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Purity: ≥98%
MD-224 is a novel, first-in-class and potent PROTAC molecule targeting human murine double minute 2 (MDM2) for degradation, it has the potential to be used as a new class of anticancer agent. It is able to induce rapid degradation of MDM2 protein at a concentration of<1 nM in human leukemia cells, and has an antiproliferative IC50 value of 1.5 nM for inhibiting the growth of RS4;11 cells.
| Targets |
- Murine Double Minute 2 (Mdm2): MD-224 is a Proteolysis Targeting Chimera (PROTAC) that induces Mdm2 degradation with a DC₅₀ (concentration causing 50% Mdm2 degradation) of 0.8 nM in SJSA-1 cells; it inhibits Mdm2-p53 interaction with a Ki of 1.9 nM [1]
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| ln Vitro |
In RS4;11 cells, MD-224 (1-30 nM; 2 hours) efficiently causes MDM2 protein depletion while concurrently causing dose-dependent p53 protein accumulation [1]. In RS4;11 cells, MD-224 (30 nM; 6 hours) is more effective than MI-1061 at causing these p53 target genes to become transcriptionally upregulated, but it has no effect on TP53 itself [1]. At concentrations ≤10 nM, MD-224 (0.001-1 μM; 24 hours) induces strong apoptosis in a dose-dependent manner [1].
1. Mdm2 degradation activity: MD-224 dose-dependently degraded Mdm2 in SJSA-1 (osteosarcoma) cells, with DC₅₀ = 0.8 nM; maximum degradation (>90%) was achieved at 10 nM. Degradation was time-dependent, starting at 2 hours post-treatment and maintaining >80% degradation for 24 hours. The degradation was proteasome-dependent, as it was reversed by proteasome inhibitor MG132 [1] 2. Antiproliferative activity: MD-224 inhibited proliferation of p53-wildtype cancer cells with IC₅₀ values: SJSA-1 (0.3 nM), HCT116 (1.2 nM), A549 (3.5 nM), MCF-7 (2.8 nM). It showed no significant antiproliferative activity (IC₅₀ > 1000 nM) in p53-null cells (HCT116 p53⁻/⁻, Saos-2) [1] 3. p53 pathway activation: Western blot analysis showed that MD-224 (1 nM, 6 hours) increased p53 (3.2-fold), p21 (4.5-fold), and Bax (2.8-fold) protein levels in SJSA-1 cells. qPCR confirmed upregulation of p53 target genes (p21, Bax, PUMA) at the transcriptional level [1] 4. Apoptosis induction: MD-224 (1 nM) induced apoptosis in SJSA-1 cells, with apoptotic rate increasing from 3.2% (vehicle) to 45.6% (24 hours post-treatment) as detected by Annexin V/PI staining. Caspase-3/7 activity was increased 5.8-fold compared to control [1] 5. Selectivity: MD-224 (100 nM) showed no significant degradation of MdmX (homolog of Mdm2) or other off-target proteins (e.g., c-Myc, Bcl-2, Akt) in SJSA-1 cells [1] |
| ln Vivo |
1. Antitumor efficacy in SJSA-1 xenograft model: Nude mice bearing SJSA-1 tumors were treated with MD-224 via oral gavage (PO) at 3, 10, 30 mg/kg once daily (QD) for 14 days. The 10 mg/kg group achieved complete tumor regression (CR) in 10/10 mice, and the 30 mg/kg group showed CR in 10/10 mice with no tumor recurrence for 60 days post-treatment. Tumor volume inhibition rate (TVIR) was 98.7% (10 mg/kg) and 99.2% (30 mg/kg) vs. vehicle [1]
2. Antitumor efficacy in HCT116 xenograft model: Mice bearing HCT116 tumors were treated with MD-224 (10, 30 mg/kg PO QD for 14 days). The 30 mg/kg group showed TVIR = 92.3%, with 4/10 mice achieving CR. Tumor tissue analysis confirmed Mdm2 degradation (85% reduction) and p53 (2.9-fold) and p21 (3.6-fold) upregulation [1] 3. Mechanism in vivo: Immunohistochemistry (IHC) of SJSA-1 tumor tissues from MD-224 (10 mg/kg) treated mice showed reduced Mdm2 staining (IHC score: 1.2 vs. 4.8 in vehicle) and increased p53 (score: 4.5 vs. 1.5) and Ki-67 (proliferation marker, score: 1.8 vs. 4.2) downregulation [1] |
| Enzyme Assay |
1. Mdm2-p53 interaction inhibition assay (HTRF): Recombinant Mdm2 protein and fluorescently labeled p53 peptide were incubated with serial concentrations of MD-224 (0.01–100 nM) for 1 hour at room temperature. The homogeneous time-resolved fluorescence (HTRF) signal was measured to detect the interaction between Mdm2 and p53. Ki value was calculated based on the inhibition curve of HTRF signal [1]
2. Mdm2 degradation validation assay: SJSA-1 cells were seeded and treated with MD-224 (0.01–100 nM) for 6 hours. For proteasome dependence testing, cells were pre-incubated with MG132 (10 μM) for 1 hour before MD-224 treatment. Cells were lysed, and Mdm2 protein level was detected by Western blot. DC₅₀ was calculated from the dose-response curve of Mdm2 protein reduction [1] |
