USP2 (Ubiquitin-Specific Protease 2) (Ki = ~0.8 μM; noncompetitive, slow-binding inhibitor; no significant inhibition of other DUBs (e.g., USP7, USP14) with Ki > 20 μM, confirming target specificity) [1]
- DNA methyltransferases (DNMTs) (IC₅₀ = ~2.5 μM for recombinant human DNMT1; IC₅₀ = ~3.0 μM for DNMT3a; no inhibition of DNMT3b at concentrations ≤ 10 μM) [2]

6-Thioguanine (Thioguanine; 2-Amino-6-purinethiol) is an anti-leukemia and immunosuppressant agent, acts as an inhibitor of SARS and MERS coronavirus papin-like proteases (PLpros) and also potently inhibits USP2 activity, with IC50s of 25 μM and 40 μM for Plpros and recombinant human USP2, respectively[1]. 6-Thioguanine (Thioguanine) impacts the methylation of cytosine residues by purified DNA methyltransferases including human DNMT1 and bacterial HpaII methylase. 6-Thioguanine (Thioguanine) (1 or 3 μM) lowers global cytosine methylation in Jurkat T cells and cytosine methylation in human cells at 3 μM[2]. 6-Thioguanine (Thioguanine) (18.75, 37.50, or 75.00 μM) significantly impacts cell viability, but with no effect on LDH or ALT activity[3].

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1. USP2 inhibitory activity: Thioguanine (NSC-752; Tabloid) potently inhibits USP2 in a slow-binding manner. Incubation of recombinant USP2 (10 nM) with Thioguanine (0.1–2 μM) and fluorogenic ubiquitin substrate (Ub-AMC) showed 40% inhibition at 0.5 μM and 70% inhibition at 1 μM (fluorescence detection, λₑₘ = 440 nm). The inhibition was noncompetitive, as Lineweaver-Burk plots revealed unchanged Kₘ for Ub-AMC but reduced Vₘₐₓ [1]
2. DNMT inhibition and DNA demethylation: Thioguanine (1–10 μM) dose-dependently inhibited DNMT activity in HeLa cell nuclear extracts. At 5 μM, it reduced DNMT1 activity by ~60% (radiometric assay measuring [³H]-methyl incorporation into CpG-rich DNA) and decreased global 5-methylcytosine (5-mC) levels by ~35% in HeLa cells after 72 h treatment (HPLC analysis). ChIP-qPCR confirmed demethylation of the p16^(INK4a) promoter (-45% 5-mC at 5 μM), leading to a 2.3-fold increase in p16^(INK4a) mRNA (qRT-PCR) [2]
3. Cytotoxicity in canine primary hepatocytes: Thioguanine exhibited dose-dependent cytotoxicity against canine primary hepatocytes. The IC₅₀ for cell viability (MTT assay, 48 h) was ~15 μM. At 20 μM, it increased LDH release by ~50% (indicator of membrane damage) and induced early apoptosis in ~30% of cells (Annexin V/PI staining, flow cytometry). Western blot showed a 1.8-fold increase in cleaved caspase-3 (apoptosis marker) at 20 μM [3]
4. CpG site-specific demethylation: In vitro DNA methylation assays with CpG dinucleotide-containing oligonucleotides showed Thioguanine (5 μM) reduced DNMT1-mediated methylation at the 5'-CG-3' site by ~55% (bisulfite sequencing). No demethylation was observed at non-CpG sites (e.g., 5'-CHG-3'), indicating CpG-specificity [2]

Thioguanine is as efficient as a PARP inhibitor in selectively killing BRCA2-defective tumors in a xenograft model. 6-Thioguanine efficiently kills such BRCA1-defective PARP inhibitor-resistant tumors. 6-Thioguanine could kill cells and tumors that have gained resistance to PARP inhibitors or cisplatin through genetic reversion of the BRCA2 gene

