Aurora A (Ki = 8 nM); Aurora B (Ki = 9.2 nM)

Moreover, CYC-116 suppresses FLT3, Src, Lck, and VEGFR2 at 44, 82, 280, and 44 nM, respectively. Broad-spectrum anticancer action is possible for CYC-116. With IC50s of 0.599, 0.59, 0.241, 0.34, 0.725, 1.375, 0.471, 0.034, 0.372, 0.681, 0.151, 1.626, 0.775, 0.308, 0.110, and 0.09 for MCF7, HeLa, Colo 205, HCT-116, HT29, K562, CCRF -CEM, MV4-11, HL60, NCI-H460, A2780, BxPC3, HuPT4, Mia-Paca-2, Saos-2, and Messa cells, CYC-116 exhibits strong antiproliferative activity against cancer cell lines. Histone H3 phosphorylation in HeLa cell lysates is completely inhibited after 7 hours of treatment with 1.25 μM CYC-116[1].

Tumor growth delays of 2.3 and 5.8 days are caused by oral administration of CYC-116 at dose levels of 75 and 100 mg/kg qd, respectively. These tumor growth delays translate into specific growth delays of 0.32 and 0.81. For the duration of the trial, the mean relative tumor volumes of mice receiving CYC-116 at both dose levels are smaller than those of animals given with a vehicle. On days 6 and 9, the growth decrease is statistically significant at 100 mg/kg po qd[1].

Kinase Assays[1]
These were carried out as described previously. IC50 values were determined using XLfit software (IDBS). Apparent inhibition constants (Ki) were calculated from IC50 values and the appropriate Km (ATP) values for each kinase using the method of Cheng and Prussoff. Recombinant human aurora A and B kinases were purchased from Upstate Discovery. Aurora A kinase assays were performed using a 25 μL reaction volume (25 mM β-glycerophosphate, 20 mM Tris/HCl, pH 7.5, 5 mM EGTA, 1 mM DTT, 1 mM Na3VO4, 10 μg of kemptide (peptide substrate)), and recombinant aurora A kinase was diluted in 20 mM Tris/HCl, pH 8, containing 0.5 mg/mL BSA, 2.5% glycerol, and 0.006% Brij-35. Reactions were started by the addition of 5 μL Mg/ATP mix (15 mM MgCl2, 100 μM ATP, with 18.5 kBq γ-32P-ATP per well) and incubated at 30 °C for 30 min before terminating by the addition of 25 μL of 75 mM H3PO4. Aurora B kinase assays were performed as for aurora A except that prior to use, aurora B was activated in a separate reaction at 30 °C for 60 min with inner centromeres protein.

Cell Cycle Analysis by Flow Cytometry[1]
To synchronize cells in early S phase, they were subjected to double thymidine block. HeLa cells were seeded at 5 × 105 cells per 10 cm dish and incubated for 16−18 h at 37 °C. Thymidine (2 mM) was added, and the cells were incubated for 18 h. The cells were released from the block by washing 3 times in 5 mL of PBS. Fresh medium was added, and the cells were incubated for 8 h. Then 2 mM thymidine was added again for a second period of 16 h. The cells were released again and fresh medium was added, together with test compound dilutions as appropriate.
To synchronize A549 cells in the M phase, they were incubated with 40 ng/mL nocodazole (Sigma) for 18 h. Where stated, cells were also treated with 50 μM MG132 proteasome inhibitor for the final 2 h of the incubation. Rounded-up mitotic cells, detached from the plate by shaking off and repeated washing, were pelleted and washed in PBS to release from the nocodazole block. The cells were replated in fresh medium with test compound dilutions as appropriate. Cell cycle analysis was performed on cells in conjunction with either TUNEL staining or cyclin B analysis. As such, all cells were harvested, washed in PBS, fixed with 0.5% PFA, and stored at −20 °C in 80% ethanol. Terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay was performed following manufacturer’s instructions. Cyclin B analysis during flow cytometry used a 1:1000 dilution of cyclin B antibody in 400 μL of 0.5% BSA PBS, with 1:400 FITC goat polyclonal to mouse secondary followed by incubation with PI for DNA staining. The 20 000 single cell events per sample were analyzed using a BD FACSCalibur flow cytometer.

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Western Blot Analysis of Histone H3 Phosphorylation[1]
For detection of phosphorylated histone H3, HeLa cells were treated with compounds for 7 h. Then extracts were prepared by acid extraction. Briefly, the cells were scraped from the plates, pelleted, washed once in PBS, then resuspended in lysis buffer (10 mM Tris/HCl, pH 8.0, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT) containing Roche complete protease inhibitor cocktail. HCl and H2SO4 were added to a final concentration of 0.2 M, and the lysates were incubated on ice for 1 h. Insoluble material was pelleted by centrifugation, and the acid-solubilized supernatant was added to 1 mL of Me2CO and stored at −20 °C for 24 h. Precipitated protein was pelleted by centrifugation, air-dried briefly, and resuspended in SDS−PAGE loading buffer. The samples were separated on a 15% SDS−polyacrylamide gel and transferred to a nitrocellulose membrane by electroblotting. Phosphorylated histone H3 was detected on the membrane with rabbit anti-phosphohistone H3 antibody, and total histone H3 was detected with mouse anti-histone H3, followed by appropriate secondary antibodies and chemiluminescent detection.

