LXH254 is a potent B and C RAF inhibitor extracted from patent WO2018051306A1, Compound A^[1].

LXH254 (Compound A) is an adenosine triphosphate (ATP)-competitive inhibitor of BRAF (also referred to herein as b-RAF or b-Raf) and CRAF (also referred to herein as c-RAF or c- Raf) protein kinases. Throughout the present disclosure, LXH254 is also referred to as a c-RAF (or CRAF) inhibitor or a C-RAF/c-Raf kinase inhibitor. In cell-based assays, LXH254 has demonstrated anti-proliferative activity in cell lines that contain a variety of mutations that activate MAPK signaling. Moreover, LXH254 is a Type 2 ATP -competitive inhibitor of both B-Raf and C-Raf that keeps the kinase pocket in an inactive conformation, thereby reducing the paradoxical activation seen with many B-Raf inhibitors, and blocking mutant RAS-driven signaling and cell proliferation^[1].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

Treatment with LXH254 (Compound A) generates tumor regression in several KRAS-mutant models including the NSCLC-derived Calu-6 (KRAS Q61K) and NCI-H358 (KRAS G12C). LXH254 exhibits efficacy in numerous MAPK-driven human cancer cell lines and in xenograft tumors representing model tumors harboring human lesions in KRAS, NRAS and BRAF oncogenes^[1].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

502.49

C₂₅H₂₅F₃N₄O₄

1800398-38-2

Room temperature in continental US; may vary elsewhere.

DMSO : 100 mg/mL (199.01 mM; Need ultrasonic)

请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。
储备液的保存方式和期限：-80°C, 6 months; -20°C, 1 month。-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。

请根据您的实验动物和给药方式选择适当的溶解方案。以下溶解方案都请先按照 In Vitro 方式配制澄清的储备液，再依次添加助溶剂：

——为保证实验结果的可靠性，澄清的储备液可以根据储存条件，适当保存；体内实验的工作液，建议您现用现配，当天使用； 以下溶剂前显示的百
分比是指该溶剂在您配制终溶液中的体积占比；如在配制过程中出现沉淀、析出现象，可以通过加热和/或超声的方式助溶

• 1.

  请依序添加每种溶剂： 10% DMSO    40% PEG300    5% Tween-80    45% saline

  Solubility: ≥ 2.5 mg/mL (4.98 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (4.98 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加入50 μL Tween-80，混合均匀；然后继续加入 450 μL生理盐水定容至 1 mL。

  将 0.9 g 氯化钠，完全溶解于 100 mL ddH₂O 中，得到澄清透明的生理盐水溶液

• 2.

  请依序添加每种溶剂： 10% DMSO    90% (20% SBE-β-CD in saline)

  Solubility: ≥ 2.5 mg/mL (4.98 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (4.98 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 900 μL 20% 的 SBE-β-CD 生理盐水水溶液中，混合均匀。

  将 2 g 磺丁基醚 β-环糊精加入 5 mL 生理盐水中，再用生理盐水定容至 10 mL，完全溶解，澄清透明
• 3.

  请依序添加每种溶剂： 10% DMSO    90% corn oil

  Solubility: ≥ 2.5 mg/mL (4.98 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (4.98 mM，饱和度未知) 的澄清溶液，此方案不适用于实验周期在半个月以上的实验。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 900 μL玉米油中，混合均匀。

• 4.

  请依序添加每种溶剂： 5% DMSO    40% PEG300    5% Tween-80    50% saline

  Solubility: 2.5 mg/mL (4.98 mM); Suspended solution; Need ultrasonic

• [1]. CAPONIGRO, Giordano, et al. THERAPEUTIC COMBINATIONS COMPRISING A RAF INHIBITOR AND A ERK INHIBITOR. WO 2018051306 A1 20180322

Mice^[1]
SCID beige female tumor-bearing NCI-H358 mice, n=8 per group, are randomized into 3 groups 14 days post tumor cell inoculation with an average tumor volume range of 259.44- 262.47mm³. Animals are administered an oral dose of either vehicle, LXH254 at 30mg/kg or 200mg/kg daily for 14 consecutive days at a dosing volume of 10 mL/kg of animal body weight during course of treatment. Tumor volumes are measured by digital caliper 3 times a week and body weights of all animals are recorded through the course of treatment.
Female nude tumor bearing Calu6 mice, n=6 per group are randomized into treatment groups on day 17 following tumor implantation, when the average tumor volume is 180 mm³. Treatments with LXH254 are initiated on Day 17 and continued for 16 days. Dosing volume is 10 mL/kg. Tumor volumes are collected at the time of randomization and twice weekly thereafter for the study duration.
Nude female mice tumor bearing NCI-H358, n=8 per group, are randomized into 2 groups with an average tumor volume range of 275.74 mm³. Animals are administered an oral dose of either vehicle or LXH254 at 100 mg/kg daily for 14 consecutive days at a dosing volume of 10 mL/kg of animal body weight during course of treatment. Tumor volumes are measured by digital caliper 3 times a week and body weights of all animals are recorded through the course of treatment^[1].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. CAPONIGRO, Giordano, et al. THERAPEUTIC COMBINATIONS COMPRISING A RAF INHIBITOR AND A ERK INHIBITOR. WO 2018051306 A1 20180322