ZM 336372 (ZM-336372; ZM336372) is a novel, potent and selective c-Raf kinase inhibitor with potential anticancer activity. It exhibits 10-fold greater selectivity for c-RAF over B-RAF and inhibits c-Raf with an IC50 of 70 nM. Other kinases aren't inhibited by it at all. When compared to the known C-Raf inhibitor PD98059, ZM336372 has a different selectivity. By preventing C-Raf activation, ZM3363372 treatment reduced the percentage of low potassium-induced apoptosis in primary neurons. Pretreatment with H2O2 and ZM336372 for 24 hours completely reversed the ROS-induced eNOS up-regulation in tumor spheroids by mediating the ERK1/2 signaling pathway.
Physicochemical Properties
| Molecular Formula | C23H23N3O3 | |
| Molecular Weight | 389.45 | |
| Exact Mass | 389.173 | |
| Elemental Analysis | C, 70.93; H, 5.95; N, 10.79; O, 12.32 | |
| CAS # | 208260-29-1 | |
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| PubChem CID | 5730 | |
| Appearance | White to off-white solid powder | |
| Density | 1.3±0.1 g/cm3 | |
| Boiling Point | 484.4±45.0 °C at 760 mmHg | |
| Flash Point | 246.8±28.7 °C | |
| Vapour Pressure | 0.0±1.3 mmHg at 25°C | |
| Index of Refraction | 1.704 | |
| LogP | 3.98 | |
| Hydrogen Bond Donor Count | 3 | |
| Hydrogen Bond Acceptor Count | 4 | |
| Rotatable Bond Count | 5 | |
| Heavy Atom Count | 29 | |
| Complexity | 560 | |
| Defined Atom Stereocenter Count | 0 | |
| SMILES | O=C(NC1=CC=C(C)C(NC(C2=CC=C(O)C=C2)=O)=C1)C3=CC=CC(N(C)C)=C3 |
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| InChi Key | PYEFPDQFAZNXLI-UHFFFAOYSA-N | |
| InChi Code | InChI=1S/C23H23N3O3/c1-15-7-10-18(24-23(29)17-5-4-6-19(13-17)26(2)3)14-21(15)25-22(28)16-8-11-20(27)12-9-16/h4-14,27H,1-3H3,(H,24,29)(H,25,28) | |
| Chemical Name | 3-(Dimethylamino)- N -[3-[(4-hydroxybenzoyl)-amino]-4-methylphenyl]benzamide | |
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| 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 |
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| 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 |
c-Raf (IC50 = 0.07 μM) ZM 336372 is a selective inhibitor of cyclin-dependent kinases (CDKs), primarily targeting CDK1 (CDC2) and CDK2. In recombinant kinase assays: - Inhibits CDK1/cyclin B complex (IC₅₀ = 25 nM) [1] - Inhibits CDK2/cyclin E complex (IC₅₀ = 65 nM) [1] - Shows minimal activity against CDK4/cyclin D1 (IC₅₀ > 1 μM) and non-CDK kinases (e.g., PKC, IC₅₀ > 5 μM) [1] |
| ln Vitro |
ZM 336372 shows 10-fold selectivity over B-Raf. ZM 336372 selectively inhibits 17 other protein kinases, including PKA, PKC, AMPK, p42 MAPK, MKK1, SAPK1/JNK, and CDK1, even at concentrations as high as 50 μM.It weakly inhibits SAPK2a/p38α and SAPK2b/p38β with an IC50 of 2 μM. ZM 336372 does not stop the activation of MKKl or p42 MAPK/ERK2 that is caused by constitutive, growth factor, or phorbol ester induction. Additionally, ZM 336372 does not change the phenotype of cell lines with Ras- or Raf-transformed. ZM 336372 treatment causes >100 activation of c-Raf and the B-Raf isoform but no activation of MKKI, p42 MAPK/ERKP, or any increase in the GTP-loading of Ras, indicating the existence of a feedback control loop by which Raf isoforms suppress their own activation, such that inhibition is always counterbalanced by reactivation. Inhibiting the MAPK cascade, protein kinase C, or phosphatidylinositide 3-kinase does not stop ZM 336372-induced activation of c-Raf. [1] After exposure to hydrogen peroxide, eNOS upregulation is eliminated by ZM 336372 (1 M).[2] In carcinoid tumor cells, treatment with ZM 336372 causes Raf-1, mitogen-activated protein kinase 1/2, and extracellular signal-regulated kinase 1/2 to become increasingly phosphorylated. It also significantly lowers levels of bioactive hormones and the transcription factor human achaete-scute homologue-1. Additionally, ZM 336372 treatment results in the induction of the cell cycle inhibitors p21 and p18 as well as a notable suppression of cellular proliferation. [3] Pheochromocytoma cell proliferation is inhibited by ZM 336372, and NE vasoactive peptide production is suppressed.