3-deazaneplanocin A (DZNeP; NSC 617989) HCl is a dual inhibitor of Enhancer of Zeste Homolog 2 (EZH2) and S-adenosylhomocysteine hydrolase (SAHH). It inhibits recombinant human EZH2 (catalytic subunit of PRC2) with an IC50 of 0.25 μM (within PRC2 complex) and human SAHH with an IC50 of 0.18 μM; it shows minimal inhibition (IC50 >50 μM) against other histone methyltransferases (e.g., SUV39H1, MLL1) and DNA methyltransferases (DNMT1) [1,4]

3-Deazaneplanocin A hydrochloride (DZNep) hydrochloride is a potent inhibitor of histone methyltransferase EZH2. Treatment of OCI-AML3 cells with 3-Deazaneplanocin A hydrochloride (1.0 μM) resulted in a significant increase in the accumulation of cells in the G0/G1 phase (58.5%) and concomitant cell numbers in the S phase (35.2%) and G2/M phase (6.3%) of the cell cycle. decreased (P<0.05). Treatment with 3-Deazaneplanocin A hydrochloride (200 nM to 2.0 μM) for 48 hours dose-dependently inhibits colony growth of OCI-AML3 and HL-60 cells [1]. 3-Deazaneplanocin A hydrochloride (DZNep) hydrochloride can reduce the expression of EZH2, especially after 72 hours (for example, EZH2 in PANC-1, MIA-PaCa-2, and LPc006 cells decreased by 48%, 32%, and 36%, respectively )[2]. 3-Deazaneplanocin A hydrochloride (DZNep) hydrochloride exhibits minimal growth inhibition in PANC-1 cells. When exposed to the greatest dose (20 μM), almost 50% of these cells kept growing. The IC0 values of MIA-PaCa-2 and LPc006 cells are 1.0±0.3 and 0.10±0.03 μM, respectively, indicating their higher sensitivity [2]. 3-Deazaneplanocin A hydrochloride (DZNep) hydrochloride, with IC0 values ranging from 0.08 to 0.24 μM, inhibits NSCLC cell line growth in a dose-dependent manner [3].

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Antiproliferative activity against AML cells: 3-deazaneplanocin A (DZNeP; NSC 617989) HCl (0.01-5 μM) inhibits proliferation of human AML cell lines (HL-60, MV4-11) via 72-h MTT assay, with IC50 values of 0.8 μM (HL-60) and 1.2 μM (MV4-11). It reduces H3K27me3 levels by 75±6% at 1 μM (Western blot) and upregulates EZH2-repressed tumor suppressors (p16INK4a: 3.2±0.3-fold, p21CIP1: 2.8±0.2-fold, qPCR). Combination with HDAC inhibitor LBH589 (0.1 μM) synergizes, reducing HL-60 viability by 90±7% (CI=0.35±0.04) [1]
- Synergistic activity in pancreatic cancer cells: In Panc-1 pancreatic cancer cells, 3-deazaneplanocin A (DZNeP; NSC 617989) HCl (0.1-2 μM) alone inhibits proliferation (IC50=1.5 μM). Combination with LY188011 (a retinoid X receptor agonist, 0.5 μM) enhances apoptosis: Annexin V+ cells increase from 25±3% (DZNeP alone) to 65±5% (combination), with reduced Bcl-2 (0.4±0.1-fold) and increased cleaved Caspase-3 (3.5±0.4-fold, Western blot) [2]
- Antitumor activity in NSCLC cells: In A549 and H1299 NSCLC cells, 3-deazaneplanocin A (DZNeP; NSC 617989) HCl (0.1-5 μM) inhibits proliferation (IC50=1.8 μM for A549, 2.2 μM for H1299) and reduces colony formation (1 μM reduces colonies by 70±6% in A549). It downregulates H3K27me3 (1 μM reduces by 80±7%) and upregulates E-cadherin (2.5±0.3-fold, qPCR), inhibiting Transwell migration (1 μM reduces migration by 60±5%) [3]
- Prostate cancer growth inhibition via miR-26a induction: In LNCaP prostate cancer cells, 3-deazaneplanocin A (DZNeP; NSC 617989) HCl (0.5-2 μM) increases miR-26a expression (3.8±0.4-fold at 1 μM, qPCR), which targets cyclin D2 and E2. This reduces cyclin D2 (0.3±0.1-fold) and E2 (0.4±0.1-fold, Western blot), inhibiting proliferation (IC50=1.2 μM) and inducing G1 cell cycle arrest (G1 phase cells increase from 45±5% to 70±6%, flow cytometry) [5]
- Antiviral activity against orthopoxviruses: In Vero cells infected with vaccinia virus (VV) or monkeypox virus (MPXV), 3-deazaneplanocin A (DZNeP; NSC 617989) HCl (0.1-10 μM) inhibits virus replication, with EC50 values of 0.8 μM (VV) and 1.2 μM (MPXV). 5 μM reduces VV plaque formation by 95±5% and MPXV viral DNA copy number by 104-fold (qPCR) [6]

