Pinometostat (formerly EPZ 5676) is a potent and SAM (S-adenosyl methionine) competitive DOT1L protein methyltransferase (PMT) inhibitor with potential antitumor activity. It inhibits DOT1L with a Ki of 80 pM in a cell-free assay, and exhibits >37,000-fold higher selectivity for DOT1L over other PMTs. It exhibits excellent antiproliferative activity and high in vivo antitumor efficacy.

Physicochemical Properties

Molecular Formula	C30H42N8O3
Molecular Weight	562.71
Exact Mass	562.338
CAS #	1380288-87-8
Related CAS #	1380288-87-8
PubChem CID	57345410
Appearance	White to light yellow solid powder
Density	1.5±0.1 g/cm3
Boiling Point	835.3±75.0 °C at 760 mmHg
Flash Point	459.0±37.1 °C
Vapour Pressure	0.0±3.2 mmHg at 25°C
Index of Refraction	1.722
LogP	4.8
Hydrogen Bond Donor Count	4
Hydrogen Bond Acceptor Count	9
Rotatable Bond Count	9
Heavy Atom Count	41
Complexity	884
Defined Atom Stereocenter Count	4
SMILES	CC(C)N(C[C@@H]1[C@H]([C@H]([C@@H](O1)N2C=NC3=C(N=CN=C32)N)O)O)C4CC(C4)CCC5=NC6=C(N5)C=C(C=C6)C(C)(C)C
InChi Key	LXFOLMYKSYSZQS-LURJZOHASA-N
InChi Code	InChI=1S/C30H42N8O3/c1-16(2)37(13-22-25(39)26(40)29(41-22)38-15-34-24-27(31)32-14-33-28(24)38)19-10-17(11-19)6-9-23-35-20-8-7-18(30(3,4)5)12-21(20)36-23/h7-8,12,14-17,19,22,25-26,29,39-40H,6,9-11,13H2,1-5H3,(H,35,36)(H2,31,32,33)/t17-,19+,22-,25-,26-,29-/m1/s1
Chemical Name	(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol
Synonyms	EPZ 5676; Pinometostat; EPZ-5676; EPZ5676
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	For MV4-11 and HL60 cells, respectively, pinometostat (EPZ-5676) has an IC50 value of 3 nM and 5 nM for inhibiting H3K79me2. Pinometostat, also known as EPZ-5676, has an IC50 value of 3.5 nM, making it a strong inhibitor of MV4-11 proliferation(1). In MLL-r cells, pinometostat (EPZ-5676) exhibits cooperative benefits when used in conjunction with AML treatment medications. It also has synergistic and durable antiproliferative effects, upregulating the expression of differentiation markers and apoptosis by itself [2].
ln Vivo	In a rat xenograft model of MLL-rearranged leukemia, pinometostat (EPZ-5676) (70 mg/kg, ip) results in total and sustained regression. Rat tumors grown from HOXA9 and MEIS1 mRNA levels are decreased by pinometostat (EPZ-5676) (70, 35 mg/kg, iv), which also lowers MLL fusion target gene expression in vivo [1].
Animal Protocol	35, 67 or 70 mg/kg/day; i.v. Nude rats bearing MV4-11 xenografts
References	[1]. Potent inhibition of DOT1L as treatment for MLL-fusion leukemia. Blood. 2013 Jun 25. [Epub ahead of print]. [2]. DOT1L inhibitor EPZ-5676 displays synergistic antiproliferative activity in combination with standard of care drugs and hypomethylating agents in MLL-rearranged leukemia cells. J Pharmacol Exp Ther. 2014 Sep;350(3):646-56.
Additional Infomation	Pinometostat (EPZ-5676) is a potent DOT1L histone methyltransferase inhibitor. Pinometostat has been used in trials studying the treatment of Leukemia, Acute Leukemias, Acute Myeloid Leukemia, Myelodysplastic Syndrome, and Acute Lymphocytic Leukemia, among others. Pinometostat is a small molecule inhibitor of histone methyltransferase with potential antineoplastic activity. Upon intravenous administration, pinometostat specifically blocks the activity of the histone lysine-methyltransferase DOT1L, thereby inhibiting the methylation of nucleosomal histone H3 on lysine 79 (H3K79) that is bound to the mixed lineage leukemia (MLL) fusion protein which targets genes and blocks the expression of leukemogenic genes. This eventually leads to an induction of apoptosis in the leukemic cells bearing the MLL gene translocations. DOT1L, a non-SET domain-containing histone methyltransferase, specifically methylates H3K79 and plays a key role in normal cell differentiation and in the development of leukemia with MLL gene rearrangement on chromosome 11 and promotes the expression of leukemia-causing genes.

Solubility Data

Solubility (In Vitro)

DMSO: 100 mg/mL (177.71mM) Water:<1 mg/mL Ethanol: 92 mg/mL (163.5 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.44 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 (4.44 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 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 (4.44 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. Solubility in Formulation 4: ≥ 2.5 mg/mL (4.44 mM) (saturation unknown) in 10% EtOH + 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 EtOH 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 5: ≥ 2.5 mg/mL (4.44 mM) (saturation unknown) in 10% EtOH + 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 25.0 mg/mL clear EtOH 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 6: ≥ 2.5 mg/mL (4.44 mM) (saturation unknown) in 10% EtOH + 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 EtOH stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 7: 2% DMSO+30% PEG 300+5% Tween 80+ddH2O:5mg/mL  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.7771 mL	8.8856 mL	17.7711 mL
	5 mM	0.3554 mL	1.7771 mL	3.5542 mL
	10 mM	0.1777 mL	0.8886 mL	1.7771 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.