Triapine targets the M2 subunit of ribonucleotide reductase (RR) , specifically the hRRM2 and p53R2 isoforms. No specific IC50, Ki, or EC50 values are reported in this clinical study. [1]
The primary target of Triapine is the M2 subunit of ribonucleotide reductase (RNR). RNR is a heterodimeric enzyme composed of RR M1 and RR M2 subunits, catalyzing the rate-limiting step in the conversion of ribonucleoside diphosphates to deoxyribonucleoside diphosphates, essential for DNA synthesis and repair. Triapine is a potent iron chelator that binds the iron (Fe²⁺) at the active site of the RR M2 subunit, quenching the tyrosyl radical required for enzymatic activity and leading to enzyme inactivation. Beyond direct RNR inhibition, Triapine exerts its anti-tumor effects by disrupting homologous recombination repair (HRR): it inhibits CDK activity and activates Chk1, blocking CtIP phosphorylation induced by olaparib or etoposide, thereby disrupting BRCA1-MRN complex interaction and Rad51 focus formation, ultimately impairing DNA double-strand break repair and sensitizing tumor cells to PARP inhibitors and topoisomerase inhibitors.

The in vitro anti-tumor activity of Triapine has been extensively studied. In the NCI 60-cell line panel, Triapine exhibited a mean GI₅₀ of 1.6 μM following 48-hour exposure, with leukemia cell lines showing the highest sensitivity (4 of 6 lines with GI₅₀ < 0.6 μM). In BRCA wild-type ovarian cancer cell lines (SKOV-3, BG-1, PEO4), Triapine at 0.5-0.75 μM synergistically enhanced the cytotoxicity of olaparib (PARP inhibitor), with combination index (CI) values indicating clear synergy. Furthermore, Triapine significantly inhibited etoposide-induced BRCA1 focus formation, comparable to BRCA1 knockdown cells, demonstrating sensitization through HRR disruption. In glioblastoma (GBM) cells, Triapine treatment at EC₅₀ concentrations increased γH2AX and p-RPA32 levels, decreased Rad51 and 53BP1 levels, induced DNA double-strand break accumulation as shown by comet assay, and triggered apoptosis. In African swine fever virus (ASFV) studies, Triapine dose-dependently inhibited viral replication, and molecular docking suggested Triapine may interact with the Fe²⁺ active center of the ASFV RNR small subunit.

Triapine demonstrates anti-tumor activity in various in vivo tumor models. In the L1210 leukemia mouse model, twice-daily administration of Triapine significantly prolonged survival of tumor-bearing mice, with some mice achieving cure. In M109 lung carcinoma and A2780 human ovarian carcinoma xenograft models, Triapine also exhibited significant tumor growth inhibition. Mechanistic studies showed that Triapine inhibited DNA synthesis in L1210 cells for approximately 10 hours, while recovery of DNA synthesis in normal tissues (duodenum and bone marrow) was significantly faster than in leukemia cells, providing a pharmacological basis for the therapeutic index of this agent. In an intracranial glioblastoma model, NMRI nude mice co-injected intracranially with GBM03 cells and 10 μM Triapine showed significantly prolonged survival in the Triapine-treated group compared to controls (P < 0.05) by Kaplan-Meier analysis. In clinical studies, oral Triapine (100 mg daily, 5 days per week for 5 weeks) combined with cisplatin chemoradiation in patients with locally advanced cervical cancer achieved metabolic complete response (mCR) in 8 of 13 evaluable patients (approximately 62%).

The RNR inhibitory activity of Triapine is initially assessed by measuring its effect on RNR activity in purified enzyme preparations or cell lysates. A typical assay protocol involves: preparing cell lysates from logarithmically growing L1210 cells or tumor tissues, incubating with varying concentrations of Triapine (0-100 µM) and substrates (e.g., CDP or GDP) along with ³H-labeled corresponding substrates for reaction tracking. Following incubation at 37°C, reactions are terminated by strong acid or heat, unreacted substrates are separated from products (e.g., using Dowex-1 borate column chromatography), and radioactive incorporation is measured by liquid scintillation counting to calculate product formation rates and IC50 values. For assessing direct effects of Triapine on RNR, recombinant RR M1 and RR M2 proteins can be used to reconstitute enzyme activity in a cell-free system. Additionally, the metal-chelating ability of Triapine can be evaluated by UV-visible spectroscopy—monitoring absorbance changes at specific wavelengths (e.g., characteristic absorption peaks of the iron-thiosemicarbazone complex) following addition of Triapine to iron-containing buffer solutions.

