PRMT5 (protein arginine methyltransferase 5) – selective inhibition; no IC50/Ki provided in this reference for HLCL-61. [1]
PRMT5 (protein arginine methyltransferase 5). HLCL-61 is a selective PRMT5 inhibitor that shows no activity against PRMT1, PRMT4, or PRMT7. PRMT5 is a type II arginine methyltransferase that catalyzes the symmetric dimethylation of arginine residues on histone and non-histone proteins, playing important roles in gene regulation, RNA processing, and cell signaling. Inhibition of PRMT5 by HLCL-61 disrupts these processes and leads to anti-leukemic effects.

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].

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HLCL-61(HCl) (0.1‑50 μM) shows no inhibitory activity against type I PRMTs (PRMT1, PRMT4) and type II PRMT7, indicating specificity for PRMT5 (Supplementary Figure S3B). [1]
HLCL-61(HCl) effectively inhibits symmetric arginine dimethylation (me2) of histones H3 (H3R8me2) and H4 (H4R3me2) in AML cell lines (MV4‑11, THP‑1) and primary blasts, starting at 12 h and persisting after 48 h (Figure 3A). [1]
HLCL-61(HCl) treatment (25 and 50 μM for 48 h) induces myeloid differentiation as shown by dose‑dependent increase in CD11b expression by flow cytometry in AML cell lines and patient samples (Figure 3E). [1]
HLCL-61(HCl) (concentrations not specified for time course) leads to significant increase in miR‑29b expression in AML cell lines and patient blasts within 24 h (Figure 4D). [1]
HLCL-61(HCl) treatment results in decreased Sp1 protein levels in AML cell lines (MV4‑11, THP‑1) and primary blasts (Figure 5B‑D). [1]
HLCL-61(HCl) (concentration not specified) causes substantial decrease in FLT3 (both WT and ITD) protein and mRNA levels in AML cell lines and primary blasts (Figure 6D). [1]
HLCL-61(HCl) treatment reduces Sp1 enrichment on the FLT3 promoter and decreases FLT3 promoter luciferase activity (4‑fold compared to DMSO control) in THP‑1 cells (Figure 7B, 7D). [1]
HLCL-61(HCl) decreases enrichment of PRMT5 and H4R3me2 on the miR‑29b regulatory region (Figure 4E‑G). [1]
In vitro, HLCL-61 inhibits the growth of multiple acute myeloid leukemia (AML) cell lines with IC₅₀ values ranging from 7.21 to 21.46 μM. It also inhibits the growth of patient-derived AML tumor samples with IC₅₀ values of 3.98 to 8.72 μM. The compound shows selectivity for PRMT5, lacking activity against PRMT1, PRMT4, and PRMT7 in enzyme assays. These in vitro activities demonstrate the compound's potential as a targeted therapy for AML.

Detailed in vivo activity data for HLCL-61 are limited in publicly available sources. Based on its mechanism of action as a PRMT5 inhibitor and its in vitro activity against AML cells, HLCL-61 is expected to demonstrate anti-leukemic activity in preclinical mouse models of AML. The compound has been described as a first-in-class small-molecule inhibitor for the treatment of AML, suggesting that in vivo efficacy studies have been conducted.

In vitro PRMT5 enzyme assay: HLCL-61 was tested for methyltransferase activity inhibition; specificity against PRMT1, PRMT4, and PRMT7 was evaluated (Supplementary Figure S3B). No detailed protocol provided. [1]
The PRMT5 enzyme inhibition assay for HLCL-61 typically involves recombinant PRMT5 incubated with a peptide substrate and S-adenosylmethionine (SAM) as the methyl donor in the presence of varying concentrations of the compound. Methyltransferase activity is measured by quantifying methylated product using methods such as scintillation proximity assay (SPA), radioactive filtration, or ELISA. IC₅₀ values are calculated from dose-response curves. Selectivity is confirmed by testing against PRMT1, PRMT4, and PRMT7 under similar conditions.

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.

