MRX-2843 (UNC2371)

FLT3 (IC50 = 0.64 nM); MERTK (IC50 = 1.3 nM)
MRX-2843 is a potent dual inhibitor of Mer tyrosine kinase (MERTK) and FMS-like tyrosine kinase 3 (FLT3). In enzymatic assays, its IC50 is 1.3 nM for MERTK and 0.64 nM for FLT3.
It shows selectivity over other TAM family kinases (AXL and TYRO-3) and other relevant tyrosine kinases, as detailed in Supplemental Table 1.
It also retains inhibitory activity against clinically relevant FLT3-ITD mutant proteins with point mutations at D835 (e.g., D835Y, IC50 = 7.2 ± 1.3 nM in BA/F3 cells) and F691 (e.g., F691L, IC50 = 20.4 ± 4.3 nM in BA/F3 cells).

在 Kasumi-1 细胞系中，用 MRX-2843 处理会导致 MERTK 磷酸化的剂量依赖性抑制。在浓度低至 10 nM 时，磷酸化作用明显降低，在 100 至 300 nM 时，MERTK 激活几乎完全消失。同样，用 MRX-2843 处理 Kasumi-1 细胞可通过对肿瘤细胞存活和增殖重要的途径介导下游信号传导的抑制。 MRX-2843治疗导致相对细胞数减少，IC50为143.5±14.1 nM，表明MRX-2843显着抑制肿瘤细胞增殖和/或存活。同样，在用 150 nM 或 300 nM MRX-2843 处理的 NOMO-1 培养物中，分别有 34.1%±5.6% 和 67.1%±2.7% 的凋亡和死亡细胞，而在用媒介物处理的培养物中，凋亡和死亡细胞的比例为 6.8%±0.7%（ P<0.001）。在 Kasumi-1 培养物中，用 50 nM 和 100 nM MRX-2843 处理分别可抑制 62.3%±6.4% 和 84.1%±7.8% 的集落形成（P<0.01）。同样，在 NOMO-1 培养物中，100 nM MRX-2843 处理后集落形成被抑制 54.8%±18.1%（P<0.001）。在 MOLM-14 细胞中，MRX-2843 处理可抑制 FLT3 的磷酸化以及通过 STAT5、ERK1/2 和 AKT 的下游信号传导。使用 50 nM MRX-2843 处理后，FLT3 及其信号通路的激活几乎完全消除，表明相对于 MERTK，针对 FLT3 的细胞效力略高[1]。

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MRX-2843 在AML细胞系（Kasumi-1、NOMO-1、MOLM-14、MV4-11）中能剂量依赖性地抑制MERTK和FLT3的磷酸化及其下游信号通路（STAT6、STAT5、AKT、ERK1/2），起效浓度可低至10-50 nM。
它抑制细胞增殖/克隆扩增，在Kasumi-1（MERTK依赖）细胞中的IC50为143.5 ± 14.1 nM，在MOLM-14（FLT3-ITD依赖）细胞中为29.5 ± 3.4 nM。
它能剂量依赖性地诱导细胞凋亡，例如在150 nM浓度下，诱导Kasumi-1细胞51.9% ± 12.4%的凋亡/死亡细胞，诱导MOLM-14细胞91.0% ± 2.7%的凋亡/死亡细胞。
在软琼脂集落形成实验中，它在25-100 nM浓度范围内几乎完全抑制多种AML细胞系的集落形成。
在原代AML患者样本（MERTK阳性，伴或不伴FLT3-ITD）中，MRX-2843 能强效抑制甲基纤维素中的集落形成，5 nM即有效，50 nM几乎完全抑制，而对正常人脐带血单个核细胞在高达500 nM浓度下影响甚微。
它对quizartinib耐药的FLT3-ITD突变细胞系（MOLM-14:D835Y和MOLM-14:F691L）保留强效活性，能抑制其增殖、信号传导和集落形成，而quizartinib则失去活性。

