DNA methyltransferases (DNMTs) (IC₅₀ = ~1.5 μM for recombinant human DNMT1; IC₅₀ = ~2.0 μM for DNMT3a; no significant inhibition of DNMT3b at concentrations ≤ 10 μM) [1]
- Cytidine deaminase (CDA) (Ki = ~0.8 μM; competitive inhibitor with respect to cytidine, no inhibition of other nucleoside deaminases (e.g., adenosine deaminase) with Ki > 20 μM) [4]

ZebuLarine accomplishes its demethylating effect by stabilizing DNMT binding to DNA, inhibiting methylation and minimizing dissociation, so trapping the enzyme and preventing turnover even at other locations. zebuLarine improves the chemo- and radiosensitivity of tumor cells and possesses antimitotic and vasculostatic action [1]. ZebuLarine inhibits DNA methylation and reactivates genes previously silenced by methylation. ZebuLarine generates a myogenic phenotype in 10T1/2 cells, a feature unique to DNA methylation inhibitors. ZebuLarine reactivates the silenced p16 gene and demethylates its promoter region in T24 bladder cancer cells [2]. ZebuLarine administration suppressed cell proliferation in a dose- and time-dependent manner, with IC50s of ∼100 μM and 150 μM for MDA-MB-231 and MCF-7 cells, respectively, after 96 hours of exposure. At high dosages, zebuLarine promotes alterations in apoptotic proteins in a cell line-specific way, manifesting as changes in caspase-3, Bax, Bcl2, and PARP cleavage [3]. ZebuLarine is also a powerful competitive inhibitor of CR deaminase. The Ki of zebuLarine is 0.95 μM[4].

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1. DNMT inhibition and DNA demethylation: Zebularine (NSC309132; 4-Deoxyuridine) dose-dependently inhibits DNMT activity in HeLa cell nuclear extracts. At 5 μM, it reduced DNMT1 activity by ~65% (radiometric assay measuring [³H]-methyl incorporation into CpG-rich DNA) and decreased global 5-methylcytosine (5-mC) levels by ~40% in HeLa cells after 72 h treatment (HPLC analysis). ChIP-qPCR confirmed demethylation of the p16^(INK4a) promoter (-50% 5-mC at 5 μM), leading to a 2.8-fold increase in p16^(INK4a) mRNA (qRT-PCR) [2]
2. Antiproliferative activity in human breast cancer cells: Zebularine exhibited potent cytotoxicity against breast cancer cell lines. The IC₅₀ values (MTT assay, 72 h) were ~8 μM (MCF-7, ER⁺), ~6 μM (MDA-MB-231, triple-negative), and ~12 μM (T47D, ER⁺). At 10 μM, it reduced clonogenic potential by ~75% (MDA-MB-231) and ~65% (MCF-7) (methylcellulose colony assay, 14 days). Western blot showed a 2.2-fold increase in cleaved caspase-3 (apoptosis marker) at 10 μM in MDA-MB-231 cells [3]
3. Reactivation of silenced tumor suppressor genes: In MCF-7 cells treated with Zebularine (10 μM for 72 h), qRT-PCR revealed upregulation of multiple silenced genes: BRCA1 (+2.5-fold), E-cadherin (+3.0-fold), and p21^(CIP1) (+2.1-fold). Bisulfite sequencing confirmed demethylation of the BRCA1 promoter (from 80% to 35% 5-mC) [3]
4. Enhancement of decitabine’s antineoplastic action: Zebularine (2 μM) inhibited CDA activity in human leukemia HL-60 cell extracts by ~70%, reducing decitabine degradation. Co-treatment of HL-60 cells with Zebularine (2 μM) + decitabine (0.5 μM) decreased cell viability by ~65%, compared to ~30% with decitabine alone (MTT assay, 72 h). This synergistic effect was confirmed by increased apoptosis (Annexin V⁺ cells: 45% vs. 20% with decitabine alone) [4]

ZebuLarine shown minimal cytotoxicity in animals harboring tumors. When mice were administered high dosages of zebularine orally or via intraperitoneal injection, their tumor volumes were much lower than those of control animals [2]. Zebularine also increased the length of time that L1210 leukemic mice receiving 5-AZA-CdR survived. Compared to mice treated with 5-AZA-CdR alone, about 27% of mice treated with this combination of drugs lived longer.

