PLK1/BRD4-IN-1 (9b) is an orally bioactive dual (bifunctional) inhibitor of PLK1 and BRD4 with IC50s of 22 nM and 109 nM for PLK1 and BRD4, respectively. PLK1/BRD4-IN-1 induces cell cycle arrest and apoptosis, downregulates the transcription of several proliferation-related oncogenes, and displays good anti-tumor activity in vivo.

Physicochemical Properties

Molecular Formula	C31H43N9O2
Molecular Weight	573.732225656509
Exact Mass	573.353
CAS #	2412707-81-2
PubChem CID	156775102
Appearance	Typically exists as solid at room temperature
LogP	4.8
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	9
Rotatable Bond Count	8
Heavy Atom Count	42
Complexity	883
Defined Atom Stereocenter Count	1
SMILES	CC[C@@H]1C2=NN=C(N2C3=CN=C(N=C3N1CC4CCCCC4)NC5=C(C=C(C=C5)C(=O)NC6CCN(CC6)C)OC)C
InChi Key	QUMCIHKVKQYNPA-RUZDIDTESA-N
InChi Code	InChI=1S/C31H43N9O2/c1-5-25-29-37-36-20(2)40(29)26-18-32-31(35-28(26)39(25)19-21-9-7-6-8-10-21)34-24-12-11-22(17-27(24)42-4)30(41)33-23-13-15-38(3)16-14-23/h11-12,17-18,21,23,25H,5-10,13-16,19H2,1-4H3,(H,33,41)(H,32,34,35)/t25-/m1/s1
Chemical Name	4-[[(4R)-5-(cyclohexylmethyl)-4-ethyl-1-methyl-4H-[1,2,4]triazolo[4,3-f]pteridin-7-yl]amino]-3-methoxy-N-(1-methylpiperidin-4-yl)benzamide
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

ln Vitro	PLK1/BRD4-IN-1 (9b) (72 h) exhibits antiproliferative activity with a broad scope [1]. A 24-hour exposure to PLK1/BRD4-IN-1 (0–9 µM) causes cell cycle arrest [1]. Apoptosis is induced by PLK1/BRD4-IN-1 (0-9 µM, 48 hours) [1]. PLK1/BRD4-IN-1 acts as an inhibitor on both PLK1 and BRD4, which prevents the growth of cancer cells [1].
ln Vivo	PLK1/BRD4-IN-1 (9b) (60 mg/kg/d; IG; 18 days) significantly decreased the average tumor size without showing any signs of toxicity [1].
Cell Assay	Cell Viability Assay[1] Cell Types: MV4-11, LnCap, HT-29, A375, SKOV-3 Tested Concentrations: Cells are maintained in RPMI 1640 or DMEM medium supplemented with 10% FBS (v/v) and 5% CO2 , except for MV4-11 cells cultured in IMDM medium. Incubation Duration: 72 hrs (hours) Experimental Results: Displayed broad-spectrum anti-proliferative activity with IC50 values of 0.13, 0.14, 1.10, 2.82 and 2.51 µM against MV4-11, LnCap, SKOV-3, A375 and HT29 cells respectively. Cell cycle analysis[1] Cell Types: MV4-11 Tested Concentrations: 0.1, 0.3, 1, 3, 9 µM Incubation Duration: 24 hrs (hours) Experimental Results: Induced obvious G2/M phase arrest in a concentration-dependent manner Apoptosis analysis[1 ] Cell Types: MV4-11 Tested Concentrations: 0.1, 0.3, 1, 3, 9 µM Incubation Duration: 48 hrs (hours) Experimental Results: The number of Annexin V/PI positive MV4-11 cells increased Dramatically in a concentration-dependent manner. RT-PCR[1] Cell Types: MV4-11 Tested Concentrations: 0.1, 0.3, 1, 3, 9 µM Incubation Duration: 24 hrs (hours) Experimental Results: Transcriptional reduction of c-MYC and MYCN and BCL-2 at c
Animal Protocol	Animal/Disease Models: Fiveweeks old male NOD-SCID (severe combined immunodeficient) mouse [1]. Doses: 60 mg/kg/d Route of Administration: po (oral gavage) for 18 days; a tumor xenograft model was established by subcutaneously (sc) (sc) injecting 100 µL of 1×108 cell/mL MV4-11 cell suspension into NOD-SCID (severe combined immunodeficient) mouse. Experimental Results: The average tumor size was Dramatically diminished, the tumor growth inhibition rate was 66%, and there was no obvious effect on the body weight of mice.
References	[1]. Design, synthesis, and biological evaluation of 4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridine derivatives as novel dual-PLK1/BRD4 inhibitors. Eur J Med Chem. 2020 Apr 1;191:112152.

Solubility Data

Solubility (In Vitro)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Solubility (In Vivo)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *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. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.7430 mL	8.7149 mL	17.4298 mL
	5 mM	0.3486 mL	1.7430 mL	3.4860 mL
	10 mM	0.1743 mL	0.8715 mL	1.7430 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.