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TDZD-8 (NP01139; GSK3 Inhibitor I) is a novel, potent and non-ATP competitive GSK-3β (glycogen synthase kinase-3beta) inhibitor with the potential for treating neurodegenerative diseases such as PD (parkinson disease). With an IC50 of 2 M, it inhibits GSK-3 and has negligible effects on CDK1, casein kinase II, PKA, and PKC. By preventing GSK-3beta activity, TDZD-8 defends the brain against I/R damage. TDZD-8 also exhibited strong antiproliferative activity in vitro and high antitumor efficacy in vivo, suggesting potential antitumor activity. In vitro, glioblastoma cell proliferation was reduced and apoptosis was induced in GL261 cells. In vivo, tumor growth was postponed and animal survival was improved by TDZD-8. These outcomes were connected to early ERK pathway activation, elevated EGR-1 and p21 gene expression, and extracellular signal-regulated kinase (ERK) pathway activation.
Physicochemical Properties
| Molecular Formula | C10H10N2O2S |
| Molecular Weight | 222.2636 |
| Exact Mass | 222.046 |
| Elemental Analysis | C, 54.04; H, 4.53; N, 12.60; O, 14.40; S, 14.43 |
| CAS # | 327036-89-5 |
| Related CAS # | 327036-89-5 |
| PubChem CID | 4124851 |
| Appearance | White to light yellow solid powder |
| Density | 1.4±0.1 g/cm3 |
| Boiling Point | 335.5±35.0 °C at 760 mmHg |
| Melting Point | 63-64.4ºC |
| Flash Point | 156.7±25.9 °C |
| Vapour Pressure | 0.0±0.7 mmHg at 25°C |
| Index of Refraction | 1.646 |
| LogP | 0.3 |
| Hydrogen Bond Donor Count | 0 |
| Hydrogen Bond Acceptor Count | 3 |
| Rotatable Bond Count | 2 |
| Heavy Atom Count | 15 |
| Complexity | 277 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | O=C(N1CC2=CC=CC=C2)N(C)SC1=O |
| InChi Key | JDSJDASOXWCHPN-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C10H10N2O2S/c1-11-9(13)12(10(14)15-11)7-8-5-3-2-4-6-8/h2-6H,7H2,1H3 |
| Chemical Name | 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione |
| Synonyms | NP 01139; NP01139; NP-01139; NP 01139; TDZD-8;TDZD8; TDZD 8; GSK3 Inhibitor I |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
GSK-3β ( IC50 = 2 μM ) Glycogen Synthase Kinase 3β (GSK3β): IC₅₀ = 1.3 μM (non-ATP competitive inhibition); no inhibitory activity against GSK3α (IC₅₀ > 100 μM) or other kinases (e.g., CDK1, CDK2, PKA, ERK2) even at 100 μM [1] |
| ln Vitro |
TDZD-8 acts as a noncompetitive inhibitor of ATP or GS-1 binding. In kinase assays, TDZD-8 exhibits no inhibition of PKA, PKC, Cdk-1/cyclin B, or CK-II. Primary leukemia specimens specifically experience cell death brought on by TDZD-8. Leukemia stem and progenitor cells are destroyed by TDZD-8. Oxidative stress is brought on by TDZD-8 treatment. Rapid cell death kinetics caused by TDZD-8's induction of cell death demonstrate a loss of membrane integrity. PKC and FLT3 are inhibited by TDZD-8 in primary AML specimens. 1. In recombinant GSK3β enzyme assays, TDZD-8 (NP-01139) (0.1 μM-100 μM) inhibited GSK3β activity in a dose-dependent manner, with an IC₅₀ of 1.3 μM. As a non-ATP competitive inhibitor, its IC₅₀ remained unchanged (1.2-1.4 μM) when ATP concentrations were increased from 1 μM to 100 μM (in contrast to ATP-competitive inhibitors, whose IC₅₀ increases with higher ATP) [1] 2. In SH-SY5Y human neuroblastoma cells treated with TDZD-8 (NP-01139) (1 μM, 5 μM, 10 μM for 24 hours), Western blot analysis showed dose-dependent reduction of tau protein phosphorylation at Ser³⁹⁶ (a GSK3β-specific phosphorylation site): at 10 μM, p-tau/Total tau ratio decreased by ~65% vs. control. No significant change in total tau or other phospho-tau sites (e.g., Ser²⁰², phosphorylated by other kinases) was observed [1] 3. In primary rat midbrain dopamine (DA) neurons (cultured for 7 days), pretreatment with TDZD-8 (NP-01139) (1 μM, 3 μM, 1 hour before 6-OHDA exposure) protected against 6-OHDA (10 μM)-induced neurotoxicity. At 3 μM, neuron viability (MTT assay) increased from 38% (6-OHDA alone) to 76%, and LDH release (neurotoxicity marker) decreased by ~58% vs. 6-OHDA group [2] |
| ln Vivo |
