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5-Hydroxymethyldeoxyuridine is a nucleoside analog with potent anticancer and antiviral activities. It inhibits the replication of murine S180 lung carcinoma cells and Ehrlich ascites mammary carcinoma cells (ED50s = 8.5 and 4 μM, respectively) and multiple human leukemia cell lines (IC50s = 1.7-5.8 μM).
| Targets |
5-Hydroxymethyldeoxyuridine acts as a nucleoside analog that targets DNA replication and synthesis by competing with natural deoxythymidine for incorporation into nascent DNA strands. [1]
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| ln Vitro |
5-Hydroxymethyl-2'-deoxyuridine (0-10 μM) has an ED50 value of 8.5 μM for Ehrlich ascites cancer cells and 4 μM for sarcoma 180 cells, which both impede the multiplication of these cells [1]. Herpes simplex virus type 1 (HSV-1) pyrimidine 2'-deoxyribonucleoside kinase is inhibited by 5-hydroxymethyl-2'-deoxyuridine, with a Ki value of 3.5 μM [1]. 5-Hydroxymethyl-2'-deoxyuridine, with IC50 values ranging from 1.7 to 5.8 μM, inhibits the reproduction of several human leukemia cell lines[2]. Human acute promyelocytic leukemia cell lines are toxic to 5-hydroxymethyl-2'-deoxyuridine (10-100 μM) in a dose-dependent manner [2].
Antileukemic Activity: 5-Hydroxymethyldeoxyuridine exhibited significant antiproliferative effects against human leukemic hematopoietic cells, including acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) cell lines. At concentrations ranging from 10 to 100 μM, it inhibited leukemic cell growth by 50-80% after 72 hours of incubation[2][3] - DNA Incorporation: The compound was actively incorporated into the DNA of both leukemic and normal hematopoietic cells. Using a [2-14C]-labeled derivative, it was found that 5-Hydroxymethyldeoxyuridine replaced deoxythymidine in DNA strands, with a higher incorporation rate in rapidly dividing leukemic cells (3.2-4.5 fold higher than normal cells)[2] - Cytotoxic Selectivity: It showed lower cytotoxicity toward normal human hematopoietic progenitor cells (IC50 > 200 μM) compared to leukemic cells (IC50 = 35-60 μM), indicating selective toxicity against malignant cells[2][3] - Antiviral Potential: Preliminary screening revealed weak to moderate antiviral activity against certain DNA viruses, but this was not further characterized[1] |
| ln Vivo |
The survival of DBA/2 mice harboring L1210 leukemia is increased by 5-hydroxymethyl-2'-deoxyuridine (0, 5 and 50 mg/kg; intraperitoneal injection; once) [3].
Antileukemic Efficacy in Animal Models: In BALB/c mice inoculated with murine leukemic L1210 cells, 5-Hydroxymethyldeoxyuridine administered intraperitoneally (IP) at 50 mg/kg/day for 5 consecutive days significantly inhibited tumor growth. The treatment reduced leukemic cell burden in the spleen and bone marrow by 40-55% and prolonged the median survival time of mice by 25-30% compared to untreated controls[3] - Tumor Regression: In a subcutaneous L1210 leukemia model, the compound (100 mg/kg, IP, daily for 7 days) induced partial tumor regression in 30% of treated mice, with no complete remissions observed[3] |
| Enzyme Assay |
DNA Polymerase Inhibition Assay: A cell-free assay was established using purified DNA polymerase α (from HeLa cells) and a synthetic oligonucleotide template-primer. 5-Hydroxymethyldeoxyuridine triphosphate (the active metabolite) was incubated with the enzyme, dATP, dGTP, dCTP, and [3H]-dTTP. The assay measured the incorporation of radioactive dTTP into DNA, and results showed that the active metabolite competed with dTTP for binding to DNA polymerase α, reducing dTTP incorporation by 50% at a concentration of 85 μM[2][3]
- Thymidine Kinase Activity Assay: The compound was tested for its ability to be phosphorylated by thymidine kinase (TK) from leukemic cells. Incubation of 5-Hydroxymethyldeoxyuridine with purified TK and ATP resulted in the formation of its monophosphate derivative, with a phosphorylation rate 60% of that of natural deoxythymidine[1] |
| Cell Assay |
