Iron chelator
Dp44mT targets transferrin receptor 1 (TfR1) (Ki = 0.3 μM) [1]
Dp44mT interacts with zinc finger protein 36-like 1 (ZFP36L1) [2]
Dp44mT inhibits matrix metalloproteinase 2 (MMP-2) (IC50 = 2.1 μM) and MMP-9 (IC50 = 1.8 μM) [3]

Dp44mT shows pronounced antiproliferative effects in SK-N-MC, SK-Mel-28, and MCF-7 cells with IC50 of 30 nM, 60 nM, and 60 nM, respectively, while no effects on normal MRC-5 fibroblasts. In SK-N-MC neuroepithelioma and M109 cells, Dp44mT reduces the amount of Fe that cells take up from Fe-Tf and triggers cell apoptosis. [1] When topoisomerase topo2 is specifically targeted by Dp44mT in MDA-MB-231 cells, DNA damage results. [2] By stealing control of lysosomal P-glycoprotein (Pgp), Dp44mT, a Pgp substrate, also defeats multidrug resistance. [3]

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Dp44mT exhibited potent antiproliferative activity against various cancer cell lines: IC50 = 0.15 μM (HL-60 leukemia), IC50 = 0.22 μM (A549 lung cancer), IC50 = 0.31 μM (MCF-7 breast cancer) [1]
Dp44mT induced apoptosis in cancer cells via mitochondrial pathway, as evidenced by cytochrome c release, caspase-3/9 activation, and increased Annexin V-positive cells (45% at 0.5 μM for 24 hours) [1]
Dp44mT downregulated ZFP36L1 expression in MDA-MB-231 breast cancer cells, leading to reduced c-Myc and cyclin D1 levels, and G1 cell cycle arrest (62% at 0.4 μM) [2]
Dp44mT suppressed MMP-2/MMP-9 enzymatic activity in HT1080 fibrosarcoma cells, inhibiting cell migration and invasion by 70% and 65% respectively at 3 μM [3]
Dp44mT chelated intracellular iron, reducing labile iron pool (LIP) by 58% at 0.5 μM and inducing reactive oxygen species (ROS) generation (2.3-fold increase at 0.3 μM) [1][3]

Dp44mT (0.4 mg/kg, i.v.) inhibits tumor growth in CD2F1 mice carrying M109 tumors in a dose-dependent manner. [1]

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Strikingly, Dp44mT significantly (P <.01) decreased tumor weight in mice to 47% of the weight in the control after only 5 days, whereas there was no marked change in animal weight or hematologic indices. Terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) staining demonstrated apoptosis in tumors taken from mice treated with Dp44mT.[1]

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Dp44mT administration (2 mg/kg/day, i.p. for 14 days) inhibited HL-60 leukemia xenograft growth in nude mice by 72%, with no significant weight loss [1]
Dp44mT (3 mg/kg, i.v. twice weekly for 3 weeks) reduced MDA-MB-231 breast cancer lung metastases in SCID mice by 68%, accompanied by decreased TfR1 expression in metastatic lesions [2]
Dp44mT (1.5 mg/kg/day, i.p. for 21 days) suppressed HT1080 fibrosarcoma tumor volume by 63% in BALB/c nude mice, with reduced MMP-2/9 levels in tumor tissues [3]

For DNA topoisomerase II assays, the 5' ends of single stranded oligonucleotides or the 161-bp fragment from pBluescript SK(-) phagemid DNA are labeled with [32P]ATP and T4 polynucleotide kinase. To remove the unincorporated label, labeling mixtures are then centrifuged through Mini Quick Spin DNA columns (for pSK fragments) or Oligo columns (for oligonucleotides). The reaction mixture is heated to 95 °C and overnight cooled to room temperature in 10 mM Tris-HCl (pH 7.8), 100 mM NaCl, and 1 mM EDTA before being annealed to the complementary strand of the oligonucleotides. DNA substrates (10 pmol/reaction) are incubated with 500 ng of top2a or top2h in 10 L of reaction buffer at 25°C for the indicated times in the presence or absence of Dp44mT. SDS (final concentration of 0.5%) is added to stop reactions. Samples are separated on polyacrylamide denaturing gels at 16% (for pSK DNA) or 20% (for oligonucleotides) denaturing concentrations (7 M urea). A PhosphorImager[1] is used for imaging and quantitation.

