ARUK-3001185

Notum (IC50 = 6.7 nM)

1,2,3-三唑 (ARUK3001185)的效力优于异构体1,3,4-三唑9a或1,2,3,4-四唑9b（表3）。这些效应似乎相当微妙，因为大多数关键抑制剂- notum相互作用可与这些可选的1-芳基唑进行，可能是由于8l与Trp128的电子相互作用更优，或者仅仅是N4原子是有害的8l (ARUK3001185)苯环对相应的吡啶9c的修饰是等效的，表明棕榈油酸袋中可以容纳一定的极性。8l在三唑的C4和C5位置的双重氘化使d2-8l成为研究母体分子代谢命运的工具。[1]

三唑8l (ARUK3001185)具有与类药物空间一致的理化和分子性质值得注意的是，8l (ARUK3001185)的实测分布系数（LogD7.4 1.3±0.05）远低于计算分配系数（clogP 3.9），尽管在生理相关的ph下未电离。这种分离可能归因于2-Cl， 3-CF3，和芳香环上的4-Cl取代基，当与它们各自的亲脂性片段常数（π）相加时，导致总体疏水表面积的减少。这些结果突出了生成与计算值相关的经验数据的重要性。使用生化试验（IC50 6.7 nM, pIC50 8.2）和测量的亲脂性（LogD7.4 1.3）的Notum抑制对8l (ARUK3001185)对标准设计指标进行评估，计算出8l (ARUK3001185)具有高LE (0.67)， LLE（6.9）和良好的脑穿透预测(CNS MPO 5.4/6.0；BBB评分5.6/6.0)。[1]

三唑8l (ARUK3001185)在磷酸盐缓冲盐水（PBS）中表现出令人满意的水溶性，在MLM和HLM中表现出良好的代谢稳定性，在MDCK-MDR1细胞转运试验中表现出高通透性，P-gp转运体识别和排出的证据很少（表3和支持信息）。相比之下，吡啶9c在渗透性测定中表现不佳，尽管这些结果与低化合物回收率相混淆。比较8l和d2-8l的MLM稳定性（Cli 3.2 vs 5.4 μL/min/mg蛋白），发现三氮唑的氘化作用没有改善，这表明cyp介导的三氮唑C-H键代谢不是该系统清除的主要途径。[1]

基于这些数据，我们从该系列中选择8l (ARUK3001185)作为候选候选药物进行进一步分析，因为它具有Notum活性和体外ADME特性（包括亲脂性）的最佳组合。[1]

然后在额外的体外药理学和ADME筛选中评估铅 (ARUK3001185)（表5）。三唑8l在Notum存在下恢复Wnt信号(EC50 110 nM；n = 4)，在基于细胞的TCF/LEF（荧光素酶）报告基因试验23,26,27中，给出了10 μM的标准s形浓度响应曲线（图S9）。在没有Notum的情况下进行这些实验显示，在所有测试浓度(高达10 μM；n = 4)。Wnt信号的激活是由于8l对Notum的直接靶向抑制，而不是由于实验干扰或细胞毒性（高达10 μM）。[1]

在小鼠体内和随后在大鼠体内生成8l (ARUK3001185)的PK数据，以评估血浆和脑暴露（表4和支持信息）。小鼠单次静脉给予8l (ARUK3001185)后，血浆清除率低于肝血流，分布体积适中，消除半衰期为2.4 h（图S3，表S11）。小鼠单次口服后，8l (ARUK3001185)被迅速吸收，并表现出良好的口服生物利用度（66%）（图S4，表S12）。脑内药物水平与血浆中的药物水平相似，证实了良好的脑穿透性（Kp 1.1）。计算游离药物浓度时，脑血浆比为0.77 （Kp,uu）。口服剂量为10 mg/kg，脑内浓度为Cmax≈300 nM（游离药物），超过了基于细胞的TCF/LEF测定的Notum EC50，脑内浓度维持在100 nM以上约4小时。尽管令人鼓舞，但该剂量和由此产生的脑暴露水平可能不足以促进持续的药理学（PD）反应，其中Notum活性在较长时间内降低。这需要根据经验来确定。因此，我们随后探索了替代给药方案，以便在确定啮齿动物疾病模型所需的有效浓度（Ceff）及其与Notum药理学（EC50）的关系方面提供一定的灵活性，即建立PK-PD关系（vide infra）。[1]

