6-Thioguanine

Synthetic Routes and Reaction Conditions

Tioguanine can be synthesized using 2,9-diacetyl guanine or 2-acetyl guanine as starting materials. The reaction involves sulfidizing these compounds with phosphorus pentasulfide in a pyridine solution, with a slight catalyst . This method offers mild reaction conditions, simple operation, and a high yield of up to 75% .

Industrial Production Methods

The industrial production of tioguanine follows similar synthetic routes but on a larger scale. The process involves the use of thiophosphoric anhydride and pyridine solution, with careful control of reaction conditions to ensure high yield and purity .

Types of Reactions

Tioguanine undergoes various chemical reactions, including:

Deamination: The deamination reaction of tioguanine with water, hydroxide ions, and protonated forms has been studied extensively.

Oxidation: Tioguanine can be oxidized to form toxic metabolites, contributing to its cytotoxic effects.

Common Reagents and Conditions

Deamination: Hydroxide ions and water are common reagents used in the deamination of tioguanine.

Oxidation: Reactive oxygen species, such as singlet oxygen and superoxide radicals, are involved in the oxidation of tioguanine.

Major Products Formed

Deamination: The deamination of tioguanine results in the formation of various intermediates and products, depending on the reaction conditions.

Oxidation: Oxidation of tioguanine leads to the formation of toxic metabolites that can be incorporated into DNA.

Pharmacological Profile

Mechanism of Action
6-Thioguanine functions as an antimetabolite, primarily inhibiting DNA synthesis by incorporating into nucleic acids, particularly DNA. This incorporation disrupts the replication process, leading to cell death in rapidly dividing tumor cells. Evidence indicates that 6-TG is more effective in younger, rapidly growing tumor populations compared to older ones, highlighting the importance of timing in treatment regimens .

Pharmacokinetics
The pharmacokinetics of 6-TG have been extensively studied. It is metabolized by guanase and xanthine oxidase, with higher blood levels observed post-intravenous administration compared to oral dosing. Studies show that repeated doses lead to significant incorporation of 6-TG into DNA, particularly in bone marrow cells .

Clinical Applications

Cancer Treatment
this compound has been utilized in various types of cancer, particularly hematological malignancies such as acute leukemia and lymphomas. Its efficacy has been noted in patients with specific genetic profiles, particularly those with homozygous deletions of the methylthioadenosine phosphorylase (MTAP) gene. These tumors are often resistant to other therapies but may respond favorably to 6-TG when combined with methylthioadenosine (MTA), which protects normal tissues from toxicity while allowing higher doses of 6-TG .

Case Study: Leukemia Treatment

A retrospective study involving 30 patients with Crohn's Disease who failed conventional immunosuppression demonstrated the efficacy of 6-TG as a third-line treatment. The median dose was 40 mg daily over 21.5 months, with an initial clinical response in 60% of patients . This study underscores the drug's potential beyond oncology into inflammatory conditions.

Combination Therapies

Research suggests that combining this compound with other agents can enhance its therapeutic effects. For instance, combinations with methotrexate or pralatrexate have shown promising results against MTAP-deficient tumors. The synergistic effects observed in xenograft models indicate a potential for developing more effective treatment protocols .

Safety and Toxicity

While 6-TG is effective, it is not without risks. Hepatotoxicity remains a concern, particularly in long-term use. In the previously mentioned study on Crohn's Disease patients, some experienced abnormal liver function tests during treatment; however, these were mostly mild and transient . Monitoring liver function during therapy is crucial for patient safety.

Summary Table of Applications

Tioguanine exerts its effects by competing with hypoxanthine and guanine for the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRTase). It is converted to 6-thioguanilyic acid, which reaches high intracellular concentrations at therapeutic doses . The compound is incorporated into DNA during the synthesis phase, leading to cytotoxic effects . Additionally, tioguanine inhibits the GTP-binding protein Rac1, which regulates the Rac/Vav pathway .