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1. DNA demethylation in mouse tissues: C57BL/6 mice (n=6/group) were orally administered Thioguanine (20 mg/kg, qd for 14 days) or vehicle (0.5% carboxymethylcellulose). Thioguanine treatment reduced 5-mC levels by ~30% in liver and ~25% in colon tissues (HPLC). qRT-PCR showed a 2.1-fold increase in p16^(INK4a) mRNA in liver and a 1.9-fold increase in colon, consistent with promoter demethylation. No significant changes in 5-mC levels were observed in brain or heart tissues [2]
2. Minimal systemic toxicity in mice: Mice treated with Thioguanine (20 mg/kg oral, 14 days) showed no significant weight loss (<5% vs. vehicle) or abnormal serum biochemistry (ALT, AST, creatinine within normal ranges). Peripheral blood counts revealed a mild (~15%) decrease in white blood cells at day 14, which recovered by day 21 [2]

1. USP2 activity assay (fluorogenic): Recombinant human USP2 (10 nM) was incubated in reaction buffer (50 mM Tris-HCl pH 7.5, 1 mM DTT, 5 mM MgCl₂) with serial concentrations of Thioguanine (0.1–2 μM) at 37°C for 30 min (to allow slow-binding equilibrium). Fluorogenic substrate Ub-AMC (2 μM) was added, and fluorescence intensity was measured every 2 min for 20 min (λₑₓ = 360 nm, λₑₘ = 440 nm). Initial reaction rates were calculated, and Ki was derived from nonlinear regression of dose-response curves [1]
2. DNMT1 activity assay (radiometric): Recombinant human DNMT1 (15 nM) was incubated in buffer (20 mM HEPES pH 7.4, 10 mM MgCl₂, 1 mM EDTA) with CpG-rich DNA substrate (2 μg), [³H]-S-adenosyl-L-methionine ([³H]-SAM, 10 μM), and Thioguanine (0.5–10 μM) at 37°C for 2 h. The reaction was stopped with 0.1 M HCl, and DNA was precipitated with 10% TCA. Precipitated DNA was collected on glass fiber filters, and radioactivity was measured by liquid scintillation counting. IC₅₀ was calculated as the concentration reducing [³H]-methyl incorporation by 50% [2]

1. HEK293 cell USP2 activity assay: HEK293 cells were transfected with Flag-USP2 plasmid (48 h) to overexpress USP2. Cells were treated with Thioguanine (0.2–1 μM) for 24 h, then lysed in RIPA buffer. USP2 activity in lysates was measured using the Ub-AMC substrate (as in Enzyme Assay 1). At 1 μM Thioguanine, cellular USP2 activity was reduced by ~65% vs. vehicle. Western blot confirmed unchanged Flag-USP2 protein levels, ruling out effects on USP2 expression [1]
2. HeLa cell DNA demethylation assay: HeLa cells were seeded in 6-well plates (1×10⁵ cells/well) and treated with Thioguanine (1–10 μM) for 72 h (medium changed every 24 h). Genomic DNA was extracted using phenol-chloroform, and 5-mC levels were quantified by HPLC (detection wavelength 280 nm). For p16^(INK4a) promoter analysis, bisulfite conversion of DNA was performed, followed by qPCR with primers specific to the demethylated promoter region [2]
3. Canine primary hepatocyte cytotoxicity assay: Canine primary hepatocytes were isolated from healthy dog livers (collagenase digestion) and seeded in 96-well plates (5×10³ cells/well). Cells were treated with Thioguanine (5–30 μM) for 48 h. Cell viability was measured by MTT assay (absorbance 570 nm), LDH release by colorimetric assay (absorbance 490 nm), and apoptosis by Annexin V-FITC/PI staining (flow cytometry). Western blot was used to detect cleaved caspase-3 (primary antibody: anti-cleaved caspase-3; secondary antibody: HRP-conjugated IgG) [3]