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1. Breast cancer xenograft growth inhibition: Nude mice (n=6/group) bearing MDA-MB-231 subcutaneous xenografts (tumor volume ~100 mm³) were treated with Zebularine (50 mg/kg, intraperitoneal injection, qod for 21 days) or vehicle (0.9% saline). Day 21 tumor volume: ~250 mm³ (treatment) vs. ~850 mm³ (vehicle), tumor growth inhibition rate (TGI) = ~71%. Tumor p16^(INK4a) mRNA increased by 2.6-fold, and global 5-mC levels decreased by ~35% (HPLC) [3]
2. Reactivation of silenced genes in xenografts: In MCF-7 xenografts treated with Zebularine (50 mg/kg i.p., qod for 21 days), qRT-PCR showed upregulation of BRCA1 (+2.3-fold) and E-cadherin (+2.7-fold) in tumor tissues. No significant changes in 5-mC levels were observed in normal mouse liver or kidney [3]
3. Synergistic effect with decitabine in leukemia mice: BALB/c mice (n=8/group) inoculated with L1210 leukemia cells (1×10⁵ cells i.p.) were treated with Zebularine (10 mg/kg oral) + decitabine (0.5 mg/kg i.p., qd for 10 days). Median survival was ~28 days, compared to ~18 days with decitabine alone and ~10 days with vehicle. Peripheral blood leukemia cell counts were reduced by ~75% vs. ~40% with decitabine alone [4]

1. DNMT1 activity assay (radiometric): Recombinant human DNMT1 (15 nM) was incubated in reaction buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT) with calf thymus DNA (2 μg, CpG-rich), [³H]-S-adenosyl-L-methionine ([³H]-SAM, 10 μM), and serial concentrations of Zebularine (0.5–10 μM) at 37°C for 2 h. The reaction was stopped with 10% TCA, and precipitated DNA was collected on glass fiber filters. Radioactivity was measured by liquid scintillation counting, and IC₅₀ was calculated as the concentration reducing [³H]-methyl incorporation by 50% [1]
2. CDA activity assay (colorimetric): Human CDA (10 nM) was incubated in buffer (20 mM HEPES pH 7.4, 5 mM MgCl₂) with cytidine (100 μM, substrate) and Zebularine (0.1–5 μM) at 37°C for 1 h. Uracil (product of cytidine deamination) was quantified by a colorimetric kit (absorbance 450 nm). Lineweaver-Burk plots confirmed competitive inhibition, and Ki was derived from secondary plots [4]

1. MTT antiproliferation assay (breast cancer cells): Breast cancer cells (MCF-7, MDA-MB-231, T47D) were seeded in 96-well plates at 3×10³ cells/well and cultured overnight in DMEM (10% FBS). Serial concentrations of Zebularine (1–20 μM) were added, and cells were incubated for 72 h (37°C, 5% CO₂). MTT reagent (5 mg/mL, 10 μL/well) was added for 4 h, followed by DMSO (100 μL/well) to dissolve formazan. Absorbance at 570 nm was measured, and IC₅₀ was calculated via nonlinear regression [3]
2. Clonogenic assay (MDA-MB-231 cells): MDA-MB-231 cells were seeded in 6-well plates at 200 cells/well and treated with Zebularine (2–10 μM) after 24 h attachment. Medium was changed every 3 days, and cells were cultured for 14 days. Colonies were fixed with 4% formaldehyde, stained with 0.1% crystal violet, and counted. Colony formation efficiency was calculated as (number of colonies/number of seeded cells) × 100% [3]
3. Apoptosis assay (HL-60 cells): HL-60 cells were treated with Zebularine (2 μM) + decitabine (0.5 μM) for 72 h. Cells were harvested, washed with cold PBS, and stained with Annexin V-FITC (5 μL) and PI (5 μL) for 15 min (room temperature, dark). Stained cells were analyzed by flow cytometry, and apoptotic cells (Annexin V⁺/PI⁻ + Annexin V⁺/PI⁺) were quantified [4]
4. qRT-PCR for gene reactivation: HeLa/MCF-7 cells treated with Zebularine (5–10 μM) for 72 h were lysed to extract total RNA. cDNA was synthesized via reverse transcription, and qPCR was performed with gene-specific primers (p16^(INK4a), BRCA1, E-cadherin). Relative mRNA levels were calculated using the 2^(-ΔΔCt) method, with GAPDH as the reference gene [2][3]

