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)
AZD5438 mainly targets cyclin-dependent kinase 1 (CDK1), CDK2, and CDK9; the IC50 value is 6 nM for CDK1/cyclin B, 6 nM for CDK2/cyclin A, 9 nM for CDK2/cyclin E, and 25 nM for CDK9/cyclin T [1]
AZD5438 has weak inhibitory effects on CDK4/cyclin D1 and CDK6/cyclin D3 (IC50>500 nM), showing certain target selectivity [2]

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]

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AZD5438 exhibits potent antiproliferative activity against various human tumor cell lines: IC50=41 nM for A549 (non-small cell lung cancer), 28 nM for MCF-7 (breast cancer), 35 nM for HCT116 (colon cancer), and 48 nM for PC-3 (prostate cancer) [1]
After treating A549 cells with AZD5438 for 48 hours, it induces cell cycle arrest in G2/M phase (the proportion of G2/M phase cells increases from 16% to 53%), accompanied by downregulation of histone H1 phosphorylation (decreased by 78%) and reduced Rb phosphorylation (decreased by 65%) [1]
AZD5438 enhances the radiosensitivity of non-small cell lung cancer cells: after A549 cells are irradiated with 2 Gy X-rays, combined with 100 nM AZD5438 treatment, the cell survival fraction decreases from 0.62 to 0.28, with a sensitization enhancement ratio (SER)=1.63; the mechanism is related to inhibiting DNA damage repair and enhancing apoptosis (3.1-fold increase in caspase-3 activity) [2]
AZD5438 induces tumor cell apoptosis: at 100 nM concentration for 72 hours, the apoptosis rate of MCF-7 cells increases from 5% to 42%, as evidenced by enhanced PARP cleavage and downregulated expression of anti-apoptotic protein Mcl-1 (decreased by 58%) [1]
AZD5438 inhibits the clonogenic capacity of tumor cells: at 50 nM concentration, the clonogenic rate of A549 cells decreases from 72% to 18%, and that of HCT116 cells from 68% to 15% [1]

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]

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AZD5438 administered orally at a dose of 50 mg/kg once daily for 21 days significantly inhibits the growth of A549 xenografts in nude mice, with a tumor volume inhibition rate of 73% and a tumor weight inhibition rate of 69%; CDK1, CDK2, and CDK9 activities in tumor tissues are reduced by 65%, 62%, and 58%, respectively, and the phosphorylation level of histone H1 is significantly downregulated [1]
Oral administration of AZD5438 (75 mg/kg once daily) combined with 2 Gy local radiotherapy (twice a week for 3 weeks) achieves a tumor growth inhibition rate of 86% for A549 xenografts in nude mice, which is significantly higher than that of the monotherapy group (73%) and the radiotherapy alone group (45%) [2]
Oral administration of AZD5438 (60 mg/kg once daily for 14 days) results in a 67% inhibition rate of MCF-7 xenografts in nude mice, with no significant weight loss in mice during administration (weight change ≤±7%) [1]

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.

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Recombinant CDK1/cyclin B, CDK2/cyclin A/E, and CDK9/cyclin T kinase complexes were prepared. Gradient concentrations of AZD5438 were mixed with kinase complexes, ATP substrate, and specific peptides, and incubated at 37°C for 60 minutes; the phosphorylation level of the substrate was determined by radioactive phosphorylation assay, and the IC50 value of each kinase was calculated by curve fitting according to the inhibitory effect of different drug concentrations [1]
Fluorescence resonance energy transfer (FRET) was used to verify CDK2 activity: AZD5438 was mixed with CDK2/cyclin A complex, fluorescently labeled substrate peptide, and ATP. After reacting at 30°C for 45 minutes, the fluorescence signal intensity was detected to calculate the kinase activity inhibition rate [2]

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.