Tioguanine is part of the thiopurine family, which includes mercaptopurine and azathioprine . Compared to these compounds, tioguanine has a higher efficacy and faster action . its use is limited by concerns about hepatotoxicity and other side effects .

Similar Compounds

Mercaptopurine: Another purine analogue used in the treatment of leukemia and other conditions.

Azathioprine: A prodrug of mercaptopurine, used as an immunosuppressant in organ transplantation and autoimmune diseases.

Tioguanine’s unique properties, such as its incorporation into DNA and inhibition of specific molecular pathways, make it a valuable compound in both research and clinical settings .

6-Thioguanine (6-TG) is a purine analog that has been utilized in the treatment of various hematological malignancies, particularly acute myeloid leukemia (AML). Since its approval in the 1960s, research has expanded to explore its biological activity beyond oncology, revealing significant effects in conditions like diabetic retinopathy and psoriasis. This article delves into the biological activity of 6-TG, supported by case studies, data tables, and comprehensive research findings.

6-TG exerts its effects primarily through the inhibition of purine metabolism and DNA synthesis. It gets incorporated into DNA, replacing guanine, which leads to the disruption of DNA replication and ultimately induces apoptosis in rapidly dividing cells, such as cancer cells and activated lymphocytes. This mechanism is particularly relevant in the treatment of leukemias and autoimmune diseases.

Key Mechanisms:

• Inhibition of DNA synthesis : 6-TG is metabolized to thioguanine nucleotides (TGNs), which interfere with DNA replication.
• Induction of apoptosis : Primarily observed in activated T lymphocytes, where it triggers programmed cell death.
• Immunosuppressive effects : Reduction in peripheral blood lymphocytes and total leukocyte counts.

1. Treatment of Psoriasis

A notable study involving 20 patients with moderate to severe plaque-type psoriasis demonstrated that 6-TG significantly improved disease severity. After six months of treatment:

• 12 patients were completely cleared of disease.
• 6 showed marked improvement .
• 2 did not respond .

Biopsy results indicated a reduction in keratinocyte proliferation and inflammation, correlating with clinical improvement. The therapy also led to a significant decrease in circulating lymphocytes by an average of 42% after two months .

2. Anti-Angiogenic Activity in Diabetic Retinopathy

Recent research highlighted the anti-angiogenic properties of 6-TG in diabetic retinopathy models. In vitro studies with human umbilical vein endothelial cells (HUVECs) exposed to high glucose levels showed:

• Marked reduction in tube formation at concentrations as low as 5 µM .
• In vivo studies demonstrated that diabetic mice treated with 6-TG exhibited reduced retinal vascular alterations, as confirmed by fluorescein angiography .

3. Efficacy in Leukemia Treatment

Comparative studies have shown that 6-TG is more effective than its counterpart, 6-mercaptopurine, particularly in reducing the risk of isolated central nervous system relapse. However, it carries a higher risk of severe adverse effects, including veno-occlusive disease .

Toxicity and Side Effects

While 6-TG has demonstrated efficacy across various conditions, its use is associated with several toxicities:

• Risk of infections due to immunosuppression.
• Development of liver complications such as veno-occlusive disease.
• Mutagenic potential leading to G→A mutations .

Summary Table: Clinical Findings

Research Findings

A variety of studies have investigated the pharmacokinetics and biological effects of 6-TG:

• Pharmacokinetics : Blood levels were higher post-intravenous administration compared to oral doses, indicating better bioavailability and incorporation into DNA .
• Mutagenicity Studies : Research indicated that both 6-TG and its metabolites are mutagenic in human cell lines, raising concerns about long-term use .
• Immunological Effects : A decrease in circulating leukocytes was noted alongside clinical improvements in autoimmune conditions .

Basic Research Questions

Q. How are 6-thioguanine nucleotide (6-TGN) levels quantified in erythrocytes, and what methodological variations affect clinical correlations?