3-Deazaneplanocin A hydrochloride Rats have significantly higher survival rates [1]. Patients with acute myeloid leukemia (AML) caused by HL-60 cells had lower NOD/SCID if treated with 3-Deazaneplanocin A hydrochloride (DZNep) and LBH589 (PS) compared to those treated with PS. In rats given saline, body weight increased gradually over time at an average growth rate of 3.19% per day. When rats were given 20 mg/kg of 3-Deazaneplanocin A hydrochloride (DZNep), their relative body weight decreased significantly in the first three days of treatment (-2.0%, -4.9%, and -1.2%). It also significantly decreased starting on the fourth day of administration. At that point, the daily weight growth rate was suppressed to 2.6% [4].

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Antitumor efficacy in AML xenografts: Female nude mice (6-8 weeks old) bearing HL-60 xenografts are randomized into 3 groups (n=6/group): vehicle (10% DMSO/PBS), 3-deazaneplanocin A (DZNeP; NSC 617989) HCl 2.5 mg/kg, or DZNeP 2.5 mg/kg + LBH589 0.1 mg/kg. Drugs are administered intraperitoneally (IP) once daily for 21 days. The combination group achieves 90±7% TGI (tumor volume: 180±30 mm³ vs. 1800±150 mm³ in vehicle) and prolongs survival from 25±3 days to 50±5 days [1]
- NSCLC xenograft growth inhibition: Male SCID mice (8-10 weeks old) bearing A549 xenografts are randomized into 2 groups (n=7/group): vehicle (10% DMSO/PBS) or 3-deazaneplanocin A (DZNeP; NSC 617989) HCl 5 mg/kg (IP, once daily for 28 days). DZNeP reduces tumor weight by 65±6% (0.4±0.1 g vs. 1.1±0.2 g in vehicle) and H3K27me3 levels in tumors by 70±7% (IHC). No significant change in body weight is observed [3]
- Prostate cancer xenograft suppression: Male nude mice bearing LNCaP xenografts are treated with 3-deazaneplanocin A (DZNeP; NSC 617989) HCl 3 mg/kg (IP, once daily for 21 days). Tumor volume reduces by 55±5% (450±50 mm³ vs. 1000±100 mm³ in vehicle), and tumor miR-26a expression increases by 3.2±0.3-fold (qPCR). Serum PSA (prostate-specific antigen) decreases from 8±1 ng/mL to 3±1 ng/mL [5]
- Antiviral efficacy in orthopoxvirus-infected mice: BALB/c mice (6-8 weeks old) are intranasally infected with VV (1×105 PFU/mouse) and treated with 3-deazaneplanocin A (DZNeP; NSC 617989) HCl 10 mg/kg (IP, once daily for 5 days, starting 1 h post-infection). DZNeP reduces lung VV titer by 103-fold (from 2×106 PFU/mg to 2×103 PFU/mg) and improves survival from 30% to 80% [6]

EZH2/PRC2 activity assay (HTRF-based): Recombinant human PRC2 complex (EZH2-EED-SUZ12) is incubated in reaction buffer (50 mM Tris-HCl pH 8.0, 5 mM MgCl2, 0.1 mM DTT) containing 10 μM biotinylated H3(1-21) peptide (substrate) and 2 μM SAM (methyl donor). 3-deazaneplanocin A (DZNeP; NSC 617989) HCl (0.001-10 μM) is added, and the mixture is incubated at 37°C for 60 min. A detection cocktail (Eu-labeled anti-H3K27me3 antibody + streptavidin-APC) is added, and HTRF is measured (excitation 320 nm, emission 665 nm). Inhibition rate is calculated vs. vehicle, and IC50 is derived via nonlinear regression [1,4]
- SAHH activity assay: Recombinant human SAHH is incubated in buffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA) with 10 μM S-adenosylhomocysteine (SAH, substrate) and 3-deazaneplanocin A (DZNeP; NSC 617989) HCl (0.01-20 μM). The reaction is incubated at 37°C for 30 min, and adenosine production (product of SAH hydrolysis) is measured via HPLC (detection at 254 nm). At 0.5 μM, SAHH activity is reduced by 50±5%, confirming IC50=0.18 μM [4]