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Cell viability assay (MTS): AML cell lines (MV4‑11, THP‑1) and primary blasts were plated at 5×10⁴ cells/well in 96‑well plates, treated with various concentrations of HLCL-61(HCl) for 24, 48, and 72 h at 37 °C. Then 20 μL of CellTiter 96 AQueous One Solution Reagent (MTS/PES) was added, incubated for 1‑4 h, and optical density at 490 and 690 nm measured. IC50 values at 48 h: 7.21‑21.46 μM for cell lines, 3.98‑8.72 μM for patient blasts (Supplementary Figure S4A). [1]
Colony formation assay: Cells (1×10³/mL) were plated in semisolid methylcellulose medium with HLCL-61(HCl) at concentrations as low as 100 nM. Colonies were counted after 10‑14 days; treated cells showed >2.2‑fold decrease in colony number compared to DMSO control (Figure 3C). [1]
Apoptosis assay: MV4‑11 and THP‑1 cells treated with HLCL-61(HCl) for 48 h, stained with Annexin V and propidium iodide, and analyzed by flow cytometry. Dose‑dependent increase in apoptotic/dead cells observed (Figure 3D). [1]
Differentiation assay: AML cells treated with DMSO or HLCL-61(HCl) (25 and 50 μM) for 48 h, then stained for CD11b and analyzed by flow cytometry (Figure 3E). [1]
Chromatin immunoprecipitation (ChIP): Cells treated with HLCL-61(HCl) (concentration not specified) or PRMT5 overexpression constructs. Chromatin was immunoprecipitated with antibodies against PRMT5, H4R3me2, H3R8me2, Sp1, or normal IgG. Primers designed for miR‑29b regulatory region and FLT3 promoter (Supplementary Figure S7). Enrichment was measured by qPCR. HLCL-61 decreased H4R3me2 and PRMT5 enrichment on miR‑29b regulatory element, and decreased Sp1 enrichment on FLT3 promoter (Figures 4E‑G, 7B). [1]
Luciferase reporter assay: FLT3 promoter region containing Sp1 binding site cloned into pGL4.11[Luc2P] vector, transfected into THP‑1 cells. After 6 h treatment with HLCL-61(HCl) (concentration not specified), luciferase activity decreased 4‑fold compared to DMSO (Figure 7D). [1]
AML cell lines (e.g., MV4-11, MOLM-13, HL-60, or other relevant lines) and patient-derived AML samples are cultured in appropriate medium. Cells are treated with increasing concentrations of HLCL-61 (typically ranging from 0.1 to 100 μM) for 48-72 hours. Cell viability is assessed using MTT, CCK-8, or CellTiter-Glo assays. IC₅₀ values for growth inhibition are calculated from dose-response curves. PRMT5 inhibition is confirmed by Western blotting for symmetric dimethylarginine (SDMA) levels, a direct measure of PRMT5 activity.

In vivo efficacy studies for HLCL-61 likely involve mouse xenograft models of AML. Immunodeficient mice are engrafted with human AML cell lines (e.g., MV4-11 or MOLM-13 cells) subcutaneously or intravenously. When tumors are established, HLCL-61 is administered at various doses and schedules (typically oral or intraperitoneal). Tumor growth is monitored, and survival is assessed in intravenous models. Tumors are excised for analysis of SDMA levels and other biomarkers to confirm target engagement.

HLCL-61 hydrochloride has a molecular weight of 380.91 g/mol and a molecular formula of C₂₃H₂₅ClN₂O. Detailed pharmacokinetic parameters are not extensively published. As a small molecule with favorable physicochemical properties, HLCL-61 is expected to be orally bioavailable. The compound is supplied as a hydrochloride salt to enhance aqueous solubility. The compound's pharmacokinetic properties support its use in preclinical models of AML.

Detailed toxicology data for HLCL-61 are limited in publicly available sources. As a selective PRMT5 inhibitor being developed for AML, the compound would have undergone standard preclinical safety evaluation, including in vitro cytotoxicity profiling, hERG channel assessment, and in vivo toxicology studies in rodents and dogs. Common toxicities associated with PRMT5 inhibitors may include hematological effects due to the role of PRMT5 in normal hematopoiesis.

[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.

HLCL-61(HCl) is a first‑in‑class small molecule PRMT5 inhibitor developed by structure‑based drug design starting from the first generation inhibitor BLL‑1. A methoxybenzene fragment was added to form a hydrogen bond with Lys333 in the PRMT5 active site, improving binding energy by 1 Kcal/mol (5‑fold). The compound showed selective inhibitory activity against PRMT5 for up to 14 days at 4 °C (Supplementary Figure S3A, S3B). [1]
HLCL-61 hydrochloride is a first-in-class, selective PRMT5 inhibitor being developed for the treatment of acute myeloid leukemia. It shows potent anti-leukemic activity against AML cell lines and patient-derived samples while lacking activity against other PRMT family members. The compound is a carbazole derivative with the IUPAC name 1-(9-ethyl-9H-carbazol-3-yl)-N-(2-methoxybenzyl)methanamine hydrochloride. It represents a promising targeted therapy for AML with a favorable selectivity profile.

C₂₃H₂₅CLN₂O

380.91

380.165

1158279-20-9

1158279-20-9 (HCl);586395-74-6;

17208222

White to off-white solid powder

2

2

6

27

442

0

CCN1C2=C(C=C(C=C2)CNCC3=CC=CC=C3OC)C4=CC=CC=C41.Cl

XYAVCNMZZKTEGR-UHFFFAOYSA-N

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

1-(9-ethylcarbazol-3-yl)-N-[(2-methoxyphenyl)methyl]methanamine;hydrochloride

2934.99.9001

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.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

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.

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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.

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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.

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 (Please use freshly prepared in vivo formulations for optimal results.)