K-Ras(G12C)

MRTX849 (0.1-10000 nM; 3天/2D条件，12天/3D条件)对基质KRAS G12C突变体细胞系的生长有明显的抑制作用，其IC50在2D条件下为10~973 nM，3D MRTX849 (0.24-1000nM; 24小时)抑制KRAS依赖的信号转导靶点，包括ERK1/2磷酸化(Thr202/Tyr204 ERK1; pERK)、S6磷酸化细胞活力测定，条件为0.2〜1042 nM[1]。 [1]细胞系：MIA PaCa-2、H1373、H358、H2122、SW1573、H2030、KYSE-410细胞（G12C）； H1299（WT）； A549 (G12S)、HCT116 (G13D) 细胞 浓度：0.1、1、10、100、1000、10000 nM 孵育时间：24 小时 结果：抑制绝大多数 KRAS G12C 突变细胞系的细胞生长，IC50 值介于2D 格式为 10 至 973 nM，3D 格式为 0.2 至 1042 nM。 Western Blot 分析[1] 细胞系：MIA PaCa-2 细胞 浓度：0.24、0.5、1.0、2.0、3.9、7.8、15.6、31.3、62.5、125、250、500、1000 nM 孵育时间：24 小时 结果：抑制KRAS 依赖性信号传导靶点，包括 ERK1/2 磷酸化（Thr202/Tyr204 ERK1；pERK）、S6 磷酸化（RSK 依赖性 Ser235/236；pS6）和 ERK 调节的 DUSP6 表达，每个靶点的 IC50 均在个位数纳摩尔范围内在细胞系中。

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抗增殖活性：Adagrasib能有效抑制KRAS G12C突变的癌细胞系生长。在17种KRAS G12C突变和3种非KRAS G12C突变的癌细胞系中，其在2D（3天贴壁细胞）培养中的IC50值介于10 nM至973 nM之间，在3D（12天球体）培养中介于0.2 nM至1042 nM之间，表明对KRAS G12C突变细胞具有较强的抗增殖活性。

MRTX849（1-100 mg/kg；口腔灌胃。；每日一次至第16天）在耐受良好的剂量范围内显示剂量怀疑抗肿瘤疗效，MRTX849的最大有效剂量在30-100 mg/kg动物模型：MIA PaCa-2模型(6-8周龄，雌性，无胸腺裸-Foxn1 nu小鼠)[1] 剂量：1、3、10、30和100 mg//天之间[1]。 kg 给药方式：口服强饲；每天一次，直至第 16 天 结果：在最早的治疗后肿瘤测量中观察到肿瘤快速消退，30 和 100 mg/kg 组中的动物在研究第 15 天表现出完全缓解的证据。在研究第 16 天停止给药，所有 4 只小鼠在研究第 70 天，100 mg/kg 组中的 7 只小鼠和 30 mg/kg 组中的 7 只小鼠中有 2 只保持无肿瘤状态。

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抗肿瘤功效：Adagrasib可在动物模型中引起肿瘤快速消退。在实验中，30 mg/kg和100 mg/kg剂量组的动物在研究第15天显示出完全缓解的迹象。在研究第16天停止给药后，100 mg/kg剂量组的4只小鼠和30 mg/kg剂量组的7只小鼠中有2只在研究第70天仍无肿瘤，表明其具有显著的体内抗肿瘤作用。

KRASG12C target engagement[2]
将肿瘤片段在6M胍-HCl、50mM N-（2-羟乙基）哌嗪-N′-乙磺酸（HEPES）（pH 7.5）和5mM TCEP中匀浆。离心后，使用Bradford测定法测定上清液的蛋白质浓度。将内标物（13C15N重组KRASG12C）和20mM碘代乙酰胺添加到200μL裂解缓冲液中的200μg肿瘤蛋白中，并将样品在37°C下黑暗孵育30分钟。烷基化后，使用96孔Zeba旋转板将100μL反应物交换为1M胍-HCl和50mM HEPES（pH 7.5）。用1μg胰蛋白酶/Lys-C混合物在37°C下消化蛋白质18小时。使用C18旋转板对肽进行脱盐，并通过蒸发去除溶剂。将肽溶解在0.1%甲酸、5%乙腈和95%水中，用于LCMS分析。使用Sciex TripleTOF仪器上的靶向方法来监测含有Cys-12的KRASG12C肽（一种内部参考肽）以及相应的同位素标记的肽。KRASG12C的参与度按照之前的报告进行计算。[2]

