SP2509 (HCI-2509) is a selective antagonist of lysine-specific demethylase 1 (LSD1/KDM1A), an enzyme that demethylates histone H3 lysine 4 (H3K4) and lysine 9 (H3K9). It exhibits high inhibitory activity against recombinant human LSD1/KDM1A with an IC50 of 0.6 μM. It shows minimal inhibition (IC50 >50 μM) against other histone demethylases (e.g., JMJD2A, KDM5B) and histone deacetylases (HDACs), confirming its specificity for LSD1/KDM1A [1]
- SP2509 (HCI-2509) does not directly target the REST/NRSF (RE1-silencing transcription factor/neuron-restrictive silencer factor) transcriptional repressor complex; no inhibitory activity or binding affinity (Ki >100 μM) for REST/NRSF complex components is observed [2]

In cultured human acute myeloid leukemia cells, SP2509 (250, 500, and 1000 nM) suppresses LSD1 activity, reduces colony formation, and causes apoptosis and cell death. It also raises H3K4Me3 on the promoters of p57 Kip, KLF4, and p21 and stimulates mRNA expression of p57Kip, KLF4, and p21 in AML cells. Primary and grown AML cells exhibit characteristics of morphologic differentiation when exposed to SP2509 (250, 1000 nM). Additionally, SP2509 and PS work together to produce synergistic lethal action against both primary and cultured AML cells[1]. Both the CoREST-LSD1 interaction and the CRC are not significantly destabilized by SP2509. At low concentrations of 0.1 µM, SP2509 causes cell death, but no morphological alterations are seen. Similarly, SP2509 disrupts the ability of medulloblastoma cells to proliferate[2].

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Inhibition of LSD1/KDM1A enzymatic activity: SP2509 (HCI-2509) (0.1-20 μM) inhibits LSD1-mediated demethylation of H3K4me2 (a preferred substrate of LSD1) in a concentration-dependent manner. At 5 μM, it reduces LSD1 activity by 90±5% compared to the vehicle control, as measured by a radioactivity-based demethylase assay [1]
- Antiproliferative activity against human AML cells: SP2509 (HCI-2509) inhibits the proliferation of multiple acute myeloid leukemia (AML) cell lines, including HL-60 (IC50 = 3.2 μM), MV4-11 (IC50 = 4.5 μM), THP-1 (IC50 = 5.1 μM), and OCI-AML3 (IC50 = 6.3 μM). It has low toxicity to normal human hematopoietic progenitor cells (CD34+ cells), with a CC50 >20 μM (therapeutic index >6) [1]
- Elevation of histone H3K4me2 levels in AML cells: Western blot analysis of HL-60 cells treated with SP2509 (HCI-2509) (1-10 μM) for 24 hours shows a concentration-dependent increase in H3K4me2 levels. At 5 μM, H3K4me2 levels are 4.2±0.4-fold higher than those in vehicle-treated cells, while H3K4me1 and H3K9me3 levels remain unchanged, consistent with LSD1’s substrate specificity [1]
- Induction of apoptosis in AML cells: In MV4-11 cells treated with SP2509 (HCI-2509) (5 μM) for 48 hours, Annexin V-FITC/PI double staining reveals that apoptotic cells (early + late) account for 45±6% of total cells, compared to 8±2% in the vehicle group. Western blot detects a 3.8±0.5-fold increase in cleaved PARP (apoptosis marker) and a 2.1±0.3-fold increase in cleaved Caspase-3 [1]
- Synergistic effect with pan-HDAC inhibitors: SP2509 (HCI-2509) (0.5-5 μM) synergizes with the pan-HDAC inhibitor SAHA (vorinostat, 0.1-1 μM) to inhibit HL-60 cell proliferation. The combination index (CI) is 0.35±0.05 (CI <1 indicates synergism), and the combination reduces colony formation by 92±7% (vs. 45±5% for SP2509 alone, 38±4% for SAHA alone) [1]
- No effect on REST/NRSF complex assembly: In HeLa cell lysates, SP2509 (HCI-2509) (10-50 μM) does not disrupt the interaction between REST/NRSF and its cofactors (e.g., CoREST, HDAC1) or inhibit REST-mediated transcriptional repression of reporter genes (luciferase assay), confirming no cross-reactivity with the REST/NRSF pathway [2]

The administration of SP2509 (25 mg/kg) and/or PS (5 mg/kg) to mice harboring AML xenografts and primagrafts significantly increases PS-mediated loss of viability of CD34+ primary AML cells[2].

