ML324 (CID44143209) is a selective inhibitor of the JMJD2 subfamily of histone demethylases (JmjC-KDMs), including JMJD2A (IC50 = 0.4 μM), JMJD2B (IC50 = 0.6 μM), and JMJD2C (IC50 = 0.8 μM). It shows minimal inhibition (IC50 >50 μM) against non-JMJD2 JmjC-KDMs (e.g., JMJD1A, KDM5B) and histone acetyltransferases (p300), confirming specificity for the JMJD2 subfamily [1]
- ML324 (CID44143209) specifically inhibits KDM4B (a member of the JMJD2 subfamily) with an IC50 of 0.5 μM, and does not affect the activity of other histone demethylases (e.g., KDM1A/LSD1) at concentrations up to 10 μM [2]

In osteoclast progenitors, ML324 significantly reduces the amount of Aa-LPS-induced osteoclastogenesis[1].

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Inhibition of JMJD2 enzymatic activity: ML324 (CID44143209) (0.01-20 μM) inhibits JMJD2-mediated demethylation of H3K9me3 (a key substrate of JMJD2) in a concentration-dependent manner. At 2 μM, it reduces JMJD2A activity by 92±5% and JMJD2B activity by 88±6% compared to vehicle, as measured by a fluorescent-based demethylase assay [1]
- Antiviral activity against DNA viruses: ML324 (CID44143209) inhibits the replication of human herpes simplex virus type 1 (HSV-1) in Vero cells with an EC50 of 1.2 μM; at 5 μM, it suppresses HSV-1 plaque formation by >90%. It also exhibits antiviral activity against human cytomegalovirus (CMV) in MRC-5 cells (EC50 = 1.8 μM) and Epstein-Barr virus (EBV) in Raji cells (EC50 = 2.3 μM) [1]
- Elevation of H3K9me3 levels in virus-infected cells: Western blot analysis of HSV-1-infected Vero cells treated with ML324 (CID44143209) (5 μM) for 24 hours shows a 3.8±0.4-fold increase in H3K9me3 levels, while H3K4me3 and H3K27me3 levels remain unchanged—consistent with JMJD2’s substrate specificity [1]
- Suppression of pro-inflammatory cytokine release: In RAW264.7 macrophages stimulated with lipopolysaccharide (LPS, 100 ng/mL), ML324 (CID44143209) (0.5-5 μM) reduces the secretion of TNF-α (EC50 = 1.1 μM) and IL-6 (EC50 = 1.3 μM). At 5 μM, TNF-α release is reduced by 75±7% and IL-6 by 70±6% compared to LPS-treated controls [2]
- Attenuation of osteoclastogenesis: In RAW264.7 cells induced to differentiate into osteoclasts with RANKL (50 ng/mL), ML324 (CID44143209) (0.5-5 μM) inhibits osteoclast formation in a concentration-dependent manner. At 5 μM, the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells is reduced by 80±8% compared to RANKL-treated controls [2]
- Activation of KDM1A: qPCR and Western blot analysis of LPS-stimulated RAW264.7 cells treated with ML324 (CID44143209) (5 μM) show a 2.5±0.3-fold increase in KDM1A mRNA levels and a 2.2±0.2-fold increase in KDM1A protein levels, indicating cross-regulation between JMJD2/KDM4B and KDM1A [2]
- Low cytotoxicity in mammalian cells: MTT assays show ML324 (CID44143209) (0.1-20 μM) has no significant cytotoxicity in Vero cells (CC50 = 28±4 μM), MRC-5 cells (CC50 = 32±5 μM), or RAW264.7 cells (CC50 = 35±6 μM) after 72 hours of treatment [1,2]

In a mouse ganglia explant model of latently infected mice, ML324 suppresses the formation of HSV plaques, and blocks HSV-1 reactivation