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8l (ARUK3001185)在大鼠体内的PK评价也显示出较低的血浆清除率和中等的体积分布，半衰期为3.3 h（表4）。大鼠口服吸收、药物水平和脑渗透良好（Kp 1.4）。口服生物利用度最高，Fo为140%。血浆（和脑）浓度-时间图清楚地显示双Cmax峰（图S7），这可能表明排泄药物从胃肠道重吸收和/或对胃排空时间有影响。综上所述，这些结果表明8l (ARUK3001185)具有与啮齿类动物疾病模型评估相一致的PK特性。[1]

ADME测定[1]
使用ChemDraw Professional v16.0.1.4（77）计算分子性质。采用摇瓶法测定分布系数LogD7.4。 所选化合物在PBS （pH 7.4）中的水溶性、在MDCK-MDR1细胞系中的转运性能（通透性）以及在MLM和HLM中的代谢稳定性（清除率）进行常规筛选。还筛选了先导化合物8l (ARUK3001185)对代表性CYP450酶的抑制作用、Caco-2细胞单层的通透性以及肝微粒体和肝细胞的代谢稳定性。检测方案和附加数据在辅助信息中给出。在这项工作中报告的ADME研究是由CRO公司独立进行的。

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Notum OPTS生化试验[1]
方法在其他地方有详细的描述，在辅助资料中提供了代表性的浓度-响应曲线。

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激酶选择性面板[1]
激酶选择性筛选由赛默飞世尔科学公司的SelectScreen生化激酶分析服务进行。检测信息和数据在辅助信息中提供。

PK Studies In vivo PK data in mouse and rat was independently generated at Charles River Laboratories (Groningen, Netherlands), GVK Biosciences (Hyderabad, India), and WuXi AppTec (Shanghai, China). PK was supported by the development of suitable formulations for the route of administration, and the measurement of plasma protein binding (PPB) and brain tissue binding for the calculation of free drug levels in these compartments. Study protocols and additional data are presented in the Supporting Information

The aqueous solubility of 8l (ARUK3001185) could be significantly enhanced to 1 mg/mL with cosolvents bringing added flexibility to study design. Triazole 8l (ARUK3001185) has good stability in liver microsomes and hepatocytes across mouse, rat, dog, and human, which predicts for low clearance across these species. Screening in HLM including eight standard substrates of cytochrome P450 (CYP450) enzymes showed moderate/weak inhibition of 1A2 (IC50 0.96 μM), 2B6 (IC50 5.2 μM), 2C9 (IC50 15 μM), and 2C19 (IC50 8.1 μM) but not 2C8, 2D6, or 3A4 (IC50 > 50 μM). Furthermore, there was no evidence for CYP time-dependent inactivation (TDI) as assessed by the preincubation of HLM with 8l (ARUK3001185) in the presence and absence of NADPH, followed by the incubation with the discrete marker substrates (IC50 shift). There was good cell permeability across a Caco-2 monolayer consistent with good absorption from the gut. The hepatocyte data for 8l (ARUK3001185) was encouraging as this deletion of the −CH2OH group from 7y seems to have removed the liability of this metabolic soft spot. Hence, 8l (ARUK3001185) was advanced to rodent PK studies. [1]

PK data for 8l (ARUK3001185) was generated in vivo in mouse, and subsequently in rat, to evaluate plasma and brain exposure (Table 4 and Supporting Information). Following single intravenous administration of 8l (ARUK3001185) to mouse, plasma clearance was low compared to liver blood flow and volume of distribution was moderate resulting in an elimination half-life of 2.4 h (Figure S3, Table S11). Following a single oral dose to mouse, 8l (ARUK3001185) was rapidly absorbed and showed good oral bioavailability (66%) (Figure S4, Table S12). Good brain penetration was confirmed with drug levels in the brain similar to drug levels in the plasma (Kp 1.1). The brain-to-plasma ratio was 0.77 (Kp,uu) when free drug concentrations were calculated. This oral dose of 10 mg/kg achieved a concentration in the brain of Cmax ≈ 300 nM (free drug) that exceeded the Notum EC50 from the cell-based TCF/LEF assay, and brain levels were maintained above 100 nM for ∼4 h. Although encouraging, this dose, and the resulting level of brain exposure, may be insufficient to promote a sustained pharmacodynamic (PD) response, where Notum activity was reduced for an extended period of time. This will need to be determined empirically. Hence, we subsequently explored alternative dosing regimes to provide some flexibility in the determination of the required efficacious concentrations (Ceff) in rodent models of disease, and its relationship to Notum pharmacology (EC50), that is, establish the PK–PD relationship (vide infra). [1]

