis and the intensity of actin band was used as a loading control and the percentage of vehicle group was designated as 100%.

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Collagen-induced arthritis model [1]
DBA1/J mice were immunized with collagen to develop the arthritis, following the onset of arthritis, mice were randomized into treatment groups. Treatment was initiated by oral dosing of ARQ 531 at 25, 50 and 75 mg/Kg and continued daily (QD at 24-hour intervals) through arthritis day 14. Clinical scores were assessed for each of the paws on study arthritis days 1–15. Dexamethasone at 3 mg/Kg was used as a control to monitor inhibition of disease symptoms ADME and pharmacokinetics studies CYP450 inhibition and P-gp substrate/inhibition potential for ARQ 531 as well as pharmacokinetics studies in monkeys and dogs were determined and analyzed by Covance Laboratories Inc.

ARQ 531 has high oral bioavailability, good ADME, pharmacokinetic and metabolic properties.
t1/2 = 9.3 hrs (dogs); 13.6 hours (monkeys)
F = 38% (dogs); 72% (monkeys)

[1]. Targeting PCI-32765-Resistant BTK-C481S Mutation with ARQ 531, a Reversible Non-Covalent Inhibitor of BTK. Clinical Lymphoma Myeloma & Leukemia, 2016, 16: S47-S48.

Nemtabrutinib is an orally available reversible inhibitor of Bruton's tyrosine kinase (BTK; Bruton agammaglobulinemia tyrosine kinase), with potential antineoplastic activity. Upon administration, nemtabrutinib non-covalently binds to and inhibits the activity of both the wild-type and the C481S mutated form of BTK, a resistance mutation in the BTK active site in which cysteine is substituted for serine at residue 481. This prevents the activation of the B-cell antigen receptor (BCR) signaling pathway and BTK-mediated activation of downstream survival pathways. This leads to an inhibition of the growth of malignant B-cells that overexpress BTK. Compared to other BTK inhibitors, nemtabrutinib does not require interaction with the BTK C481 site and inhibits the proliferation of cells harboring the BTK C481S mutation. BTK, a member of the Src-related BTK/Tec family of cytoplasmic tyrosine kinases, is overexpressed or mutated in B-cell malignancies; it plays an important role in the development, activation, signaling, proliferation and survival of B-lymphocytes.

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Drug Indication
Treatment of mature B-cell malignancies

C25H23CLN4O4

478.927524805069

478.14

C, 62.70; H, 4.84; Cl, 7.40; N, 11.70; O, 13.36

2095393-15-8

129045720

White to off-white solid powder

1.4±0.1 g/cm3

731.6±60.0 °C at 760 mmHg

396.2±32.9 °C

0.0±2.5 mmHg at 25°C

1.687

3.87

109

3

7

7

34

681

2

ClC1C=C(C=CC=1C(C1=CNC2=C1C(=NC=N2)N[C@H]1CO[C@H](CO)CC1)=O)OC1C=CC=CC=1

JSFCZQSJQXFJDS-QAPCUYQASA-N

InChI=1S/C25H23ClN4O4/c26-21-10-17(34-16-4-2-1-3-5-16)8-9-19(21)23(32)20-11-27-24-22(20)25(29-14-28-24)30-15-6-7-18(12-31)33-13-15/h1-5,8-11,14-15,18,31H,6-7,12-13H2,(H2,27,28,29,30)/t15-,18+/m1/s1

(2-chloro-4-phenoxyphenyl)(4-(((3R,6S)-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)methanone

ARQ531; MK1026; ARQ-531; MK-1026; MK-1026; JTZ51LIXN4; (2-Chloro-4-phenoxyphenyl)(4-(((3R,6S)-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)-7h-pyrrolo[2,3-d]pyrimidin-5-yl)methanone; ARQ 531;

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)

DMSO : ≥ 50 mg/mL (~104.40 mM)

配方 1 中的溶解度: ≥ 2.08 mg/mL (4.34 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 20.8 mg/mL澄清DMSO储备液加入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: ≥ 2.08 mg/mL (4.34 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.08 mg/mL (4.34 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 20.8 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；
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。