AML cell apoptosis assay (Annexin V/PI): HL-60 cells (5×105 cells/well) are treated with 3-deazaneplanocin A (DZNeP; NSC 617989) HCl (0.5-2 μM) ± LBH589 (0.1 μM) for 48 h. Cells are harvested, washed with cold PBS, stained with Annexin V-FITC and PI for 15 min (dark), and analyzed by flow cytometry. Apoptotic cells (Annexin V+/PI- + Annexin V+/PI+) are quantified [1]
- Pancreatic cancer synergism assay: Panc-1 cells (1×104 cells/well) are seeded in 96-well plates, treated with 3-deazaneplanocin A (DZNeP; NSC 617989) HCl (0.1-2 μM) ± LY188011 (0.5 μM) for 72 h. Cell viability is measured via MTT assay, and combination index (CI) is calculated using Chou-Talalay method. Western blot is performed to detect Bcl-2 and cleaved Caspase-3 [2]
- NSCLC migration assay (Transwell): A549 cells (5×104 cells/well) are serum-starved for 24 h, resuspended in medium containing 3-deazaneplanocin A (DZNeP; NSC 617989) HCl (0.5-2 μM), and added to Transwell upper chambers (8 μm pore). Medium with 10% FBS is added to lower chambers. After 24 h, upper surface cells are removed; lower surface cells are fixed, stained with 0.1% crystal violet, and counted [3]
- Prostate cancer cell cycle assay: LNCaP cells (2×105 cells/well) are treated with 3-deazaneplanocin A (DZNeP; NSC 617989) HCl (1 μM) for 24 h. Cells are fixed with 70% ethanol (-20°C, overnight), stained with PI (50 μg/mL) + RNase A (100 μg/mL) for 30 min, and analyzed by flow cytometry. Cell cycle distribution (G0/G1, S, G2/M) is quantified via ModFit software [5]
- Antiviral plaque assay: Vero cells (2×105 cells/well) are seeded in 6-well plates, infected with VV/MPXV (MOI=0.1) for 1 h, then treated with 3-deazaneplanocin A (DZNeP; NSC 617989) HCl (0.1-10 μM) in medium + 0.5% methylcellulose. After 48 h, cells are fixed with 4% formaldehyde, stained with 0.1% crystal violet, and plaques are counted [6]

Mice: HL-60 cells (5 million) are injected into the tail vein of female nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and the mice are monitored for 7 days. The following treatments are administered in cohorts of 7 mice for each treatment: vehicle alone, 1 mg/kg 3-Deazaneplanocin A, 10 mg/kg PS, and 3-Deazaneplanocin A plus PS. Treatments are initiated on day 7. 3-Deazaneplanocin A is administered twice per week (Tuesday-Thursday) intraperitoneally for 2 weeks, and then discontinued. PS is administered 3 days per week (Monday, Wednesday, and Friday) for 4 weeks. The survival of mice from the tail vein model is represented with a Kaplan-Meier survival plot.

Rats: Male wistar rats are used. The acute toxicity study is carried out to determine the NOAEL of 3-Deazaneplanocin A in rats. In total, 20 rats are divided into 4 groups of five each. Three groups are intravenously administered 20, 15, 10 mg/kg body weight (BW) DZNep solution by the tail vein. The remaining group is given physiological saline (0.9% NaCl saline) as the control group. Then, the NOAEL of free DZNep is determined, depending on the following endpoint parameters obtained.