MRX-2843 在 3 mg/kg 剂量下的口服生物利用度为 78%，Cmax 为 1.3 μM，t1/2 为 4.4 小时。在 MOLM-14 亲代异种移植物中，与媒介物治疗的小鼠相比，quizartinib 和 MRX-2843 均增加了中位生存期（分别为 172.5 天与 40 天和 121 天与 36 天，P<0.001）。在此模型中，quizartinib 比 MRX-2843 更有效（P<0.005），尽管没有评估更高剂量的 MRX-2843。在 MOLM-14:D835Y 异种移植物中，与媒介物治疗的小鼠相比，quizartinib 延长了生存期，但效果很小（中位生存期 45 天与 36 天，P<0.001）。在 MOLM-14:F691L 异种移植物中，MRX-2843 治疗使 NSG 和 NSGS 小鼠的生存期延长了近 2 倍（中位生存期分别为 87 天与 44.5 天和 87 天与 48 天，P<0.005）。与 quizartinib 相比，MRX-2843 治疗的存活率有所增加，但差异仅在 NSG 小鼠中显着[1]。

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在NOD-SCID-γ (NSG) 或NSGS小鼠的异位移植瘤模型中，每日一次口服MRX-2843 能显著延长生存期。
在NOMO-1（MERTK依赖）移植瘤中，65 mg/kg/天的MRX-2843 将中位生存期从对照组的37天延长至51天。
在MOLM-14（FLT3-ITD）移植瘤中，50 mg/kg/天将中位生存期从38天延长至126天。
在AML患者来源的异种移植（PDX）模型中，MRX-2843（30-75 mg/kg/天）能显著延长生存期并降低外周疾病负荷。
在quizartinib耐药的FLT3-ITD突变移植瘤模型（MOLM-14:D835Y和MOLM-14:F691L）中，MRX-2843（50 mg/kg/天）能显著延长生存期，而quizartinib疗效甚微或降低。

如前所述合成MRX-2843和对照TKI（20）。如前所述，使用Michaelis-Menton动力学方程估算体内90%抑制所需的激酶抑制剂的量。奎扎替尼（AC220）与羟基丁烯基-β-环糊精（CD）的比例为1:4。对于体外研究，在DMSO中制备储备溶液，DMSO载体对照浓度相当于实验中最高剂量的测试试剂。对于体内研究，试验药物要么溶解（MRX-2843），要么在生理盐水中制备成均匀的悬浮液（喹扎替尼和CD）[1]。

细胞系在 0.35% Noble 琼脂基础层上的 0.35% Noble 琼脂中培养（10,000 个细胞/样品），并用含有激酶抑制剂（包括 MRX-2843）或载体的 cRPMI 覆盖。每周更换上层培养基 2 至 3 次，并重复评估载体处理。 14 天或 21 天后（仅限 Kasumi-1 细胞），将菌落用 1 mg/mL 硝基四唑蓝染色 4 小时，并使用菌落计数器进行计数。单核细胞是从人类脐带血和急性髓系白血病 (AML) 患者的样本中分离出来的。将患者样本以 1×106 个细胞/mL 的密度一式三份地在含有 MRX-2843 或载体的 MethoCult H4434 经典甲基纤维素培养基以及人类细胞重组细胞因子中培养。 10 天后使用菌落计数器对菌落进行计数。脐带血细胞在补充有 BIT 9500 血清替代品、低密度脂蛋白和 2-ME 的无血清 Iscove's 改良 Dulbecco 培养基 (IMDM) 中孵育 1 小时，然后以 2×106 个细胞/的密度一式三份培养。 mL 的 Methocult H4434 甲基纤维素溶液，含有 MRX-2843 或载体。 14 天后以盲法对菌落进行手动计数[1]。

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下游信号免疫印迹分析：指数生长期的细胞与溶剂、对照TKI或MRX-2843 培养1.5小时。制备细胞裂解液，定量蛋白，经SDS-PAGE分离后转膜，用针对信号蛋白（STAT5、STAT6、AKT、ERK1/2）磷酸化和总形式的特异性抗体进行检测。以Actin作为上样对照。使用化学发光法检测。
MERTK或FLT3磷酸化分析：处理类似，但在裂解前加入过钒酸盐磷酸酶抑制剂以稳定磷酸化蛋白。裂解液用抗MERTK或抗FLT3抗体进行免疫沉淀，然后用磷酸化特异性抗体和总蛋白抗体进行免疫印迹。
克隆扩增测定：细胞与测试药物培养48小时，然后使用基于化学发光法的ATP测定（指示活细胞数）来评估细胞活力。
凋亡测定：细胞与药物培养72小时，然后用YO-PRO-1碘化物和碘化丙啶染色，通过流式细胞术分析。
细胞系集落形成实验：细胞在软琼脂中培养，上覆含测试药物的培养基，培养14-21天后，对集落进行染色和计数。
原代患者样本或脐带血集落形成实验：分离单个核细胞，在含测试药物的甲基纤维素培养基中培养10-14天，然后计数集落。