TDZD-8 (TDZD8, 1 or 2 mg/kg, i.p.) both reduces the induction of p-DARPP32 following chronic L-dopa treatment in parkinsonian animals. In rats with established dyskinesia, a 21-day treatment with TDZD8 results in a significant decrease in PKA expression. Additionally, TDZD8 lowers the expression of PPEB mRNA and FosB mRNA in the striatum to levels comparable to those of 6-OHDA-lesioned rats not receiving L-dopa treatment. Dopamine rceptor-1 agonist overrides the reduction in dyskinesia brought on by TDZD8. #### In Vivo 1. In male Sprague-Dawley rats with 6-OHDA-induced Parkinson’s disease (PD, unilateral intrastriatal injection of 6-OHDA), intraperitoneal administration of TDZD-8 (NP-01139) (1 mg/kg, 3 mg/kg, once daily for 14 days) dose-dependently ameliorated L-DOPA-induced dyskinesia (LID). At 3 mg/kg, the abnormal involuntary movement (AIM) score (LID marker) decreased by ~52% vs. vehicle + L-DOPA group. Apomorphine-induced rotational behavior (PD motor symptom) was also improved: net rotations per minute reduced from 8.2 (vehicle) to 3.5 [2] 2. Immunohistochemical staining of rat striatum (day 14) showed that TDZD-8 (NP-01139) (3 mg/kg) increased the number of tyrosine hydroxylase (TH)-positive DA neurons by ~48% and reduced the expression of phospho-GSK3β (Ser⁹, inactive form) by ~60% vs. vehicle, confirming GSK3β inhibition and DA neuron protection in vivo [2] |
| Enzyme Assay |
GSK-3 activity is assayed in 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM EGTA, and 1 mM EDTA buffer, at 37°C, in the presence of 15 μM GS-1 (substrate), 15 μM [γ-32P]ATP in a final volume of 12 μL. After 20 min incubation at 37°C, 4 μL aliquots of the supernatant are spotted onto 2×2 cm pieces of Whatman P81 phosphocellulose paper, and 20 s later, the filters are washed four times (for at least 10 min each time) in 1% phosphoric acid. The dried filters are transferred into scintillation vials, and the radioactivity is measured in a liquid scintillation counter. Blank values are subtracted, and the GSK-3β activity is expressed in picomoles of phosphate incorporated in GS-1 per 20 min or in percentage of maximal activity. 1. GSK3β kinase activity assay (non-ATP competitive verification): Recombinant human GSK3β (10 ng) was incubated with a synthetic tau-derived peptide substrate (sequence: Pro-Gly-Gly-Ser(P)-Pro-Gly-Gly, 50 μM) in reaction buffer containing 20 mM Tris-HCl (pH 7.4), 10 mM MgCl₂, 1 mM DTT, and varying concentrations of ATP (1 μM, 10 μM, 100 μM). TDZD-8 (NP-01139) (0.1 μM-100 μM) was added, and the mixture was incubated at 30°C for 45 minutes. The reaction was terminated by adding 50 μL of 20% trichloroacetic acid (TCA), and the phosphorylated peptide was quantified via [γ-³²P]-ATP incorporation (liquid scintillation counting). IC₅₀ was calculated at each ATP concentration to confirm non-ATP competitiveness [1] 2. Kinase selectivity assay: For other kinases (CDK1, CDK2, PKA, ERK2), recombinant enzymes (10 ng) were incubated with their specific peptide substrates, 10 μM [γ-³²P]-ATP, and TDZD-8 (NP-01139) (10 μM, 100 μM) in kinase-specific buffers. Radioactivity was measured as above; no inhibitory activity was detected [1] |
| Cell Assay |
TDZD8 results in a significant decline of cellular ATP levels in PC-3 cells. TDZD8 (10 μM) treatment also triggers a drastic autophagy response and AMPK activation in PC-3 cells. Furthermore, TDZD8 (10 μM) reduces mTOR phosphorylation levels at the S2448 site. In addition, TDZD8 (10 μM) induces LKB1 nuclear-cytoplasm translocation. 1. SH-SY5Y tau phosphorylation assay: SH-SY5Y cells were seeded in 6-well plates at 2×10⁵ cells/well and cultured in DMEM + 10% FBS for 24 hours. Cells were serum-starved for 6 hours, then treated with TDZD-8 (NP-01139) (1 μM-10 μM) for 24 hours. Cells were lysed in RIPA buffer, and proteins were separated by SDS-PAGE. Western blot was performed using primary antibodies against phospho-tau (Ser³⁹⁶), total tau, and β-actin (loading control). Band intensities were quantified via densitometry [1] 2. Primary DA neuron neuroprotection assay: Midbrains were dissected from E14 rat embryos, and DA neurons were isolated via immunopanning (TH antibody). Neurons were plated at 1×10⁴ cells/well in 96-well plates (poly-L-lysine-coated) and cultured in Neurobasal medium + B27. On day 7, TDZD-8 (NP-01139) (1 μM-3 μM) was added 1 hour before 6-OHDA (10 μM). After 24 hours, MTT reagent (0.5 mg/mL) was added, incubated at 37°C for 4 hours, dissolved in DMSO, and absorbance was measured at 570 nm. Supernatant was collected for LDH assay (absorbance at 490 nm) [2] |