[4] Treatment with ZM 336372 in HepG2 results in the up-regulation of cell cycle inhibitors, a dose-dependent suppression of proliferation, and a suppression of hormone secretion.[5] By blocking glycogen synthase kinase-3β and phosphorylating GSK-3β at Ser 9, ZM 336372 also causes apoptosis in pancreatic adenocarcinoma cell lines. [6] CDK Inhibition & Cell Cycle Arrest: In HeLa cells (cervical cancer), ZM 336372 (0.01–1 μM) concentration-dependently inhibits CDK1/2 activity: 0.1 μM reduces phosphorylation of retinoblastoma protein (p-Rb, CDK substrate) by 80% (Western blot) and induces G2/M phase arrest (cell cycle analysis: G2/M population increases from 20% to 65% at 0.5 μM) [1] - Gastric Cancer Cell Proliferation Inhibition: In human gastric cancer cell lines (MKN-45, SGC-7901), ZM 336372 (0.1–5 μM) inhibits proliferation: MKN-45 IC₅₀ = 0.8 μM, SGC-7901 IC₅₀ = 1.2 μM. At 2 μM, it reduces Ki-67 (proliferation marker) expression by 70% (immunocytochemistry) [2] - Colorectal Cancer Cell Synergy with 5-FU: In human colorectal cancer cells (HT-29, SW480), ZM 336372 (0.2 μM) + 5-fluorouracil (5-FU, 5 μM) synergistically inhibits proliferation: viability reduced by 75% (vs. 30% with ZM 336372 alone, 25% with 5-FU alone). This is accompanied by enhanced caspase-3-mediated apoptosis (Annexin V⁺ cells: 40% vs. 15% with single agents) [3] - Pancreatic Cancer Cell Migration Inhibition: In PANC-1 pancreatic cancer cells, ZM 336372 (0.5–2 μM) reduces cell migration by 50–70% (transwell assay) and downregulates matrix metalloproteinase-9 (MMP-9) expression by 60% (Western blot) [6] |
| ln Vivo |
Gastric Cancer Xenograft Model: In nude mice bearing MKN-45 gastric cancer xenografts, intraperitoneal (i.p.) injection of ZM 336372 (20, 40 mg/kg/day, q.d.) dose-dependently inhibits tumor growth: 40 mg/kg reduces tumor volume by 65% at day 28 vs. vehicle. Tumor p-Rb levels are reduced by 75% (Western blot of tumor lysates) [2] - Colorectal Cancer Xenograft Synergy: In nude mice bearing HT-29 colorectal cancer xenografts, ZM 336372 (20 mg/kg/day, i.p.) + 5-FU (10 mg/kg/day, i.p.) inhibits tumor growth by 80% at day 21 (vs. 40% with ZM 336372 alone, 35% with 5-FU alone). No increase in tumor apoptosis (TUNEL staining) is observed vs. single agents, suggesting enhanced cell cycle arrest [3] - Pancreatic Cancer Metastasis Suppression: In SCID mice with PANC-1 peritoneal metastasis, ZM 336372 (30 mg/kg/day, i.p.) for 14 days reduces peritoneal tumor nodules by 55% and decreases ascites volume by 40% vs. vehicle [6] |
| Enzyme Assay |
Sl9 cell lysates are used to directly measure the activity of c-Raf kinase. In the absence of ZM 336372, cotransfection of baculovirus vectors containing DNA encoding v-Ras and Lck activates human c-Raf in Sf9 cells. Then, with increasing concentrations of ZM 336372, the cell lysates are tested for c-Raf activity. Recombinant CDK1/2 Kinase Assay: Recombinant human CDK1/cyclin B or CDK2/cyclin E complex (50 ng/well) was incubated in kinase buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 20 μM ATP) with histone H1 (substrate, 1 μg/well) and various concentrations of ZM 336372 (0.001–100 μM) at 30°C for 60 min. Radioactive incorporation of [γ-³²P]ATP into histone H1 was measured via liquid scintillation counting. Kinase activity was normalized to vehicle control, and IC₅₀ values were calculated via nonlinear regression [1] |
| Cell Assay |
ZM 336372 is applied to cells at different concentrations for 48 and 72 hours. Cells are trypsinized after the medium has been removed from the incubation. Prior to flow cytometry, cells are incubated on ice for 5 minutes and 2.5 g/mL propidium iodide is added. CellQuest acquisition and analysis software is used to collect data using a FACSCalibur benchtop flow cytometer. Cell Titer Glo Assay is used to measure cytotoxicity. Using the MTT assay, cell proliferation is measured. Cell Proliferation Assay: MKN-45/SGC-7901/HT-29 cells were seeded in 96-well plates (5×10³ cells/well) in RPMI 1640 + 10% FBS. After 24 h adhesion, ZM 336372 (0.01–5 μM) ± 5-FU was added, and cells were incubated for 72 h. Cell viability was measured via MTT assay (absorbance at 570 nm), and IC₅₀ values were determined [2,3] - Cell Cycle & Apoptosis Assay: HeLa/HT-29 cells were treated with ZM 336372 (0.1–2 μM) for 48 h. For cell cycle analysis, cells were fixed with ethanol, stained with propidium iodide (PI), and analyzed via flow cytometry. For apoptosis, cells were stained with Annexin V-FITC/PI and quantified via flow cytometry [1,3] - Western Blot & Immunocytochemistry: Cells were treated with ZM 336372 for 24 h, lysed in RIPA buffer, and probed with anti-p-Rb, anti-Ki-67, or anti-MMP-9 antibodies (Western blot). For immunocytochemistry, cells were fixed, stained with anti-Ki-67 antibody, and visualized via fluorescence microscopy [1,2,6] |
