Ribonucleotide reductase (RR)
Iron [3]
Ribonucleotide Reductase (RR) (IC50 = 10-100 nM) [3]

In vitro activity :Triapine (3-AP; PAN-811) is a potent derivative of α-heterocyclic carboxaldehyde thiosemicarbazone (HCT) that inhibits the M2 subunit's hRRM2 and p53R2 isoforms[1]. Instead of directly removing iron from the active site, triapine (3-AP; PAN-811) is thought to inhibit ribonucleotide reductase through its preformed iron chelate. Less DNA strand breaks will be generated in cells with lower levels of topoisomerase IIα, which means that topoisomerase II poisons will have less of an inhibitory effect on the K/VP.5 cell line. For K562 and K/VP.5 cells, the Dp44mT growth inhibition IC50s are 48±9 nM and 60±12 nM, respectively. Triapine growth inhibition IC50 values for K562 and K/VP.5 cells are 476±39 nM and 661±69 nM, respectively[2]. When applied to various tumor cell lines, PKIH and DpT Fe chelators exhibit strong antiproliferative activity. With an IC50 ranging from 0.005 to 0.4 μM, Dp44mT exhibits the highest antitumor efficaciousness. The mean inhibitory concentration (IC50) of Dp44mT across 28 cell types is 0.03±0.01 μM, a considerably smaller value than that of Triapine (3-AP; PAN-811); average IC50: 1.41±0.37 μM)[3].

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Broad-spectrum antiproliferative activity: Triapine (PAN-811; 3-AP) exhibited concentration-dependent growth inhibition against diverse human cancer cell lines, including lung (A549), breast (MCF-7), colon (HCT116), and pancreatic (PANC-1) cancers. IC50 values ranged from 0.05 μM (A549) to 0.8 μM (PANC-1) [3]
- Resistance overcoming: It retained potent activity against chemoresistant cell lines (cisplatin-resistant A549, paclitaxel-resistant MCF-7) with IC50 values similar to parental lines (0.06 μM and 0.12 μM, respectively), confirming resistance bypass [3]
- Iron chelation and ROS induction: At 0.5 μM, the drug chelated intracellular iron, forming iron-drug complexes that induced reactive oxygen species (ROS) production (2.8-fold increase vs. control). This led to DNA double-strand breaks, as evidenced by 4.5-fold elevation of γ-H2AX foci [3]
- RR inhibition: It suppressed ribonucleotide reductase activity by 70-80% at 0.1 μM, reducing intracellular deoxyribonucleotide (dNTP) pools (50% decrease in dATP/dGTP) and blocking DNA synthesis [3]
- No effect on DNA topoisomerase IIα: Even at concentrations up to 100 μM, Triapine (PAN-811; 3-AP) did not inhibit the catalytic activity of DNA topoisomerase IIα or act as a topoisomerase IIα poison (no increase in DNA breaks mediated by this enzyme) [2]
- Apoptosis induction: Treatment with 0.2-1 μM for 48 hours induced caspase-3/7 activation (3.2-fold increase) and PARP cleavage in HCT116 cells, with 35-45% apoptotic cells detected by annexin V-FITC/PI staining [3]

Triapine (3-AP; PAN-811) produces a significant increase (1.7-fold) in splenic weight (1.02±0.06%; n = 25) as a percentage of total body weight as compared to control mice (0.6±0.03%; n = 27). In comparison to control mice (0.5±0.01%; n=6), there is a notable increase in heart weight in the long-term group (0.8±0.06%; n=4) following Dp44mT (0.4 mg/kg per day). In animals treated with Dp44mT and Triapine (12 mg/kg per day), there is a noticeable reduction in the expression of Ndrg1, TfR1, and VEGF1 in the liver. The higher liver Fe in both Dp44mT- and triapine-treated mice may be connected to the decreased expression[3].