Rat Pharmacokinetics. [1]
The PK parameters for test compounds were determined in male Wistar rats. For each compound, 3 rats were dosed either by intravenous bolus injection or by oral gavage. Dose volume was 10 mL/kg for oral gavage administration and 12 mL/kg for intravenous administration (1mL/min). Three serum samples were collected from each rat by jugular vein cannulation at 0, 5, 15, and 30 min, 1, 2, 4, and 6 h following i.v. dosing; and at 0, 0.5, 1, 2, 4, 6, 8, and 24 h after p.o. dosing. All blood samples were centrifuged immediately following collection. The plasma was harvested and stored at –20 °C until analysis. The samples were analyzed by LC-MS/MS methods. The PK parameters were derived by noncompartmental methods using WinNonlin 5.2 software program. The oral bioavailability (% F) was calculated by taking the ratio of dose-normalized AUC values from oral versus i.v. dosing.

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Murine P388/D1 Leukemia Model. [1]
Female Balb/c × DBA/2J F1 mice were implanted intraperitoneally with 2.1 × 105 P388/D1 leukemia cells on day 0. Starting on day 1 the animals were administered compound 18 by oral gavage at the indicated doses (0.1 mL / 10 g body weight) twice a day on days 1–3 and 7–9. The effectiveness of treatment was assessed by comparison of the median post-inoculation lifespan (ILS) of each group of treated mice with that of the vehicle control group. The ratio of ILS values for the treated versus the control groups was expressed as a percentage value (% ILS) and used as an indicator of relative efficacy.

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NCI-H460 Xenograft. [1]
Human NCI-H460 non-small cell lung tumor cells were harvested from sub-confluent cultures grown in vitro and the number of viable cells was determined. Cells were then resuspended in sterile PBS at a concentration of ca. 7 × 107 cells/mL. Nude (athymic) mice were injected subcutaneously in the right flank with approximately 7 × 106 cells. When measurable tumors had established (80-100 mm3), animals were assigned into the treatment and the control groups with 10 mice per group. Tumor size was measured at least twice weekly. Animals were terminated at any time during the study if the tumor size became excessive or any adverse effects were noted. The treatments were administrated orally, by gavage, daily, starting on day 1, and continuing for 5 days. In the control group, animals were treated with the vehicle orally, by gavage, once a day starting on day 1, and continuing for 5 days. The tumor dimensions measured over the period of the study were recorded. Calculations of relative tumor volumes and plots of mean tumor growth curves were S8 performed. The relative tumor volume data from each group were compared using a one-way analysis of variance (ANOVA) and statistical significance was determined using a Dunnett’s t-test.

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Dissolved in DMSO and then diluted in water; 75, 100 mg/kg; Oral gavage

NCI-H460 cells are implanted intraperitoneally into the mice

[1]. Wang S, et al. Discovery of N-phenyl-4-(thiazol-5-yl)pyrimidin-2-amine aurora kinase inhibitors. J Med Chem. 2010 Jun 10;53(11):4367-78

CYC116 is a novel anticancer compound with a unique target profile involving both cell cycle and angiogenesis inhibition mechanisms. In preclinical studies, CYC116 has demonstrated antitumor activity in both solid tumors and hematological cancers. Cyclacel's small molecule investigational drug, CYC116, is the third orally-available Cyclacel drug to enter development, which demonstrated anticancer activity with a mechanism consistent with inhibition of Aurora kinase.

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Aurora kinases are enzymes that help dividing cells share their materials between two daughter cells. In many people with cancer Aurora kinase malfunctions and normal control of cell division is lost resulting in abnormal growth. CYC116 inhibits Aurora kinase may slow down the growth of cancer cells and lead to their death by apoptosis.CYC116, an orally-available inhibitor of Aurora kinases A and B, and VEGFR2, in patients with advanced solid tumors.

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Through cell-based screening of our kinase-directed compound collection, we discovered that a subset of N-phenyl-4-(thiazol-5-yl)pyrimidin-2-amines were potent cytotoxic agents against cancer cell lines, suppressed mitotic histone H3 phosphorylation, and caused aberrant mitotic phenotypes. It was subsequently established that these compounds were in fact potent inhibitors of aurora A and B kinases. It was shown that potency and selectivity of aurora kinase inhibition correlated with the presence of a substituent at the aniline para-position in these compounds. The anticancer effects of lead compound 4-methyl-5-(2-(4-morpholinophenylamino)pyrimidin-4-yl)thiazol-2-amine (18; K(i) values of 8.0 and 9.2 nM for aurora A and B, respectively) were shown to emanate from cell death following mitotic failure and increased polyploidy as a consequence of cellular inhibition of aurora A and B kinases. Preliminary in vivo assessment showed that compound 18 was orally bioavailable and possessed anticancer activity. Compound 18 (CYC116) is currently undergoing phase I clinical evaluation in cancer patients.

C18H20N6OS

368.46

368.14193

C, 58.68; H, 5.47; N, 22.81; O, 4.34; S, 8.70

693228-63-6

6420138

Typically exists as light yellow to yellow solids at room temperature

1.4±0.1 g/cm3

648.8±65.0 °C at 760 mmHg

346.2±34.3 °C

0.0±1.9 mmHg at 25°C

1.689

1.4

118.16

NC1=NC(C)=C(C2=NC(NC3=CC=C(N4CCOCC4)C=C3)=NC=C2)S1

GPSZYOIFQZPWEJ-UHFFFAOYSA-N

InChI=1S/C18H20N6OS/c1-12-16(26-17(19)21-12)15-6-7-20-18(23-15)22-13-2-4-14(5-3-13)24-8-10-25-11-9-24/h2-7H,8-11H2,1H3,(H2,19,21)(H,20,22,23)

4-methyl-5-(2-(4-morpholinophenylamino)pyrimidin-4-yl)thiazol-2-amine

CYC 116; CYC-116; CYC116.

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: 1.5 mg/mL (4.07 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 15.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: ≥ 1.5 mg/mL (4.07 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 15.0 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: 1% DMSO+30% polyethylene glycol+1% Tween 80:30mg/mL

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