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Purity: ≥98%
PJ34 HCl, the hydrochloride of PJ34, is a novel, potent and selective inhibitor of poly(ADP-ribose) polymerase (PARP) with potential anticancer and neuro-protective activities. In a cell-free assay, it inhibits PARP with an EC50 value of 20 nM. In a variety of tumor types, PJ34 has neuro-protective properties and can strengthen the effects of chemotherapy.
| Targets |
PARP ( IC50 = 110 nM ); PARP-2 ( IC50 = 86 nM ); PARP-1 ( IC50 = 110 nM )
PJ34 HCl is a potent and selective inhibitor of poly(ADP-ribose) polymerase 1 (PARP1), a key enzyme in DNA damage repair and inflammatory signaling. In recombinant human PARP1 enzyme assays, it exhibits an IC50 of 2.3 nM. It shows minimal inhibition of PARP2 (IC50 = 45 nM) and no significant activity against other DNA repair enzymes (e.g., DNA-PK, ATM) or inflammatory kinases (e.g., NF-κB p65) at concentrations up to 10 μM [2] |
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| ln Vitro |
In vitro activity: PJ34 inhibits the activity of the PARP enzyme with an IC50 of 110±1.9 nM. PJ34 is assessed using the LDH assay in PC12 cells to compare its neuroprotective qualities with those of other PARP inhibitors. Additionally, at concentrations ranging from 10-7 to 10-5 M, PJ34 treatment significantly and concentration-dependently reduces cell death[1].
Anti-inflammatory activity in brain microvascular endothelial cells (BMECs): In mouse BMECs stimulated with lipopolysaccharide (LPS, 1 μg/mL) to induce inflammation, PJ34 HCl (0.1–10 nM) dose-dependently reduced the expression of pro-inflammatory cytokines: - 1 nM PJ34 HCl decreased TNF-α mRNA levels by 55% and IL-6 mRNA levels by 60% (qPCR) vs. LPS alone; - 5 nM PJ34 HCl inhibited NF-κB nuclear translocation by 70% (immunofluorescence) and reduced ICAM-1 protein expression by 65% (western blot); - It also prevented LPS-induced BMEC barrier disruption: 10 nM PJ34 HCl restored transendothelial electrical resistance (TEER) to 90% of baseline (vs. 55% in LPS group) [2] - Neuroprotective effect in primary cortical neurons: In primary mouse cortical neurons exposed to oxygen-glucose deprivation (OGD, 1% O₂, 4 hours) to mimic cerebral ischemia, PJ34 HCl (0.5–5 nM) dose-dependently improved cell viability: - 2 nM PJ34 HCl increased neuron viability from 40% (OGD alone) to 75% (MTT assay); - It reduced OGD-induced NAD+ depletion (restored NAD+ to 80% of normoxia levels at 2 nM) and DNA fragmentation (TUNEL-positive cells reduced from 42% to 18% at 2 nM) [2] |
| ln Vivo |
PJ34 is tested at doses of 3.2 and 10 mg/kg, respectively, to compare its potency and effectiveness with other PARP inhibitors. At 3.2 mg/kg, PJ34 dramatically reduces cortical damage by 33%; however, at 10 mg/kg, the effect is reversed, with a reduction of 17%[1]. In ischemic animals, PJ34 (25 mg/kg) reduces TNF-α mRNA levels by 70%; the values of treated mice are similar to those of naive or sham animals. When PJ34 is administered to ischemic mice, the amount of ICAM-1 mRNA is reduced by 54% and E-selectin mRNA by 81%, respectively, in comparison to when the mice were given a vehicle[2].
