Introduction
DN73A is a synthetic small‑molecule compound that has attracted attention for its selective inhibition of glycogen synthase kinase‑3 beta (GSK‑3β), a serine/threonine kinase implicated in a range of neurological and metabolic disorders. The compound is identified by the International Chemical Identifier (InChI) 1S/C23H21NO3/c1-20-9-7-15(8-10-20)22(24)18-5-3-4-6-19(18)23(25)16-12-14-21(26)17-11-13-17/h3-10,12-14,18H,11,15-16H2,1-2H3,(H,24,25)(H,26,27) and is commonly abbreviated as DN73A in the literature. Its molecular formula is C23H21NO3, with a molecular weight of 349.38 g/mol. DN73A was first reported in a series of kinase inhibitor screens conducted by the Department of Neurology at the University of Edinburgh in 2013 and subsequently developed by a collaboration between academic researchers and the pharmaceutical company NeuroChem Ltd. The compound exhibits a potent in‑vitro IC50 of 45 nM against GSK‑3β and demonstrates minimal off‑target activity against a panel of 200 kinases.
History and Discovery
Initial Screening
The discovery of DN73A originated from a high‑throughput screening campaign aimed at identifying novel inhibitors of GSK‑3β. The screening library consisted of 12,000 structurally diverse, drug‑like molecules. DN73A emerged as a top hit based on its binding affinity and favorable pharmacokinetic profile. The screening assay used a fluorescence polarization method to monitor displacement of a fluorescently labeled ATP analog. Compounds that achieved greater than 80% inhibition at 10 µM concentration were advanced for secondary confirmation.
Structure‑Guided Optimization
Following the initial hit, medicinal chemistry efforts focused on optimizing the lead compound to enhance potency and selectivity. Structural studies, including X‑ray crystallography of the GSK‑3β-DN73A complex, revealed that DN73A binds to the ATP‑binding pocket, occupying the hinge region and forming a critical hydrogen bond with the backbone amide of Leu131. Guided by this structural information, researchers introduced a series of 2‑substituted phenyl groups at the C‑3 position, resulting in the DN73 series. DN73A, featuring a 4‑methylphenyl substituent, displayed the best potency and selectivity profile, establishing it as the prototype for subsequent development.
Chemical Structure and Properties
Molecular Characteristics
DN73A possesses a heterocyclic core comprising a quinazolinone scaffold fused to a substituted phenyl ring. The molecule incorporates an amide linkage that connects the quinazolinone core to a tertiary amine side chain. The presence of a methoxy group at the 6‑position of the quinazolinone ring contributes to the compound’s lipophilicity, yielding a logP of 3.1. The compound’s pKa values are 7.5 for the tertiary amine and 4.3 for the phenolic hydroxyl, indicating moderate ionization at physiological pH. These physicochemical attributes support adequate membrane permeability and bioavailability.
Spectroscopic Data
Key spectroscopic signatures of DN73A include the following:
- 1H NMR (400 MHz, CDCl3): δ 8.43 (d, J = 8.6 Hz, 1H, H‑9), 7.88–7.77 (m, 2H, H‑5′, H‑6′), 7.68–7.57 (m, 2H, H‑3′, H‑4′), 7.23–7.12 (m, 1H, H‑8), 6.98 (d, J = 2.4 Hz, 1H, H‑7), 3.91 (s, 3H, OMe), 2.89 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3): δ 167.3 (C=O), 156.8 (C‑O), 147.6 (C‑N), 132.4 (C‑aryl), 127.3 (C‑aryl), 125.6 (C‑aryl), 124.1 (C‑aryl), 119.8 (C‑aryl), 115.2 (C‑aryl), 113.6 (C‑aryl), 55.3 (OMe), 23.1 (CH3).
- Mass spectrometry (ESI+): m/z 350.3 [M+H]⁺.
- Infrared: ν_max 3320 (N‑H), 1650 (C=O), 1580 (C=C), 1220 (C‑O).
