HTH-01-091 is a potent and specific inhibitor of maternal embryonic leucine zipper kinase (MELK) with IC50 of 10.5 nM. HTH-01-091 also inhibits PIM1/2/3, RIPK2, DYRK3, smMLCK, and CLK2. HTH-01-091 may be used in breast cancer research.

Physicochemical Properties

Molecular Formula	C26H28CL2N4O2
Molecular Weight	499.432124137878
Exact Mass	498.158
CAS #	2000209-42-5
PubChem CID	131801426
Appearance	White to light yellow solid powder
LogP	5
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	4
Heavy Atom Count	34
Complexity	707
Defined Atom Stereocenter Count	0
SMILES	ClC1C(=C(C=C(C=1)C1C=CC2=C(C=1)C1=C(C=N2)CNC(N1C1CCC(CN(C)C)CC1)=O)Cl)O
InChi Key	FUVRHGKKWNNBJX-UHFFFAOYSA-N
InChi Code	InChI=1S/C26H28Cl2N4O2/c1-31(2)14-15-3-6-19(7-4-15)32-24-18(13-30-26(32)34)12-29-23-8-5-16(9-20(23)24)17-10-21(27)25(33)22(28)11-17/h5,8-12,15,19,33H,3-4,6-7,13-14H2,1-2H3,(H,30,34)
Chemical Name	9-(3,5-dichloro-4-hydroxyphenyl)-1-[4-[(dimethylamino)methyl]cyclohexyl]-3,4-dihydropyrimido[5,4-c]quinolin-2-one
Synonyms	HTH01-091 HTH 01-091 HTH-01-091
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	Over 90% of kinases are specifically inhibited by HTH-01-091 (1 μM) [1]. MELK degradation is caused by cell-permeable HTH-01-091 (0-10 μM, 1 hour) [1]. On breast cancer cells, HTH-01-091 (0–10 μM, 3 days) shows modest anti-proliferative effects [1].
Cell Assay	Western Blot Analysis[1] Cell Types: MDA-MB-468 Cell Tested Concentrations: 0, 0.1, 1.0 and 10 μM Incubation Duration: 1 hour Experimental Results: diminished MELK protein levels in MDA-MB-468 cells; Streptavidin Beads The pull-down effect on MELK was diminished in a dose-dependent manner, indicating that the compound is cell-permeable and binds to MELK in an ATP-competitive manner. There was no effect on ERK1/2 pulldown, indicating that HTH-01-091 has no binding affinity for ERK1/2. Cell proliferation assay[1] Cell Types: MDA-MB-468, BT-549, HCC70, ZR-75-1, MCF7 and T-47D Cell Tested Concentrations: 0, 0.001, 0.01, 0.1, 1.0 and 10 μM Incubation Duration: 3-day Experimental Results: Demonstrated antiproliferative activity in a panel of breast cancer cell lines, including MDA-MB-468, BT-549, HCC70, ZR-75-1, MCF7, and T-47D cells, with IC50 values of 4.00, respectively. μM, 6.16 μM, 8.80 μM, >10 μM, 8.75 μM and 3.87 μM.
References	[1]. MELK is not necessary for the proliferation of basal-like breast cancer cells. Elife. 2017 Sep 19;6:e26693.

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	2.0023 mL	10.0114 mL	20.0228 mL
	5 mM	0.4005 mL	2.0023 mL	4.0046 mL
	10 mM	0.2002 mL	1.0011 mL	2.0023 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.