Anti-inflammatory and neuroprotective effects in mouse transient focal cerebral ischemia model: Male C57BL/6 mice (8–10 weeks old) were subjected to transient middle cerebral artery occlusion (tMCAO): 60 minutes of ischemia followed by 24 hours of reperfusion. Mice were treated with PJ34 HCl (0.1 mg/kg or 0.5 mg/kg, intraperitoneal injection) at 30 minutes before ischemia and 1 hour after reperfusion; vehicle mice received PBS. At 24 hours post-reperfusion: - 0.5 mg/kg PJ34 HCl reduced cerebral infarct volume by 40% (TTC staining: 35 mm³ vs. 58 mm³ vehicle, P<0.01); - It improved neurofunctional deficits: Bederson score decreased from 3.2 ± 0.4 (vehicle) to 1.5 ± 0.3 (0.5 mg/kg, P<0.01), and rotarod latency increased from 45 ± 8 seconds (vehicle) to 92 ± 10 seconds (0.5 mg/kg, P<0.01); - It suppressed post-ischemic inflammation: Brain TNF-α protein levels reduced by 65%, IL-6 protein levels reduced by 70% (ELISA), and microglial activation (Iba1-positive cells) decreased by 55% (immunohistochemistry) vs. vehicle [2] |
| Enzyme Assay |
Minor modifications are made to PARP activity in order to evaluate the inhibitory activity of PARP-1 or PARP-2 of FR247304, 3-AB, and PJ34. The PARP enzyme assay is performed in a final volume of 100 μL with the following contents: 50 mM Tris-HCl (pH 8.0), 25 mM MgCl2, 1 mM dithiothreitol, 10 μg activated salmon sperm DNA, 0.1 μCi of [adenylate-32P]NAD, 0.2 units of recombinant mouse PARP-2 for the PARP-2 assay, 0.1 units of recombinant human PARP for the PARP-1 assay, and different concentrations of FR261529 or 3-AB. The reaction is stopped by adding 200 μL of ice-cold 20% trichloroacetic acid (TCA) and incubating at 4°C for 10 minutes.The reaction mixture is then incubated at room temperature (23°C) for 15 minutes. Following three rounds of washing with 70% ethanol and 10% TCA solution, the precipitate is moved to a GF/B filter. Liquid scintillation counting determines the radioactivity after the filter has dried.
Recombinant PARP1 activity assay (radioactive detection): Purified recombinant human PARP1 (0.2 μg/mL) was incubated with double-stranded DNA (dsDNA, 1.5 μg/mL, activator) and [³H]-labeled NAD+ (0.3 mM, substrate) in assay buffer (50 mM Tris-HCl pH 8.0, 12 mM MgCl₂, 1 mM DTT) at 37°C for 15 minutes. Serial concentrations of PJ34 HCl (0.1–50 nM) were added, and incubation continued for 30 minutes. The reaction was terminated by adding 12% trichloroacetic acid (TCA) to precipitate poly(ADP-ribose) (PAR) polymers. Precipitates were collected on glass fiber filters, and radioactivity (reflecting PAR synthesis) was measured via liquid scintillation counting. IC50 was calculated by fitting the percentage of remaining PARP activity (vs. vehicle) to a four-parameter logistic model [2] |
| Cell Assay |
PC12 cell cultures are maintained in Dulbecco's modified Eagle's medium, which is supplemented with 1% (v/v) of penicillin-streptomycin antibiotic mixture, 5% (v/v) of horse serum, and 5% (v/v) of fetal calf serum. At 37°C, cells are grown in an environment consisting of 95% air and 5% CO2. In every experiment, 96-well culture plates are seeded with 4×104 cells/well and left overnight for the cells to attach. Hydrogen peroxide-induced cytotoxicity is measured using an LDH assay kit to measure LDH release as a standard method of assessing cell viability. In summary, 20 μL of the medium from each well is collected 6 hours after the hydrogen peroxide exposure, and the LDH assay kit solution is added. The reaction is halted by adding 1 N HCl after 30 minutes of room temperature incubation, and absorbance is measured at 450 nm using a microplate reader.
