RO3280 (also known as Ro-5203280; RO 3280; Ro5203280; RO-3280) is a novel, potent and highly selective inhibitor of Polo-like kinase 1 (PLK1) with potential anticancer activity. With an IC50 of 3 nM and a Kd of 0.09 nM, respectively, it inhibits PLK1. There is minimal impact of RO3280 on PLK2 and PLK3. Strong antitumor activity was demonstrated by RO3280 in xenograft mouse models. In primary acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML) cells, the IC50 of RO3280 ranged from 35.49 to 110.76 nM and 52.80 to 147.50 nM, respectively. In leukemia cells, RO3280 caused apoptosis and a disruption in the cell cycle. Numerous genes related to apoptosis were regulated by RO3280 treatment.

Physicochemical Properties

Molecular Formula	C27H35F2N7O3
Molecular Weight	543.61
Exact Mass	543.276
Elemental Analysis	C, 59.65; H, 6.49; F, 6.99; N, 18.04; O, 8.83
CAS #	1062243-51-9
Related CAS #	1062243-51-9
PubChem CID	25015677
Appearance	Off-white to yellow solid powder
Density	1.4±0.1 g/cm3
Index of Refraction	1.623
LogP	1.19
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	10
Rotatable Bond Count	6
Heavy Atom Count	39
Complexity	868
Defined Atom Stereocenter Count	0
SMILES	COC1=C(NC2=NC(N(C3CCCC3)CC(F)(F)C(N4C)=O)=C4C=N2)C=CC(C(NC5CCN(C)CC5)=O)=C1
InChi Key	DJNZZLZKAXGMMC-UHFFFAOYSA-N
InChi Code	InChI=1S/C27H35F2N7O3/c1-34-12-10-18(11-13-34)31-24(37)17-8-9-20(22(14-17)39-3)32-26-30-15-21-23(33-26)36(19-6-4-5-7-19)16-27(28,29)25(38)35(21)2/h8-9,14-15,18-19H,4-7,10-13,16H2,1-3H3,(H,31,37)(H,30,32,33)
Chemical Name	4-[(9-cyclopentyl-7,7-difluoro-5-methyl-6-oxo-8H-pyrimido[4,5-b][1,4]diazepin-2-yl)amino]-3-methoxy-N-(1-methylpiperidin-4-yl)benzamide
Synonyms	Ro5203280; RO3280; Ro5203280; RO-3280; Ro-5203280;RO 3280; Ro 5203280
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	PLK1 (Kd = 0.09 nM); ALK (Kd = 230 nM); CAMKK1 (Kd = 1100 nM); CAMKK2 (Kd = 87 nM); DAPK1 (IC50 = 100 nM); DAPK3 (Kd = 70 nM); FER (Kd = 53 nM); GAK (Kd = 87 nM); MYLK (Kd = 170 nM); PTK2 (IC50 = 84 nM); PTK2B (Kd = 130 nM); RPS6KA6 (KinDom.2) (IC50 = 560 nM); TTK (Kd = 51 nM)
ln Vitro	RO3280 demonstrates potent anti-proliferative activity with IC50s of 5, 10, 19, 12, and 70 nM against the lung cancer cell line H82, the colorectal cancer cell HT-29, the breast cancer cell MDA-MB-468, the prostate cancer cell PC3, and the skin cancer cell A375.
ln Vivo	RO3280 shows significant anticancer action in nude mice bearing HT-29 human colorectal tumors, with doses as low as 40 mg/kg once a week resulting in 72% tumor growth inhibition and as high as 40 mg/kg once a week causing complete tumor regression.[1]
Cell Assay	Primary leukemia cells (2 × 104) or leukemia cells seeded overnight are incubated with DMSO or increasing concentrations of RO3280 (0.05-120 μM) for a full day. The vehicle treated wells with an equal volume of DMSO added. Four duplicates of each drug concentration are made. Following the addition of 10 μL CCK8 solution to each well, the wells are incubated for 2–4 hours at 37°C. A scanning multi-well spectrophotometer is then used to measure the optical density (OD) values at 450 nm. The absorbance values are compared with the control group to determine the relative survival rate. After plotting proliferation values on a logarithmic curve, the percentage of DMSO-treated control wells with 50% inhibitory concentration (IC50) values is used to calculate the proliferation of cells. The PLK1 inhibitor's IC50 is determined using the Graph Prism program.
Animal Protocol	In short, the assay uses female mice that are 4-5 weeks old. Every BALB/c nude mouse has its flank subcutaneously injected with 5 × 106 cells in 150 μL of RPMI 1640 suspension. On day five, the size of the tumor is measured, the animals are divided into two groups (n = 15 per group), and intraperitoneal injections of RO3280 (40 mg/kg, once every five days) are administered to start the treatment. A vehicle solution containing 1.5% DMSO in PBS is administered to the control group. The 40-day course of medication (or vehicle) treatment is administered. Every three days, callipers are used to measure the length and width (in millimeters) of the resulting tumours. The volume (length × width2 × 0.52) is calculated after the tumor diameter is measured. On day 45, the tumors are removed and weighed before the mice are humanely put to death. These sections are also used for Western blot and immunohistochemistry assays. After that, the tumors are embedded, fixed, and cut into sections that are 3 μm thick. These sections are then stained with eosin and haematoxylin to make the tumor margin visible.
References	[1]. Bioorg Med Chem Lett . 2012 Jan 15;22(2):1247-50.

Solubility Data

Solubility (In Vitro)

DMSO: ~100 mg/mL (~184.0 mM) Water: <1 mg/mL Ethanol: ~100 mg/mL (~184.0 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.60 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 (4.60 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. 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 (4.60 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	1.8396 mL	9.1978 mL	18.3955 mL
	5 mM	0.3679 mL	1.8396 mL	3.6791 mL
	10 mM	0.1840 mL	0.9198 mL	1.8396 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.