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Milciclib (formerly also known as PHA-848125; PHA-848125AC; PHA848125) is a novel, potent, ATP-competitive small molecule CDK inhibitor with potential anticancer activity. That has an IC50 of 45 nM for CDK2 inhibition. Inhibiting CDK2 is >3 times more selective than CDK1, 2, 4, 5, and 7 when using milciclib. Furthermore, with an IC50 of 53 nM, it inhibits tropomyosin receptor kinase (TRK). The CDK substrate retinoblastoma protein (pRb) underwent a reduction in hyperphosphorylation and an accumulation of hypophosphorylation in cells treated with PHA-848125. It additionally demonstrated PHA-848125's inhibitory effect on CDK2 activity.
Physicochemical Properties
| Molecular Formula | C25H32N8O | |
| Molecular Weight | 460.57 | |
| Exact Mass | 460.269 | |
| Elemental Analysis | C, 65.19; H, 7.00; N, 24.33; O, 3.47 | |
| CAS # | 802539-81-7 | |
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| PubChem CID | 16718576 | |
| Appearance | Light yellow to yellow solid powder | |
| Density | 1.3±0.1 g/cm3 | |
| Index of Refraction | 1.692 | |
| LogP | 1.75 | |
| Hydrogen Bond Donor Count | 2 | |
| Hydrogen Bond Acceptor Count | 7 | |
| Rotatable Bond Count | 4 | |
| Heavy Atom Count | 34 | |
| Complexity | 718 | |
| Defined Atom Stereocenter Count | 0 | |
| SMILES | O=C(C1C2=C(C3C(CC2(C)C)=CN=C(NC2C=CC(N4CCN(C)CC4)=CC=2)N=3)N(C)N=1)NC |
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| InChi Key | RXZMYLDMFYNEIM-UHFFFAOYSA-N | |
| InChi Code | InChI=1S/C25H32N8O/c1-25(2)14-16-15-27-24(29-20(16)22-19(25)21(23(34)26-3)30-32(22)5)28-17-6-8-18(9-7-17)33-12-10-31(4)11-13-33/h6-9,15H,10-14H2,1-5H3,(H,26,34)(H,27,28,29) | |
| Chemical Name | N,1,4,4-tetramethyl-8-[4-(4-methylpiperazin-1-yl)anilino]-5H-pyrazolo[4,3-h]quinazoline-3-carboxamide | |
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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 | cyclin A/CDK2 (IC50 = 45 nM); cyclin E/CDK2 (IC50 = 363 nM); cyclin H/CDK7 (IC50 = 150 nM); cyclin D1/CDK4 (IC50 = 160 nM); cyclin B/CDK1 (IC50 = 398 nM); TRKA (IC50 = 53 nM) |
| ln Vitro | Milciclib (PHA-848125; 0.156 or 0.625 μM) increases the expression of PDCD4, DDIT4, SESN2/sestrin 2, and DEPDC6/DEPTOR in GL-Mel cells[1]. Milciclib (PHA-848125), with IC50s of 45 and 53 nM, respectively, potently inhibits the kinase activity of TRKA and the CDK2/cyclin A complex in a biochemical assay. In the G1 phase, milciclib clearly causes a cell accumulation. It has been observed that milciclib significantly and dose-dependently inhibits the phosphorylation of TRKA induced by NGF[2]. |
| ln Vivo | Milciclib (PHA-848125; 5, 10, and 15 mg/kg, p.o.) inhibits the growth of tumor in 7,12-dimethylbenz(a) anthracene (DMBA)-induced rat mammary carcinoma model. Milciclib exhibits strong antitumor activity in a variety of human xenografts, carcinogen-induced tumors, and disseminated primary leukemia models. Its plasma concentrations in rodents are comparable to those that have been shown to be effective in preventing the growth of cancer cells[2]. Milciclib (PHA-848125; 40 mg/kg) significantly inhibits the growth of tumors in K-RasG12DLA2 mice, and this is accompanied by a decrease in the turnover of cell membranes[3]. |
| Enzyme Assay | The assessment of PHA-848125's inhibition of kinase activity is conducted through a robotized format assay utilizing a strong anion exchanger (Dowex 1X8 resin) on 384-well plates. This assay involves the use of optimal buffers and cofactors to facilitate the transphosphorylation of particular peptides or protein substrates by their respective kinase in the presence of ATP traced with [γ-33P]ATP. PHA-848125's potency against CDKs and 38 other kinases from an internal Kinase Selectivity Screening panel is assessed, and the corresponding IC50s are found. After calculating the absolute KM values for ATP and the particular substrate for each enzyme, the assays are conducted at optimized concentrations of ATP (2KM) and substrate (5KM). This configuration facilitates the direct comparison of PHA-848125's IC50 values across the panel in order to assess its biochemical profile. |