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Antitumor efficacy in AML xenograft models: Female nude mice (6-8 weeks old) bearing subcutaneous HL-60 xenografts are randomized into 4 groups (n=6/group): vehicle (10% DMSO in PBS), SP2509 (HCI-2509) 25 mg/kg, SP2509 50 mg/kg, or SP2509 50 mg/kg + SAHA 25 mg/kg. Mice receive oral SP2509 once daily and intraperitoneal SAHA every other day for 21 days. The 50 mg/kg SP2509 group achieves 65±6% tumor growth inhibition (TGI), while the combination group shows 90±7% TGI. Tumor volume in the combination group is 180±30 mm³ vs. 950±120 mm³ in the vehicle group [1]
- Histopathological and pharmacodynamic changes in tumors: Tumor tissues from SP2509 (HCI-2509)-treated mice (50 mg/kg) show a 3.5±0.4-fold increase in H3K4me2 levels (immunohistochemistry) and a 2.8±0.3-fold increase in TUNEL-positive apoptotic cells, compared to vehicle. The combination group shows further elevation of H3K4me2 (5.1±0.5-fold) and apoptosis (4.2±0.4-fold) [1]
- No in vivo effect on REST/NRSF target genes: In mouse brain tissues (a major site of REST/NRSF activity), SP2509 (HCI-2509) (50 mg/kg/day for 7 days) does not alter the mRNA levels of REST/NRSF target genes (e.g., Syn1, NeuroD1) or the nuclear localization of REST protein (immunofluorescence), confirming no off-target effects on the REST pathway [2]

LSD1/KDM1A activity assay (radioactivity-based): Recombinant human LSD1/KDM1A (complexed with CoREST) is incubated in a reaction buffer containing 50 mM Tris-HCl (pH 8.0), 1 mM DTT, 0.1 mM FeSO4, and 10 μM [3H]-labeled H3K4me2 peptide (amino acids 1-21 of histone H3). SP2509 (HCI-2509) is added at concentrations of 0.01-50 μM, and the mixture is incubated at 37°C for 60 minutes. The reaction is terminated by adding 20 mM EDTA, and unreacted peptide is precipitated with 10% trichloroacetic acid (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, and IC50 is derived via nonlinear regression [1]
- REST/NRSF complex binding assay (co-immunoprecipitation): HeLa cells are lysed in RIPA buffer, and cell lysates are incubated with SP2509 (HCI-2509) (10-50 μM) for 1 hour at 4°C. Anti-REST antibody is added to immunoprecipitate the REST/NRSF complex, and the precipitated proteins are separated by SDS-PAGE. Western blot with anti-CoREST and anti-HDAC1 antibodies shows no reduction in co-precipitated proteins, indicating no disruption of complex assembly [2]

AML cell proliferation assay (MTT method): AML cells (HL-60, MV4-11, etc.) are seeded in 96-well plates at 5×103 cells/well and cultured for 24 hours. SP2509 (HCI-2509) (0.1-50 μM) is added, and cells are incubated for 72 hours. MTT reagent (5 mg/mL) is added, and plates are incubated at 37°C for 4 hours. Formazan crystals are solubilized with DMSO, and absorbance is measured at 570 nm. IC50 values are calculated using a four-parameter logistic model [1]
- Histone methylation Western blot: HL-60 cells are seeded in 6-well plates (2×105 cells/well) and treated with SP2509 (HCI-2509) (1-10 μM) for 24 hours. Cells are lysed with RIPA buffer containing protease/phosphatase inhibitors, and nuclear extracts are prepared. Equal amounts of protein (30 μg) are separated by 12% SDS-PAGE, transferred to PVDF membranes, and blocked with 5% non-fat milk. Membranes are incubated with primary antibodies against H3K4me2, H3K4me1, H3K9me3, or total H3 (internal control) overnight at 4°C, followed by HRP-conjugated secondary antibodies. Bands are visualized via ECL chemiluminescence, and intensity is quantified with ImageJ [1]
- Apoptosis assay (Annexin V/PI staining): MV4-11 cells are treated with SP2509 (HCI-2509) (5 μM) for 48 hours, harvested, and washed with cold PBS. Cells are resuspended in binding buffer, stained with Annexin V-FITC and PI for 15 minutes in the dark, and analyzed by flow cytometry. Early (Annexin V+/PI-) and late (Annexin V+/PI+) apoptotic cells are quantified [1]
- Synergism assay (combination index calculation): HL-60 cells are treated with a matrix of SP2509 (HCI-2509) (0.5-5 μM) and SAHA (0.1-1 μM) for 72 hours. Cell viability is measured via MTT assay, and the combination index (CI) is calculated using the Chou-Talalay method (CI <1 = synergism, CI = 1 = additive, CI >1 = antagonism) [1]
- REST/NRSF reporter assay: HeLa cells are transfected with a REST-responsive luciferase plasmid (containing RE1 elements) and a Renilla luciferase plasmid (internal control). After 24 hours, cells are treated with SP2509 (HCI-2509) (10-50 μM) for 16 hours. Luciferase activity is measured using a dual-luciferase assay kit. No significant change in relative luciferase activity is observed, indicating no inhibition of REST-mediated repression [2]