• Methodology : 6-TGN levels are typically measured via high-performance liquid chromatography (HPLC) after acid hydrolysis of erythrocyte lysates. Key variations include hydrolysis conditions (e.g., 1 M perchloric acid vs. 0.5 M sulfuric acid) and reducing agents (e.g., dithiothreitol concentration), which impact nucleotide-to-base conversion efficiency . Standardization is critical, as differences in protocols lead to 2.6-fold discrepancies in reported 6-TGN levels, complicating cross-study comparisons .

Q. What experimental approaches are used to identify tautomeric forms of 6-TG in photochemical studies?

• Methodology : Resonance-enhanced multiphoton ionization (REMPI) and pump-probe spectroscopy are employed to study 6-TG's excited-state dynamics. Jet-cooled 6-TG samples allow differentiation of thiol tautomers, revealing distinct photophysical behaviors (e.g., lifetimes, energy transfer pathways) . IR-UV double resonance spectroscopy further resolves tautomer-specific vibrational modes .

Q. How is 6-TG incorporated into preclinical models to assess cytotoxicity and mutagenicity?

• Methodology : In vitro assays use cell lines (e.g., HCT116 colorectal cancer cells) to quantify apoptosis (via Annexin V/PI staining) and autophagy (LC3-II Western blotting). Mutagenicity is evaluated in V79 Chinese hamster lung fibroblasts using clonogenic survival and HPRT gene mutation assays . Dosing protocols often involve 24–72 hr exposures to 0.1–10 µM 6-TG .

Advanced Research Questions

Q. How do discrepancies in 6-TGN assay methodologies impact meta-analyses of inflammatory bowel disease (IBD) outcomes?

• Methodology : Heterogeneity in meta-analyses (e.g., pooled odds ratio [OR] = 3.3 in vs. OR = 3.15 in ) arises from inconsistent hydrolysis protocols and thresholds (230–260 pmol/8×10⁸ RBC). Studies using the Lennard reference method (acid hydrolysis + DTT) show reduced variability, emphasizing the need for standardized metabolite quantification .

Q. What CRISPR-based screening strategies identify genes conferring 6-TG resistance?

• Methodology : Genome-wide sgRNA libraries (e.g., pooled lentiviral constructs) are transduced into 4T1-luc or similar cell lines. Post-6-TG treatment, next-generation sequencing (NGS) of surviving cells reveals enriched sgRNAs targeting pathways like purine metabolism (IMPDH1/2) or DNA repair (MSH2) . Data analysis tools (e.g., MAGeCK) rank candidate resistance genes .

Q. How does 6-TG induce DNA demethylation, and what structural mechanisms underlie its interaction with methyltransferases?

• Methodology : 6-TG incorporates into DNA, forming S6-methylthioguanine adducts via SAM-dependent methylation. These adducts disrupt DNMT1 binding, as shown in crystallography studies. Demethylation is quantified via bisulfite sequencing in HCT116 cells treated with 1–5 µM 6-TG for 48 hr .

Q. What role do disulfide crosslinks play in 6-TG’s DNA interaction, and how are they characterized structurally?

• Methodology : X-ray crystallography of DNA duplexes with consecutive 6-TG residues reveals kinked helices at disulfide-bonded sites (6-thioG-6-thioG). Redox conditions (e.g., Cu²⁺/glutathione) reversibly modulate crosslinking, assessed via gel electrophoresis and circular dichroism .

Q. Data Contradiction and Resolution

Q. How can researchers resolve conflicting reports on 6-TG’s association with clinical remission in IBD?

• Methodology : Stratify studies by assay method (e.g., Lennard vs. Dervieux-Boulieu protocols) to isolate technical variability. Sensitivity analyses excluding outliers (e.g., studies with non-standard thresholds) reduce heterogeneity (I² from 75% to 30%) . Pooled ORs from homogenized subgroups provide more reliable effect estimates .

Q. Safety and Handling

Q. What precautions are critical for handling 6-TG in laboratory settings?

• Methodology : Use local exhaust ventilation and PPE (nitrile gloves, N95 masks) to avoid inhalation/contact. Store at 4°C in airtight, light-resistant containers. Decontaminate waste with 10% sodium hypochlorite before disposal .