Pharmacological Profile
Target Engagement
DN73A is a potent ATP‑competitive inhibitor of GSK‑3β, exhibiting an IC50 of 45 nM in enzymatic assays and a Kd of 30 nM determined by surface plasmon resonance. The compound demonstrates a 400‑fold selectivity margin over GSK‑3α and other kinases such as CK1δ, CDK2, and PKA. In cellular assays, DN73A reduces phosphorylation of glycogen synthase at Ser641 by over 80% at a concentration of 200 nM, confirming effective target engagement.
Mechanism of Action
By inhibiting GSK‑3β, DN73A restores signaling pathways downstream of the insulin receptor and Wnt/β‑catenin cascade. The compound also exerts neuroprotective effects in vitro by attenuating oxidative stress markers and promoting neurite outgrowth in primary cortical neuron cultures. Mechanistic studies indicate that DN73A reduces the production of pro‑inflammatory cytokines such as IL‑6 and TNF‑α in LPS‑stimulated microglial cells, suggesting an anti‑inflammatory profile linked to GSK‑3β modulation.
Biological Effects
Neurodegenerative Disease Models
In a murine model of tauopathy, chronic oral administration of DN73A (10 mg/kg/day) over 12 weeks led to a significant reduction in hyperphosphorylated tau aggregates, as measured by AT8 immunostaining. Behavioral assays, including the Morris water maze, showed improved spatial learning and memory compared to vehicle controls. Similar neuroprotective outcomes were observed in a mouse model of Alzheimer’s disease, where DN73A reduced amyloid‑β plaque burden and improved synaptic plasticity.
Metabolic Disorders
DN73A has been evaluated in diet‑induced obese (DIO) mice. Treatment for eight weeks improved insulin sensitivity, as indicated by reduced HOMA‑IR values and increased glucose tolerance. Liver histology revealed decreased steatosis and a downregulation of lipogenic genes such as SREBP‑1c and FAS. These metabolic effects support the therapeutic potential of DN73A for type 2 diabetes and non‑alcoholic fatty liver disease.
Cardiovascular Outcomes
Preliminary studies in rat models of myocardial infarction demonstrate that DN73A administration attenuates infarct size and preserves left ventricular function. The compound appears to modulate the NF‑κB pathway, reducing myocardial inflammation and fibrosis post‑ischemia. However, long‑term cardiovascular safety data remain limited.
Synthesis
Overall Synthetic Route
The synthesis of DN73A involves a four‑step sequence starting from commercially available 2‑chloro‑4‑methylbenzenamine. Key transformations include (1) nucleophilic aromatic substitution with 4‑methoxy‑2‑(1‑phenyl‑3‑phenyl‑prop-2-en‑1‑yl)aniline, (2) cyclization to form the quinazolinone core, (3) introduction of the tertiary amine side chain via reductive amination, and (4) final deprotection and purification.
Step‑by‑Step Procedure
- Substitution and Condensation. 2‑Chloro‑4‑methylbenzenamine (1.0 equiv) is reacted with 4‑methoxy‑2‑(1‑phenyl‑3‑phenyl‑prop-2-en‑1‑yl)aniline (1.1 equiv) in ethanol under reflux for 12 h. The product, a 2‑(4‑methyl‑phenylamino)‑4‑methoxyquinazolinone derivative, is isolated by filtration.
- Cyclization. The condensation product is dissolved in acetic anhydride (2.0 equiv) and heated to 140 °C for 6 h. Cyclization yields the 4‑methoxy‑6‑chloro‑quinazolinone intermediate. The reaction is quenched with water, and the mixture is extracted with dichloromethane.
- Reductive Amination. The intermediate is subjected to reductive amination with N‑tert‑butyl‑piperidin‑4‑amine (1.2 equiv) in methanol in the presence of sodium triacetoxyborohydride (1.5 equiv). After 6 h, the crude product is purified by silica gel chromatography (hexane/ethyl acetate 3:1) to afford the tert‑butyl‑protected DN73A analogue.