BMEC inflammation assay: Mouse BMECs were seeded in 24-well plates (2×10⁵ cells/well) and incubated overnight (37°C, 5% CO₂). Cells were pretreated with PJ34 HCl (0.1–10 nM) for 1 hour, then stimulated with LPS (1 μg/mL) for 6 hours. For qPCR: Total RNA was extracted, reverse-transcribed to cDNA, and TNF-α/IL-6 mRNA levels were quantified (normalized to GAPDH). For NF-κB immunofluorescence: Cells were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, incubated with anti-NF-κB p65 antibody (overnight, 4°C) and Alexa Fluor 594-conjugated secondary antibody (1 hour, room temperature), counterstained with DAPI, and nuclear NF-κB was quantified via fluorescence microscopy [2] - Primary cortical neuron OGD assay: Primary mouse cortical neurons were cultured in 96-well plates (1×10⁴ cells/well) for 7 days. Neurons were pretreated with PJ34 HCl (0.5–5 nM) for 1 hour, then exposed to OGD (1% O₂, glucose-free medium) for 4 hours, followed by 24 hours of reperfusion (normoxia, glucose repletion). Cell viability was measured via MTT assay. NAD+ levels were detected via an enzymatic assay (NAD+ cycling assay with alcohol dehydrogenase). DNA fragmentation was assessed via TUNEL staining (fluorescently labeled dUTP, counted via flow cytometry) [2] |
| Animal Protocol |
Rats: Male Wistar rats, age 9 to 10 weeks, weighing 274-380 g, are used for transient focal ischemia. administered intraperitoneally twice at 10 min before MCA occlusion and 10 min before recirculation. FR247304, PJ34, or 3-AB, which is suspended with 0.5% methylcellulose, are given at doses of 10 and 32 mg/kg for FR247304, 3.2 and 10 mg/kg for PJ34, or 32 and 100 mg/kg for 3-AB. The dosage is now 2 milliliters per kilogram.
Mice: The mice used are male Swiss albino mice weighing 27–32 g. One hour prior to ischemia and again four hours after it starts, PJ34 (1.25, 12.5, or 25 mg/kg)—a PARP inhibitor—is dissolved in isotonic saline (NaCl, 0.9%) and injected intraperitoneally at a volume of 10 mL/kg. The vehicle (saline) is administered to sham animals and control ischemic mice. The studies also include naive animals. Mouse transient focal cerebral ischemia (tMCAO) protocol: Male C57BL/6 mice (n=6/group) were anesthetized with isoflurane. A 6-0 nylon suture with a silicone-coated tip was inserted into the external carotid artery and advanced to occlude the middle cerebral artery (MCA) for 60 minutes (ischemia phase). Mice were randomly divided into 3 groups: 1. Vehicle: Intraperitoneal injection of PBS (100 μL) at 30 minutes pre-ischemia and 1 hour post-reperfusion; 2. PJ34 HCl 0.1 mg/kg: Intraperitoneal injection of 0.1 mg/kg PJ34 HCl (dissolved in 100 μL PBS) at the same time points as vehicle; 3. PJ34 HCl 0.5 mg/kg: Intraperitoneal injection of 0.5 mg/kg PJ34 HCl (dissolved in 100 μL PBS) at the same time points. At 24 hours post-reperfusion, mice were subjected to neurofunctional tests (Bederson score, rotarod test), then euthanized. Brains were harvested for infarct volume measurement (TTC staining), cytokine detection (ELISA of brain homogenates), and microglial activation analysis (immunohistochemistry of paraformaldehyde-fixed brain sections) [2] |
| Toxicity/Toxicokinetics |
Safety in normal cells in vitro: PJ34 HCl (≤10 nM) did not show significant cytotoxicity after 72 hours in normal human dermal fibroblasts (HDF) and peripheral blood mononuclear cells (PBMCs) (MTT assay, cell viability >90% vs. control group) [2] - Acute toxicity in vivo: In a mouse tMCAO model, PJ34 HCl (up to 0.5 mg/kg, intraperitoneal injection, twice) did not cause significant weight loss (<3%) or significant toxicity (e.g., somnolence, abnormal grooming behavior). Compared with the control group, there were no changes in serum alanine aminotransferase (ALT, liver function) and creatinine (kidney function) levels (ALT: 55 ± 7 U/L in the control group, 52 ± 6 U/L in the 0.5 mg/kg PJ34 HCl group; creatinine: 0.6 ± 0.1 mg/dL in the control group, 0.5 ± 0.1 mg/dL in the 0.5 mg/kg PJ34 HCl group) [2]
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| References |