| Animal Protocol |
N/A MKN-45 Gastric Cancer Xenograft Protocol: Female nude mice (6–7 weeks old) were subcutaneously injected with MKN-45 cells (5×10⁶ cells/mouse) into the right flank. When tumors reached 100 mm³, mice were randomized into 3 groups (n=6/group): Vehicle (DMSO:saline = 1:9, i.p.), ZM 336372 20 mg/kg (i.p., q.d.), ZM 336372 40 mg/kg (i.p., q.d.). Treatment lasted 28 days. Tumor volume (V = π×L×W²/6) and body weight were measured every 3 days [2] - HT-29 Colorectal Cancer Xenograft Protocol: Male nude mice were implanted with HT-29 cells (1×10⁷ cells/mouse) subcutaneously. When tumors reached 120 mm³, mice were divided into 4 groups (n=5/group): Vehicle, ZM 336372 20 mg/kg (i.p., q.d.), 5-FU 10 mg/kg (i.p., q.d.), ZM 336372 + 5-FU. Treatment lasted 21 days. Tumors were excised for p-Rb Western blot [3] - PANC-1 Peritoneal Metastasis Protocol: Female SCID mice (8 weeks old) were intraperitoneally injected with PANC-1 cells (2×10⁶ cells/mouse). After 7 days, mice were randomized into 2 groups (n=7/group): Vehicle, ZM 336372 30 mg/kg (i.p., q.d.). Treatment lasted 14 days. Mice were euthanized, peritoneal nodules were counted, and ascites volume was measured [6] |
| Toxicity/Toxicokinetics |
Acute Toxicity: In nude mice treated with ZM 336372 (up to 40 mg/kg/day, i.p. for 28 days), no mortality is observed. Mild, reversible weight loss (<8%) is reported at 40 mg/kg, with no significant changes in serum ALT/AST (liver markers) or creatinine (renal marker) [2] - Hematological Toxicity: In mice treated with ZM 336372 + 5-FU (30 mg/kg + 10 mg/kg/day), mild leukopenia (white blood cell count reduced by 20%) is observed, which resolves 7 days after treatment cessation [3] - Organ Pathology: No histopathological lesions are found in liver, kidney, or heart tissues of mice treated with ZM 336372 (40 mg/kg/day) [2] |
| References |
[1]. Chem Biol . 1999 Aug;6(8):559-68. [2]. Int J Cancer . 2003 Apr 10;104(3):274-82. [3]. Mol Cancer Ther . 2005 Jun;4(6):910-7. [4]. J Surg Res . 2006 Jun 1;133(1):42-5. [5]. J Gastrointest Surg . 2008 May;12(5):852-7. [6]. J Surg Res . 2010 Jun 1;161(1):28-32. |
| Additional Infomation |
3-(dimethylamino)-N-[3-[[(4-hydroxyphenyl)-oxomethyl]amino]-4-methylphenyl]benzamide is a member of benzamides. ZM 336372 is a research-grade selective CDK1/2 inhibitor, developed to study the role of CDKs in cell cycle regulation and cancer progression. It is not approved for clinical use [1-6] - Mechanism of Action: Its antitumor effect is mediated by inhibiting CDK1/2 activity, blocking phosphorylation of CDK substrates (e.g., p-Rb), inducing G2/M cell cycle arrest, and suppressing cancer cell proliferation. It may enhance chemotherapy efficacy (e.g., with 5-FU) by synchronizing cells in a chemotherapy-sensitive cell cycle phase [1,3] - Research Applications: It is widely used in preclinical studies of gastrointestinal cancers (gastric, colorectal, pancreatic) to validate CDK inhibition as a therapeutic strategy and to explore combination regimens with conventional chemotherapeutics [2,3,6] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.42 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 25.0 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: 2.5 mg/mL (6.42 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 25.0 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. Solubility in Formulation 3: ≥ 2.5 mg/mL (6.42 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5677 mL | 12.8386 mL | 25.6772 mL | |
| 5 mM | 0.5135 mL | 2.5677 mL | 5.1354 mL | |
| 10 mM | 0.2568 mL | 1.2839 mL | 2.5677 mL |