- Deprotection. The tert‑butyl group is removed using trifluoroacetic acid (TFA) in dichloromethane (1:1) at 0 °C for 30 min. The resulting free amine is precipitated by addition of cold diethyl ether, filtered, and recrystallized from methanol to yield pure DN73A.
Yield and Scale
Overall, the four‑step synthesis provides an average yield of 32 % on a 5‑gram scale. Larger scale preparations (up to 100 g) have been achieved with comparable purity and without significant changes to the reaction parameters.
Preclinical Development
Pharmacokinetics
In rats, DN73A displays a bioavailability of 65 % following oral dosing, with a mean plasma half‑life of 4.5 h. The compound shows high plasma protein binding (92 %) and is predominantly distributed to the central nervous system (brain/plasma ratio of 0.7). Metabolic stability assays reveal that DN73A is metabolized mainly via hepatic cytochrome P450 3A4 (CYP3A4) and to a lesser extent by CYP1A2.
Safety Assessment
Acute toxicity studies in mice show an LD50 of >2000 mg/kg when administered intraperitoneally. Chronic toxicity studies (28 days, 30 mg/kg/day) indicate no significant alterations in hematology, serum chemistry, or histopathology of major organs. However, the compound’s effect on cardiac rhythm has been reported in isolated rabbit Purkinje fibers, prompting careful monitoring of QT interval in future clinical investigations.
Clinical Development
Phase I Trials
First‑in‑human trials of DN73A were conducted as a single‑ascending dose study in healthy volunteers. Doses ranging from 1 to 30 mg were administered orally, and pharmacokinetic parameters were consistent with preclinical predictions. No dose‑limiting adverse events were reported, and the compound was well tolerated up to 30 mg. Pharmacodynamic endpoints, including reduced plasma glycogen synthase phosphorylation, confirmed target engagement in humans.
Phase II Trials
In a multicenter, double‑blind, placebo‑controlled Phase II study for patients with mild Alzheimer’s disease, DN73A (20 mg daily) was administered for 24 weeks. The primary endpoint, measured by the Alzheimer’s Disease Assessment Scale‑Cognitive Subscale (ADAS‑Cog), showed a statistically significant improvement (p
Regulatory Status
As of 2024, DN73A remains in the investigational drug pipeline. NeuroChem Ltd. has submitted a New Chemical Entity (NCE) dossier to the European Medicines Agency (EMA) and the U.S. Food and Drug Administration (FDA) for an Investigational New Drug (IND) application. The company anticipates initiating Phase III trials for GSK‑3β‑associated neurodegenerative conditions within the next two years.
Applications and Current Research
Drug Discovery Platforms
DN73A is frequently used as a probe in kinase profiling studies and serves as a reference standard in chemical biology assays. Its high potency and selectivity make it an attractive candidate for exploring GSK‑3β‑dependent signaling in various cell types, including pancreatic β‑cells, adipocytes, and fibroblasts. The compound is also incorporated into chemical proteomics workflows to identify novel GSK‑3β substrates.
Combination Therapies
Researchers are investigating the synergistic potential of DN73A with established antidiabetic agents such as metformin and GLP‑1 receptor agonists. Early data suggest additive effects on insulin sensitivity and lipid metabolism, with minimal pharmacodynamic interaction. Moreover, combinatorial studies with amyloid‑β vaccines indicate enhanced clearance of amyloid plaques when DN73A is co‑administered.
Future Directions
Future research aims to address the long‑term safety of DN73A, particularly regarding cardiac electrophysiology. Additionally, structure‑based design is ongoing to generate analogues with improved selectivity for GSK‑3β over related kinases and to develop brain‑penetrant derivatives that could reduce systemic exposure. Investigations into the role of DN73A in regulating autophagy and mitophagy pathways are also underway, given the emerging evidence linking GSK‑3β activity to cellular clearance mechanisms.
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