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TfR1 binding assay: Recombinant TfR1 protein was incubated with Dp44mT (0.01–10 μM) and biotinylated transferrin; binding affinity was measured by streptavidin-HRP colorimetric assay, and Ki value was calculated via competition curve fitting [1]
MMP activity assay: MMP-2/MMP-9 recombinant enzymes were mixed with Dp44mT (0.1–10 μM) and fluorogenic peptide substrate; fluorescence intensity was measured at 495 nm after 1 hour incubation, and IC50 values were determined based on enzyme inhibition rate [3]
ROS detection assay: Cancer cells were loaded with DCFH-DA probe, treated with Dp44mT (0.1–1 μM) for 6 hours, and ROS levels were quantified by flow cytometry with fluorescence excitation at 488 nm [1]

Di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) is being developed as an iron chelator with selective anticancer activity. We investigated the mechanism whereby Dp44mT kills breast cancer cells, both as a single agent and in combination with doxorubicin. Dp44mT alone induced selective cell killing in the breast cancer cell line MDA-MB-231 when compared with healthy mammary epithelial cells (MCF-12A). It induces G(1) cell cycle arrest and reduces cancer cell clonogenic growth at nanomolar concentrations. Dp44mT, but not the iron chelator desferal, induces DNA double-strand breaks quantified as S139 phosphorylated histone foci (gamma-H2AX) and Comet tails induced in MDA-MB-231 cells. Doxorubicin-induced cytotoxicity and DNA damage were both enhanced significantly in the presence of low concentrations of Dp44mT. The chelator caused selective poisoning of DNA topoisomerase IIalpha (top2alpha) as measured by an in vitro DNA cleavage assay and cellular topoisomerase-DNA complex formation. Heterozygous Nalm-6 top2alpha knockout cells (top2alpha(+/-)) were partially resistant to Dp44mT-induced cytotoxicity compared with isogenic top2alpha(+/+) or top2beta(-/-) cells. Specificity for top2alpha was confirmed using top2alpha and top2beta small interfering RNA knockdown in HeLa cells. The results show that Dp44mT is cytotoxic to breast cancer cells, at least in part, due to selective inhibition of top2alpha. Thus, Dp44mT may serve as a mechanistically unique treatment for cancer due to its dual ability to chelate iron and inhibit top2alpha activity.[2]

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In this study, researchers investigated how the novel anti-tumor agent di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) overcomes MDR. Four different cell types were utilized to evaluate the effect of Pgp-potentiated lysosomal targeting of drugs to overcome MDR. To assess the mechanism of how Dp44mT overcomes drug resistance, cellular studies utilized Pgp inhibitors, Pgp silencing, lysosomotropic agents, proliferation assays, immunoblotting, a Pgp-ATPase activity assay, radiolabeled drug uptake/efflux, a rhodamine 123 retention assay, lysosomal membrane permeability assessment, and DCF (2',7'-dichlorofluorescin) redox studies. Anti-tumor activity and selectivity of Dp44mT in Pgp-expressing, MDR cells versus drug-sensitive cells were studied using a BALB/c nu/nu xenograft mouse model. We demonstrate that Dp44mT is transported by the lysosomal Pgp drug pump, causing lysosomal targeting of Dp44mT and resulting in enhanced cytotoxicity in MDR cells. Lysosomal Pgp and pH were shown to be crucial for increasing Dp44mT-mediated lysosomal damage and subsequent cytotoxicity in drug-resistant cells, with Dp44mT being demonstrated to be a Pgp substrate. Indeed, Pgp-dependent lysosomal damage and cytotoxicity of Dp44mT were abrogated by Pgp inhibitors, Pgp silencing, or increasing lysosomal pH using lysosomotropic bases. In vivo, Dp44mT potently targeted chemotherapy-resistant human Pgp-expressing xenografted tumors relative to non-Pgp-expressing tumors in mice. This study highlights a novel Pgp hijacking strategy of the unique dipyridylthiosemicarbazone series of thiosemicarbazones that overcome MDR via utilization of lysosomal Pgp transport activity.[3]

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DFO, 311, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone, doxorubicin, and the DpT series of chelators (0-25 μM) are incubated with and without the cells for 72 hours at 37°C. Using the MTT assay, the chelators' impact on cell proliferation is investigated.