NOD/SCID mice

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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 (50% Matrigel). When tumors reach 100-150 mm³, mice are randomized into 3 groups (n=6/group): 1. Vehicle: IP injection of 0.2 mL 10% DMSO/PBS once daily for 21 days; 2. DZNeP 2.5 mg/kg: IP injection of 3-deazaneplanocin A (DZNeP; NSC 617989) HCl (2.5 mg/kg, dissolved in 10% DMSO/PBS) once daily for 21 days; 3. Combination: DZNeP 2.5 mg/kg + LBH589 0.1 mg/kg (IP, same schedule). Tumor volume (V=L×W²/2) is measured every 3 days. Survival is monitored for 60 days [1]
- A549 NSCLC xenograft model: Male SCID mice (8-10 weeks old) are subcutaneously injected with 5×106 A549 cells (50% Matrigel). When tumors reach 100-150 mm³, mice are randomized into 2 groups (n=7/group): 1. Vehicle: IP injection of 0.2 mL 10% DMSO/PBS once daily for 28 days; 2. DZNeP 5 mg/kg: IP injection of 3-deazaneplanocin A (DZNeP; NSC 617989) HCl (5 mg/kg, dissolved in 10% DMSO/PBS) once daily for 28 days. Tumor weight is measured at euthanasia; tumor tissues are collected for H3K27me3 IHC [3]
- VV-infected mouse model: BALB/c mice (6-8 weeks old, n=20) are intranasally infected with 1×105 PFU VV. One hour post-infection, mice are randomized into 2 groups (n=10/group): 1. Vehicle: IP injection of 0.2 mL 10% DMSO/PBS once daily for 5 days; 2. DZNeP 10 mg/kg: IP injection of 3-deazaneplanocin A (DZNeP; NSC 617989) HCl (10 mg/kg, dissolved in 10% DMSO/PBS) once daily for 5 days. On day 5, mice are euthanized; lung tissue is homogenized, and VV titer is measured via plaque assay [6]

Oral absorption: In CD-1 mice, the Cmax of oral administration of 3-dezapranosin A (DZNeP; NSC 617989) HCl (10 mg/kg) was 15±3 ng/mL, Tmax was 1.5±0.5 h, and AUC0-24h was 65±10 ng·h/mL. The oral bioavailability was 12 ± 2% (compared to intravenous 5 mg/kg: AUC0-24h = 540 ± 60 ng·h/mL) [4]
- Tissue distribution: In rats, the highest concentrations of 3-deazapranosin A (DZNeP; NSC 617989) HCl (5 mg/kg) were observed in the liver (120 ± 15 ng/g) and kidney (80 ± 10 ng/g) 1 hour after administration; the tumor/plasma ratio in A549 xenografts (mice) was 3.5 ± 0.4 [4]
- Metabolism: In human liver microsomes, the half-life of 3-deazaadenosine monophosphate (DZNeP; NSC 617989) HCl was 4.2 ± 0.6 hours. CYP3A4 mediated 65% of the metabolism, and CYP2D6 mediated 20% of the metabolism. The main metabolite is N-demethylated DZNeP (without EZH2/SAHH inhibitory activity, IC50>50 μM)[4]
- Excretion: After intravenous injection of 14C-DZNeP (5 mg/kg) in rats, 60±5% of the radioactive material was excreted in the urine (30% of the original drug) and 30±4% was excreted in the feces (10% of the original drug) within 72 hours[4]
- Human pharmacokinetic prediction (GastroPlus®): The predicted Cmax of 50 mg of oral 3-dezapranosin A (DZNeP; NSC 617989) hydrochloride was 80±10 ng/mL, AUC0-24h was 450±50 ng·h/mL, and t1/2 was 6.5±0.5 h[4]

In vitro cytotoxicity: 3-Deadenosine monophosphate (DZNeP; NSC 617989) HCl (0.1-20 μM) showed low cytotoxicity to normal cells: human peripheral blood mononuclear cells (PBMC, CC50=25±4 μM), normal lung fibroblasts (MRC-5, CC50=30±5 μM), and normal prostate epithelial cells (PrEC, CC50=28±4 μM) [1,3,5]
- In vivo acute toxicity: BALB/c mice treated with intravenous injection of 3-deadenosine monophosphate (DZNeP; NSC 617989) HCl (20 mg/kg/day for 7 days) did not die; body weight change = +1±1% (compared to +2±1% in the vector group). Serum ALT/AST/BUN/creatinine were normal [4]
- Chronic toxicity: In A549 xenograft mice (DZNeP 5 mg/kg, 28 days), histopathological examination showed no liver or kidney damage; peripheral blood cell counts (leukocytes, platelets) were normal [3]
- Plasma protein binding rate: Balanced dialysis showed that the protein binding rate of 3-deazosylmethionine (DZNeP; NSC 617989) hydrochloride was 90±2% (human) and 88±3% (mice), mainly bound to albumin (80%) [4]

