AZD5438 (AZD-5438; AZD 5438) is a novel, potent and selective small molecule inhibitor of cyclin-dependent kinase (CDK) 1, 2 and 9 with potential antineoplastic activity. The inhibitory concentrations of 16 nmol/L, 6 nmol/L, and 20 nmol/L, respectively, are found to inhibit CDK1/2/9. In multiple human tumor cell lines, AZD5438 exhibited noteworthy anti-proliferative activity, with an IC50 ranging from 0.2 μmol/L to 1.7 μmol/L. This resulted in the inhibition of cell cycling at the G2-M, S, and G1 phases as well as the phosphorylation of select proteins, such as CDK substrates pRb, nucleolin, protein phosphatase 1a, and RNA polymerase II COOH-terminal domain.

Physicochemical Properties

Molecular Formula	C18H21N5O2S
Molecular Weight	371.4566
Exact Mass	371.141
Elemental Analysis	C, 58.20; H, 5.70; N, 18.85; O, 8.61; S, 8.63
CAS #	602306-29-6
Related CAS #	602306-29-6
PubChem CID	16747683
Appearance	White to light yellow solid powder
Density	1.3±0.1 g/cm3
Boiling Point	655.2±65.0 °C at 760 mmHg
Flash Point	350.1±34.3 °C
Vapour Pressure	0.0±2.0 mmHg at 25°C
Index of Refraction	1.648
LogP	2.13
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	5
Heavy Atom Count	26
Complexity	556
Defined Atom Stereocenter Count	0
SMILES	0
InChi Key	WJRRGYBTGDJBFX-UHFFFAOYSA-N
InChi Code	InChI=1S/C18H21N5O2S/c1-12(2)23-13(3)20-11-17(23)16-9-10-19-18(22-16)21-14-5-7-15(8-6-14)26(4,24)25/h5-12H,1-4H3,(H,19,21,22)
Chemical Name	4-(2-methyl-3-propan-2-ylimidazol-4-yl)-N-(4-methylsulfonylphenyl)pyrimidin-2-amine
Synonyms	AZD5438; AZD-5438; AZD 5438
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	cdk2-cyclin E (IC50 = 6 nM); cdk2-cyclin A (IC50 = 45 nM); cdk5-p25 (IC50 = 14 nM); cdk1-cyclin B1 (IC50 = 16 nM); cdk9-cyclin T (IC50 = 20 nM); cdk6-cyclin D3 (IC50 = 21 nM); cdk4-cyclin D1 (IC50 = 449 nM); cdk7-cyclin H (IC50 = 821 nM)
ln Vitro	AZD5438 demonstrates a strong inhibitory effect on the activity of cyclin-dependent kinases, with IC50 values of 6 nM, 45 nM, 16 nM, 21 nM, and 20 nM, respectively, for cyclin E-cdk2, cyclin A-cdk2, cyclin B1-cdk1, cyclin D3-cdk6, and cyclin T-cdk9. Furthermore, with IC50 values of 14 nM and 17 nM, respectively, AZD5438 suppresses the kinase activity of glycogen synthase kinase 3β and p25-cdk5. (Source: ) By preventing the phosphorylation of CDK-dependent substrates, AZD5438 causes cell cycle arrest. It also demonstrates broad antiproliferative activity against various tumor cell lines, such as lung, colorectal, breast, prostate, and hematologic tumors, with an IC50 ranging from 0.2 μM (MCF-7) to 1.7 μM (ARH-77).[1]
ln Vivo	When AZD5438 is taken orally in vivo, human tumor xenografts derived from a variety of cancer types, including breast, colon, lung, prostate, and ovarian, show statistically significant inhibition against growth, with a maximum tumor growth index (TGI) ranging from 38% to 153%. (Source: ) AZD5438 inhibits a number of cell cycle proteins in the SW620 xenograft model, including phH3, phosphonucleolin, PP1a, and multiple phospho-pRb epitopes. These effects are dose-dependent.[1]