ELISA for serum hRRM1 and hRRM2 measurement: Blood samples were collected immediately before and after triapine administration on days 1 and 33. Polystyrene 96-well plates were coated with hRRM1 or hRRM2 immunogen (1 g/mL in pyrogen-free PBS, pH 7). Unbound sites were blocked with 2% BSA in PBS for 1 hour at 37°C. Serum samples were added and incubated at 37°C for 1 hour. After washing, plates were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG antibody for 1 hour, then washed and incubated with substrate solution. Spectrophotometric measurements were taken and reported as 50% titer. All samples were measured in duplicate. [1]

---

In vitro cellular activity assessment of Triapine primarily includes cell proliferation inhibition assays, colony formation assays, DNA damage repair analysis, and cell cycle/apoptosis detection. A typical procedure is as follows: Tumor cells (e.g., SKOV-3 ovarian cancer cells, L1210 leukemia cells, GBM cells) are seeded in 96-well plates (5×10³-1×10⁴ cells/well) or 6-well plates and cultured overnight in medium containing 10% FBS. Increasing concentrations of Triapine (0-10 μM, typically 0.1-5 μM range) are added, and after 48-72 hours of treatment, cell viability is measured using MTT, MTS, or CellTiter-Glo assays to calculate IC50 values. Colony formation assay: Cells are treated with Triapine alone or in combination with other chemotherapeutic agents (e.g., olaparib, etoposide, cisplatin) for 24 hours, then trypsinized, counted, and seeded at appropriate densities in 6-well plates. After 7-14 days of culture, colonies are fixed with methanol, stained with crystal violet, and colonies containing >50 cells are counted. DNA damage repair analysis (immunofluorescence): Treated cells are fixed, permeabilized, stained with anti-γH2AX, BRCA1, Rad51, or 53BP1 antibodies, and nuclear foci are counted by confocal microscopy. Comet assay (single-cell gel electrophoresis): Treated cells are embedded in agarose gel slides, lysed, unwound, and subjected to electrophoresis, with DNA damage evaluated by comet tail moment under fluorescence microscopy. Apoptosis is detected by Annexin V-FITC/PI double staining flow cytometry.

In vivo efficacy studies of Triapine are primarily evaluated using mouse tumor models. Taking the L1210 leukemia model as an example: L1210 cells (approximately 1×10⁵-1×10⁶ cells/mouse) are intraperitoneally inoculated into 6-8 week old female DBA/2 or BDF1 mice. Triapine is prepared in saline and typically administered starting on day 1 after inoculation, via intraperitoneal or intravenous injection. Dosing regimens vary by experimental design (e.g., twice daily, once daily, or continuous infusion). Efficacy endpoints include median survival time (MST), increased life span percentage (ILS%), and proportion of long-term survivors (survival >60 days). For solid tumor models, M109 lung carcinoma cells or A2780 human ovarian carcinoma cells are inoculated subcutaneously or intraperitoneally into nude mice, and treatment begins when tumor volumes reach approximately 100 mm³. The dosing regimen for Triapine has been optimized to twice-daily (bid) administration to achieve optimal efficacy, based on in vivo pharmacodynamic findings that Triapine inhibits DNA synthesis for approximately 10 hours. In combination studies, Triapine exhibits synergistic anti-tumor activity when combined with DNA-damaging agents including cisplatin, etoposide, and doxorubicin. In an intracranial glioblastoma model, GBM cells are mixed with Triapine (10 μM) and stereotactically injected into the striatum of NMRI nude mice, with efficacy assessed by Kaplan-Meier survival curves.

The pharmacokinetic profile of Triapine in humans has been determined through multiple Phase I clinical trials. In a Phase I study of patients with advanced solid tumors, Triapine was administered as a 2-h intravenous infusion daily for 5 days, with courses repeated every 4 weeks. At the dose of 96 mg/m²/day, the mean peak concentration (Cmax) was approximately 8 µM, and the mean elimination half-life (t₁/₂) ranged from 35 minutes to 3 hours, with a median value of approximately 1 hour. Cumulative urinary recovery averaged 1-3% of the administered dose, suggesting that Triapine is primarily eliminated through metabolism. In leukemia patients receiving 96-hour continuous intravenous infusion, steady-state concentrations of Triapine were maintained between 0.6 and 1 µM. A recent study examining the pharmacokinetics of oral Triapine (100 mg daily, 5 days per week for 5 weeks) reported an oral bioavailability of approximately 59%. This study also found that smoking status affects Triapine clearance—clearance mediated by CYP1A2 was nearly doubled in smokers compared to non-smokers, suggesting smoking may reduce systemic exposure to Triapine. Additionally, methemoglobin levels correlated positively with Triapine exposure and can serve as a biomarker of drug exposure.

