Aurora A (Ki = 8 nM); Aurora B (Ki = 9.2 nM)
Dual inhibitor of Aurora A kinase and Aurora B kinase with potent activity. For recombinant Aurora A: IC₅₀ = 1.2 nM (kinase activity assay); for recombinant Aurora B: IC₅₀ = 3.5 nM. It showed high selectivity over other kinases, with IC₅₀ > 1000 nM for CDK1/cyclin B, EGFR, and VEGFR2, confirming >800-fold selectivity for Aurora kinases over non-Aurora kinases [1]
- In HCT116 colorectal cancer cells, inhibition of Aurora A-mediated TPX2 phosphorylation showed an EC₅₀ = 4.8 nM, and inhibition of Aurora B-mediated histone H3 (Ser10) phosphorylation showed an EC₅₀ = 6.2 nM [1]

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].

---

Antiproliferative activity against human cancer cell lines: CYC116 exhibited broad and potent antiproliferative effects across multiple solid tumor cell lines, with IC₅₀ values ranging from 15 nM to 30 nM. Specific examples include: - HCT116 (colorectal cancer): IC₅₀ = 18 nM - MCF-7 (breast cancer): IC₅₀ = 25 nM - SK-OV-3 (ovarian cancer): IC₅₀ = 15 nM - A549 (lung cancer): IC₅₀ = 30 nM [1]
- Induction of G2/M cell cycle arrest: Treatment of HCT116 cells with CYC116 (20 nM) for 24 hours resulted in a significant increase in G2/M phase accumulation—from 14% (vehicle control) to 58% (treated group)—as detected by propidium iodide (PI) staining and flow cytometry. This arrest was associated with abnormal mitotic spindle formation (observed in 65% of treated cells via α-tubulin immunofluorescence) [1]
- Inhibition of Aurora substrate phosphorylation: Western blot analysis of HCT116 cells treated with CYC116 (5–50 nM) for 6 hours showed dose-dependent reductions in: - Aurora A-mediated TPX2 phosphorylation (80% reduction at 20 nM vs. control) - Aurora B-mediated histone H3 (Ser10) phosphorylation (70% reduction at 30 nM vs. control) No significant changes in total TPX2 or total histone H3 levels were observed [1]
- Induction of cancer cell apoptosis: MCF-7 cells treated with CYC116 (30 nM) for 48 hours showed a 38% increase in annexin V-positive apoptotic cells (early + late apoptosis) compared to vehicle control. This was accompanied by a 3.0-fold increase in cleaved caspase-3 and a 2.6-fold increase in cleaved PARP (apoptotic markers) via western blot [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].

---

HCT116 colorectal cancer xenograft model (nude mice): Female nude mice (6–7 weeks old, n=8 per group) bearing HCT116 xenografts were treated with CYC116 at 50 mg/kg via oral gavage once daily for 14 days. This treatment resulted in 72% tumor growth inhibition (TGI) compared to vehicle control. At study end, tumor volume in the treated group was 200 ± 28 mm³, versus 710 ± 42 mm³ in the control group (p < 0.001). No significant body weight loss (<5%) was observed in the treated mice [1]
- SK-OV-3 ovarian cancer xenograft model: Oral administration of CYC116 (60 mg/kg daily) for 18 days in nude mice bearing SK-OV-3 xenografts achieved 68% TGI. Immunohistochemical analysis of excised tumors showed: - 85% reduction in phospho-histone H3 (Ser10) staining (Aurora B activity marker) - 75% reduction in phospho-TPX2 staining (Aurora A activity marker) [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.

---

Aurora A kinase activity assay (HTRF format): Recombinant human Aurora A kinase (complexed with TPX2 to enhance catalytic activity) was incubated with CYC116 (serial concentrations: 0.01 nM to 500 nM), ATP (10 μM), and a biotinylated TPX2-derived peptide substrate (containing the Aurora A phosphorylation site at Ser466) in kinase buffer (50 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT, 0.01% BSA, pH 7.5) at 30°C for 60 minutes. The reaction was terminated by adding 50 mM EDTA. Phosphorylated substrate was detected using a streptavidin-conjugated europium cryptate (fluorescence donor) and a phospho-specific antibody labeled with XL665 (fluorescence acceptor). Fluorescence resonance energy transfer (FRET) signals were measured using a microplate reader, and IC₅₀ values were calculated by fitting dose-response curves to a four-parameter logistic model [1]
- Aurora B kinase activity assay: Recombinant human Aurora B kinase (complexed with INCENP) was incubated with CYC116 (0.01 nM to 500 nM), ATP (10 μM), and a biotinylated histone H3 (Ser10) peptide substrate in the same kinase buffer as above. Incubation was carried out at 30°C for 60 minutes, and the reaction was stopped with 50 mM EDTA. Phosphorylated substrate was detected using the same HTRF-based method as the Aurora A assay, and IC₅₀ values were determined from dose-response curves [1]

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.

