 Chemical Structure.png)
MRX-2843 (also known as MRX2843; UNC-2371) is a novel, potent and orally bioactive small-molecule inhibitor of MERTK and FLT3 with anticancer activity. In cases of acute myeloid leukemia, MRX-2843 overcomes FLT3 mutations that confer resistance. A novel approach to treating patients with wtEGFR NSCLC is to combine MRX-2843 with an irreversible EGFR TKI. With a broad therapeutic window compared to normal human cord blood cells, MRX-2843 treatment induces apoptosis and inhibits colony formation in AML cell lines and primary patient samples expressing MERTK and/or FLT3-ITD.
Physicochemical Properties
| Molecular Formula | C29H40N6O |
| Molecular Weight | 488.6675 |
| Exact Mass | 488.326 |
| Elemental Analysis | C, 71.28; H, 8.25; N, 17.20; O, 3.27 |
| CAS # | 1429882-07-4 |
| Related CAS # | 1429882-07-4; |
| PubChem CID | 89495685 |
| Appearance | Off-white to light yellow solid powder |
| Density | 1.3±0.1 g/cm3 |
| Boiling Point | 697.3±65.0 °C at 760 mmHg |
| Flash Point | 375.5±34.3 °C |
| Vapour Pressure | 0.0±2.3 mmHg at 25°C |
| Index of Refraction | 1.696 |
| LogP | 2.92 |
| Hydrogen Bond Donor Count | 2 |
| Hydrogen Bond Acceptor Count | 6 |
| Rotatable Bond Count | 8 |
| Heavy Atom Count | 36 |
| Complexity | 681 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | OC1CCC(CC1)N1C=C(C2C=CC(=CC=2)CN2CCN(C)CC2)C2=CN=C(N=C12)NCCC1CC1 |
| InChi Key | LBEJYFVJIPQSNX-UHFFFAOYSA-N |
| InChi Code | 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) |
| Chemical Name | 4-[2-(2-cyclopropylethylamino)-5-[4-[(4-methylpiperazin-1-yl)methyl]phenyl]pyrrolo[2,3-d]pyrimidin-7-yl]cyclohexan-1-ol |
| Synonyms | 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 |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
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). |
| ln Vitro |
MRX-2843 treatment induces a dose-dependent suppression of MERTK phosphorylation in the Kasumi-1 cell line. As little as 10 nM shows signs of decreased phosphorylation, and between 100 and 300 nM almost entirely abolishes MERTK activation. In a similar vein, MRX-2843 treatment of Kasumi-1 cells results in downstream signaling pathways crucial for tumor cell survival and proliferation being inhibited. With an IC50 of 143.5±14.1 nM, MRX-2843 treatment causes a decrease in relative cell numbers, suggesting that it significantly inhibits tumor cell survival and/or proliferation. In NOMO-1 cultures treated with 150 nM or 300 nM MRX-2843, respectively, there are 34.1%±5.6% and 67.1%±2.7% apoptotic and dead cells, compared with 6.8%±0.7% in cultures treated with vehicle (P<0.001). When Kasumi-1 cultures are treated with 50 nM and 100 nM MRX-2843, respectively, colony formation is inhibited by 62.3%±6.4% and 84.1%±7.8% (P<0.01). In NOMO-1 cultures, treatment with 100 nM MRX-2843 inhibits colony formation by 54.8%±18.1% (P<0.001). Treatment with MRX-2843 inhibits downstream signaling via STAT5, ERK1/2, and AKT as well as FLT3 phosphorylation in MOLM-14 cells. Treatment with 50 nM MRX-2843 almost completely inhibits FLT3 activation and its signaling pathways, suggesting that it has a slightly stronger cellular potency against FLT3 than MERTK[1]. MRX-2843 dose-dependently inhibited MERTK and FLT3 phosphorylation and their downstream signaling pathways (STAT6, STAT5, AKT, ERK1/2) in AML cell lines (Kasumi-1, NOMO-1, MOLM-14, MV4-11) at concentrations as low as 10-50 nM. It inhibited cell proliferation/clonal expansion with IC50 values of 143.5 ± 14.1 nM in Kasumi-1 (MERTK-dependent) and 29.5 ± 3.4 nM in MOLM-14 (FLT3-ITD-dependent) cells. It induced apoptosis in a dose-dependent manner, e.g., at 150 nM, it induced 51.9% ± 12.4% apoptotic/dead cells in Kasumi-1 and 91.0% ± 2.7% in MOLM-14 cells. It inhibited colony formation in soft agar assays, with near-complete inhibition at 25-100 nM in various AML cell lines. In primary AML patient samples (MERTK-positive, with or without FLT3-ITD), MRX-2843 potently inhibited colony formation in methylcellulose, with effects evident at 5 nM and near-complete inhibition at 50 nM, while showing minimal effect on normal human cord blood mononuclear cells up to 500 nM. It retained potent activity against quizartinib-resistant FLT3-ITD mutant cell lines (MOLM-14:D835Y and MOLM-14:F691L), inhibiting their proliferation, signaling, and colony formation, whereas quizartinib lost activity. |
| ln Vivo |
MRX-2843 is 78% orally bioavailable at a dose of 3 mg/kg with a Cmax of 1.3 μM and a t1/2 of 4.4 hours. Quizartinib and MRX-2843 both improve the median survival in MOLM-14 parental xenografts when compared to mice treated with a vehicle (172.5 days versus 40 days and 121 days versus 36 days, respectively, P<0.001). Although higher doses of MRX-2843 are not evaluated, quizartinib is more effective than MRX-2843 in this model (P<0.005). Quizaratinib increases survival in MOLM-14:D835Y xenografts relative to mice treated with a vehicle, although the difference is not statistically significant (median survival 45 days vs. 36 days, P<0.001). MRX-2843 treatment increases survival in MOLM-14:F691L xenografts by nearly two times in both NSG and NSGS mice (median survival 87 vs. 44.5 days and 87 vs. 48 days, respectively, P<0.005). MRX-2843 treatment is associated with improved