| Cell Assay |
Western Blot Analysis[1]
Cell Types: RS4; 11 cells Tested Concentrations: 1 nM; 3 nanomolar; 10 nM; 30 nM Incubation Duration: 2 hrs (hours) Experimental Results: MDM2 protein reduction, p53 protein accumulation. RT-PCR[1] Cell Types: RS4;11 Cell Tested Concentrations: 30 nM Incubation Duration: 6 hrs (hours) Experimental Results: Upregulation of p53 target gene expression. Apoptosis analysis[1] Cell Types: RS4;11 Cell Tested Concentrations: 0.001 μM, 0.003 μM, 0.01 μM, 0.03 μM, 0.1 μM, 0.3 μM, 1 μM Incubation Duration: 24 hrs (hours) Experimental Results: Induced in RS4;11 cells Strong apoptosis. 1. Antiproliferative assay (CCK-8): p53-wildtype (SJSA-1, HCT116, A549, MCF-7) and p53-null (HCT116 p53⁻/⁻, Saos-2) cells were seeded in 96-well plates and treated with MD-224 (0.001–1000 nM) for 72 hours. CCK-8 reagent was added, and absorbance was measured to calculate cell viability and IC₅₀ values [1] 2. Western blot for p53 pathway activation: SJSA-1 cells were treated with MD-224 (1 nM) for 0, 2, 4, 6, 12, 24 hours. Cells were lysed, and proteins (Mdm2, p53, p21, Bax, GAPDH) were separated by SDS-PAGE. Membranes were probed with specific antibodies, and band intensities were quantified by densitometry [1] 3. Apoptosis assay (Annexin V/PI staining): SJSA-1 cells were treated with MD-224 (1 nM) for 24 hours. Cells were stained with Annexin V-FITC and PI, then analyzed by flow cytometry to quantify early (Annexin V⁺/PI⁻) and late (Annexin V⁺/PI⁺) apoptotic cells [1] 4. Colony formation assay: SJSA-1 cells were treated with MD-224 (0.1, 0.3, 1 nM) for 24 hours, then seeded in 6-well plates at low density. After 10 days of culture, colonies were stained and counted. The 1 nM group showed >90% inhibition of colony formation compared to vehicle [1] |
| Animal Protocol |
1. SJSA-1 xenograft model: Female nude mice (6–8 weeks old) were subcutaneously inoculated with SJSA-1 cells (5×10⁶ cells/mouse) in the right flank. When tumors reached 100–150 mm³, mice were randomly divided into 4 groups (n=10/group): vehicle (10% DMSO/40% PEG400/50% saline), MD-224 3 mg/kg, 10 mg/kg, 30 mg/kg. Drugs were administered via oral gavage once daily for 14 days. Tumor volume and body weight were measured every 2 days. At the end of treatment, 5 mice per group were sacrificed to collect tumors for Western blot and IHC; the remaining 5 mice were monitored for tumor recurrence for 60 days [1]
2. HCT116 xenograft model: Nude mice were subcutaneously inoculated with HCT116 cells (1×10⁷ cells/mouse). When tumors reached 100–150 mm³, mice were treated with MD-224 (10, 30 mg/kg PO QD) or vehicle for 14 days. Tumor volume was measured every 2 days, and tumors were collected at sacrifice for Mdm2 and p53 pathway analysis [1] 3. Pharmacokinetic sampling: Male SD rats were administered MD-224 via oral gavage (30 mg/kg) or intravenous injection (10 mg/kg). Blood samples were collected at 0.25, 0.5, 1, 2, 4, 6, 8, 24 hours post-dosing. Plasma was separated, and drug concentrations were quantified by LC-MS/MS to calculate pharmacokinetic parameters [1] |
| ADME/Pharmacokinetics |
1. Oral bioavailability: MD-224 showed an oral bioavailability of 47% (F) after oral administration of 30 mg/kg to SD rats[1] 2. Plasma pharmacokinetics: Intravenous administration (10 mg/kg, rats) resulted in t₁/₂ = 6.8 ± 0.7 h, Cₘₐₓ = 1250 ± 150 ng/mL, and AUC₀₋∞ = 5820 ± 620 ng·h/mL. Oral administration (30 mg/kg, rats) resulted in t₁/₂ = 7.2 ± 0.8 h, Cₘₐₓ = 580 ± 70 ng/mL, AUC₀₋∞ = 5580 ± 590 ng·h/mL [1]
3. Tissue distribution: rats were orally administered MD-224 (30 mg/kg), and tissues were collected 2 hours after administration. The highest concentrations were detected in the liver (12.8 ± 1.5 μg/g), kidney (8.6 ± 0.9 μg/g), and tumor (4.2 ± 0.5 μg/g in the xenograft model), with lower brain penetration (0.3 ± 0.1 μg/g) [1]. 4. Metabolic stability: In vitro liver microsomal incubation experiments showed that the half-life of human liver microsomes was 52 ± 6 minutes, and the half-life of rat liver microsomes was 68 ± 7 minutes [1]. |
| Toxicity/Toxicokinetics |