In this phase I study (12 patients with LAPCA), triapine was administered intravenously over 2 hours, three times weekly every other week (weeks 1, 3, 5) within 30 minutes of radiation, at dose levels of 24, 48, and 72 mg/m². No dose-limiting toxicities (DLTs) were observed, and the maximum tolerated dose (MTD) was not reached. [1]
The most common toxicities (all grades) across all dose levels were: lymphopenia (92%), nausea (56%), anemia (56%), vomiting (44%), and fatigue (44%). Grade 3-4 uncomplicated lymphopenia was observed in all patients at the 72 mg/m² dose level. Other grade 3-4 toxicities were uncommon: anemia (8%), thrombocytopenia (8%). There were no incidences of methemoglobinemia >15% or persistent hypoxia requiring hospitalization. Triapine was discontinued in one patient due to grade 2 fever attributed to an infusion reaction, but this did not qualify as a DLT. One patient required a therapy delay due to unrelated cholangitis. [1]

---

Triapine demonstrates an acceptable safety profile in clinical trials, with hematologic toxicity as the primary dose-limiting toxicity. In the Phase I study of patients with advanced solid tumors, with intravenous administration at 96 mg/m²/day (once daily for 5 days, every 2 weeks), grade 4 leukopenia occurred in 93% of patients in at least one course, grade 2 anemia in 71%, grade 3 anemia in 22%, and grade 3-4 thrombocytopenia in 22%. Non-hematologic toxicities were predominantly grade 1-2, including asthenia, fever, nausea and vomiting, mucositis, decreased serum bicarbonate, and hyperbilirubinemia. Hepatotoxicity was dose-limiting at higher dose levels. In the Phase I study of leukemia patients receiving 96-hour continuous infusion, the maximum tolerated dose (MTD) was 160 mg/m²/day, with dose-limiting hepatotoxicity. In the study of oral Triapine (100 mg once daily, 5 days per week for 5 weeks) combined with cisplatin chemoradiation, dose-limiting toxicities included grade 4 neutropenia, leukopenia, lymphopenia, and hypokalemia; grade ≥3 treatment-related adverse events included lymphopenia (n=12), anemia (n=10), leukopenia (n=8), neutropenia (n=4), and thrombocytopenia (n=2). According to ECHA classification information, the substance is not classified as hazardous; however, it is for research use only and is not intended for human diagnostic or therapeutic use.

[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.

Clinical trial design: Phase I standard 3+3 dose-escalation study of triapine combined with radiation (50.4 Gy in 28 fractions) in patients with untreated locally advanced pancreatic cancer (stage III). Triapine was administered thrice weekly (Monday, Wednesday, Friday) every other week (weeks 1, 3, 5) for a total of 9 doses. [1]
Efficacy results: Among treated patients (n=12 evaluable), 2 patients (17%) achieved partial response (PR), 6 patients (50%) had stable disease (SD), and 1 patient underwent R0 surgical resection following therapy. 92% of patients (100% at the 72 mg/m² dose level) experienced freedom from local tumor progression. 75% of patients who eventually progressed developed metastases without local progression. [1]
Correlative studies: DCE-MRI was evaluated as a potential predictor of early response in 4 patients, with findings suggesting potential predictive value. Serum hRRM1 and hRRM2 levels did not appear to predict clinical outcome, though more consistent inhibition was observed at higher dose levels. [1]
Exclusion criteria: Patients with known G6PD deficiency were excluded due to the potential for triapine to cause severe methemoglobinemia. [1]

C7H10CLN5S

231.71

231.035

C, 36.29; H, 4.35; Cl, 15.30; N, 30.23; S, 13.84

216240-62-9

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

176419013

Typically exists as solids at room temperature

4

2

14

205

0

C1=CC(=C(N=C1)C=NNC(=S)N)N.Cl

VCIFBCIFIYJMDO-UHFFFAOYSA-N

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

[(3-amino-2-pyridinyl)methylideneamino]thiourea;hydrochloride

3-AP hydrochloride; PAN-811 hydrochloride; OCX191 hydrochloride

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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 Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

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
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

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