PK evaluation of 8l (ARUK3001185) in rat also showed low plasma clearance and moderate volume of distribution with a half-life of 3.3 h (Table 4). Oral administration to rat showed good absorption, drug levels, and brain penetration (Kp 1.4). The oral bioavailability was supramaximal with Fo 140%. The plasma (and brain) concentration–time plots clearly show double Cmax peaks (Figure S7), which could indicate some contribution from excreted drug being reabsorbed from the gastrointestinal tract and/or an effect on gastric emptying times. On balance, these results established that 8l (ARUK3001185) has PK properties compatible with evaluation in rodent models of disease. [1]

[1]. Design of a Potent, Selective, and Brain-Penetrant Inhibitor of Wnt-Deactivating Enzyme Notum by Optimization of a Crystallographic Fragment Hit. J Med Chem. 2022 May 26;65(10):7212-7230.

Exploration of the substituents on the aryl ring that binds in the palmitoleate pocket of Notum gave potent early lead 7y (IC50 9.5 nM) and further optimization of the triazole group delivered 8l (ARUK3001185) (IC50 6.7 nM). It is noteworthy that advanced lead 8l can be characterized as a fragment-sized molecule (MW 282; HAC 17) with high LE and LLE, and that inhibition of Notum activity was increased by over 15,000-fold by optimization of the substituents on the aryl ring (8l vs 7a). These findings highlight the benefits of FBDD when aligned with a suitable drug target. The Notum-8l X-ray structure clearly showed that 8l (ARUK3001185) has near complete occupancy of the palmitoleate pocket through the 2-Cl-3-CF3-4-Cl substituents on the aryl ring. Triazole 8l restored Wnt signaling in the presence of Notum (EC50 110 nM) in a cell-based TCF/LEF reporter assay confirming functional activity. Assessment in broader off-target and safety pharmacology screens showed 8l to be selective against serine hydrolases, kinases, and representative drug targets. PK studies with 8l in mouse and rat showed good plasma exposure and brain penetration, and was well tolerated. In summary, triazole 8l (ARUK3001185) is a potent and selective inhibitor of Notum activity with good brain penetration suitable for oral dosing in rodent models of disease.[1]

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Notum is a carboxylesterase that suppresses Wnt signaling through deacylation of an essential palmitoleate group on Wnt proteins. There is a growing understanding of the role Notum plays in human diseases such as colorectal cancer and Alzheimer's disease, supporting the need to discover improved inhibitors, especially for use in models of neurodegeneration. Here, we have described the discovery and profile of 8l (ARUK3001185) as a potent, selective, and brain-penetrant inhibitor of Notum activity suitable for oral dosing in rodent models of disease. Crystallographic fragment screening of the Diamond-SGC Poised Library for binding to Notum, supported by a biochemical enzyme assay to rank inhibition activity, identified 6a and 6b as a pair of outstanding hits. Fragment development of 6 delivered 8l that restored Wnt signaling in the presence of Notum in a cell-based reporter assay. Assessment in pharmacology screens showed 8l to be selective against serine hydrolases, kinases, and drug targets.

C9H4CL2F3N3

282.049369812012

280.973

C, 38.33; H, 1.43; Cl, 25.14; F, 20.21; N, 14.90

2411969-39-4

146438044

Off-white to pink solid powder

3.5

30.7

0

5

1

17

277

0

N1(C2=CC=C(Cl)C(C(F)(F)F)=C2Cl)C=CN=N1

XVFXHCLMGYJAQI-UHFFFAOYSA-N

InChI=1S/C9H4Cl2F3N3/c10-5-1-2-6(17-4-3-15-16-17)8(11)7(5)9(12,13)14/h1-4H

1-[2,4-dichloro-3-(trifluoromethyl)phenyl]triazole

ARUK3001185; 2411969-39-4; 1-[2,4-Dichloro-3-(trifluoromethyl)phenyl]triazole; 1-[2,4-bis(chloranyl)-3-(trifluoromethyl)phenyl]-1,2,3-triazole; CHEMBL5178575; SCHEMBL21792325; XVFXHCLMGYJAQI-UHFFFAOYSA-N; BDBM601033;

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 : ~100 mg/mL (~354.55 mM)

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;
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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;
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