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kinact/KI的测量[2]
在室温下，将重组KRASG12C“Lite”（C51S/C80L/C118S）与一定浓度的MRTX849在25 mM HEPES（pH 7.0）、150 mM NaCl、5 mM MgCl2、10 mM辛基β-吡喃葡糖苷和0.5 mM TCEP中反应0–45 s。在每个时间点，用50mM HCl猝灭反应，并加入0.25μg胃蛋白酶。将KRASG12C在37°C下消化4小时，并通过LCMS分析所得的含有Cys-12的肽。对于每种浓度的MRTX849，从0s对照样品计算每个时间点的修饰KRASG12C的百分比，随后根据ln（POC）相对于时间数据的斜率计算kobs。速率与浓度的数据符合Michaelis–Menten方程。

所有细胞系均保存在37℃、5%CO2的加湿培养箱中，并常规检查其支原体水平。使用 CellTiter-Glo 测定对 7 个 KRAS G12C 突变细胞系和 3 个非 KRAS G12C 突变细胞系进行细胞活力评估，这些细胞系在 12 天测定中使用 96 孔 ULA 板在 3D 条件下生长，或在 2D 组织中生长3 天测定中的培养条件。

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Cell Viability Assay[1]

Cell Types: MIA PaCa-2, H1373, H358, H2122, SW1573, H2030, KYSE-410 cells (G12C); H1299 (WT); A549 (G12S), HCT116 (G13D) cells
Tested Concentrations: 0.1, 1, 10, 100, 1000, 10000 nM
Incubation Duration: 24 h
Experimental Results: Inhibits cell growth in the vast majority of KRAS G12C-mutant cell lines with IC₅₀ values ranging between 10 and 973 nM in the 2D format and between 0.2 and 1042 nM in the 3D format.

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Western Blot Analysis[1]

Cell Types: MIA PaCa-2 cells
Tested Concentrations: 0.24, 0.5, 1.0, 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000 nM
Incubation Duration: 24 h
Experimental Results: Inhibits KRAS-dependent signaling targets including ERK1/2 phosphorylation (Thr202/Tyr204 ERK1; pERK), S6 phosphorylation (RSK-dependent Ser235/236; pS6) and expression of the ERK-regulated DUSP6, each with IC₅₀s in the single-digit nanomolar range in cell lines.

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细胞活力测定：培养17种KRAS G12C突变和3种非KRAS G12C突变的癌细胞系，设置2D（贴壁细胞培养3天）和3D（球体培养12天）培养体系。向培养基中加入不同浓度的Adagrasib，在规定的培养时间后，采用合适的方法检测细胞活力，从而评估Adagrasib对KRAS G12C突变癌细胞生长的抑制作用。

Animal/Disease Models: MIA PaCa-2 model (6-8-week-age, female, athymic nude-Foxn1 nu mice)[1]
Doses: 1, 3, 10, 30, 100 mg/kg
Route of Administration: p.o., for 16 days, daily
Experimental Results: Rapid tumor regression was observed at the earliest posttreatment tumor measurement and animals in the 30 and 100 mg/kg cohorts exhibited evidence of a complete response at study Day 15. Dosing was stopped at study Day 16 and all 4 mice in the 100 mg/kg cohort and 2 out of 7 mice in the 30 mg/kg cohort remained tumor-free through study Day 70.

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Administer Adagrasib to animal models by gavage (the specific solvent and formulation are not described in the literature). Set up 30 mg/kg and 100 mg/kg dosage groups, and observe the tumor volume change of animals at different time points. The results show that rapid tumor regression can be observed at the earliest post - treatment tumor measurement, and some animals have a complete response, and part of them can maintain a tumor - free state for a long time after the drug is stopped.

Absorption, Distribution and Excretion
The AUC and Cmax of adagrasib increase in a dose-proportional manner between 400 mg and 600 mg (0.67 to 1 times the approved recommended dose). At the recommended dose, adagrasib reached steady-state within 8 days, with a 6-fold accumulation. The Tmax of adagrasib is approximately 6 hours. The administration of a high-fat and high-calorie meal (900-1000 calories, 50% from fat) did not have a clinically significant effect on the pharmacokinetics of adagrasib. Adagrasib has high oral bioavailability and is able to penetrate the central nervous system.
Adagrasib is eliminated through feces and urine. In patients given a single dose of radiolabeled adagrasib, 75% of the dose was recovered in feces (14% as unchanged), while 4.5% was recovered in urine (2% as unchanged).
Adagrasib has an apparent volume of distribution of 942 L.
Adagrasib has an apparent oral clearance (CL/F) of 37 L/h.