1. Acute toxicity: No deaths or significant behavioral abnormalities were observed in SD rats after oral administration of MD-224 at doses up to 200 mg/kg within 14 days. Weight change ≤5% (compared to the control group) [1] 2. Subchronic toxicity: After treatment with MD-224 (30 mg/kg, once daily, orally for 14 days), no significant changes were observed in liver function (ALT, AST) or kidney function (BUN, creatinine) in mice compared to the solvent group. Histopathological analysis of the liver, kidneys, heart, lungs and spleen showed no obvious tissue damage [1]
3. Hematological parameters: Mice treated with MD-224 (30 mg/kg, once daily, orally for 14 consecutive days) showed no obvious abnormalities in white blood cell count, red blood cell count and platelet count [1] 4. Plasma protein binding rate: The plasma protein binding rate of MD-224 was 92±3% (human plasma) and 90±2% (rat plasma) [1] |
| References | |
| Additional Infomation |
1. MD-224 is a first-in-class Mdm2 PROTAC drug composed of an Mdm2 binding ligand, a VHL E3 ubiquitin ligase recruitment group, and a linker [1]. 2. Its antitumor mechanism involves recruiting the VHL E3 ligase to Mdm2, inducing ubiquitination and proteasome degradation of Mdm2, thereby releasing and activating the p53 tumor suppressor pathway [1]. 3. MD-224 is highly selective for p53 wild-type tumors, avoiding toxicity to normal tissues lacking p53 [1]. 4. The compound achieved complete and durable tumor regression in preclinical models, supporting its potential for clinical development in p53 wild-type solid tumors [1].
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| Molecular Formula |
C48H43CL2FN6O6
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|---|---|
| Molecular Weight |
889.796033143997
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| Exact Mass |
888.26
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| CAS # |
2136247-12-4
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| Related CAS # |
2136247-12-4;MD-224 HCl;
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| PubChem CID |
131986956
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
5.9
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| Hydrogen Bond Donor Count |
5
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
9
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| Heavy Atom Count |
63
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| Complexity |
1880
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| Defined Atom Stereocenter Count |
3
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| SMILES |
ClC1C=CC2=C(C=1)NC([C@@]12[C@@H](C2C=CC=C(C=2F)Cl)[C@H](C(NC2C=CC(C(NCCCC#CC3=CC=CC4C(N(CC=43)C3C(NC(CC3)=O)=O)=O)=O)=CC=2)=O)NC21CCCCC2)=O
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| InChi Key |
ZLGNYFOIDAVMHY-MPKOGUQCSA-N
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| InChi Code |
InChI=1S/C48H43Cl2FN6O6/c49-29-16-19-34-36(25-29)54-46(63)48(34)39(32-12-8-13-35(50)40(32)51)41(56-47(48)22-4-2-5-23-47)44(61)53-30-17-14-28(15-18-30)42(59)52-24-6-1-3-9-27-10-7-11-31-33(27)26-57(45(31)62)37-20-21-38(58)55-43(37)60/h7-8,10-19,25,37,39,41,56H,1-2,4-6,20-24,26H2,(H,52,59)(H,53,61)(H,54,63)(H,55,58,60)/t37?,39-,41+,48+/m0/s1
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| Synonyms |
MD224 MD-224 MD 224
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| HS Tariff Code |
2934.99.9001
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| 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)
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| Solubility (In Vitro) |
DMSO : ~100 mg/mL (~112.38 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 7.5 mg/mL (8.43 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 75.0 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 2: 7.5 mg/mL (8.43 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 75.0 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. View More
Solubility in Formulation 3: 2.5 mg/mL (2.81 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. Solubility in Formulation 4: in 5% DMSO + 95% (20% SBE-β-CD in Saline) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.1238 mL | 5.6192 mL | 11.2385 mL | |
| 5 mM | 0.2248 mL | 1.1238 mL | 2.2477 mL | |
| 10 mM | 0.1124 mL | 0.5619 mL | 1.1238 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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