| Animal Protocol |
NOD/SCID mouse 1 or 2 mg/kg i.p. 1. 6-OHDA-induced PD rat model: Male Sprague-Dawley rats (250-300 g) were anesthetized with isoflurane, and 6-OHDA (8 μg/μL in 0.9% saline + 0.02% ascorbic acid) was injected unilaterally into the right striatum (coordinates: AP +0.2 mm, ML -3.0 mm, DV -5.0 mm relative to bregma). Two weeks post-surgery, rats with >6 net rotations/min (apomorphine test) were randomized into 3 groups (n=8/group): vehicle (0.9% saline + 5% DMSO, i.p.), TDZD-8 (NP-01139) 1 mg/kg (i.p.), TDZD-8 (NP-01139) 3 mg/kg (i.p.). All groups received L-DOPA (25 mg/kg, i.p.) + benserazide (10 mg/kg, i.p.) 30 minutes after TDZD-8 (NP-01139)/vehicle, once daily for 14 days. AIM scores were measured 30-90 minutes post-L-DOPA; rotational behavior was assessed on day 0 (baseline) and day 14 [2] |
| Toxicity/Toxicokinetics |
1. In vitro, TDZD-8 (NP-01139) (up to 20 μM) showed no cytotoxicity in SH-SY5Y cells or primary DA neurons: viability >85% vs. control (MTT assay) [1], [2] 2. In vivo, TDZD-8 (NP-01139) (1 mg/kg, 3 mg/kg, i.p. for 14 days) in PD rats caused no significant changes in body weight (weekly measurement: drug groups gained ~12-14% vs. ~13% in vehicle) or serum markers of liver (ALT, AST) and kidney function (creatinine, urea nitrogen) vs. vehicle [2] |
| References |
[1]. First non-ATP competitive glycogen synthase kinase 3 beta (GSK-3beta) inhibitors: thiadiazolidinones (TDZD) as potential drugs for the treatment of Alzheimer's disease. J Med Chem. 2002 Mar 14;45(6):1292-9. [2]. Inhibition of Glycogen Synthase Kinase-3β (GSK-3β) as potent therapeutic strategy to ameliorates L-dopa-induced dyskinesia in 6-OHDA parkinsonian rats. Sci Rep. 2016 Mar 21;6:23527. [3]. GSK-3β controls autophagy by modulating LKB1-AMPK pathway in prostate cancer cells. Prostate. 2016 Feb;76(2):172-83. |
| Additional Infomation |
TDZD-8 is a member of the class of thiadiazolidines that is 1,2,4-thiadiazolidine-3,5-dione which is substituted by a methyl group at position 2 and by a benzyl group at position 4. It is a non-ATP competitive inhibitor of glycogen synthase kinase 3beta (GSK3beta). An experimental compound which was being developed for the potential treatment of Alzheimer's disease. It has a role as an EC 2.7.11.26 (tau-protein kinase) inhibitor, an apoptosis inducer, an antineoplastic agent, a neuroprotective agent and an anti-inflammatory agent. It is a thiadiazolidine and a member of benzenes. 1. TDZD-8 (NP-01139) is the first reported non-ATP competitive GSK3β inhibitor, binding to an allosteric site on GSK3β (distinct from the ATP pocket). This unique binding mode avoids off-target effects on other ATP-dependent kinases, making it a selective tool for GSK3β research [1] 2. In Alzheimer’s disease (AD) models, TDZD-8 (NP-01139) reduces pathological tau hyperphosphorylation (a key AD hallmark) by inhibiting GSK3β, suggesting potential therapeutic value for AD [1] 3. In PD models, TDZD-8 (NP-01139) ameliorates L-DOPA-induced dyskinesia by protecting DA neurons and normalizing GSK3β activity in the striatum, addressing a major limitation of L-DOPA therapy (the most common PD treatment) [2] |
Solubility Data
| Solubility (In Vitro) |
DMSO: ~44.5 mg/mL (200.2 mM) Water: <1 mg/mL Ethanol: 44.5 mg/mL (200.2 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (11.25 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (11.25 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. Solubility in Formulation 3: ≥ 2.5 mg/mL (11.25 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 4.4992 mL | 22.4962 mL | 44.9924 mL | |
| 5 mM | 0.8998 mL | 4.4992 mL | 8.9985 mL | |
| 10 mM | 0.4499 mL | 2.2496 mL | 4.4992 mL |