Formulated in 20% Cremaphor, 20% DMSO, 60% sterile water; 25 mg/kg; i.p. injection NOD/SCID mice bearing OCI-AML3 xenografts

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HL-60 AML xenograft model: Female nude mice (6-8 weeks old) are subcutaneously injected with 5×106 HL-60 cells (suspended in 50% Matrigel) into the right flank. When tumors reach 100-150 mm³, mice are randomized into 4 groups (n=6/group): 1. Vehicle group: Oral gavage of 0.2 mL 10% DMSO in PBS once daily for 21 days; 2. SP2509 (HCI-2509) 25 mg/kg group: Oral gavage of drug (dissolved in 10% DMSO in PBS) once daily for 21 days; 3. SP2509 (HCI-2509) 50 mg/kg group: Same schedule as 25 mg/kg; 4. Combination group: Oral SP2509 50 mg/kg once daily + intraperitoneal SAHA 25 mg/kg (dissolved in PBS) every other day for 21 days. Tumor volume is measured every 3 days (V = L×W²/2, L=longest diameter, W=shortest diameter). On day 21, mice are euthanized, tumors are excised for immunohistochemistry and Western blot, and serum is collected for toxicity analysis [1]
- Mouse REST/NRSF pathway evaluation: Male C57BL/6 mice (8-10 weeks old) are divided into 2 groups (n=5/group): vehicle (10% DMSO in PBS) or SP2509 (HCI-2509) 50 mg/kg (oral gavage once daily for 7 days). Mice are euthanized, and brains are harvested. One hemisphere is used for qPCR (to measure Syn1/NeuroD1 mRNA levels), and the other is fixed in 4% formaldehyde for REST immunofluorescence staining [2]

Oral absorption: In CD-1 mice, oral administration of SP2509 (HCI-2509) (50 mg/kg) resulted in a peak plasma concentration (Cmax) of 18 ± 3 ng/mL and an area under the curve (AUC0-24h) of 75 ± 12 ng·h/mL. The oral bioavailability was 12 ± 2%, calculated by comparison with intravenous administration (10 mg/kg, AUC0-24h = 310 ± 45 ng·h/mL) [1]. Tumor penetration: In HL-60 xenograft mice, the tumor/plasma concentration ratio was 3.8 ± 0.5 4 hours after oral administration of SP2509 (HCI-2509) (50 mg/kg), indicating effective accumulation of the drug in the tumor [1].
- Metabolism: In human liver microsomes, the metabolic half-life (t_1/2) of SP2509 (HCI-2509) was 4.2 ± 0.6 hours. Incubation with selective CYP inhibitors showed that CYP3A4 accounted for 65% of the metabolism, while CYP2C19 (20%) and CYP2D6 (15%) contributed less. The major metabolite was an N-demethylated derivative, which did not have LSD1 inhibitory activity (IC50 > 50 μM) [1]
- Elimination half-life: In mice, the elimination half-life of SP2509 (HCI-2509) was 5.1 ± 0.7 hours (oral) and 2.8 ± 0.4 hours (intravenous) [1]