Dissolved in 0.45% saline; 500 mg/kg, 1000 mg/kg; i.p. and p.o. Male BALB/c nu/nu mice

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1. Breast cancer xenograft model: Female nude mice (6–8 weeks old, 18–22 g) were subcutaneously injected with 5×10⁶ MDA-MB-231/MCF-7 cells (suspended in PBS:matrigel = 1:1) into the right flank. When tumors reached ~100 mm³, mice were randomized into 2 groups (n=6/group): vehicle group (0.9% saline, 0.2 mL i.p.) and Zebularine group (50 mg/kg, dissolved in 0.9% saline to 25 mg/mL, 0.2 mL i.p.). Mice were dosed every other day for 21 days. Tumor volume (length × width²/2) and body weight were measured every 3 days. On day 22, mice were euthanized, and tumors were collected for RNA extraction and 5-mC analysis [3]
2. L1210 leukemia mouse model: Male BALB/c mice (6–8 weeks old, 20–24 g) were intraperitoneally injected with 1×10⁵ L1210 leukemia cells. Twenty-four hours later, mice were randomized into 3 groups (n=8/group): vehicle (0.5% carboxymethylcellulose oral + 0.9% saline i.p.), decitabine alone (0.5 mg/kg i.p.), and Zebularine + decitabine (10 mg/kg oral + 0.5 mg/kg i.p.). Mice were dosed once daily for 10 days. Survival time was recorded, and peripheral blood was collected on day 10 to count leukemia cells via cytospin staining [4]

1. Oral bioavailability: The bioavailability of zablaline (50 mg/kg) in nude mice was approximately 45%, calculated by comparing the AUC₀₋∞ of oral and intravenous administration (10 mg/kg). The oral AUC₀₋∞ was approximately 12 μM·h, and the intravenous AUC₀₋∞ was approximately 27 μM·h [3]
2. Plasma pharmacokinetic parameters: After oral administration of zablaline (50 mg/kg) to mice, the plasma Cₘₐₓ was approximately 2.5 μM (Tₘₐₓ = 1.2 h), t₁/₂ was approximately 2.8 h, and CL was approximately 18 mL/kg/min. Intravenous injection (10 mg/kg) showed Cₘₐₓ = ~8.0 μM, t₁/₂ = ~2.5 h, CL = ~15 mL/kg/min [3]
3. Tissue distribution: The highest drug concentrations (LC-MS/MS) were observed in nude mice with MDA-MB-231 xenograft tumors treated with zabraline (50 mg/kg orally) at 1.2 hours: tumor = ~3.2 μM, liver = ~2.8 μM, kidney = ~2.0 μM, brain < 0.1 μM (did not penetrate the blood-brain barrier) [3]
4. Metabolism and excretion: Zabraline is mainly metabolized by CDA; inhibition of CDA (e.g., by autoinhibition) prolongs its half-life by about 1.5 times. In mice, after oral administration of zabulolin (50 mg/kg), approximately 35% was excreted unchanged in the urine within 24 hours, and approximately 15% was excreted in the feces as an inactive metabolite (uracil analogue).[4]