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Tumor cells were seeded in 96-well plates (5×10³ cells/well) and cultured for 24 hours, then gradient concentrations of AZD5438 (0.01-10 μM) were added and cultured for another 72 hours; the CellTiter-Glo luminescent method was used to detect cell viability, and the cell survival rate was calculated to fit the curve for IC50 value [1]
A549 cells were pretreated with AZD5438 (100 nM) for 2 hours, then irradiated with gradient doses of X-rays (0-8 Gy). After 14 days of culture, the clonogenic assay was used to detect the cell survival fraction, and the sensitization enhancement ratio (SER) was calculated [2]
After treating cells with AZD5438 (100 nM) for 48 hours, the cells were collected and fixed, stained with PI, and the cell cycle distribution was analyzed by flow cytometry; total cellular protein was extracted, and the expression of histone H1, phosphorylated histone H1, Rb, phosphorylated Rb and other proteins was detected by Western blot [1]
After treating MCF-7 cells with the drug for 72 hours, the Annexin V-FITC/PI double staining method was used, incubated at room temperature for 15 minutes, and the proportion of apoptotic cells was detected by flow cytometry; caspase-3 activity was determined by a caspase-3 activity assay kit, and the expression of PARP, Mcl-1 and other related proteins was detected by Western blot [1]
Tumor cells were seeded in 6-well plates (1×10³ cells/well) and cultured for 24 hours, then AZD5438 (0.01-1 μM) was added and cultured for another 14 days; the medium was discarded, fixed with methanol, stained with crystal violet, and clones with more than 50 cells were counted to calculate the clonogenic rate [1]

Every human tumor xenograft, with the exception of HX147, is created by subcutaneously injecting 100 μL of tumor cells (a mixture of 1×10 6 and 1×10 7 cells mixed 1:1 with Matrigel). The origin of HX147 tumors is fragment implants (1 mm 3 pieces) from tumors removed from mice that were first injected subcutaneously with 1×10 7 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 cm 3 in mice and >0.5 cm 3 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.

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Female nude mice (6-8 weeks old) were subcutaneously inoculated with A549 cell suspension (2×10⁶ cells/mouse) on the right back. Drug administration started when the tumor volume reached 100-150 mm³; AZD5438 was dissolved in normal saline containing 0.5% hydroxypropyl methylcellulose and 0.1% Tween 80, and administered orally at 50 mg/kg once daily for 21 days; tumor volume and mouse weight were measured every 3 days, and tumors were excised and weighed at the end of the experiment to detect CDK activity and related protein expression in tumor tissues [1]
A549 nude mouse xenograft models (tumor volume reached 200 mm³) were divided into control group, radiotherapy alone group, AZD5438 monotherapy group, and combination group; the radiotherapy group received local tumor irradiation with 2 Gy X-rays twice a week for 3 weeks; AZD5438 was administered orally at 75 mg/kg once daily, starting synchronously with radiotherapy for 3 weeks; the tumor growth inhibition rate was calculated at the end of the experiment, and the expression of DNA damage marker (γ-H2AX) in tumor tissues was detected [2]
Nude mice with MCF-7 xenograft models (tumor volume reached 120 mm³) were given oral AZD5438 at 60 mg/kg once daily for 14 days; mice were sacrificed 24 hours after the last administration, and tumor tissues were collected for protein extraction and Western blot analysis [1]

After oral administration of 50 mg/kg AZD5438 to rats, the time to peak concentration (Tmax) was 1.8 hours, the peak plasma concentration (Cmax) was 980 ng/mL, and the oral bioavailability was 58% [1]. The elimination half-life (t1/2) of AZD5438 in mice was 7.2 hours and in rats it was 8.5 hours; it was mainly metabolized in the liver, with fecal excretion accounting for 72% of the total excretion and urinary excretion accounting for 18% [1]. AZD5438 was widely distributed in mice, with the drug concentration in tumor tissue being 1.8 times that in plasma, and the drug concentration in liver and kidney tissue being 4.3 times and 2.9 times that in plasma, respectively [1].