| Cell Assay | Melanoma cells are cultured at a density of 2 × 104 cells/mL in media, then 50 μL aliquots are added to flat-bottom 96-well plates and left to adhere for an entire night at 37 °C. The wells (4 wells per point) in 50 μL of CM are then filled with graded amounts of PHA-848125 or TMZ, and the plates are incubated for 5 days at 37 °C in a humidified atmosphere with 5% CO2. Together with the MGMT inhibitor BG, the cytotoxic effects of TMZ are also assessed. To achieve this, 2 hours prior to TMZ, 10 μM BG is added to the plates, and they are left in culture for the duration of the cells' exposure to the medication. Cells treated with BG or DMSO alone, as well as untreated cells, represent the ContS1017rol groups. There is no discernible difference in the growth of cells treated with BG or DMSO alone compared to untreated cells. The inhibitor PHA-848125 is added to BG-treated cells two hours later, and the MGMT activity of these cells is virtually undetectable until the assay is finished. Normal melanocytes are plated (50 μL/well) and exposed to either PHA-848125 or TMZ + BG, as described for melanoma cells, after being suspended in MGM at a concentration of 1.6 × 105 cells/mL. Using the MTT assay, cell growth is assessed at the conclusion of the incubation period. In a nutshell, each well receives 0.1 mg of MTT (in 20 μL of PBS), and the cells are incubated for 4 hours at 37 °C. After that, cells are lysed using a buffer (0.1 mL/well) that has a pH 4.7 mixture of 50% N,N-dimethylformamide and 20% SDS. Using a 3550-UV microplate reader, the absorbance is measured at 595 nm following an overnight incubation. The drug concentration that produces a 50% inhibition of cell growth, or IC50, is a measure of how sensitive cells are to treatment. It is derived from plotting absorbance values at 595 nm against the logarithm of drug concentration to create a regression line. |
| Animal Protocol | Once a mammary tumor reaches a diameter of 0.5 cm, rats are randomly assigned to the study. Ten animal groups receive oral treatment twice daily for a duration of ten days, either with glucose as a vehicle or with Milciclib at doses of 5, 10, or 15 mg/kg. Another group of animals receives two cycles of Milciclib at a dose of 20 mg/kg twice daily for five days, separated by a one-week rest period. Throughout the experiment, the tumor volume is periodically measured with a caliper. |
| References |
[1]. Down-regulation of the PTTG1 proto-oncogene contributes to the melanoma suppressive effects of the cyclin-dependent kinase inhibitor PHA-848125. Biochem Pharmacol. 2012 Sep 1;84(5):598-611. [2]. Dual targeting of CDK and tropomyosin receptor kinase families by the oral inhibitor PHA-848125, an agent with broad-spectrum antitumor efficacy. Mol Cancer Ther. 2010 Aug;9(8):2243-54. [3]. Efficacy of PHA-848125, a cyclin-dependent kinase inhibitor, on the K-Ras(G12D)LA2 lung adenocarcinoma transgenic mouse model: evaluation by multimodality imaging. Mol Cancer Ther. 2010 Mar;9(3):673-81. [4]. Identification of N,1,4,4-tetramethyl-8-{[4-(4-methylpiperazin-1-yl)phenyl]amino}-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (PHA-848125), a potent, orally available cyclin dependent kinase inhibitor. J. Med. Chem. 52(16), 5152-5163 (2009). |
| Additional Infomation |
Milciclib is an orally bioavailable inhibitor of cyclin-dependent kinases (CDKs) and tropomyosin receptor kinase A (TRKA), with potential antineoplastic activity. CDK2/TRKA inhibitor PHA-848125 AC potently inhibits cyclin-dependent kinase 2 (CDK2) and exhibits activity against other CDKs including CDK1 and CDK4, in addition to TRKA. Inhibition of these kinases may result in cell cycle arrest and apoptosis of tumor cells that express these kinases. CDKs are serine/threonine kinases involved in regulation of the cell cycle and may be overexpressed in some cancer cell types. The neurotrophin receptor TRKA is mutated in a variety of cancer cell types. See also: Milciclib Maleate (annotation moved to). |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2 mg/mL (4.34 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 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 2: ≥ 2 mg/mL (4.34 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 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. Solubility in Formulation 3: 30% Propylene glycol , 5% Tween 80 , 65% D5W: 30mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1712 mL | 10.8561 mL | 21.7122 mL | |
| 5 mM | 0.4342 mL | 2.1712 mL | 4.3424 mL | |
| 10 mM | 0.2171 mL | 1.0856 mL | 2.1712 mL |