---

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.

---

Antiproliferation assay (CellTiter-Glo method): Human cancer cell lines (HCT116, MCF-7, SK-OV-3, A549) were seeded in 96-well plates at a density of 2×10³ cells/well and incubated overnight at 37°C (5% CO₂). CYC116 was added at serial concentrations (1 nM to 200 nM), and cells were cultured for 72 hours. CellTiter-Glo reagent (which generates luminescence proportional to viable cell ATP content) was added to each well, and luminescence was measured after 10 minutes of incubation at room temperature. IC₅₀ values were defined as the concentration of CYC116 that inhibited 50% of viable cells, calculated using GraphPad Prism software [1]
- Cell cycle analysis (PI staining): HCT116 cells were seeded in 6-well plates at 5×10⁵ cells/well and treated with CYC116 (20 nM) or vehicle for 24 hours. Cells were harvested by trypsinization, washed with cold PBS, and fixed in 70% ethanol at -20°C overnight. Fixed cells were washed again with PBS, resuspended in PI staining solution (50 μg/mL PI, 100 μg/mL RNase A, 0.1% Triton X-100 in PBS), and incubated at 37°C for 30 minutes. Cell cycle distribution (G0/G1, S, G2/M phases) was analyzed using a flow cytometer, and the percentage of cells in each phase was quantified using ModFit software [1]
- Apoptosis assay (annexin V-FITC/PI double staining): MCF-7 cells were treated with CYC116 (30 nM) or vehicle for 48 hours. Cells were harvested, washed with cold PBS, and resuspended in annexin V binding buffer. Annexin V-FITC and PI were added to the cell suspension, which was incubated in the dark at room temperature for 15 minutes. Apoptotic cells (early apoptosis: annexin V-positive/PI-negative; late apoptosis: annexin V-positive/PI-positive) were detected and counted using a flow cytometer [1]
- Western blot for Aurora substrates: HCT116 cells were treated with CYC116 (5, 10, 20, 30, 50 nM) for 6 hours, then lysed in RIPA buffer (supplemented with protease and phosphatase inhibitors). Protein extracts (30 μg per lane) were separated by 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk in TBST for 1 hour, then probed with primary antibodies against phospho-TPX2 (Ser466), phospho-histone H3 (Ser10), total TPX2, total histone H3, and β-actin (loading control) overnight at 4°C. After washing with TBST, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. Signals were detected using enhanced chemiluminescence (ECL) reagent, and band intensities were quantified using ImageJ software [1]

Dissolved in DMSO and then diluted in water; 75, 100 mg/kg; Oral gavage NCI-H460 cells are implanted intraperitoneally into the mice \nRat Pharmacokinetics. [1]
\nThe 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.

---

\nMurine P388/D1 Leukemia Model. [1]
\nFemale 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.

---

\nNCI-H460 Xenograft. [1]
\nHuman 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.

---

\n

---

\nHCT116 colorectal cancer xenograft model: Female nude mice (6–7 weeks old) were subcutaneously injected with 5×10⁶ HCT116 cells (suspended in a 1:1 mixture of PBS and Matrigel) into the right flank. When tumors reached a volume of 100–150 mm³, mice were randomly assigned to two groups (n=8 per group): vehicle control (0.5% carboxymethylcellulose sodium + 0.1% Tween 80 in distilled water) and CYC116 treatment. CYC116 was dissolved in the vehicle at a concentration of 10 mg/mL and administered via oral gavage at 50 mg/kg once daily for 14 days. Tumor volume was measured every 2 days using calipers, calculated as (length × width²)/2. Mouse body weight was also measured every 2 days to monitor potential toxicity [1]
\n- SK-OV-3 ovarian cancer xenograft model: Female nude mice were subcutaneously implanted with 1×10⁷ SK-OV-3 cells (mixed with Matrigel). When tumors reached ~120 mm³, mice were grouped (n=8 per group). CYC116 was prepared in the same vehicle as above at a concentration of 12 mg/mL and administered orally at 60 mg/kg once daily for 18 days. At the end of the study, tumors were excised, weighed, and fixed in 10% neutral buffered formalin for immunohistochemical analysis of phospho-histone H3 (Ser10) and phospho-TPX2 [1]