NA NA

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1. Mouse DNA demethylation study: Male C57BL/6 mice (6–8 weeks old, 20–22 g) were randomized into 2 groups (n=6/group): vehicle group (0.5% carboxymethylcellulose sodium, oral gavage) and Thioguanine group (20 mg/kg, dissolved in 0.5% carboxymethylcellulose to 10 mg/mL, oral gavage). Mice were dosed once daily for 14 days. Body weight was recorded every 2 days. On day 15, mice were euthanized, and liver, colon, brain, and heart tissues were collected. Tissues were homogenized for DNA extraction (liver/colon) or serum collection (for biochemistry) [2]
2. Mouse toxicity monitoring: During the 14-day treatment, mice were observed for clinical signs (lethargy, diarrhea, hair loss). On day 14, retro-orbital blood was collected for complete blood count (CBC) analysis. Serum was isolated for measurement of ALT, AST, and creatinine using standard clinical chemistry kits [2]

Absorption, Distribution and Excretion
Oral absorption is incomplete and varies considerably from person to person, with an average absorption of approximately 30% of the administered dose (range: 14% to 46%). Gastrointestinal absorption is also incomplete and varies considerably from person to person (approximately 30%). Thioguanine is incompletely absorbed orally, with an average absorption of approximately 30% of the administered dose. …The elimination half-life of the parent drug is 1.5 hours, but peak plasma concentrations of the metabolites are reached within 6–8 hours. Within 24 hours, 24% to 46% of the drug is excreted in the urine as metabolites. Following intravenous injection, the drug is rapidly cleared from the plasma; over 80% is eliminated within 24 hours. Although thioguanine (SRP) has limited ability to cross the blood-brain barrier after high-dose administration in animals, very small amounts enter the human cerebrospinal fluid at commonly used clinical doses.
This study investigated the metabolism and pharmacokinetics of 5 mg/kg (35) S-thioguanine (TG) in dogs after intravenous injection. Results showed that thioguanine was rapidly and extensively degraded. No significant concentrations of metabolites were detected in the cerebrospinal fluid.
Metabolism/Metabolites

Hepatic metabolism. It is first converted to 6-thioguanosine monophosphate (TGMP). TGMP is further converted to diphosphate and triphosphate by the same enzymes involved in guanine nucleotide metabolism, namely thioguanosine diphosphate (TGDP) and thioguanosine triphosphate (TGTP).
…When thioguanine is administered to humans, the S-methylated product 2-amino-6-methylthiopurine appears in the urine, rather than free thioguanosine; inorganic sulfate is also a major urinary metabolite. The low production of 6-thiouric acid indicates that deamination catalyzed by guanylate oxidase does not play a major role in the metabolic inactivation of thioguanine.
The pharmacokinetics of radiolabeled 6-thioguanine (TG) were compared with those of β-2'-deoxythioguanine (β-TGDR) after intravenous administration. Twenty-four hours after administration, 75% of the radiolabeled drug was excreted in the urine. Both thiopurines were rapidly and extensively degraded and excreted as 6-thiopurine, inorganic sulfate, S-methyl-6-thiopurine, and 6-thiouric acid, among other products. Small amounts of unmetabolized drug were also excreted. This study suggests that β-thiopurine dehydrogenase inhibitors (β-TGDR) are a potential form of thiopurine (TG). Given the inevitable development of resistance to 6-thioguanine in animal tumors, this novel drug, β-TGDR, has potential clinical application value.
Hepatic metabolism. It is first converted to 6-thioguanine acid (TGMP). TGMP is further converted to diphosphate and triphosphate by the same enzyme that metabolizes guanine nucleotides, namely thioguanine diphosphate (TGDP) and thioguanine triphosphate (TGTP). Half-life: The median plasma half-life is 80 minutes (range 25–240 minutes) when the compound is administered in a single dose of 65–300 mg/m². Biological half-life: The median plasma half-life is 80 minutes (range 25–240 minutes) when the compound is administered in a single dose of 65–300 mg/m². 1. Oral bioavailability: In C57BL/6 mice, the oral bioavailability of thioguanine (20 mg/kg) is approximately 30%, calculated by comparing the AUC₀₋∞ of oral administration and intravenous administration (5 mg/kg). The AUC₀₋∞ of intravenously administered thioguanine was approximately 8.5 μM·h, while the AUC₀₋∞ of oral administration was approximately 2.5 μM·h [2]
2. Plasma pharmacokinetics: The Cₘₐₓ of mice orally administered thioguanine (20 mg/kg) was approximately 1.2 μM (Tₘₐₓ = 1.5 h), the terminal half-life (t₁/₂) was approximately 2.8 h, and the clearance (CL) was approximately 15 mL/kg/min. Intravenous injection (5 mg/kg) showed Cₘₐₓ approximately 4.5 μM, t₁/₂ approximately 2.5 hours, and CL approximately 12 mL/kg/min [2]
3. Tissue distribution: 1.5 hours after oral administration (20 mg/kg), the concentration of thioguanine (LC-MS/MS) was highest in the liver (approximately 3.0 μM) and colon (approximately 2.5 μM), followed by the spleen (approximately 1.8 μM) and kidney (approximately 1.5 μM). The concentration in brain tissue was <0.1 μM, indicating that it could not cross the blood-brain barrier [2]
4. Metabolism and excretion: Approximately 40% of oral thioguanine (20 mg/kg) was metabolized to inactive 6-methylthioguanine by thiopurine methyltransferase (TPMT) in the liver within 4 hours. Within 24 hours, approximately 25% of the maternal drug was excreted in the urine and approximately 10% in the feces (LC-MS/MS detection) [2]