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Metabolism / Metabolites
Following single-dose administration, adagrasib is mainly metabolized by CYP3A4. However, since adagrasib inhibits CYP3A4 following multiple dosing, other enzymes such as CYP2C8, CYP1A2, CYP2B6, CYP2C9, and CYP2D6 contribute to its metabolism at steady-state.

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Biological Half-Life
Adagrasib has a terminal elimination half-life of 23 hours.

Hepatotoxicity
In the prelicensure clinical trials of adagrasib in patients with solid tumors harboring KRAS G12C mutations, liver test abnormalities were frequent although usually self-limited and mild. Some degree of ALT elevations arose in 28% to 46% of adagrasib treated patients and elevations above 5 times the upper limit of normal (ULN) were seen in 5% to 7%. In these trials that enrolled approximately 366 patients, adagrasib was discontinued early due to increased AST or ALT in 8% of patients. In addition, a small proportion of patients developed clinically apparent hepatotoxicity requiring adagrasib discontinuation. The liver test abnormalities had a median onset of 3 weeks after initiation of therapy. While serum aminotransferase elevations were occasionally quite high (5 to 20 times ULN), there were no accompanying elevations in serum bilirubin and no patient developed clinically apparent liver injury with jaundice. The product label for adagrasib recommends monitoring for routine liver tests before, at 3 week intervals during the first 3 months of therapy, and thereafter as clinically indicated.
Likelihood score: D (possible but infrequent cause of clinically apparent liver injury).

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Protein Binding
_In vitro_, adagrasib has a human plasma protein binding of 98%.

[1]. Cancer Discov. 2020 Jan;10(1):54-71.

[2]. J Med Chem. 2020 Jul 9;63(13):6679-6693.

Adagrasib (MRTX849) is an oral, small-molecule KRAS inhibitor developed by Mirati Therapeutics. KRAS mutations are highly common in cancer and account for approximately 85% of all RAS family mutations. However, the development of KRAS inhibitors has been challenging due to their high affinity for guanosine triphosphate (GTP) and guanosine diphosphate (GDP), as well as the lack of a clear binding pocket. Adagrasib targets KRASG12C, one of the most common KRAS mutations, at the cysteine 12 residue and inhibits KRAS-dependent signalling. In a phase I/IB clinical study that included patients with KRASG12C-mutated advanced solid tumors (NCT03785249), adagrasib exhibited anti-tumor activity. The phase II of the same study showed that in patients with KRASG12C-mutated non-small-cell lung cancer (NSCLC), adagrasib was efficient without new safety signals. In February 2022, the FDA accepted a new drug application (NDA) for adagrasib for the treatment of patients with previously treated KRASG12C–positive NSCLC. In December 2022, the FDA granted accelerated approval to adagrasib for the treatment of KRASG12C-mutated locally advanced or metastatic NSCLC who have received at least one prior systemic therapy. Adagrasib joins [sotorasib] as another KRASG12C inhibitor approved by the FDA.
Adagrasib is a small molecule inhibitor of the KRAS G12C mutant protein which is found in up to 13% of refractory cases of non-small cell lung cancer. Serum aminotransferase elevations are common during therapy with adagrasib, and a proportion of patients develop clinically apparent liver injury that can be severe.
Adagrasib is an orally available, small molecule inhibitor that targets the oncogenic KRAS substitution mutation, G12C, with potential antineoplastic activity. Upon oral administration adagrasib covalently binds to cytosine 12 within the switch II pocket of GDP-bound KRAS G12C, thereby inhibiting mutant KRAS-dependent signaling. KRAS, a member of the RAS family of oncogenes, serves an important role in cell signaling, division and differentiation. Mutations of KRAS may induce constitutive signal transduction leading to tumor cell growth, proliferation, invasion, and metastasis.