Oral bioavailability: In male Sprague-Dawley rats, the oral bioavailability of CYC116 (20 mg/kg) was 30%. Plasma concentration-time curves showed that the peak plasma concentration (Cmax) was 1.1 μg/mL 1.8 hours after administration, and the terminal half-life (t₁/₂) was 4.5 hours [1] - Intravenous pharmacokinetics (rat): After intravenous injection of CYC116 (5 mg/kg) in rats, the clearance (CL) was 15 mL/min/kg, the steady-state volume of distribution (Vss) was 5.2 L/kg, and the t₁/₂ was 4.2 hours [1] - Plasma protein binding rate: CYC116 showed high plasma protein binding rates in human (95%), rat (94%) and mouse (93%) plasmas as determined by equilibrium dialysis. At 37°C, dialysis was performed for 4 hours using a 10 kDa molecular weight cutoff membrane, and the plasma concentration of CYC116 was 1 μg/mL [1]. Metabolic stability: In human liver microsomes, the half-life of CYC116 was 3.8 hours (moderate metabolic stability); in rat liver microsomes, t₁/₂ was 4.3 hours. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified the major metabolites as monohydroxylated derivatives (accounting for 55% of the total metabolites), mainly generated through CYP3A4-mediated oxidation [1].

Protein Binding
CYC116 is an orally potent inhibitor of Aurora kinases A and B and VEGFR2 for the treatment of patients with advanced solid tumors. Acute oral toxicity (mice): Single oral doses of up to 2000 mg/kg of CYC116 in female CD-1 mice did not result in death. At doses ≥1500 mg/kg, mice exhibited transient decreases in kinetic activity, which recovered within 24 hours. At doses ≤1000 mg/kg, no significant changes in body weight were observed [1]. Chronic oral toxicity (rats): Male Sprague-Dawley rats were treated with CYC116 (50 mg/kg, once daily, orally) for 28 days. Mild myelosuppression was observed: white blood cell count decreased by 18% compared to the vector control group, while red blood cell and platelet counts remained within the normal range. Serum levels of liver function indicators (ALT, AST) and kidney function indicators (BUN, creatinine) were not significantly different from those in the control group, and pathological examination of liver, kidney and heart tissues revealed no treatment-related lesions [1].

[1]. 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 spectrum involving cell cycle and angiogenesis inhibition mechanisms. In preclinical studies, CYC116 has demonstrated antitumor activity in both solid tumors and hematological malignancies. CYC116 is the third oral drug developed by Cyclacel, and its anticancer activity mechanism is consistent with Aurora kinase inhibition.

---

Indications
Advanced Solid TumorsMechanism of Action
Aurora kinase is an enzyme that helps dividing cells share substances between two daughter cells. In many cancer patients, Aurora kinase dysfunction leads to loss of normal regulation of cell division, resulting in abnormal growth. CYC116 inhibits Aurora kinase, potentially slowing cancer cell growth and inducing apoptosis.

---

Chemical Classification and Design: CYC116 belongs to the class of N-phenyl-4-(thiazol-5-yl)pyrimidine-2-amine derivatives, whose chemical skeleton is optimized to simultaneously inhibit Aurora A and B kinases. Its design balances the inhibitory efficacy against both Aurora isoforms while minimizing off-target kinase activity, thus overcoming the limitations of single-isoform inhibitors (single-isoform inhibitors may allow tumor cells to evade immunosuppression through compensatory Aurora activity)[1].
- Mechanism of Action: CYC116 exerts its antitumor activity by simultaneously inhibiting Aurora A and B kinases (key regulators of mitotic processes). Inhibition of Aurora A disrupts the assembly of the mitotic spindle and the maturation of the centrosome, while inhibition of Aurora B impairs chromosome segregation and cytokinesis. This combined effect leads to G2/M phase cell cycle arrest, mitotic catastrophe, and subsequent apoptosis in cancer cells, particularly those with elevated Aurora kinase expression, a common feature of colorectal, breast, and ovarian cancers [1]. - Preclinical therapeutic potential: CYC116 shows no cross-resistance with standard chemotherapeutic agents (e.g., 5-fluorouracil, paclitaxel) in HCT116 and SK-OV-3 cell lines, supporting its potential for treating chemotherapy-refractory solid tumors. Furthermore, its high oral bioavailability and favorable pharmacokinetic profile make it suitable for oral administration in a clinical setting [1].

C18H20N6OS

368.46

368.141

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

1.44

2

8

4

26

443

0

S1C(N([H])[H])=NC(C([H])([H])[H])=C1C1C([H])=C([H])N=C(N=1)N([H])C1C([H])=C([H])C(=C([H])C=1[H])N1C([H])([H])C([H])([H])OC([H])([H])C1([H])[H]

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.

---

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.

---

View More

Solubility in Formulation 3: 1% DMSO+30% polyethylene glycol+1% Tween 80:30mg/mL

---

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