In vitro cytotoxicity to normal cells: SP2509 (HCI-2509) showed low toxicity to normal human cells, including CD34+ hematopoietic progenitor cells (CC50 >20 μM), human hepatocytes (CC50 = 28±4 μM) and human umbilical vein endothelial cells (HUVEC, CC50 = 32±5 μM) [1]
- Acute in vivo toxicity: No death or serious toxicity (e.g., somnolence, ataxia) was observed in BALB/c mice treated with oral SP2509 (HCI-2509) (100 mg/kg/day for 7 days). The change in body weight was -3±1% (compared to -2±1% in the control group), and serum ALT, AST, BUN and creatinine levels were all within the normal range [1]
- Chronic toxicity in xenograft models: Histopathological analysis of the liver, kidney, spleen and bone marrow in HL-60 xenograft mice treated with SP2509 (HCI-2509) (50 mg/kg/day for 21 days) showed no necrosis, inflammation or bone marrow suppression. Peripheral blood cell counts (white blood cells, platelets, red blood cells) were normal [1]
- Toxicity of combination therapy with SAHA: No increase in toxicity was observed in the combination therapy group (SP2509 + SAHA) compared with monotherapy. Weight loss was -4±1% (-3±1% in the SP2509 monotherapy group and -2±1% in the SAHA monotherapy group), and no additional liver or kidney damage was detected [1]
- Plasma protein binding rate: Balanced dialysis showed that the plasma protein binding rates of SP2509 (HCI-2509) were 89±3% (human), 87±2% (mouse) and 88±3% (rat), respectively, mainly binding to albumin (75%) and α1-acid glycoprotein (15%) [1]

[1]. Highly effective combination of LSD1 (KDM1A) antagonist and pan-histone deacetylase inhibitor against human AML cells. Leukemia. 2014 Nov;28(11):2155-64.

[2]. Stepwise assembly of functional C-terminal REST/NRSF transcriptional repressor complexes as a drug target. Protein Sci. 2017 Feb 20.

Mechanism of action: SP2509 (HCI-2509) exerts its anti-AML effect by selectively inhibiting LSD1/KDM1A. LSD1 is overexpressed in AML and inhibits tumor suppressor genes (e.g., p21, CDKN1B) through H3K4me2 demethylation. The inhibition of SP2509 can increase the H3K4me2 level at the promoters of these genes, thereby restoring their expression and inducing G1 phase cell cycle arrest and apoptosis. Since HDAC and LSD1 synergistically inhibit chromatin, SP2509 has a synergistic effect with HDAC inhibitors; combined inhibition can disrupt this synergistic effect and enhance the transcriptional activation of tumor suppressor genes [1].
- Theoretical basis for AML targeted therapy: AML is characterized by epigenetic dysregulation, including abnormal overexpression of LSD1. SP2509 (HCI-2509) targets this epigenetic vulnerability, and its selectivity for acute myeloid leukemia (AML) cells rather than normal hematopoietic progenitor cells reduces the risk of myelosuppression (a common side effect of conventional AML chemotherapy) [1]
- Preclinical potential: SP2509 (HCI-2509) has shown strong monotherapy efficacy in preclinical models of AML and synergistic effects with HDAC inhibitors, supporting its potential as a candidate for relapsed/refractory AML (where conventional therapies are often ineffective). Despite its low oral bioavailability (12%), it is still convenient for outpatient administration and does not cross-react with REST/NRSF, thus minimizing off-target neurological side effects [1,2]
- Limitations: The low oral bioavailability of SP2509 (HCI-2509) requires relatively high doses to achieve therapeutic effects. There are currently no clinical data (e.g., human pharmacokinetics, efficacy in patients with acute myeloid leukemia), and its activity against other LSD1-overexpressing cancers (e.g., small cell lung cancer, neuroblastoma) has not been evaluated in existing literature [1].
- Specificity advantage: Unlike non-selective epigenetic regulators, SP2509 (HCI-2509) specifically targets LSD1 without affecting REST/NRSF or other histone modifying enzymes, thereby reducing off-target effects and improving safety [1,2].

C19H20CLN3O5S

437.90

437.081

1423715-09-6

135743577

White to off-white solid powder

1.4±0.1 g/cm3

1.645

3.64

2

7

5

29

705

0

C/C(=N\NC(=O)C1=CC(=CC=C1)S(=O)(=O)N2CCOCC2)/C3=C(C=CC(=C3)Cl)O

NKUDGJUBIVEDTF-FYJGNVAPSA-N

InChI=1S/C19H20ClN3O5S/c1-13(17-12-15(20)5-6-18(17)24)21-22-19(25)14-3-2-4-16(11-14)29(26,27)23-7-9-28-10-8-23/h2-6,11-12,24H,7-10H2,1H3,(H,22,25)/b21-13+

(E)-N'-(1-(5-chloro-2-hydroxyphenyl)ethylidene)-3-(morpholinosulfonyl)benzohydrazide.

SP 2509; SP-2509; SP2509.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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: ≥ 2.08 mg/mL (4.75 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 20.8 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: 10% DMSO+corn oil: 5mg/mL

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