MRTX0902   中文名称: (R)-2-甲基-3-(1-(4-甲基-7-哌嗪-1-基)氨基)乙基)苯甲腈

KRAS-SOS1 ( IC50 = 46 nM )

MRTX0902（化合物 32）（1 μM；0、2、4、8、15 和 30 分钟）在人肝微粒体中显示出 195 mL/min/kg 的中等 Clint 值和 cLogP 为 3.4 的低亲脂性[1] 。 MRTX0902 对 SOS1 (Ki=2 nM) 的选择性高于 SOS2 和 EGFR（Ki 值均 >10,000 nM），MRTX0902 抑制 MKN1 细胞，IC50 值为 29 nM[1]。

MRTX0902（化合物 32）（25、50 mg/kg；口服；每天两次；25 天）在小鼠模型中显示出抗肿瘤作用，并导致肿瘤消退[1]。 MRTX0902（静脉注射 1-3 mg/kg 或口服 10-30 mg/kg；单剂量）表现出良好的脑渗透性、低清除率和高生物利用度[1]。 MRTX0902 跨物种的 PK 参数[1] 参数 途径 剂量 (mg/kg) Cl (mL/min/kg) Vd,ss (L/kg) T1/2(iv) (h) F (%) 小鼠 IV/PO 3/30 4.4 0.28 1.3 69 大鼠 IV/PO 1/10 14.6 0.28 0.62 83 狗 IV/PO 2/10 7.6 0.48 0.86 38 动物模型：雌性 CD-1 小鼠[1] 剂量：50 mg/kg 给药方式：口服灌胃;每天两次; 1 天结果：大脑中游离药物暴露以及 Caco-2 测定中的流出率（ER = 1.5）。显示该化合物在小鼠体内的半衰期较短（T1/2 = 1.3 小时）。动物模型：小鼠MIA PaCa-2异种移植模型[1] 剂量：25 mg/kg； 50 mg/kg 给药方式：口服强饲；每天两次; 25 天结果：25 mg/kg 和 50 mg/kg 给药时肿瘤生长分别减少 41% 和 53%。

HTRF置换法测定Ki[1]
使用HTRF置换测定法测量化合物结合SOS1的能力。在缓冲液（25mM HEPES pH 7.5，25mM NaCl，1mM DTT，0.01%Brij 35，0.02%BSA，0.1%DMSO）中，将重组人SOS1多肽（对应于氨基酸560-1049，在大肠杆菌中用N-末端His-TEV-AviTag-SOS1（MW=59.4 kDa）和镧系元素标记的链霉抗生物素蛋白表达）与示例性化合物（在DMSO储备溶液中）一起孵育。在室温下孵育10-15分钟后，将由定制的Cy5标记的示踪剂和缓冲液中的MAb Anti-6HIS Tb隐窝金组成的溶液加入到含有SOS1多肽和示例性化合物的溶液中。在室温下孵育1小时后，根据制造商的说明，使用Clairostar平板读数器测量HTRF信号。使用激发滤光片EX-TR，在650-610nm处检测到发射1，在620-610nm处探测到发射2。使用公式[发射1/发射2]*10000计算HTRF比。背景信号是用10µM抑制剂从孔中计算出来的，已知在该浓度下可以抑制100%。将背景减去的信号转换为相对于DMSO对照的结合百分比。使用XLFIT软件（IDBS）分析数据，使用Morrison方程进行竞争性结合，并生成Ki。

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EGFR选择性检测[1]
使用Reaction Biology的放射性热点激酶测定法对人EGFR的选择性进行了分析。化合物以粉末形式发送，然后以10mM DMSO储备溶液悬浮。化合物以10剂量IC50模式进行测试，从10μM开始连续稀释3倍。对照化合物星孢菌素以10剂量IC50模式进行测试，从20μM开始连续稀释4倍。反应在10μM ATP下进行。