survival compared to quizartinib treatment; however, this difference is only statistically significant in NSG mice[1]. In orthotopic xenograft models using NOD-SCID-γ (NSG) or NSGS mice, once-daily oral administration of MRX-2843 significantly prolonged survival. In NOMO-1 (MERTK-dependent) xenografts, 65 mg/kg/day MRX-2843 increased median survival from 37 days (vehicle) to 51 days. In MOLM-14 (FLT3-ITD) xenografts, 50 mg/kg/day increased median survival from 38 days (vehicle) to 126 days. In patient-derived xenograft (PDX) models of AML, MRX-2843 (30-75 mg/kg/day) significantly prolonged survival and reduced peripheral disease burden. In quizartinib-resistant FLT3-ITD mutant xenograft models (MOLM-14:D835Y and MOLM-14:F691L), MRX-2843 (50 mg/kg/day) significantly extended survival, whereas quizartinib showed minimal or reduced efficacy. |
| Enzyme Assay | MRX-2843 and the control TKI were synthesized as previously described (20). The amount of kinase inhibitor required for 90% inhibition in vivo was estimated using Michaelis-Menton kinetic equations as previously described. Quizartinib (AC220) was obtained as a 1:4 formulation with hydroxybutenyl-β-cyclodextrin (CD). For in vitro studies, stock solutions were prepared in DMSO, and the DMSO vehicle control concentration was equivalent to the highest dose of test agent for the experiment. For in vivo studies, test agents were either dissolved (MRX-2843) or prepared in a homogeneous suspension (quizartinib and CD) in saline[1]. |
| Cell Assay |
Cell lines are cultured (10,000 cells/sample) in 0.35% Noble agar on a 0.5% Noble agar base layer and overlaid with cRPMI containing kinase inhibitor (including MRX-2843) or vehicle. The overlying medium is replaced 2 to 3 times per week, and vehicle treatment is assessed in duplicate. After 14 days or 21 days (Kasumi-1 cells only), colonies are stained with 1 mg/mL nitrotetrazolium blue for 4 hours and counted using a colony counter. Mononuclear cells are isolated from human cord blood and samples from acute myeloid leukemia (AML) patients. Patient samples are cultured in triplicate at a density of 1×106 cells/mL in MethoCult H4434 Classic Methylcellulose-Based Medium with Recombinant Cytokines for Human Cells containing MRX-2843 or vehicle. Colonies are counted after 10 days using the colony counter. Cord blood cells are incubated for 1 hour in serum-free Iscove’s modified Dulbecco’s medium (IMDM) supplemented with BIT 9500 Serum Substitute, low-density lipoproteins, and 2-ME, and then cultured in triplicate at a density of 2×106 cells/mL in Methocult H4434 methylcellulose containing MRX-2843 or vehicle. Colonies are manually counted in a blinded manner after 14 days[1]. For immunoblot analysis of downstream signaling, exponentially growing cells were cultured with vehicle, control TKI, or MRX-2843 for 1.5 hours. Cell lysates were prepared, proteins were quantified, separated by SDS-PAGE, transferred to membranes, and probed with specific antibodies against phosphorylated and total forms of signaling proteins (STAT5, STAT6, AKT, ERK1/2). Actin was used as a loading control. Chemiluminescence detection was used for visualization. For phosphorylation analysis of MERTK or FLT3, cells were treated similarly, but a pervanadate phosphatase inhibitor was added prior to lysis to stabilize phosphorylated proteins. Lysates were immunoprecipitated with anti-MERTK or anti-FLT3 antibodies, followed by immunoblotting with phospho-specific and total protein antibodies. Clonal expansion was assessed by culturing cells with test agents for 48 hours, followed by measurement of ATP levels (indicative of viable cell number) using a luminescence-based assay. Apoptosis was assessed by culturing cells with agents for 72 hours, then staining with YO-PRO-1 iodide and propidium iodide, followed by flow cytometry analysis. For colony-forming assays with cell lines, cells were cultured in soft agar with test agents in the overlay medium for 14-21 days, then colonies were stained and counted. For colony-forming assays with primary patient samples or cord blood, mononuclear cells were isolated and cultured in methylcellulose medium containing test agents for 10-14 days, after which colonies were counted. |
| Animal Protocol |
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. 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] 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). |
| ADME/Pharmacokinetics |
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. |
| Toxicity/Toxicokinetics |
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. |
| References |
[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. |
| Additional Infomation |
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. 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. |
Solubility Data
| Solubility (In Vitro) |
DMSO: 20~98 mg/mL (200.5 mM) Ethanol: ~25 mg/mL (51.2 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.26 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 2: 2 mg/mL (4.09 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 3: 2 mg/mL (4.09 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0464 mL | 10.2319 mL | 20.4637 mL | |
| 5 mM | 0.4093 mL | 2.0464 mL | 4.0927 mL | |
| 10 mM | 0.2046 mL | 1.0232 mL | 2.0464 mL |