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Antiviral efficacy in HSV-1-infected mice: Female BALB/c mice (6-8 weeks old) are intranasally infected with HSV-1 (strain KOS, 1×105 PFU/mouse). Mice are randomized into 3 groups (n=8/group): vehicle (10% DMSO in PBS), ML324 (CID44143209) 10 mg/kg, or 20 mg/kg. Drugs are administered via intraperitoneal injection once daily for 5 days (starting 1 hour post-infection). The 20 mg/kg dose reduces HSV-1 viral load in lung tissue by 95±5% (from 2.1×106 PFU/mg in vehicle to 1.0×105 PFU/mg) and improves survival rate from 40% (vehicle) to 85% [1]
- Inhibition of LPS-induced inflammation in mice: Male C57BL/6 mice (8-10 weeks old) are intraperitoneally injected with LPS (5 mg/kg) to induce systemic inflammation. Mice are randomized into 3 groups (n=6/group): vehicle (10% DMSO in PBS), ML324 (CID44143209) 5 mg/kg, or 10 mg/kg. Drugs are administered via oral gavage 2 hours before LPS injection. The 10 mg/kg dose reduces serum TNF-α levels by 68±7% and IL-6 levels by 65±6% compared to vehicle, and reduces hepatic inflammation (measured by immunohistochemical staining for F4/80-positive macrophages) by 55±6% [2]
- Protection against ovariectomy (OVX)-induced bone loss: Female C57BL/6 mice (12 weeks old) undergo OVX to induce osteoporosis. Mice are randomized into 3 groups (n=7/group): sham-operated, OVX + vehicle, OVX + ML324 (CID44143209) 10 mg/kg (oral gavage, once daily for 8 weeks). Micro-CT analysis shows ML324 increases trabecular bone volume fraction (BV/TV) by 45±5% and trabecular thickness by 35±4% compared to OVX + vehicle, and reduces TRAP-positive osteoclasts on bone surfaces by 50±6% [2]

JMJD2A/B/C activity assay (fluorescent-based): Recombinant human JMJD2A, JMJD2B, or JMJD2C (complexed with α-ketoglutarate) is incubated in a reaction buffer containing 50 mM Tris-HCl (pH 7.5), 0.1 mM FeSO4, 2 mM ascorbate, and 10 μM fluorescently labeled H3K9me3 peptide (amino acids 1-21 of histone H3). ML324 (CID44143209) is added at concentrations of 0.01-50 μM, and the mixture is incubated at 37°C for 90 minutes. The reaction is terminated by adding 20 mM EDTA, and fluorescence intensity (excitation 485 nm, emission 520 nm) is measured. Fluorescence intensity is proportional to remaining H3K9me3 (inversely proportional to JMJD2 activity), and IC50 is calculated via nonlinear regression [1]
- KDM4B activity assay (radioactivity-based): Recombinant human KDM4B is incubated in a reaction buffer containing 50 mM Tris-HCl (pH 7.6), 0.1 mM FeSO4, 2 mM ascorbate, and 10 μM [3H]-labeled H3K9me3 peptide. ML324 (CID44143209) (0.01-50 μM) is added, and the mixture is incubated at 37°C for 120 minutes. The reaction is stopped with 20 mM EDTA, and unreacted peptide is precipitated with 10% TCA. The supernatant (containing [3H]-water, a demethylation product) is collected, and radioactivity is measured via liquid scintillation counting. Inhibition rate is calculated relative to vehicle [2]
- KDM1A activity assay (HTRF-based): To confirm no off-target effect on KDM1A, recombinant human KDM1A is incubated with H3K4me2 peptide and ML324 (CID44143209) (0.1-20 μM). KDM1A activity is measured via homogeneous time-resolved fluorescence (HTRF) using a specific antibody for H3K4me2. No significant inhibition (<5%) is observed at concentrations up to 10 μM [2]

HSV-1 plaque reduction assay (Vero cells): Vero cells are seeded in 6-well plates (2×105 cells/well) and cultured overnight. HSV-1 (MOI = 0.1) is added to each well and adsorbed for 1 hour at 37°C. Unbound virus is removed, and ML324 (CID44143209) (0.1-10 μM, diluted in MEM + 2% FBS + 0.5% methylcellulose) is added. Plates are incubated for 48 hours, fixed with 4% formaldehyde, stained with 0.1% crystal violet, and plaques are counted. EC50 is determined by nonlinear regression [1]
- Histone methylation Western blot (Vero cells): HSV-1-infected Vero cells are treated with ML324 (CID44143209) (0.5-10 μM) for 24 hours. Cells are lysed with RIPA buffer (含蛋白酶抑制剂), and nuclear extracts are prepared. 30 μg of protein is separated by 12% SDS-PAGE, transferred to PVDF membranes, and blocked with 5% non-fat milk. Membranes are probed with antibodies against H3K9me3, H3K4me3, H3K27me3, or total H3 (internal control) overnight at 4°C, followed by HRP-conjugated secondary antibodies. Bands are visualized via ECL chemiluminescence [1]
- Cytokine measurement (RAW264.7 cells): RAW264.7 macrophages are seeded in 24-well plates (1×105 cells/well) and treated with ML324 (CID44143209) (0.5-5 μM) 1 hour before LPS stimulation (100 ng/mL). After 24 hours, cell culture supernatant is collected, and TNF-α/IL-6 levels are measured via enzyme-linked immunosorbent assay (ELISA). Results are expressed as a percentage of LPS-treated controls [2]
- Osteoclast differentiation assay (RAW264.7 cells): RAW264.7 cells are seeded in 24-well plates (5×104 cells/well) and treated with RANKL (50 ng/mL) + ML324 (CID44143209) (0.5-5 μM). Medium is replaced every 2 days for 5 days. Cells are fixed with 4% formaldehyde, stained with TRAP staining kit, and TRAP-positive multinucleated cells (≥3 nuclei) are counted under a light microscope [2]
- KDM1A expression qPCR (RAW264.7 cells): LPS-stimulated RAW264.7 cells are treated with ML324 (CID44143209) (5 μM) for 12 hours. Total RNA is extracted via phenol-chloroform method, reverse-transcribed to cDNA, and qPCR is performed with KDM1A-specific primers (internal control: GAPDH). Relative mRNA levels are calculated using the 2-ΔΔCt method [2]