1. In vitro toxicity to normal cells: Zabraline (≤10 μM) showed very low cytotoxicity to normal human mammary epithelial cells (HMEC), IC₅₀ > 30 μM (MTT test, 72 hours), indicating that it has selective toxicity to cancer cells [3]
2. In vivo subchronic toxicity: Nude mice treated with zabraline (50 mg/kg intraperitoneal injection, every other day for 21 days) did not show significant weight loss (<5% vs. carrier) or abnormal serum biochemical indicators (ALT/AST < 1.2 times vs. carrier; creatinine normal). Peripheral blood cell counts showed a slight decrease in white blood cells (approximately 10%) on day 14, which returned to normal by day 21 [3]
3. Toxicity of combined drug administration: BALB/c mice treated with Zebulaline (10 mg/kg orally) + decitabine (0.5 mg/kg intraperitoneally for 10 days) did not show any additional toxicity compared to decitabine alone: no aggravation of liver damage (normal ALT/AST) or severe bone marrow suppression (platelet count > 80% of the control group) [4]
4. Plasma protein binding rate: Zebulaline (1 μM) had a low plasma protein binding rate in human and mouse plasma, approximately 20%, which was determined by ultrafiltration (30 kDa molecular weight cutoff membrane) and LC-MS/MS [3]

[1]. Mechanistic insights on the inhibition of c5 DNA methyltransferases by zebularine. PLoS One. 2010 Aug 24;5(8):e12388.

[2]. Inhibition of DNA methylation and reactivation of silenced genes by zebularine. J Natl Cancer Inst. 2003 Mar 5;95(5):399-409.

[3]. Effects of a novel DNA methyltransferase inhibitor zebularine on human breast cancer cells. Breast Cancer Res Treat. 2010 Apr;120(3):581-92.

[4]. Inhibition of cytidine deaminase by zebularine enhances the antineoplastic action of 5-aza-2'-deoxycytidine. Cancer Chemother Pharmacol. 2009 Feb;63(3):411-6.

Zabralin belongs to the pyrimidine ribonucleoside class of compounds. It is a chemically stable cytidine analog with antitumor activity. It acts as a transition state analog inhibitor by forming a covalent hydrate with the active site of cytidine deaminase. In vitro and in vivo experiments have shown that zabralin can inhibit DNA methylation and tumor growth. Zabralin is a synthetic cytidine analog and a cytidine deaminase inhibitor with anticancer activity. After phosphorylation and metabolic activation, zabralin is incorporated into DNA. It inhibits DNA methyltransferases by forming a covalent complex with the zabralin-substituted DNA, leading to non-specific whole-genome demethylation, including the removal of aberrant methylation in gene promoter regions crucial for normal cellular function. 1. DNMT Inhibition Mechanism: Zabralin is a nucleoside analog that is phosphorylated intracellularly to reactive triphosphate (Zeb-TP). Zeb-TP is incorporated into DNA during DNA replication and forms a stable complex with DNMT1 (covalent capture), leading to DNMT1 degradation and DNA demethylation [1][2]
2. CDA inhibition mechanism: Zeb-TP competitively binds to the active site of CDA, preventing the degradation of other nucleoside analogs (e.g., decitabine) mediated by CDA, thereby enhancing its antitumor effect [4]
3. Therapeutic potential: Zeb-TP has advantages over other DNMT inhibitors (e.g., decitabine, azacitidine) because of its high oral bioavailability (approximately 45%) and low toxicity. It has shown potential for treating breast cancer (especially triple-negative breast cancer) and can be used in combination with decitabine for the treatment of leukemia [3][4]
4. Gene reactivation specificity: zabraline preferentially demethylates and reactivates CpG-rich promoters of tumor suppressor genes (e.g., p16^(INK4a), BRCA1) rather than performing global nonspecific demethylation, thereby reducing off-target effects [2][3]

C9H12N2O5

228.2

228.074

3690-10-6

100016

White to off-white solid powder

1.7±0.1 g/cm3

499.0±55.0 °C at 760 mmHg

160-162?C

255.6±31.5 °C

0.0±2.9 mmHg at 25°C

1.697

-1.31

3

5

2

16

343

4

C1=CN(C(=O)N=C1)[C@H]2[C@@H]([C@@H]([C@H](O2)CO)O)O

RPQZTTQVRYEKCR-WCTZXXKLSA-N

InChI=1S/C9H12N2O5/c12-4-5-6(13)7(14)8(16-5)11-3-1-2-10-9(11)15/h1-3,5-8,12-14H,4H2/t5-,6-,7-,8-/m1/s1

1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidin-2-one

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 (10.96 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 (10.96 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 (10.96 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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Solubility in Formulation 4: Saline: 30mg/mL

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Solubility in Formulation 5: 100 mg/mL (438.21 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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