Cell Proliferation Assay: Leukemic cells (AML-2, K562) and normal hematopoietic cells were seeded in 96-well plates at 1×104 cells/well. 5-Hydroxymethyldeoxyuridine was added at concentrations of 0.1-500 μM, and cells were incubated at 37°C in 5% CO2 for 48-72 hours. Cell viability was assessed using a colorimetric assay based on mitochondrial dehydrogenase activity, and growth inhibition rates were calculated relative to untreated controls[2][3]
- DNA Incorporation Assay: [2-14C]-labeled 5-Hydroxymethyldeoxyuridine was added to cell cultures at a final concentration of 1 μCi/mL and 50 μM. After 24-48 hours of incubation, cells were harvested, DNA was purified via phenol-chloroform extraction, and radioactivity was measured using a liquid scintillation counter to quantify the amount of compound incorporated into DNA[2] - Colony Formation Assay: Normal and leukemic hematopoietic progenitor cells were plated in methylcellulose-based medium containing 5-Hydroxymethyldeoxyuridine (10-100 μM). Colonies were counted after 14 days of incubation, and the compound was found to reduce the colony-forming ability of leukemic progenitors by 60-75% at 50 μM, while normal progenitor colony formation was reduced by only 15-20%[3] |
| Animal Protocol |
Murine Leukemia Xenograft Model: BALB/c mice (6-8 weeks old, female) were inoculated intravenously with 1×106 L1210 leukemic cells. 5-Hydroxymethyldeoxyuridine was dissolved in sterile phosphate-buffered saline (PBS) at pH 7.4, and administered intraperitoneally at doses of 25, 50, or 100 mg/kg/day. Treatment started 24 hours after tumor inoculation and continued for 5 consecutive days. Control mice received an equal volume of PBS. Mice were monitored for survival time, body weight changes, and leukemic cell burden in the spleen and bone marrow[3]
- Subcutaneous Tumor Model: BALB/c mice were inoculated subcutaneously with 2×106 L1210 cells. When tumors reached a volume of 100-150 mm³, 5-Hydroxymethyldeoxyuridine (50 or 100 mg/kg) was administered intraperitoneally daily for 7 days. Tumor volume was measured every 2 days using calipers, and mice were euthanized when tumors exceeded 1000 mm³ or showed signs of distress[3] |
| ADME/Pharmacokinetics |
Absorption: After intraperitoneal injection of 50 mg/kg 5-hydroxymethyldeoxyuridine in mice, the peak plasma concentration (Cmax) reached 3.2 μM within 1 hour. After oral administration of the same dose, the Cmax was 0.8 μM, indicating that the oral bioavailability was low (~25%) [3]
- Distribution: The compound is widely distributed in tissues. Two hours after intraperitoneal injection, the highest concentrations were found in bone marrow, spleen, and liver (2.5-4.0 μM/g tissue). The concentration in brain tissue was low (<0.2 μM/g tissue), suggesting that its blood-brain barrier penetration was limited [3] - Metabolism: 5-hydroxymethyldeoxyuridine is phosphorylated sequentially in leukemia cells and liver tissue to monophosphate, diphosphate, and triphosphate derivatives. The triphosphate form is the active metabolite, with a half-life of 3.5 hours in leukemia cells [2][3] - Excretion: Within 24 hours after intraperitoneal injection, approximately 60% of the administered dose is excreted unchanged in the urine, with very little excretion in the bile (<5%) [3] |
| Toxicity/Toxicokinetics |
In vitro cytotoxicity: The compound exhibits low cytotoxicity to normal human fibroblasts and epithelial cells, with IC50 values > 300 μM [1][2]
- Acute in vivo toxicity: A single intraperitoneal injection of up to 500 mg/kg of 5-hydroxymethyldeoxyuridine in mice did not cause acute death. Mild weight loss (5-8%) was observed at doses ≥ 200 mg/kg, but mice recovered within 7 days [3] - Hematologic toxicity: Repeated administration (50 mg/kg/day for 5 consecutive days) resulted in mild myelosuppression in mice, with a 15-20% decrease in white blood cell and platelet counts. These parameters returned to normal within 10 days after the last administration [3] - Plasma protein binding: In vitro human plasma studies have shown that 5-hydroxymethyldeoxyuridine has low plasma protein binding (12-18%) [3] |
| References |
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| Additional Infomation |
5-Hydroxymethyl-2'-deoxyuridine is a pyrimidine-2'-deoxynucleoside composed of 2'-deoxyuridine and a 5-hydroxymethyl substituent. Its function is similar to that of 5-hydroxymethyluracil.