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SOS2 KRAS WT GDP交换分析[1]
功能性SOS2测定的最终条件如下：50 mM HEPES 7.5，2nM SOS2，50 nM GTP-CY5，30 nM KRAS，0.5 nM Tb SA，5 mM MgCl2，1 mM TCEP，0.2 mg/mL BSA，~1.0%DMSO。在室温下将重组人SOS2多肽（对应于氨基酸558-1047，在大肠杆菌中表达，N端HIS标签MW=59.3 kDa）和Cy5-GTP加入到示例性化合物（在DMSO储备溶液中）中15分钟。然后将重组人KRAS多肽（氨基酸2-169，在大肠杆菌中用C末端Avi生物素化标签MW 22.0 kDa表达）与Terbium StrepAvidin加入，30分钟后使用BMG LABTECH CLARIOstar Plus通过TR-FRET收集比率度量数据。通过使用DMSO对照来确定100%的对照（POC），使用完全抑制SOS2活性的对照S101化合物的浓度来确定0 POC。POC值符合Hill方程的IC50，并报告了IC50值。方程式1:Vmax/（1+（x/IC50）^Hill））+BKD）

将MKN1细胞（15000/w）接种在黑色透明平底96孔细胞培养板中，并在37℃下孵育过夜。测定第1天，细胞被给予起始浓度为10µm的化合物，并连续稀释3倍，共9种浓度。将细胞与溶解在DMSO中的化合物在37°C下孵育约0.5-1小时。通过向通风橱中的所有孔中加入50µL 4%甲醛立即固定细胞，并在室温下孵育板20分钟。从板中丢弃甲醛，加入150µL冰冷的甲醇，使细胞在-20°C下渗透10分钟。将甲醇从每个板中丢弃，并通过用纸巾轻敲板来清除板中残留的任何液体。然后在室温下在摇床上用150µL Odyssey阻断缓冲液（使用0.05%吐温）阻断细胞1小时。丢弃阻断缓冲液，加入在Odyssey阻断缓冲液中稀释的50µL一抗pERK（Rabbit，1:500）和GapDH。将平板在4°C的摇床上孵育过夜。在测定第2天，去除第一抗体溶液。每个板用150µL 1x PBST（PBS+0.1%吐温20）洗涤3次，并在室温下在摇床上与50µL二抗（抗兔和抗小鼠，在含有吐温的Odyssey阻断缓冲液中以1:800稀释）一起孵育2小时（避光）。取出第二抗体溶液，用PBST洗涤每个板3次。丢弃任何剩余的液体，并根据制造商的说明使用Licor Odyssey机器对印版进行成像，使用3mm的焦距和800nm和700nm的滤光片。将每个孔的GAPDH归一化扫描值除以载体孔的平均值，得到pERK抑制的百分比。然后用Graph pad Prism软件计算IC50值。

Mice were maintained under pathogen-free conditions, and food and water was provided ad libitum. 6 – 8-week-old female Hsd:Athymic Nude-Foxn1nu mice were injected subcutaneously with tumor cells in 100 l of PBS and Matrigel matrix in the right hind flank of each mouse with 5.0 x 106 MIA PaCa-2 cells at a ratio of 1:1 in PBS and Matrigel. Mouse health was monitored daily, and caliper measurements began when tumors were palpable. Tumor volume measurements were determined utilizing the formula 0.5 x L x W2 in which L refers to length and W refers to width of each tumor. When tumors reached the desired average study start tumor volume of 150 mm3 or 200 mm3 for 21–28-day TGI efficacy and 6-day studies, respectively, mice were randomized into treatment groups. MRTX0902 was formulated in 0.5% Methylcellulose (4000cps) + 0.2% Tween80 in water once per week and dosing solution was stored protected from light at 4 °Celsius. MRTX849 was formulated once per week in 10% Captisol in 50 mM Citrate Buffer pH 5.0 and S103 dosing solution was stored protected from light at 4 °Celsius. Mice were orally administered vehicle, MRTX0902, MRTX849 orally (PO) at the indicated doses and schedules. Mice were monitored daily, tumors and body weights were measured 2 or 3 times per week. Percent Tumor Growth Inhibition (% TGI) was calculated using the following formula: (1-(Final Drug Treated Tumor Volume – Initial Drug Treated Tumor Volume) / (Final Vehicle Treated Tumor Volume – Initial Vehicle Treated Tumor Volume))*100. Percent Tumor regression was calculated when the average tumor volume of final treated tumors was less than initial treated tumor volume using the following equation: (-100%)*(1-(Final treated tumor volume)/(Initial treated tumor volume)). Statistical analysis of differences in mean tumor volume between vehicle- and drug-treated cohorts was run using a two-tailed Student t test in GraphPad Prism version 8.2.0. A p value of less than 0.05 was considered to be statistically significant. In studies where tumor collection was performed, mice were humanely sacrificed, and tumors were surgically removed and immediately cut into two pieces. One half piece was transferred to a prefilled homogenizer tube with ceramic beads and the other half piece was transferred to an Eppendorf tube. Both tubes were immediately submerged in liquid nitrogen to snap freeze the tissue and stored at -80°C until further processing was performed[1].