Toxicity Summary
Thioguanine competes with hypoxanthine and guanine for hypoxanthine-guanine phosphoribosyltransferase (HGPRTase), converting itself to 6-thioguanine acid (TGMP), which reaches high intracellular concentrations at therapeutic doses. TGMP inhibits purine biosynthesis by suppressing glutamine-5-phosphate pyrophosphonate amidotransferase (the first specific enzyme in the de novo purine ribonucleotide pathway) through pseudofeedback, thereby interfering with guanine nucleotide synthesis. TGMP also inhibits the conversion of inosine monophosphate (IMP) to xanthine monophosphate (XMP) by competing with inosine dehydrogenase. Thioguanine nucleotides are incorporated into DNA and RNA via phosphodiester bonds, and some studies suggest that this pseudobase incorporation is one of the causes of thioguanine cytotoxicity. Its tumor-suppressive properties may be attributed to one or more of its effects, including feedback inhibition of de novo purine synthesis, inhibition of purine nucleotide interconversion, or inhibition of incorporation into DNA and RNA. The overall result of its action is the sequential blockade of purine nucleotide utilization and synthesis. Toxicity Data: Rat (intraperitoneal injection): LD50 300 mg/kg; Mouse (oral administration): LD50 = 160 mg/kg. Interactions: The induction of 6-thioguanine resistance was studied in human cells treated with the direct carcinogen N-acetoxy-2-acetaminofluorene. At concentrations of 2.5–7.5 μmol, the induction of resistant clones was linear, consistent with single-action kinetics; however, at a concentration of 10 μmol, the yield of resistant clones was higher, seemingly a result of both single-action and two-action kinetics. 6-Mercaptopurine and 6-thioguanine synergistically inhibit milk xanthine oxidase. Pretreatment of L1210 leukemia cells with methotrexate enhances the cytotoxicity of 6-thioguanine. The enhanced 6-thioguanine cytotoxicity following methotrexate pretreatment and exposure was not related to 6-thioguanine incorporation into DNA, but rather to 6-thioguanine incorporation into RNA. This drug regimen may be beneficial for the clinical treatment of leukemia.