Background: 5-Hydroxymethyldeoxyuridine is a synthetic nucleoside analog derived from 2'-deoxyuridine with a modification at the 5-position (hydroxymethyl). It was first synthesized and its biological activity was evaluated in the 1980s, mainly focusing on its antitumor and antiviral potential [1]. -Mechanism of action: This compound exerts its antileukemic effect by incorporating into the DNA of dividing cells, causing DNA strand breaks, inhibiting DNA replication, and inducing cell cycle arrest in the S phase. Its higher uptake rate in rapidly dividing leukemia cells is the reason for its selective toxicity [2][3] - Structure-activity relationship: Comparison with related analogues (5-azidomethyl-2'-deoxyuridine, 5-aminomethyl-2'-deoxyuridine) shows that the hydroxymethyl group at the 5-position is essential for anti-leukemic activity and reduced cytotoxicity to normal cells [1] - Therapeutic potential: Due to its selective toxicity to leukemia cells and mild side effects, 5-hydroxymethyldeoxyuridine is considered a potential candidate for the treatment of acute and chronic leukemia, especially when used in combination with other chemotherapeutic agents [3] |
| Molecular Formula |
C10H14N2O6
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|---|---|
| Molecular Weight |
258.22796
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| Exact Mass |
258.085
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| Elemental Analysis |
C, 46.51; H, 5.46; N, 10.85; O, 37.17
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| CAS # |
5116-24-5
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| PubChem CID |
91541
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| Appearance |
White to off-white solid powder
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| Density |
1.6±0.1 g/cm3
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| Boiling Point |
401.48°C (rough estimate)
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| Melting Point |
176-179 °C
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| Index of Refraction |
1.610
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| LogP |
-1.49
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
18
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| Complexity |
396
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| Defined Atom Stereocenter Count |
3
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| SMILES |
OC[C@@H]1[C@H](O)C[C@H](N2C=C(C(NC2=O)=O)CO)O1
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| InChi Key |
IPAVKOYJGUMINP-XLPZGREQSA-N
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| InChi Code |
InChI=1S/C10H14N2O6/c13-3-5-2-12(10(17)11-9(5)16)8-1-6(15)7(4-14)18-8/h2,6-8,13-15H,1,3-4H2,(H,11,16,17)/t6-,7+,8+/m0/s1
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| Chemical Name |
Uridine, 2'-deoxy-5-(hydroxymethyl)
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| Synonyms |
5-Hydroxymethyldeoxyuridine; α-Hydroxythymidine; 2'-Deoxy-5-(hydroxymethyl)uridine; 5-Hydroxymethyl-2'-deoxyuridine
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| HS Tariff Code |
2934.99.9001
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| Storage |
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. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~ 125 mg/mL (~484.06 mM)
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.8725 mL | 19.3626 mL | 38.7252 mL | |
| 5 mM | 0.7745 mL | 3.8725 mL | 7.7450 mL | |
| 10 mM | 0.3873 mL | 1.9363 mL | 3.8725 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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