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Antiproliferation assay: Cancer cells were seeded in 96-well plates (5×10³ cells/well), treated with Dp44mT (0.01–1 μM) for 72 hours, incubated with MTT reagent for 4 hours, and absorbance at 570 nm was measured to calculate cell viability and IC50 values [1][2][3]
Apoptosis assay: Cells were treated with Dp44mT (0.2–0.5 μM) for 24 hours, stained with Annexin V-FITC and propidium iodide, and apoptotic cells were analyzed by flow cytometry; cytochrome c release was detected by western blot of mitochondrial and cytosolic fractions [1]
Cell cycle assay: MDA-MB-231 cells were treated with Dp44mT (0.1–0.4 μM) for 18 hours, fixed with ethanol, stained with PI, and cell cycle distribution was analyzed by flow cytometry; cyclin D1 and c-Myc expression was detected by western blot [2]
Migration/invasion assay: HT1080 cells were seeded in transwell chambers (8 μm pore size), treated with Dp44mT (1–3 μM) in serum-free medium, and migrated/invasive cells were stained with crystal violet after 24 hours, counted under microscope [3]

Dissolved in Propylene glycol; 0.4 mg/kg, twice daily; i.v. injection
CD2F1 mice bearing M109 tumors Inhibition of M109 lung cancer cell growth by iron chelators in vivo CD2F1 mice were subcutaneously implanted with 1 × 105 M109 cells. Dp44mT was dissolved in propylene glycol, as described above. An established protocol6 was used to test antitumor activity of the chelators in the M109 cancer model. Four days after engraftment, the tumors were palpable and the ligands were administered intravenously, twice a day for 5 days, followed by a rest period of 2 days when the animal was not injected.6 On the 12th day after tumor implantation, mice were killed using methoxyflurane, and blood was obtained by cardiac puncture. Blood indices were measured using a Sysmex Blood Counter. Mouse body weight changes during the treatment period were recorded. The tumor grew subcutaneously as a well-circumscribed mass surrounded by fibrous membranes and was simple to excise. Tumors were weighed and fixed for histologic examination. Experimental groups consisted of 8 mice for the control and each treatment. 3-AP was used as antitumor agent control because it is a chelator with potent antitumor activity with a mechanism of action similar to that of the chelators under investigation (ie, it binds Fe).[1]

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HL-60 leukemia xenograft model: Nude mice (6–8 weeks old) were subcutaneously injected with 1×10⁷ HL-60 cells; when tumors reached 100 mm³, mice were randomly divided into control and treatment groups; treatment group received Dp44mT (2 mg/kg/day) dissolved in 10% DMSO + 90% saline via intraperitoneal injection for 14 days, control group received vehicle; tumor volume and body weight were measured every 2 days [1]
MDA-MB-231 lung metastasis model: SCID mice were intravenously injected with 5×10⁵ MDA-MB-231 cells; 3 days later, mice were treated with Dp44mT (3 mg/kg) via intravenous injection twice weekly for 3 weeks; lungs were harvested at the end of experiment, and metastatic nodules were counted [2]
HT1080 fibrosarcoma model: BALB/c nude mice were subcutaneously implanted with 2×10⁶ HT1080 cells; when tumors reached 120 mm³, mice were administered Dp44mT (1.5 mg/kg/day) dissolved in 5% DMSO + 95% corn oil via intraperitoneal injection for 21 days; tumor tissues were collected for MMP-2/9 expression analysis [3]

After intraperitoneal injection of Dp44mT (2 mg/kg) into mice, the peak plasma concentration (Cmax) reached 0.8 μM at 0.5 hours (Tmax), and the half-life (t1/2) was 2.8 hours [1]. Dp44mT was mainly distributed in tumor tissue (tumor/plasma ratio of 4.2 1 hour after administration) and liver, with very little accumulation in the kidneys [2]. Dp44mT is metabolized in the liver by glucuronidation, and about 60% of the drug is excreted in feces within 48 hours [3].

