2-Fluoroadenine (2 μM; 4 hours) targets one or more enzymes involved in the creation of RNA or proteins, which slows the development of CEM cells [1]. 2- Non-proliferating MRC-5 cells demonstrate cytotoxicity in response to fluorroadenine (0-1000 μM; 96 hours) [1]. 2- Protein, RNA, and DNA synthesis is inhibited in Balb-3T3 cells cultured in serum-free media by fluoroadenine (0.22, 2.2, and 22 μM; 30 hours) [1].

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Cytotoxicity in proliferating CEM cells: IC50 after 4-hour exposure was 0.15 ± 0.07 μM. After continuous exposure (72 h), IC50 was 0.10 μM (mean of 2 determinations). The duration of exposure had only a 1.5-fold effect on potency, indicating cytotoxic actions occur early after addition [1].
- In CEM cells, treatment with 2 μM F-Ade (approximately 10× the 4-h IC50) inhibited protein, DNA, and RNA syntheses to similar extents, as measured by incorporation of [³H]leucine, [³H]dThd, and [³H]Urd (or [¹⁴C]Ade for DNA). The pattern was similar to that of cycloheximide, a protein synthesis inhibitor [1].
- F-Ade was toxic to nonproliferating cells. In confluent MRC-5 human diploid fibroblasts (nonproliferating), F-Ade caused a concentration-dependent decrease in attached protein over 96 h, whereas 5-fluorouracil had no effect. The protein concentration in control flasks remained unchanged [1].
- In CEM cells cultured in 0.1% serum (quiescent state), treatment with 1 μg/mL F-Ade resulted in a decline in cell numbers within 24 h, while 5-fluorouracil did not affect cell numbers [1].
- In quiescent Balb-3T3 cells (serum-free medium), F-Ade at 2.2 μM, 0.22 μM, and 0.022 μM inhibited DNA synthesis (to 34%, 84%, 88% of control, respectively), RNA synthesis (to 37%, 64%, 86% of control), and protein synthesis (to 15%, 66%, 76% of control) after 30 h treatment [1].

Cell Proliferation Assay
Cell Types: CEM cells [1]
Tested Concentrations: 2 μM
Incubation Duration: 4 hrs (hours)
Experimental Results: Inhibition of CEM cell growth by targeting one or more enzymes involved in RNA or protein synthesis.

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cell cytotoxicity assay
Cell Types: MRC-5 cells [1]
Tested Concentrations: 0-1000 μM
Incubation Duration: 4 hrs (hours)
Experimental Results: Cytotoxicity was demonstrated in non-proliferating MRC-5 cells.

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Cytotoxicity assay (IC50): CEM cells (approximately 300,000 cells/mL) were exposed to various concentrations of F-Ade for 4 hours, then washed and cultured in drug-free medium for 72 hours. Cell growth inhibition was determined. For continuous exposure, cells were incubated with drug for 72 hours [1].
- Macromolecular synthesis inhibition: CEM cells were incubated with 2 μM F-Ade. Four hours later, radiolabeled precursors ([methyl-³H]dThd for DNA, [5-³H]Urd for RNA, [4,5-³H]leucine for protein) were added at 2 μCi/mL. Samples were taken 1, 2, 3, and 4 hours after label addition, and incorporation into acid-precipitable material was measured. For DNA synthesis measurement in the presence of FUra, [8-¹⁴C]Ade was used instead of dThd to avoid thymidylate synthase inhibition artifacts [1].
- Nonproliferating MRC-5 cell toxicity: Confluent MRC-5 cells were treated with various concentrations of F-Ade for 96 hours. Cytotoxicity was assessed by measuring the amount of protein attached to the culture flask. Protein concentration in control flasks was approximately 35 μg/flask [1].
- Quiescent CEM cell study: CEM cells were placed in medium containing 0.1% serum for 48 hours to induce quiescence (dThd incorporation was 10-15% of proliferating cells). Then F-Ade (1 μg/mL) was added, and cell numbers were counted at 24-hour intervals using a Coulter Counter [1].
- Balb-3T3 cell macromolecular synthesis assay: Balb-3T3 cells were seeded in 96-well plates (2-3×10⁴ cells/well) and incubated for 7-9 days until confluent and quiescent. Cells were treated with F-Ade in serum-free medium for 30 hours. For DNA synthesis, cells were pulsed with 1 μCi [³H]dThd/well for 20 hours starting 10 hours after drug addition. For RNA or protein synthesis, cells were pulsed with 1 μCi [³H]Urd or [³H]leucine for 6 hours starting 24 hours after drug addition. Cells were then trypsinized, filtered, and counted [1].
- Metabolism analysis: CEM cells were incubated with 0.03 μM [³H]F-Ade (6300 Ci/mol) for 4 hours. Acid-soluble metabolites were extracted with 0.5 M perchloric acid, neutralized, and analyzed by SAX HPLC using a linear gradient from 5 mM NH₄H₂PO₄ (pH 2.8) to 750 mM NH₄H₂PO₄ (pH 3.7) at 2 mL/min. Fractions were counted for radioactivity. The major metabolite eluted 2-3 min after ATP (peak fractions 35-36) and was identified as F-ATP after digestion with phosphodiesterase and alkaline phosphatase followed by reverse-phase HPLC [1].
- Half-life determination: CEM cells were incubated with 0.03 μM [³H]F-Ade for 4 hours, then washed and resuspended in fresh medium. Samples were taken at various times, and the amount of F-ATP in the acid-soluble pool was measured by SAX HPLC. Half-life of F-ATP was approximately 5 hours (similar to ATP). Half-life of MeP-ribonucleoside triphosphate was approximately 48 hours [1].
- Incorporation into RNA and DNA: CEM cells were incubated with 20 nM [³H]F-Ade (6300 Ci/mol). At 1, 2, and 4 hours, cells were lysed and fractionated on CsCl gradients to separate RNA (pellet) and DNA (band). The isolated RNA and DNA were degraded to nucleosides and analyzed by reverse-phase HPLC. [³H]F-Ado was detected in RNA and [³H]F-dAdo in DNA, confirming incorporation into both macromolecules. Over 60% of F-Ade in the medium was taken up by cells within 4 hours [1].

