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]

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]

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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]

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]

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.

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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]

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.

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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]

Acute toxicity: No deaths were observed in nude mice treated with ZM 336372 (up to 40 mg/kg/day, intraperitoneal injection, for 28 days). Mild, reversible weight loss (<8%) was reported in mice at a dose of 40 mg/kg, with no significant changes in serum ALT/AST (liver marker) or creatinine (kidney marker) [2]
- Hematologic toxicity: Mild leukopenia (20% decrease in white blood cell count) was observed in mice treated with ZM 336372 + 5-FU (30 mg/kg + 10 mg/kg/day), which subsided 7 days after discontinuation of the drug [3]
- Organ pathology: No histopathological lesions were found in the liver, kidneys, or heart tissues of mice treated with ZM 336372 (40 mg/kg/day) [2]

[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.

3-(dimethylamino)-N-[3-[[(4-hydroxyphenyl)-oxymethyl]amino]-4-methylphenyl]benzamide belongs to the benzamide class of compounds. ZM 336372 is an investigational-grade selective CDK1/2 inhibitor used to study the role of CDKs in cell cycle regulation and cancer progression. It has not yet been approved for clinical use [1-6]. Mechanism of action: Its antitumor effect is achieved by inhibiting CDK1/2 activity, blocking the phosphorylation of CDK substrates (e.g., p-Rb), inducing G2/M phase cell cycle arrest, and inhibiting cancer cell proliferation. It can enhance the efficacy of chemotherapy by synchronizing cells to a chemosensitive cell cycle stage (e.g., in combination with 5-fluorouracil) [1,3]
- Research applications: It is widely used in preclinical studies of gastrointestinal cancers (gastric cancer, colorectal cancer, pancreatic cancer) to validate CDK inhibition as a therapeutic strategy and to explore combination therapy regimens with conventional chemotherapy drugs [2,3,6]

C23H23N3O3

389.45

389.173

C, 70.93; H, 5.95; N, 10.79; O, 12.32

208260-29-1

5730

White to off-white solid powder

1.3±0.1 g/cm3

484.4±45.0 °C at 760 mmHg

246.8±28.7 °C

0.0±1.3 mmHg at 25°C

1.704

3.98

3

4

5

29

560

0

O=C(NC1=CC=C(C)C(NC(C2=CC=C(O)C=C2)=O)=C1)C3=CC=CC(N(C)C)=C3

PYEFPDQFAZNXLI-UHFFFAOYSA-N

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)

3-(Dimethylamino)- N -[3-[(4-hydroxybenzoyl)-amino]-4-methylphenyl]benzamide

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)

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.

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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.

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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.

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 (Please use freshly prepared in vivo formulations for optimal results.)