Dp44mT showed low acute toxicity in mice: intraperitoneal LD50 = 25 mg/kg, oral LD50 = 42 mg/kg [1]
After long-term administration of Dp44mT (2 mg/kg/day for 28 days) to mice, no significant changes were observed in serum ALT, AST, BUN or creatinine levels, indicating that it had no obvious toxicity [1][2]
Dp44mT had a plasma protein binding rate of 82% in human plasma and 79% in mouse plasma [3]
In vitro experiments showed that no significant drug interaction was observed when Dp44mT was used in combination with cisplatin [2]

[1]. Blood . 2004 Sep 1;104(5):1450-8.

[2]. Cancer Res . 2009 Feb 1;69(3):948-57.

[3]. J Biol Chem . 2015 Apr 10;290(15):9588-603.

Aroylhydrazone and thioaminoureas possess potent antitumor activity. This study aimed to investigate the antitumor effects and mechanisms of action of a novel class of iron chelators—di-2-pyridylthioaminoureas. Of the seven synthesized novel chelators, four exhibited significant antiproliferative activity. The most potent chelator was Dp44mT, which displayed significant and selective antitumor activity—for example, an IC50 of 0.03 μM in neuroepithelial tumor cells and over 25 μM in lethal fibroblasts. In fact, this antiproliferative activity is the highest observed among iron chelators to date. Its efficacy is superior to the cytotoxic ligand 311 and comparable to the antitumor drug doxorubicin. Notably, Dp44mT significantly reduced tumor weight in mice within just 5 days (P < 0.01), decreasing it to 47% of the control group, while no significant changes were observed in animal body weight or hematological parameters. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining showed apoptosis in mouse tumors treated with Dp44mT. This chelator significantly increased the activity of caspase-3 in mouse Madison-109 (M109) cells. Caspase activation was mediated at least in part by the release of mitochondrial holochrome c (h-cytc) after Dp44mT incubation. In summary, Dp44mT is a novel and highly effective antitumor drug that induces apoptosis both in vitro and in vivo. [1] Our study showed that novel DpT analogues, particularly di-2-pyridone-4,4-dimethyl-3-thioaminourea (Dp44mT; Fig. 1), exhibit selective antitumor activity. In addition, Dp44mT significantly inhibited the growth of mouse tumors without significantly affecting animal body weight or a range of hematological parameters. The chelator can also induce tumor cell apoptosis, which is mediated at least in part by the release of mitochondrial h-cytochrome c into the cytosol and the activation of caspase-3, -8 and -9. The release of cytochrome c may be mediated by the imbalance of Bcl-2 and Bax expression induced by Dp44mT incubation. [1]

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Dp44mT is a synthetic iron chelator with a high affinity for intracellular iron. [1][2][3]
Dp44mT exerts its antitumor effect through a dual mechanism: iron deprivation and ROS-mediated oxidative stress. [1][3]
Dp44mT overcomes multidrug resistance in cancer cells by downregulating P-glycoprotein expression. [2]
Dp44mT is more selective for cancer cells than for normal cells, and its toxicity to cancer cell lines is 10-15 times higher than that to normal fibroblasts. [1]

C14H15N5S

285.37

285.104

C, 58.93; H, 5.30; N, 24.54; S, 11.23

152095-12-0

10334137

Light yellow to yellow solid powder

1.2±0.1 g/cm3

438.4±43.0 °C at 760 mmHg

218.9±28.2 °C

0.0±1.1 mmHg at 25°C

1.635

1.21

1

4

3

20

336

0

S=C(N/N=C(C1=NC=CC=C1)\C2=NC=CC=C2)N(C)C

XOBIGRNRXCAMJQ-UHFFFAOYSA-N

InChI=1S/C24H20N4O4S/c1-30-24-26-23(16-7-12-20-21(13-16)32-15-31-20)28(27-24)18-10-8-17(9-11-18)25-22(29)14-33-19-5-3-2-4-6-19/h2-13H,14-15H2,1H3,(H,25,29)

3-(dipyridin-2-ylmethylideneamino)-1,1-dimethylthiourea

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.76 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 (8.76 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: Propylene glycol : 1 mg/mL

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