HLCL-61 hydrochloride is a potent, selective, first-in-class small-molecule inhibitor of PRMT5 (protein arginine methyltransferase) that has the potential for treatment of acute myeloid leukemia. HLCL-61 resulted in significantly increased expression of miR-29b and consequent suppression of Sp1 and FLT3 in AML (acute myeloid leukemia) cells. As a result, significant antileukemic activity was achieved. Inhibition of PRMT5 via sh/siRNA or a first-in-class small-molecule inhibitor (HLCL-61) resulted in significantly increased expression of miR-29b and consequent suppression of Sp1 and FLT3 in AML cells. As a result, significant antileukemic activity was achieved. Collectively, the data support a novel leukemogenic mechanism in AML where PRMT5 mediates both silencing and transcription of genes that participate in a 'yin-yang' functional network supporting leukemia growth. As FLT3 is often mutated in AML and pharmacologic inhibition of PRMT5 appears feasible, the PRMT5-miR-29b-FLT3 network should be further explored as a novel therapeutic target for AML.

Physicochemical Properties

Molecular Formula	C₂₃H₂₅CLN₂O
Molecular Weight	380.91
Exact Mass	380.165
CAS #	1158279-20-9
Related CAS #	1158279-20-9 (HCl);586395-74-6;
PubChem CID	17208222
Appearance	White to off-white solid powder
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	2
Rotatable Bond Count	6
Heavy Atom Count	27
Complexity	442
Defined Atom Stereocenter Count	0
InChi Key	XYAVCNMZZKTEGR-UHFFFAOYSA-N
InChi Code	InChI=1S/C23H24N2O.ClH/c1-3-25-21-10-6-5-9-19(21)20-14-17(12-13-22(20)25)15-24-16-18-8-4-7-11-23(18)26-2;/h4-14,24H,3,15-16H2,1-2H3;1H
Chemical Name	1-(9-ethylcarbazol-3-yl)-N-[(2-methoxyphenyl)methyl]methanamine;hydrochloride
Synonyms	HLCL61 HCl; HLCL 61; HLCL-61.
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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.
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	With dose-dependent effects on cell growth, HLCL-61 hydrochloride (1–100 μM; 24-72 hours) has IC50 values of 14.12, 16.74, 6.3, and 8.72 μM for MV4-11 cells, THP-1 cells, FLT3-WT blast, and FLT3-ITD blast, respectively[1]. In AML samples, symmetric arginine dimethylation (me2) of histones H3 and H4 is effectively inhibited by HLCL-61 hydrochloride. This inhibition begins at 12 hours post-treatment and continues for 48 hours[1].
ln Vivo
Cell Assay	Cell Viability Assay[1] Cell Types: MV4-11 cells, THP-1 cells, FLT3-WT blast (primary blasts from patients), FLT3-ITD blast (primary blasts from patients) Tested Concentrations: 1, 5, 10, 25, 50, 100 μM Incubation Duration: 24, 48, 72 hrs (hours) Experimental Results: Dose-dependent reduction in cell viability with IC50s of 14.12, 16.74, 6.3, 8.72 μM for MV4-11 cells, THP-1 cells, FLT3-WT blast, and FLT3-ITD blast, respectively.
Animal Protocol
References	[1]. The dual epigenetic role of PRMT5 in acute myeloid leukemia: gene activation and repression via histone arginine methylation. Leukemia. 2016 Apr;30(4):789-99.

Solubility Data

Solubility (In Vitro)

DMSO:≥ 30 mg/mL Water:N/A Ethanol:N/A

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.56 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 (6.56 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 (6.56 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	2.6253 mL	13.1265 mL	26.2529 mL
	5 mM	0.5251 mL	2.6253 mL	5.2506 mL
	10 mM	0.2625 mL	1.3126 mL	2.6253 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.