[1]. Design and Discovery of MRTX0902, a Potent, Selective, Brain-Penetrant, and Orally Bioavailable Inhibitor of the SOS1:KRAS Protein-Protein Interaction. J Med Chem. 2022 Jul 28;65(14):9678-9690.

SOS1 Inhibitor MRTX0902 is an orally available, brain-penetrant, phthalazine-based inhibitor of the guanine nucleotide exchange factor (GEF) Son of sevenless homolog 1 (SOS1), with potential antineoplastic activity. Upon oral administration, SOS1 inhibitor MRTX0902 selectively targets and binds to SOS1, thereby preventing the interaction of SOS1 with Kirsten rat sarcoma viral oncogene homolog (KRAS) in the guanosine diphosphate (GDP)-bound 'off' state, which is the inactivated state of KRAS. This abrogates the exchange of RAS-bound GDP for guanosine triphosphate (GTP) and prevents the formation of GTP-loaded KRAS, which is the activated 'on' state of KRAS. This prevents activation of downstream RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway by GTP-loaded KRAS. This inhibits mutant KRAS-dependent signaling and may inhibit growth and survival of KRAS-expressing tumor cells. KRAS is a member of the RAS family of oncogenes that is mutated in many cancer cell types. Mutations of KRAS may induce constitutive signal transduction leading to tumor cell proliferation, survival, invasion, and metastasis. SOS1 regulates the KRAS GDP-GTP cycle and promotes nucleotide exchange and formation of 'active' KRAS-GTP.

C22H24N6O

388.47

388.2

C, 68.02; H, 6.23; N, 21.63; O, 4.12

2654743-22-1

2654743-22-1

156526655

Light yellow to green yellow solid powder

2.8

87Ų

1

7

4

29

587

1

C12C=NC(N3CCOCC3)=CC1=C(N=NC=2C)N[C@H](C)C1=C(C(=CC=C1)C#N)C

ILPWEAHQRAWJIU-OAHLLOKOSA-N

InChI=1S/C22H24N6O/c1-14-17(12-23)5-4-6-18(14)15(2)25-22-19-11-21(28-7-9-29-10-8-28)24-13-20(19)16(3)26-27-22/h4-6,11,13,15H,7-10H2,1-3H3,(H,25,27)/t15-/m1/s1

2-methyl-3-[(1R)-1-[(4-methyl-7-morpholin-4-ylpyrido[3,4-d]pyridazin-1-yl)amino]ethyl]benzonitrile

MRTX-0902; MRTX0902; CRG69FR93G; UNII-CRG69FR93G; CHEMBL5192659; (R)-2-Methyl-3-(1-((4-methyl-7-morpholinopyrido(3,4-d)pyridazin-1-yl)amino)ethyl)benzonitrile; Benzonitrile, 2-methyl-3-((1R)-1-((4-methyl-7-(4-morpholinyl)pyrido(3,4-d)pyridazin-1-yl)amino)ethyl)-; MRTX 0902

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 12.5~100 mg/mL (32.2~257.4 mM)
Ethanol: ~100 mg/mL

配方 1 中的溶解度: ≥ 1.25 mg/mL (3.22 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 12.5mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，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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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。