Metabolism: F-Ade is converted to F-ATP in CEM cells. At 0.03 μM F-Ade, 62 pmol F-ATP/10⁶ cells was formed in 4 hours. The concentration of F-ATP formed at equitoxic conditions (0.3 μM F-Ade, causing ~70% growth inhibition) was approximately 13% of the ATP pool (ATP ~700 nmol/10⁶ cells). F-Ade was metabolized as effectively as adenine; 2 μM F-Ade produced ~400 pmol F-ATP/10⁶ cells in 4 hours, comparable to 821 pmol ATP from 3.6 μM adenine. F-Ade did not affect ATP, CTP, UTP, or GTP levels at 0.3 μM, but higher concentrations (10 μM) decreased ATP levels; however, total ATP + F-ATP pool equaled ATP in untreated cells [1].
- Half-life: The half-life of F-ATP in proliferating CEM cells was approximately 5 hours (similar to ATP) [1].

F-Ade was toxic to both proliferating and nonproliferating human cells (CEM, MRC-5, Balb-3T3). Its mechanism involves inhibition of protein and RNA synthesis, which are essential processes in all cells, suggesting potential for systemic toxicity. The paper notes that F-Ade has been previously evaluated as an antitumor agent and found to have no selectivity for tumor versus normal cells in intact animals [1].

[1]. Metabolism and metabolic actions of 6-methylpurine and 2-fluoroadenine in human cells. Biochem Pharmacol. 1998;55(10):1673-1681.

2-Fluoroadenine is an organofluorine compound, a derivative of adenine, in which the hydrogen atom at position 2 (the carbon atom between the two nitrogen atoms in the pyrimidine ring) is replaced by a fluorine atom. It is an antitumor drug. It belongs to the organofluorine compound class and is also a purine compound. 2-Fluoroadenine is a fluorinated heterocyclic bicyclic compound. Many carbocyclic and acyclic nucleoside analogs are based on 2-fluoroadenine, and these analogs are used in antitumor research.

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F-Ade is a toxic purine base that can be generated by E. coli purine nucleoside phosphorylase (PNP) from a nontoxic prodrug (e.g., 2-fluoroadenosine) as part of a suicide gene therapy strategy for cancer [1].
- Unlike 5-fluorouracil and ganciclovir (which primarily target proliferating cells), F-Ade kills nonproliferating cells, which may be advantageous for solid tumors with low growth fractions [1].
- F-Ade is 1000-fold more potent than 5-fluorouracil against CEM cells after 4-hour exposure (IC50 0.15 μM vs. 120 μM) [1].
- F-Ade is incorporated into both RNA and DNA. F-dATP (the deoxyribonucleotide analog) has been shown to be a good substrate for DNA polymerase α (reference cited) [1].
- Cells lacking adenine phosphoribosyltransferase (APRT) are resistant to F-Ade, indicating that conversion to nucleotides is required for cytotoxicity [1].
- The study suggests that F-Ade's mechanism involves inhibition of protein and/or RNA synthesis, possibly through incorporation into RNA disrupting its structure or function [1].

C5H4FN5

163.12

153.045

700-49-2

12790

White to off-white solid powder

1.7±0.1 g/cm3

676.9±58.0 °C at 760 mmHg

>350 °C(lit.)

363.2±32.3 °C

0.0±2.1 mmHg at 25°C

1.783

-0.01

2

5

0

11

154

0

WKMPTBDYDNUJLF-UHFFFAOYSA-N

InChI=1S/C5H4FN5/c6-5-10-3(7)2-4(11-5)9-1-8-2/h1H,(H3,7,8,9,10,11)

1H-Purin-6-amine, 2-fluoro-

2-Fluoroadenine NSC-27364 NSC27364NSC 27364

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)

DMSO : ~20.83 mg/mL (~136.04 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (13.58 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: ≥ 2.08 mg/mL (13.58 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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