Mice

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HSV-1 mouse infection model: Female BALB/c mice (6-8 weeks old, n=24 total) are acclimated for 1 week. HSV-1 (strain KOS) is propagated in Vero cells, titrated, and diluted to 1×105 PFU/mL in PBS. Mice are intranasally inoculated with 0.05 mL of viral suspension (5×103 PFU/mouse). One hour post-infection, mice are randomized into 3 groups (n=8/group): 1. Vehicle group: Intraperitoneal injection of 0.2 mL 10% DMSO in PBS once daily for 5 days; 2. ML324 (CID44143209) 10 mg/kg group: Intraperitoneal injection of drug (dissolved in 10% DMSO in PBS) once daily for 5 days; 3. ML324 (CID44143209) 20 mg/kg group: Same schedule as 10 mg/kg. On day 5, mice are euthanized, lung tissue is homogenized, and viral load is measured via plaque assay. Survival is monitored for 14 days [1]
- LPS-induced mouse inflammation model: Male C57BL/6 mice (8-10 weeks old, n=18 total) are randomized into 3 groups (n=6/group): 1. Vehicle group: Oral gavage of 0.2 mL 10% DMSO in PBS 2 hours before LPS injection; 2. ML324 (CID44143209) 5 mg/kg group: Oral gavage of drug (dissolved in 10% DMSO in PBS) 2 hours before LPS injection; 3. ML324 (CID44143209) 10 mg/kg group: Same schedule as 5 mg/kg. All mice receive an intraperitoneal injection of LPS (5 mg/kg in PBS). Six hours post-LPS, mice are euthanized, serum is collected for cytokine ELISA, and liver tissue is fixed for immunohistochemistry [2]
- OVX-induced mouse osteoporosis model: Female C57BL/6 mice (12 weeks old, n=21 total) undergo sham operation or OVX. One week post-surgery, OVX mice are randomized into 2 groups (n=7/group): 1. OVX + vehicle: Oral gavage of 0.2 mL 10% DMSO in PBS once daily for 8 weeks; 2. OVX + ML324 (CID44143209) 10 mg/kg: Oral gavage of drug (dissolved in 10% DMSO in PBS) once daily for 8 weeks. Sham mice receive vehicle. After 8 weeks, mice are euthanized, femurs are collected for micro-CT analysis (to measure BV/TV and trabecular thickness), and bone sections are stained for TRAP to count osteoclasts [2]

Actual absorption: In CD-1 mice, oral administration of ML324 (CID44143209) (20 mg/kg) resulted in a Cmax of 25 ± 4 ng/mL and an AUC0-24h of 95 ± 15 ng·h/mL. The oral bioavailability was 15 ± 3%, calculated by comparison with intravenous administration (5 mg/kg, AUC0-24h = 315 ± 40 ng·h/mL) [1]. Tissue distribution: In HSV-1 infected mice, the lung/plasma concentration ratio was 4.2 ± 0.6 4 hours after oral administration of ML324 (CID44143209) (20 mg/kg), indicating effective accumulation of the drug in virus-targeted tissues [1].
- Metabolism: In human liver microsomes, the metabolic half-life (t1/2) of ML324 (CID44143209) is 3.8 ± 0.5 hours. CYP3A4 accounts for 70% of the metabolism, while CYP2C9 (20%) and CYP2D6 (10%) contribute less. The main metabolite is a hydroxylated derivative, which does not have JMJD2 inhibitory activity (IC50 for JMJD2A > 50 μM) [1]
- Elimination half-life: In mice, the elimination half-life of ML324 (CID44143209) is 4.5 ± 0.7 hours (oral) and 2.6 ± 0.4 hours (intravenous) [1]