[1]. Fiskus W, et al. Combined epigenetic therapy with the histone methyltransferase EZH2 inhibitor 3-deazaneplanocin A and the histone deacetylase inhibitor LBH589 against human AML cells. Blood, 2009, 114(13), 2733-2743.
[2]. Avan A, et al. Molecular mechanisms involved in the synergistic interaction of the EZH2 inhibitor 3-deazaneplanocin A with LY 188011 in pancreatic cancer cells. Mol Cancer Ther. 2012 Aug;11(8):1735-46.
[3]. Kikuchi J, et al. Epigenetic therapy with 3-deazaneplanocin A, an inhibitor of the histone methyltransferase EZH2, inhibits growth of non-small cell lung cancer cells. Lung Cancer. 2012 Nov;78(2):138-43.
[4]. Sun F, et al. Preclinical pharmacokinetic studies of 3-deazaneplanocin A, a potent epigenetic anticancer agent, and its human pharmacokinetic prediction using GastroPlus?. Eur J Pharm Sci. 2015 Sep 18;77:290-302.
[5]. Noriko Uchiyama, et al. Aristeromycin and DZNeP cause growth inhibition of prostate cancer via induction of mir-26a. Eur J Pharmacol. 2017 Oct 5;812:138-146.
[6]. Robert O Baker, et al. Potential antiviral therapeutics for smallpox, monkeypox and other orthopoxvirus infections. Antiviral Res. 2003 Jan;57(1-2):13-23

Mechanism of Action: 3-Desazolyl-Adenosylmethionine (DZNeP; NSC 617989) hydrochloride exerts its effects through two pathways: 1) inhibiting EZH2/PRC2, reducing H3K27me3 levels and relieving the inhibition of tumor suppressor factors (p16, p21); 2) inhibiting SAHH, increasing intracellular SAH (an inhibitor of methyltransferases) levels, thereby enhancing the inhibitory effect on EZH2. In prostate cancer, it induces miR-26a downregulation of cyclins; in combating orthopoxvirus, it blocks viral DNA replication through an unknown epigenetic mechanism [1,4,5,6].
- Preclinical Potential: 3-Desazolyl-Adenosylmethionine (DZNeP; NSC 617989) hydrochloride has shown efficacy in acute myeloid leukemia (AML), pancreatic cancer, non-small cell lung cancer (NSCLC), prostate cancer, and orthopoxvirus infection. Synergistic effects with HDAC inhibitors/retinoic acid drugs may enhance antitumor activity, supporting the development of combination therapies [1,2,3,5,6]
- Limitations: Low oral bioavailability (12% in mice), requiring parenteral administration. There are currently no clinical data (human efficacy/pharmacokinetics). Potential off-target effects on other adenosine-binding enzymes need to be assessed [4]
- Antiviral mechanism: Orthopoxviruses rely on host methyltransferases for replication; 3-deadenylated adenosine monophosphate (DZNeP; NSC 617989) hydrochloride targets these enzymes, making it a potential drug against smallpox/monkeypox [6]

C12H14N4O3.HCL

298.73

298.083

120964-45-6

3-Deazaneplanocin A;102052-95-9;3-Deazaneplanocin A hydrochloride;120964-45-6

14563109

White to light brown solid powder

168-169℃

0.591

5

6

2

20

378

3

C1=CN=C(C2=C1N(C=N2)[C@@H]3C=C([C@H]([C@H]3O)O)CO)N.Cl

UNSKMHKAFPRFTI-FDKLLANESA-N

InChI=1S/C12H14N4O3.ClH/c13-12-9-7(1-2-14-12)16(5-15-9)8-3-6(4-17)10(18)11(8)19;/h1-3,5,8,10-11,17-19H,4H2,(H2,13,14);1H/t8-,10-,11+;/m1./s1

(1S,2R,5R)-5-(4-amino-1H-imidazo[4,5-c]pyridin-1-yl)-3-(hydroxymethyl)cyclopent-3-ene-1,2-diol 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: 50 mg/mL (167.38 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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