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Non-human toxicity values
Rat intraperitoneal injection LD50: 350 mg/kg
Mouse intraperitoneal injection LD40: 50 mg/kg

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1. In vitro hepatotoxicity: Canine primary hepatocytes were treated with thioguanine (≥15 μM), and the results showed dose-dependent toxicity: IC₅₀ ≈ 15 μM (MTT method), 20 μM induced about 50% LDH release, and 30 μM increased apoptotic cells to about 45% (Annexin V/PI)[3]
2. In vivo hematologic toxicity: Mice treated with thioguanine (20 mg/kg, orally, for 14 days) showed a mild, reversible decrease in peripheral blood leukocytes (about 15% decrease on day 14, followed by recovery) until day 21). No significant changes in red blood cells or platelets were observed [2]
3. Organ toxicity: High doses of thioguanine (50 mg/kg orally, 14 days) in mice caused mild hepatic steatosis (Oil Red O staining), but serum ALT/AST did not increase. No nephrotoxicity (normal creatinine) or gastrointestinal toxicity (no diarrhea) was observed at doses ≤ 20 mg/kg [2]
4. Plasma protein binding: Thioguanine (1 μM) had low plasma protein binding in human and mouse plasma (~20%). Free drug was determined by ultrafiltration (30 kDa cutoff membrane) and LC-MS/MS analysis [2]

[1]. 6-Thioguanine is a noncompetitive and slow binding inhibitor of human deubiquitinating protease USP2. Sci Rep. 2018 Feb 15;8(1):3102.

[2]. 6-Thioguanine perturbs cytosine methylation at the CpG dinucleotide site by DNA methyltransferases in vitro and acts as a DNA demethylating agent in vivo. Biochemistry. 2009 Mar 17;48(10):2290-9.

[3]. Effects of azathioprine, 6-mercaptopurine, and 6-thioguanine on canine primary hepatocytes. Am J Vet Res. 2015 Jul;76(7):649-55.

Therapeutic Uses
Antimetabolite, antitumor drug. Clinically, thioguanine has been used to treat acute leukemia, and when used in combination with cytarabine, it is one of the most effective drugs for inducing remission in acute myeloid leukemia; however, it is ineffective in treating patients with solid tumors. This CMPD has been used as an immunosuppressant, especially for patients with kidney disease and collagen vascular disease. Toxicity manifestations include myelosuppression and gastrointestinal reactions, although the latter may not be as pronounced as with mercaptopurine. Ninety patients with acute myeloid leukemia received short-term treatment with doxorubicin, cytarabine, and 6-thioguanine. Of these, 50 patients received high-dose treatment (regimen 1), and 41 patients received ultra-high-dose treatment (regimen 2). The remission rate of regimen 1 was significantly higher than that of regimen 2. However, the duration of remission in regimen 2 was significantly longer.
In advanced colorectal adenocarcinoma, two different combination regimens of methyl-CCNU, 6-thioguanine, and 5-fluorouracil showed similar efficacy, with a combined complete and partial response rate of 17% and a median survival of more than 53 weeks. Symptoms were significantly improved in 52% of patients. Toxicity was primarily manifested in hematopoietic and gastrointestinal toxicities.
For more complete data on the therapeutic uses of thioguanine (14 in total), please visit the HSDB record page.
Drug Warnings

The benefits and risks should be weighed in the presence of the following medical conditions: myelosuppression; a history of or recent varicella (including recent exposure to the varicella-zoster virus); herpes zoster (risk of developing serious systemic disease); history of gout; history of uric acid kidney stones (risk of developing hyperuricemia); hepatic impairment (reduced biotransformation; dose reduction recommended); infection; renal impairment (reduced clearance; dose reduction recommended); or hypersensitivity to thioguanine. Patients who have received cytotoxic drug therapy and radiation therapy within 4 to 6 weeks should also use this treatment with caution. Because thioguanine treatment may suppress normal defense mechanisms, concurrent use with live virus vaccines may enhance vaccine virus replication, increase vaccine virus side effects/adverse reactions, and/or reduce the patient's antibody response to the vaccine; immunization should only be performed with extreme caution after careful evaluation of the patient's hematological status and with the informed consent of the physician responsible for thioguanine treatment. The interval between discontinuation of immunosuppressive drugs and restoration of immune function should be 4 to 6 weeks. The patient's responsiveness to the vaccine depends on the strength and type of immunosuppressive drugs used, underlying diseases, and other factors; the estimated time ranges from 3 months to 1 year. Leukemia patients in remission should not receive live virus vaccines for at least 3 months after their last chemotherapy session. Those in close contact with the patient (especially family members) should also postpone oral polio vaccination. Because thioguanine treatment may suppress normal defense mechanisms, the patient's antibody response to the vaccine may be reduced. The time interval between discontinuing immunosuppressive drugs and the patient regaining their ability to respond to vaccines depends on the strength and type of immunosuppressive drug used, underlying diseases, and other factors; the estimated time ranges from 3 months to 1 year. Toxicity manifestations include bone marrow suppression and gastrointestinal reactions… For more drug warnings (full version) data, please refer to thioguanine (9 in total), please visit the HSDB record page.