Murine xenograft models.[1]
NOD.Cg-PrkdcscidIl2rgtm1WjlTg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (NSGS) mice and NOD.Cg-PrkdcscidIl2rgtm1Wj1/SzJ (NSG) mice were purchased from The Jackson Laboratory or bred in-house and maintained under sterile conditions. Established leukemia cell lines or mononuclear cells isolated from samples from patients with AML (1 × 106 to 2.5 × 106 per mouse) were suspended in PBS and injected into the tail veins of NSG or NSGS mice to establish xenografts. All mice were 4–6 months of age at the time of injection and were male, with the exception of the NOMO-1, MOLM-14:D835Y, and MOLM-14:F691L NSG xenografts, which were established in female mice. Myeloblasts were detected in peripheral blood (patient-derived xenografts) or bone marrow (MOLM-14 xenografts) samples after staining with a FITC-conjugated anti-human CD45 Ab. Samples were analyzed by flow cytometry using a Gallios flow cytometer and Kaluza software. After engraftment, the mice were weighed and treated once daily with MRX-2843, quizartinib, or vehicle administered by oral gavage in a volume of 10 ml/kg. When mice appeared ill or lost more than 20% of their body weight, they were euthanized.

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Mertk-null (B6;129-Mertktm1Grl/J) mice were backcrossed with C57BL/6J mice for more than 12 generations (Mertk–/–), and Mertk genotype was verified prior to use. WT C57BL/6J and NSG mice were purchased from the Jackson Laboratory and bred in-house. Young mice (aged 2–4 months) were used, given that Mertk–/– mice have the potential to develop autoimmunity after 6 months of age. Arf-null (Arf–/–) BCR-ABL p185+ murine ALL cells expressing GFP were injected via tail vein, and MRX-2843 or vehicle were administered via oral gavage beginning 1 day or 5 days after transplant. Mice with advanced leukemia (>20% weight loss, tachypnea, hind-limb paralysis, minimal activity) were euthanized, and survival was monitored. Alternatively, when mice developed symptoms of leukemia (17–39 days after transplant), they and their cagemates (1 mouse per treatment group) were euthanized, and bone marrow and spleen were harvested for analysis by flow cytometry. Leukemic burden and immune cell infiltration were quantitated on an LSR II flow cytometer and analyzed using FlowJo version X software.[2]

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For orthotopic xenograft models, established leukemia cell lines (e.g., NOMO-1, MOLM-14, or mutant derivatives) or mononuclear cells from primary AML patient samples were suspended in PBS and injected into the tail veins of NSG or NOD-SCID-γ mice expressing transgenic human cytokines (NSGS) mice.
MRX-2843 was dissolved in saline. Quizartinib was prepared as a homogeneous suspension in saline with hydroxybutenyl-β-cyclodextrin.
Drug or vehicle was administered once daily by oral gavage at a volume of 10 ml/kg. Treatment began after engraftment was confirmed (e.g., 21-53 days post-transplantation), when a certain percentage of blasts was detected in bone marrow or peripheral blood.
Doses used: 30, 50, 65, or 75 mg/kg/day for MRX-2843; 10 mg/kg/day for quizartinib.
Mice were monitored for survival, body weight, and peripheral/blood disease burden (by flow cytometry for human CD45+ cells). Treatment continued for specified durations (up to 140-145 days) or until mice met euthanasia criteria (appeared ill or lost >20% body weight).

In mice, MRX-2843 is 78% orally bioavailable at a dose of 3 mg/kg. The maximum plasma concentration (Cmax) is 1.3 μM, and the half-life (t1/2) is 4.4 hours.
The activity of MRX-2843 was retained in the presence of human plasma with minimal shift in potency in a plasma inhibitory assay.

Daily oral treatment with MRX-2843 was well tolerated in mice for up to 120 days, with no significant body weight loss reported in the studies.
MRX-2843 showed a 10-fold differential effect on colony formation between primary AML blasts and normal human cord blood mononuclear cells, indicating a substantial therapeutic window and selective toxicity towards leukemia cells.
Compared to some FLT3 inhibitors, MRX-2843 has reduced activity against c-KIT, potentially mitigating associated myelosuppression.

[1]. The MERTK/FLT3 inhibitor MRX-2843 overcomes resistance-conferring FLT3 mutations in acute myeloid leukemia. JCI Insight. 2016 Mar;1(3):e85630.