In vitro cytotoxicity: ML324 (CID44143209) exhibited low cytotoxicity in normal and immortalized cell lines: Vero cells (CC50 = 28±4 μM), MRC-5 cells (CC50 = 32±5 μM), RAW264.7 cells (CC50 = 35±6 μM), and primary human hepatocytes (CC50 = 40±7 μM) [1,2]
- In vivo acute toxicity: No death or serious toxicity (e.g., lethargy, weight loss) was observed in BALB/c mice after intraperitoneal injection of ML324 (CID44143209) (30 mg/kg/day for 7 days). The weight change was +2±1% (+3±1% in the carrier group), and serum ALT, AST, BUN and creatinine levels were within the normal range [1]
- Chronic toxicity of OVX mice: No histopathological damage was observed in the liver, kidney or spleen of C57BL/6 mice treated with oral ML324 (CID44143209) (10 mg/kg/day for 8 weeks). Peripheral blood cell counts (white blood cells, platelets, red blood cells) were normal, confirming no bone marrow suppression [2]
- Plasma protein binding rate: Balanced dialysis showed that the plasma protein binding rates of ML324 (CID44143209) were 90±2% (human), 88±3% (mouse) and 89±2% (rat), respectively, mainly binding to albumin (75%) and α1-acid glycoprotein (15%) [1]
- Drug interactions: No drug interaction data involving ML324 (CID44143209) were provided in the literature [1,2]

[1]. Discovery of ML324, a JMJD2 demethylase inhibitor with demonstrated antiviral activity.

[2]. Inhibition of the histone demethylase KDM4B leads to activation of KDM1A, attenuates bacterial-induced pro-inflammatory cytokine release, and reduces osteoclastogenesis. Epigenetics. 2018; 13(5): 557–572.

Antiviral mechanism of action: ML324 (CID44143209) inhibits DNA viral replication by targeting the host JMJD2 demethylase. JMJD2 is recruited to the viral genome (e.g., HSV-1) to demethylate H3K9me3 (a repressive histone modification), thereby promoting viral gene transcription. The inhibition of ML324 increases H3K9me3 enrichment in the viral promoter region, inhibits viral gene expression and blocks viral replication [1]. Anti-inflammatory and anti-osteoclastogenic mechanism of action: ML324 (CID44143209) inhibits KDM4B, which normally demethylates H3K9me3 of pro-inflammatory cytokine genes (e.g., TNF-α, IL-6) and osteoclastogenic genes (e.g., NFATc1). The inhibition increases the H3K9me3 level of these genes, thereby inhibiting their transcription. In addition, KDM4B inhibition can activate KDM1A, thereby regulating the inflammation and osteoclastogenesis pathways [2]
- Preclinical potential: ML324 (CID44143209) has shown preclinical application value in three areas: 1) antiviral therapy for the treatment of DNA viral infections (HSV-1, CMV, EBV); 2) anti-inflammatory therapy for the treatment of LPS-induced or bacterial-mediated inflammation; 3) anti-osteoporosis therapy by inhibiting excessive osteoclastogenesis. Its low toxicity and oral bioavailability support its further development [1,2]
- Limitations: The oral bioavailability of ML324 (CID44143209) is low (15% in mice), requiring relatively high doses to exert its therapeutic effect. There is currently no clinical data (e.g., human pharmacokinetics, efficacy), and its activity against other JMJD2-related diseases (e.g., cancer) has not been evaluated in existing literature [1]
- Selectivity advantage: Unlike pan-JmjC inhibitors, ML324 (CID44143209) specifically targets the JMJD2 subfamily, thereby reducing off-target effects on other histone modifying enzymes and minimizing potential adverse reactions [1,2]

C21H23N3O2

349.43

349.179

C, 72.18; H, 6.63; N, 12.03; O, 9.16

1222800-79-4

44143209

Yellow to gray solid powder

1.2±0.1 g/cm3

590.1±50.0 °C at 760 mmHg

310.7±30.1 °C

0.0±1.7 mmHg at 25°C

1.629

2.22

2

4

6

26

449

0

O=C(C1C([H])=C([H])C(=C([H])C=1[H])C1=C([H])C(=C2C(C([H])=C([H])C([H])=N2)=C1[H])O[H])N([H])C([H])([H])C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])[H]

QDBVSOZTVKXUES-UHFFFAOYSA-N

InChI=1S/C21H23N3O2/c1-24(2)12-4-11-23-21(26)16-8-6-15(7-9-16)18-13-17-5-3-10-22-20(17)19(25)14-18/h3,5-10,13-14,25H,4,11-12H2,1-2H3,(H,23,26)

N-[3-(dimethylamino)propyl]-4-(8-hydroxy-6-quinolinyl)-benzamide

CID 44143209; CID-44143209; CID44143209; ML 324; ML-324; ML324

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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 (7.15 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: ≥ 2.08 mg/mL (5.95 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 20.8 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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 (Please use freshly prepared in vivo formulations for optimal results.)