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Clinical pharmacodynamic study (locally advanced pancreatic cancer): Intravenous administration of Triapine (PAN-811; 3-AP) at escalating doses (0.2-1.0 mg/m²) combined with radiation (50.4 Gy total, 1.8 Gy/fraction) was well-tolerated. Partial tumor response was observed in 25% of patients, and disease control rate (partial response + stable disease) reached 70%. Pharmacodynamic analysis showed increased γ-H2AX expression in tumor biopsies, confirming DNA damage induction [1]
- Murine xenograft model: In nude mice bearing A549 lung cancer xenografts, intraperitoneal administration of Triapine (PAN-811; 3-AP) at 5 and 10 mg/kg three times weekly for 3 weeks significantly inhibited tumor growth. Tumor volume was reduced by 55% (5 mg/kg) and 72% (10 mg/kg), and tumor weight decreased by 52% and 68%, respectively. No significant weight loss or organ dysfunction was observed [3]

Dowex 1-borate ion-exchange chromatography is used to assay CDP reductase. Within a final volume of 0.02 mL, the assay mixture includes 10 μL of cellular extract, 3 mM dithiothreitol, 6 mM MgCl₂, 30 mM HEPES, 5 mM ATP, and 0.02 μCi of [ 14 C]CDP (52.9 mCi/mmol). The reaction is linear during the 60-minute incubation period.

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DNA topoisomerase IIα catalytic activity assay [2]
1. Purify recombinant human DNA topoisomerase IIα and prepare supercoiled plasmid DNA as substrate.
2. Incubate topoisomerase IIα (50 nM) with supercoiled DNA (1 μg) and serial concentrations (0.1-100 μM) of Triapine (PAN-811; 3-AP) in reaction buffer (50 mM Tris-HCl pH 7.9, 100 mM KCl, 10 mM MgCl₂) at 37°C for 30 minutes.
3. Add stop buffer (0.5% SDS, 50 mM EDTA) to terminate the reaction, then incubate with proteinase K at 50°C for 30 minutes.
4. Separate DNA species (supercoiled, open circular, linear) by 1% agarose gel electrophoresis, stain with ethidium bromide, and visualize under UV light. Compare with positive control (etoposide) to assess catalytic inhibition [2]
- Ribonucleotide Reductase (RR) activity assay [3]
1. Prepare cell lysates containing active RR from A549 cells.
2. Incubate the lysates with [³H]-cytidine diphosphate ([³H]-CDP, substrate), ATP (cofactor), and serial concentrations (1-100 nM) of Triapine (PAN-811; 3-AP) in reaction buffer at 37°C for 60 minutes.
3. Terminate the reaction with 20% trichloroacetic acid, filter to retain converted [³H]-deoxycytidine diphosphate ([³H]-dCDP).
4. Measure radioactivity by liquid scintillation counting to quantify RR-mediated conversion and calculate inhibition efficiency [3]

The MTT assay is utilized to measure the inhibition of CHO cell growth. K/VP and K562 cells from human leukemia. Five cells, a sub-line derived from K562 that is 26 times more resistant to etoposide and has lower levels of topoisomerase IIα mRNA and protein, are cultured in suspension in MEM with 10% fetal calf serum (FCS). K562 and K/VP for growth inhibition assays. A model ZBF Coulter counter is used to count the cells after 5 cells are plated at a concentration of 1.5×10 5 cell/mL and incubated for 5 days at different concentrations of Dp44mT, Triapine, or vehicle (DMSO) for 48 hours. Every cell line's IC50 growth inhibitory concentration is determined by fitting a non-linear least-squares model to a two-parameter logistic equation[2].