[2]. MERTK inhibition alters the PD-1 axis and promotes anti-leukemia immunity. JCI Insight . 2018 Nov 2;3(21):e97941.
[3]. MERTK activation drives osimertinib resistance in EGFR-mutant non-small cell lung cancer. J Clin Invest . 2022 Aug 1;132(15):e150517.

Flt3/MerTK Inhibitor MRX-2843 is an orally bioavailable inhibitor of two receptor tyrosine kinases (RTKs), FMS-like tyrosine kinase-3 (Flt3; CD135; fetal liver kinase-2; Flk2) and tyrosine-protein kinase Mer (MerTK; proto-oncogene c-Mer; Mer), with potential antineoplastic activity. Upon administration, MRX-2843 targets and binds to both Flt3 and MerTK. This prevents ligand-dependent phosphorylation and activation of Flt3 and MerTK, which inhibits the activation of their downstream signaling pathways. This induces apoptosis and inhibits proliferation of Flt3- and/or MerTK-overexpressing tumor cells. Flt3 and MerTK, are overexpressed in certain tumor cell types and play key roles in tumor cell proliferation and survival.

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MRX-2843 is an orally available, ATP-competitive type I tyrosine kinase inhibitor.
It was developed to overcome limitations of previous inhibitors, specifically to target both MERTK and FLT3 with high potency and to retain activity against resistance-conferring FLT3 mutations (D835 and F691) that emerge under therapy with inhibitors like quizartinib.
Its binding mode as a type I inhibitor allows it to be equally potent against both active ("DFG-in") and inactive ("DFG-out") conformations of FLT3, contributing to its activity against activation loop mutants like D835Y.
The dual inhibition of MERTK (overexpressed in 80-90% of AML) and FLT3 (mutated in 20-30% of AML) is proposed as a promising therapeutic strategy for a broad range of AML patients, potentially delaying resistance and improving efficacy.
The compound has been validated in multiple preclinical models, supporting its clinical development for AML.

C29H40N6O

488.6675

488.326

C, 71.28; H, 8.25; N, 17.20; O, 3.27

1429882-07-4

1429882-07-4;

89495685

Off-white to light yellow solid powder

1.3±0.1 g/cm3

697.3±65.0 °C at 760 mmHg

375.5±34.3 °C

0.0±2.3 mmHg at 25°C

1.696

2.92

69.4Ų

2

6

8

36

681

0

OC1CCC(CC1)N1C=C(C2C=CC(=CC=2)CN2CCN(C)CC2)C2=CN=C(N=C12)NCCC1CC1

LBEJYFVJIPQSNX-UHFFFAOYSA-N

InChI=1S/C29H40N6O/c1-33-14-16-34(17-15-33)19-22-4-6-23(7-5-22)27-20-35(24-8-10-25(36)11-9-24)28-26(27)18-31-29(32-28)30-13-12-21-2-3-21/h4-7,18,20-21,24-25,36H,2-3,8-17,19H2,1H3,(H,30,31,32)

4-[2-(2-cyclopropylethylamino)-5-[4-[(4-methylpiperazin-1-yl)methyl]phenyl]pyrrolo[2,3-d]pyrimidin-7-yl]cyclohexan-1-ol

MRX-2843; UNC 2371; UNC2371A; UNC-2371; UNC-2371A; MRX2843; UNC2371; UNC 2371A; UNC-2371A; 2MT30EHI63; UNII-2MT30EHI63; CHEMBL3326007; Cyclohexanol, 4-(2-((2-cyclopropylethyl)amino)-5-(4-((4-methyl-1-piperazinyl)methyl)phenyl)-7H-pyrrolo(2,3-d)pyrimidin-7-yl)-, trans-; trans-4-(2-((2-Cyclopropylethyl)amino)-5-(4-((4-methyl-1-piperazinyl)methyl)phenyl)-7H-pyrrolo(2,3-d)pyrimidin-7-yl)cyclohexanol; MRX 2843

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: 20~98 mg/mL (200.5 mM)
Ethanol: ~25 mg/mL (51.2 mM)

配方 1 中的溶解度: ≥ 2.08 mg/mL (4.26 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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配方 2 中的溶解度: 2 mg/mL (4.09 mM) 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; 超声助溶.
例如，若需制备1 mL的工作液，可将 100 μL 20.0 mg/mL 澄清的 DMSO 储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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 mg/mL (4.09 mM) 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), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 20.0mg/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。