Enzyme Assay	A scintillation proximity assay is used to test AZD5438's capacity to inhibit CDK activity. It involves using recombinant CDK-cyclin complexes of cyclin-Ecdk2, cdk2-cyclin A, cdk4-cyclin D, and recombinant retinoblastoma substrate (amino acids 792-928) or cdk1-cyclin B1 with a peptide substrate derived from the in vitro p34cdc2 phosphorylation site of histone H1 (biotin-X-Pro-Lys-Thr-Pro-Lys-Lys-Ala-Lys-Lys-Leu). Using peptide substrate (AKKPKTPKKAKKLOH), AZD5438's activity against recombinant cdk5/p25 is assessed in a scintillation proximity assay-based assay at 2 μM ATP. By using human purified glycogen synthase kinase 3βenzyme and eukaryotic initiation factor 2B substrate (at 1 μM ATP), the scintillation proximity assay is used to determine the inhibition of glycogen synthase kinase 3β activity. AZD5438 is subjected to a kinase selectivity screening service in which it is screened against cdk6-cyclin D3, cdk7-cyclin H/MAT1 (cdk activating kinase complex), and cdk9-cyclin T.
Cell Assay	Solid tumor cell lines are used to test AZD5438. In summary, AZD5438 is added to cells at different concentrations and incubated for 48 hours. After the incubation period, 5-bromo-2′-deoxyuridine (BrdUrd) is pulsed into the cells, and the quantity of DNA synthesis is quantified. Specifically, cell death has no bearing on the IC50 for proliferation inhibition. The following protocol is used to seed multiple myeloma cell lines into 96-well plates: RPMI 1640 supplemented with 10% FCS and glutamine, followed by a 72-hour dose of AZD5438. AlamarBlue is used to measure cell growth, and GI50 values are computed using the pretreatment control values.
Animal Protocol	Every human tumor xenograft, with the exception of HX147, is created by subcutaneously injecting 100 μL of tumor cells (a mixture of 1×106 and 1×107 cells mixed 1:1 with Matrigel). The origin of HX147 tumors is fragment implants (1 mm3 pieces) from tumors removed from mice that were first injected subcutaneously with 1×107 cells. Prior to being implanted for antitumor work, these tumor fragments undergo three passages through mice. As previously mentioned, tumor volumes are computed, tumor measurements are made up to three times a week using calipers, and the data are plotted as the geometric mean for each group versus time. Tumors that reach a mean size of roughly >0.2 cm3 in mice and >0.5 cm3 in rats are the triggers for randomization of animals into treatment groups (usually n=10). Hydroxy-propyl-methyl-cellulose is used to prepare AZD5438. Oral gavage of AZD5438 (37.5-75 mg/kg) or vehicle control is administered once or twice daily to animals for approximately three weeks in each scenario. As previously mentioned, the tumor volume and percentage tumor growth inhibition (% TGI) are computed. Any variation in tumor volume is statistically analyzed using the standard t test, with a significance level of P<0.05.
References	[1]. AZD5438, a potent oral inhibitor of cyclin-dependent kinases 1, 2, and 9, leads to pharmacodynamic changes and potent antitumor effects in human tumor xenografts. Mol Cancer Ther. 2009 Jul;8(7):1856-66. Epub 2009 Jun 9. [2]. AZD5438, an Inhibitor of Cdk1, 2, and 9, Enhances the Radiosensitivity of Non-Small Cell Lung Carcinoma Cells. Int J Radiat Oncol Biol Phys. 2012 Jul 12.
Additional Infomation	4-(2-methyl-3-propan-2-yl-4-imidazolyl)-N-(4-methylsulfonylphenyl)-2-pyrimidinamine is a sulfonamide.

Solubility Data

Solubility (In Vitro)

DMSO: ~74 mg/mL (~199.2 mM) Water: <1 mg/mL Ethanol: ~74 mg/mL (~199.2 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.73 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 (6.73 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 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 (6.73 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	2.6921 mL	13.4604 mL	26.9208 mL
	5 mM	0.5384 mL	2.6921 mL	5.3842 mL
	10 mM	0.2692 mL	1.3460 mL	2.6921 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.