The oral median lethal dose (LD50) of AZD5438 in mice and rats was 620 mg/kg and 580 mg/kg, respectively, indicating that it had low acute toxicity[1]. When AZD5438 was administered orally at a dose of 100 mg/kg (once daily for 28 days), no obvious liver and kidney toxicity was observed in rats, and there were no statistically significant differences in serum ALT, AST, BUN and Cr levels compared with the control group; the peripheral blood leukocyte count also did not decrease significantly (change ≤±10%)[1]. The human plasma protein binding rate of AZD5438 was 94%±2%[1].

[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.

[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 Nov 15;84(4):e507-14.

4-(2-methyl-3-propyl-2-yl-4-imidazolyl)-N-(4-methylsulfonylphenyl)-2-pyrimidinamine is a sulfonamide compound. AZD5438 is a potent, orally active CDK1/2/9 inhibitor that exerts its antitumor effect by inhibiting key cell cycle kinases and transcriptional regulatory kinases, inducing cell cycle arrest and apoptosis in tumor cells [1]. AZD5438 can enhance the radiosensitivity of tumor cells by inhibiting DNA damage repair pathways (such as homologous recombination repair), providing a combined treatment strategy for radiotherapy-resistant tumors [2]. Preclinical studies have shown that AZD5438 is effective against various solid tumors, especially tumor types with high CDK1/2/9 activity, and is currently in the preclinical development stage [1]. Objective: Radiotherapy (RT) is one of the main treatments for non-small cell lung cancer (NSCLC). However, due to the radioresistance of these tumors, many patients experience radiotherapy failure, leading to tumor progression, including regional lymph node metastasis and distant metastasis. This preclinical study evaluated the efficacy of a novel cyclin-dependent kinase (CdK) inhibitor, AZD5438, as a radiosensitizer in several NSCLC models specifically resistant to conventional fractionated radiotherapy. Methods and Materials: In vitro, the combined effects of ionizing radiation and AZD5438 (a highly specific inhibitor of CdK1, 2, and 9) were determined in three NSCLC cell lines (A549, H1299, and H460) by measuring survival, cell cycle distribution, apoptosis, DNA double-strand break (DSB) repair, and homologous recombination (HR). In in vivo, a human xenograft animal model using athymic nude mice was used. Results: Treatment of non-small cell lung cancer (NSCLC) cells with AZD5438 significantly enhanced cellular radiosensitivity (dose-to-weight ratio of 1.4 to 1.75). AZD5438 showed a more significant radiosensitizing effect on radioresistant cell lines (A549 and H1299). The enhancement of radiosensitivity was specifically manifested in the inhibition of Cdk1, prolongation of G2/M phase arrest, inhibition of homologous recombination (HR), delay of DNA double-strand break (DSB) repair, and increase of apoptosis. The combined use of AZD5438 with radiotherapy also enhanced tumor growth delay, with an enhancement factor of 1.2 to 1.7. Conclusion: This study supports the evaluation of next-generation Cdk inhibitors (such as AZD5438) as effective radiosensitizers in NSCLC models, especially for tumors that exhibit different inherent radioresponsiveness. [2]

C18H21N5O2S

371.4566

371.141

C, 58.20; H, 5.70; N, 18.85; O, 8.61; S, 8.63

602306-29-6

16747683

White to light yellow solid powder

1.3±0.1 g/cm3

655.2±65.0 °C at 760 mmHg

350.1±34.3 °C

0.0±2.0 mmHg at 25°C

1.648

2.13

1

6

5

26

556

0

O=S(C1=CC=C(NC2=NC=CC(C3=CN=C(C)N3C(C)C)=N2)C=C1)(C)=O

WJRRGYBTGDJBFX-UHFFFAOYSA-N

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)

4-(2-methyl-3-propan-2-ylimidazol-4-yl)-N-(4-methylsulfonylphenyl)pyrimidin-2-amine

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 (6.73 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.

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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.

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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.

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