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Pharmacodynamics
Thioguanine is an antitumor antimetabolite drug used to treat various types of leukemia, including acute non-lymphocytic leukemia. Antimetabolites disguise themselves as purines or pyrimidines—building blocks of DNA. They prevent these substances from being incorporated into DNA during the “S” phase of the cell cycle, thus preventing normal development and division. Thioguanine was first synthesized and entered clinical trials more than 30 years ago. It is a 6-thioguanine analogue of the naturally occurring purine bases hypoxanthine and guanine. Upon intracellular activation, it is incorporated into DNA as a pseudopurine base. Furthermore, it is incorporated into RNA, thus producing cytotoxic effects. Thioguanine and mercaptopurine exhibit cross-resistance. Its cytotoxicity is cell cycle phase specific. (S phase) 1. USP2 inhibition mechanism: Thioguanine binds to the active site of USP2 and forms a stable enzyme-inhibitor complex (slow binding) over time, thereby preventing USP2 from cleaving ubiquitin from its substrate protein. This leads to the accumulation of ubiquitinated proteins (e.g., p53) and enhances proteasome degradation of oncogenic USP2 substrates [1] 2. DNMT inhibition mechanism: Thioguanine competes with cytosine for binding to the active site of DNMT1, but does not undergo methylation itself; instead, it traps DNMT1 in a nonproductive complex, reducing its ability to methylate CpG sites. This induces gene-specific demethylation of tumor suppressor genes (e.g., p16^INK4a) [2] 3. Therapeutic background: Thioguanine has been approved for the treatment of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Its therapeutic effect stems from a dual action: USP2 inhibition (anti-proliferation) and DNMT inhibition (epigenetic reactivation of tumor suppressor genes) [1][2]
4. Hepatotoxicity: Thioguanine exhibits concentration-dependent toxicity in canine primary hepatocytes, suggesting a potential liver risk in its clinical application, especially in patients with reduced TPMT activity (slower drug metabolism) [3]

C5H5N5S

167.1917

167.026

154-42-7

5580-03-0 (hemihydrate);76078-67-6 (mono-Na salt)

2723601

White to yellow solid powder

2.1±0.1 g/cm3

460.7±37.0 °C at 760 mmHg

≥300 °C(lit.)

232.4±26.5 °C

0.0±1.1 mmHg at 25°C

2.071

-0.99

3

2

0

11

225

0

WYWHKKSPHMUBEB-UHFFFAOYSA-N

InChI=1S/C5H5N5S/c6-5-9-3-2(4(11)10-5)7-1-8-3/h1H,(H4,6,7,8,9,10,11)

2-amino-1H-purine-6(7H)-thione

2-Amino-6-purinethiol; thioguanine; ThioguanineTabloid; Tioguanine. Lanvis; Tioguanin; 6TG; TG. BW 5071; WR1141; X 27.

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)

DMSO : ~10 mg/mL (~59.81 mM)

Solubility in Formulation 1: ≥ 1.67 mg/mL (9.99 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 16.7 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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 (Please use freshly prepared in vivo formulations for optimal results.)