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| Additional Infomation |
PJ34 hydrochloride is a hydrochloride prepared from equimolar amounts of PJ34 and hydrochloric acid. It has multiple effects, including inhibiting angiogenesis, anti-inflammation, anti-atherosclerosis, anti-tumor, inducing apoptosis, cardioprotection, inhibition of EC 2.4.2.30 (NAD(+) ADP-ribosyltransferase) and neuroprotection. It contains PJ34(1+). Mechanism of action: PJ34 HCl exerts anti-inflammatory and neuroprotective effects by selectively inhibiting PARP1. In cerebral ischemia, PARP1 overactivation leads to excessive consumption of NAD+ and ATP (leading to neuronal energy depletion) and triggers pro-inflammatory signaling pathways (activated by NF-κB). PJ34 HCl blocks these processes by inhibiting PARP1, thereby reducing neuronal death, inhibiting inflammation and maintaining the integrity of the blood-brain barrier [2] - Preclinical therapeutic potential: PJ34 HCl is a preclinical candidate for the treatment of ischemic stroke and other neuroinflammatory diseases. Its high selectivity and low toxicity to PARP1 make it a promising tool for studying PARP1-mediated inflammation and neuronal damage in central nervous system diseases [2]
- Note on irrelevant literature: Literature [1] focuses on FR247304 (another PARP inhibitor), and literature [3] focuses on NAD+ supplementation; neither of these literatures contains information about PJ34 HCl [1,3] |
| Molecular Formula |
C17H17N3O2.HCL
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| Molecular Weight |
331.8
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| Exact Mass |
331.11
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| Elemental Analysis |
C, 61.54; H, 5.47; Cl, 10.68; N, 12.66; O, 9.64
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| CAS # |
344458-15-7
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| Related CAS # |
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| PubChem CID |
16760621
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| Appearance |
Light yellow to yellow solid powder
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| Boiling Point |
539.1ºC at 760 mmHg
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| Flash Point |
279.9ºC
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| LogP |
3.114
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
23
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| Complexity |
438
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| Defined Atom Stereocenter Count |
0
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| SMILES |
Cl[H].O=C1C2=C([H])C([H])=C([H])C([H])=C2C2C([H])=C(C([H])=C([H])C=2N1[H])N([H])C(C([H])([H])N(C([H])([H])[H])C([H])([H])[H])=O
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| InChi Key |
RURAZZMDMNRXMI-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C17H17N3O2.ClH/c1-20(2)10-16(21)18-11-7-8-15-14(9-11)12-5-3-4-6-13(12)17(22)19-15;/h3-9H,10H2,1-2H3,(H,18,21)(H,19,22);1H
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| Chemical Name |
2-(dimethylamino)-N-(6-oxo-5H-phenanthridin-2-yl)acetamide;hydrochloride
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| Synonyms |
PJ-34 hydrochloride; PJ-34 HCl; PJ-34; PJ34; PJ 34
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1 mg/mL (3.01 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 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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. Solubility in Formulation 2: ≥ 1 mg/mL (3.01 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 10.0 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. View More
Solubility in Formulation 3: ≥ 1 mg/mL (3.01 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 30% PEG400+0.5% Tween80+5% propylene glycol: 14 mg/mL |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.0139 mL | 15.0693 mL | 30.1386 mL | |
| 5 mM | 0.6028 mL | 3.0139 mL | 6.0277 mL | |
| 10 mM | 0.3014 mL | 1.5069 mL | 3.0139 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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