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Drug Indication
Adagrasib is indicated for the treatment of adult patients with KRAS G12C-mutated locally advanced or metastatic non-small cell lung cancer (NSCLC), as determined by an FDA-approved test, who have received at least one prior systemic therapy. This indication is approved under accelerated approval based on objective response rate (ORR) and duration of response (DOR). Continued approval for this indication may be contingent upon verification and description of a clinical benefit in a confirmatory trial(s).
Treatment of all solid and haematological malignancies

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Mechanism of Action
In normal cells, KRAS is activated by binding to guanosine triphosphate (GTP), and this promotes the activation of the MAP kinase pathway and intracellular signal transduction. When GTP is hydrolyzed to guanosine diphosphate (GDP), KRAS is inactivated. This mechanism works as an "on"/"off" system that regulates cell growth. The substitution of Gly12 by cysteine in KRAS (KRAS^G12C) impairs GTP hydrolysis, and maintains KRAS in its active form. Therefore, the presence of this mutation leads to uncontrolled cellular proliferation and growth, as well as malignant transformation. Adagrasib is a covalent inhibitor of KRAS^G12C that irreversibly and selectively binds and locks KRAS^G12C in its inactive, guanosine diphosphate–bound state. Therefore, the use of adagrasib inhibits tumor cell growth and viability in cancers with KRAS^G12C mutations with minimal off-target activity.

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Pharmacodynamics
The exposure-response relationship and pharmacodynamic response time course of adagrasib have not been elucidated. The use of adagrasib can cause QTc interval prolongation. The increase in QTc is concentration-dependent. In patients given 600 mg of adagrasib twice daily, the mean QTcF change from baseline (ΔQTcF) was 18 ms at the mean steady-state maximum concentration. The use of adagrasib can also lead to severe gastrointestinal adverse reactions, hepatotoxicity and interstitial lung disease/pneumonitis.

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- Mechanism of Action: Adagrasib is a potent, selective, and covalent KRAS G12C inhibitor. It can selectively modify the mutant cysteine 12 in GDP - bound KRAS G12C, thereby inhibiting KRAS - dependent signaling, and then playing an anti - tumor role. - Therapeutic Effect: It has shown certain curative effects in the treatment of KRAS G12C - mutant non - small cell lung cancer. In clinical trials, it can make some patients produce a partial response, but the response is not durable, and acquired resistance will occur. For example, a patient with KRAS G12C - mutant non - small cell lung cancer initially had a partial response to Adagrasib, but later developed resistance, and the resistance was related to the emergence of a secondary KRAS Y96D mutation, which affected the binding of the drug to the KRAS switch - II pocket. - Indications: It is mainly used for the treatment of adults with KRAS G12C - mutated locally advanced or metastatic non - small cell lung cancer who have received at least one prior systemic therapy.

C32H35CLFN7O2

604.1174

603.25

C, 63.62; H, 5.84; Cl, 5.87; F, 3.14; N, 16.23; O, 5.30

2326521-71-3

MRTX849 analog 24; 2490716-96-4; LC-2; 2502156-03-6

138611145

White to yellow solid powder

From > 262 mg/mL to < 0.010 mg/mL

5

88.8Ų

0

9

7

43

1060

2

CN1CCC[C@H]1COC2=NC3=C(CCN(C3)C4=CC=CC5=C4C(=CC=C5)Cl)C(=N2)N6CCN([C@H](C6)CC#N)C(=O)C(=C)F

PEMUGDMSUDYLHU-ZEQRLZLVSA-N

InChI=1S/C32H35ClFN7O2/c1-21(34)31(42)41-17-16-40(18-23(41)11-13-35)30-25-12-15-39(28-10-4-7-22-6-3-9-26(33)29(22)28)19-27(25)36-32(37-30)43-20-24-8-5-14-38(24)2/h3-4,6-7,9-10,23-24H,1,5,8,11-12,14-20H2,2H3/t23-,24-/m0/s1

2-[(2S)-4-[7-(8-chloronaphthalen-1-yl)-2-[[(2S)-1-methylpyrrolidin-2-yl]methoxy]-6,8-dihydro-5H-pyrido[3,4-d]pyrimidin-4-yl]-1-(2-fluoroprop-2-enoyl)piperazin-2-yl]acetonitrile

Adagrasib; MRTX 849; MRTX849; KRAZATI; Kras G12C inhibitor MRTX849; 8EOO6HQF8Y; Adagrasib [USAN]; MRTX-849; brand name: Krazati

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: 25~100 mg/mL (41.4~165.5 mM)
Ethanol: ~100 mg/mL

配方 1 中的溶解度: ≥ 2.62 mg/mL (4.34 mM) (饱和度未知) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*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 (4.14 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 3 中的溶解度: ≥ 2.5 mg/mL (4.14 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900μL玉米油中，混合均匀。
*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 4 中的溶解度: in 5%DMSO+ 40%PEG300+ 5%Tween 80+ 50%ddH2O: 5.0mg/ml (8.28mM) (这些助溶剂从左到右依次添加，逐一添加),

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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。