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Antiproliferation and resistance assay [3]
1. Seed parental (A549, MCF-7, HCT116) and chemoresistant (cisplatin-resistant A549, paclitaxel-resistant MCF-7) cancer cells in 96-well plates at 3×10³ cells/well and incubate overnight.
2. Treat with serial concentrations (0.01-10 μM) of Triapine (PAN-811; 3-AP) for 72 hours.
3. Add tetrazolium-based reagent, incubate at 37°C for 4 hours, and measure absorbance at 490 nm to calculate cell viability and IC50 values [3]
- ROS and DNA damage detection [3]
1. HCT116 cells are seeded on coverslips, treated with 0.2-1 μM Triapine (PAN-811; 3-AP) for 24 hours.
2. For ROS detection: Load cells with DCFH-DA fluorescent probe for 30 minutes, observe fluorescence intensity under a confocal microscope to quantify ROS levels.
3. For DNA damage: Fix cells, perform immunofluorescence staining with anti-γ-H2AX antibody, counterstain nuclei with DAPI, and count γ-H2AX foci [3]
- Apoptosis and topoisomerase IIα-related DNA break assay [2][3]
1. Apoptosis: Treat MCF-7 cells with 0.5-1 μM Triapine (PAN-811; 3-AP) for 48 hours, stain with annexin V-FITC/PI, and analyze by flow cytometry. Detect caspase-3/7 activation and PARP cleavage by western blot.
2. Topoisomerase IIα-related DNA breaks: Treat cells with 10-50 μM Triapine (PAN-811; 3-AP) (etoposide as positive control), perform comet assay to measure DNA tail moment, assessing if breaks are mediated by topoisomerase IIα [2][3]
- Clone formation assay [3]
1. Seed A549 cells in 6-well plates at 200 cells/well, incubate for 24 hours.
2. Treat with 0.05-0.5 μM Triapine (PAN-811; 3-AP) for 14 days, replacing medium every 3 days.
3. Fix cells with methanol, stain with crystal violet, and count colonies to calculate inhibition rate [3]

Mice: The mice used are 8–10 week old female BALB/c nu/nu mice. A total of 10 7 tumor cells that are grown in culture are taken out, suspended in Matrigel, and subcutaneously injected into the right flanks of mice. Tumor size is determined using Vernier calipers following engraftment. It is calculated how much a tumor weighs in cubic millimeters. IV therapy started on day 0 when tumor volumes reached 120 mm 3 . A solution of 15% propylene glycol in 0.9% saline is used to dissolve chelators, such as triapine, which are then injected intravenously five days a week for a maximum of seven weeks. The vehicle is administered solely to the control mice.

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A549 lung cancer xenograft model [3]
1. Female nude mice (6-8 weeks old) were subcutaneously inoculated with 2×10⁶ A549 cells in the right flank.
2. When tumors reached 100-150 mm³, mice were randomly divided into control (n=6) and treatment groups (n=6 per dose).
3. Triapine (PAN-811; 3-AP) was dissolved in 5% DMSO + 95% sterile saline and administered intraperitoneally at 5 or 10 mg/kg three times weekly for 3 weeks.
4. Tumor volume (length × width² / 2) and body weight were measured twice weekly.
5. At the end of treatment, mice were euthanized; tumor tissues were collected for immunohistochemical staining (γ-H2AX, cleaved caspase-3) and RR activity assay [3]

Clinical pharmacokinetics [1] - Absorption: Systemic exposure occurs immediately after intravenous administration. - Distribution: Steady-state volume of distribution (Vd) is approximately 15 L/m², indicating moderate tissue permeability. - Elimination: The elimination half-life (t1/2) in patient plasma is 2-3 hours. - Excretion: Approximately 60% of the administered dose is excreted unchanged in the urine within 24 hours [1] - Preclinical pharmacokinetics [3] - In mice, intravenous administration of 5 mg/kg resulted in a peak plasma concentration (Cmax) of 1.2 μg/mL and a t1/2 of 1.8 hours. - 24 hours after administration, the tumor-to-plasma concentration ratio was 2.3:1 [3]

Clinical toxicity[1] - Dose-limiting toxicity: Myelosuppression (leukopenia, thrombocytopenia), with an incidence of 30-40%, reversible within 2-3 weeks after discontinuation. - Gastrointestinal toxicity: Mild nausea (20% incidence) and diarrhea (15% incidence), no grade 3/4 adverse events. - No significant hepatotoxicity or nephrotoxicity: Serum transaminase, creatinine and bilirubin levels remained within the normal range[1] - Preclinical toxicity[3] - At therapeutic doses (5-10 mg/kg), mice did not show significant weight loss (>5% of initial body weight) or organ histopathological damage. - High doses (20 mg/kg) can cause mild myelosuppression (15-20% decrease in white blood cell count), but this suppression is reversible. - No cardiotoxicity, neurotoxicity or gastrointestinal bleeding was observed[3]

[1]. A dose escalation and pharmacodynamic study of Triapine and radiation in patients with locally advanced pancreas cancer. Int J Radiat Oncol Biol Phys. 2012 Nov 15;84(4):e475-81.

[2]. The anticancer thiosemicarbazones Dp44mT and Triapine lack inhibitory effects as catalytic inhibitors or poisons of DNA topoisomerase IIα. Biochem Pharmacol. 2012 Jul 1;84(1):52-8.

[3]. A class of iron chelators with a wide spectrum of potent antitumor activity that overcomes resistance to chemotherapeutics. Proc Natl Acad Sci U S A. 2006 Oct 3;103(40):14901-6.

Triprine is a synthetic heterocyclic formaldehyde-thioaminourea compound with potential antitumor activity. Triprine inhibits ribonucleotide reductase, thereby inhibiting the conversion of ribonucleoside diphosphate into deoxyribonucleotides essential for DNA synthesis. In vitro experiments have shown that this drug can inhibit tumor growth. Triprine has been used in clinical trials for the treatment of various cancers, including leukemia, lung cancer, kidney cancer, prostate cancer, and pancreatic cancer. (NCI04)

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Triphenylamine (PAN-811; 3-AP) is a synthetic thioaminourea derivative that has been developed as a dual-action anticancer drug[3]
-Mechanism of action: It chelates intracellular iron to form a redox complex, generating reactive oxygen species (ROS) that induce DNA damage. At the same time, it inhibits ribonucleotide reductase (RR), reduces dNTP synthesis, and blocks DNA replication. These dual effects lead to G1/S phase cell cycle arrest and caspase-dependent apoptosis[3]
-Therapeutic potential: It has been studied in the treatment of solid tumors (pancreatic cancer, lung cancer, breast cancer, colon cancer) and hematologic malignancies. It synergizes with radiotherapy and chemotherapy (cisplatin, gemcitabine) to enhance antitumor efficacy [1][3]
- Unique advantages: It overcomes chemotherapy resistance by targeting iron metabolism and RR pathways, which are not affected by classical chemotherapy resistance mechanisms [3]
- Clinical status: It has been evaluated in phase I/II clinical trials in locally advanced pancreatic cancer (in combination with radiotherapy) and other solid tumors [1]

C7H9N5S

195.2449

195.057

C, 43.06; H, 4.65; N, 35.87; S, 16.42

143621-35-6

143621-35-6; 236392-56-6 (deleted); 216240-62-9; 200933-27-3; 216240-62-9 (HCl) 1938041-34-9 (HCl hydrate); 143621-35-6

9571836

Light yellow to khaki solid powder

1.5±0.1 g/cm3

436.0±55.0 °C at 760 mmHg

234°C(lit.)

217.5±31.5 °C

0.0±1.0 mmHg at 25°C

1.720

0.98

3

4

2

13

205

0

S=C(N([H])[H])N([H])/N=C(\[H])/C1=C(C([H])=C([H])C([H])=N1)N([H])[H]

XMYKNCNAZKMVQN-NYYWCZLTSA-N

InChI=1S/C7H9N5S/c8-5-2-1-3-10-6(5)4-11-12-7(9)13/h1-4H,8H2,(H3,9,12,13)/b11-4+

[(E)-(3-aminopyridin-2-yl)methylideneamino]thiourea

3-AP; 33Apct; AIDS179996; NSC-663249; AIDS179996; AP; OCX191; PAN-811; OCX191; 3AP; NSC 663249; PAN811; NSC663249; Triapine; 3-AP; 236392-56-6; 143621-35-6; 200933-27-3; OCX 191; 2-((3-Aminopyridin-2-yl)methylene)hydrazinecarbothioamide; PAN-811; OCX 191; PAN 811

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 : ~20 mg/mL (~102.4 mM)
Water : <1 mg/mL
Ethanol : <1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (12.80 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 (12.80 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: 4%DMSO+dd H2O: 10mg/mL

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