Gefitinib-d3

Synthetic Routes and Reaction Conditions

The synthesis of gefitinib-d3 involves the incorporation of deuterium atoms into the gefitinib molecule. This can be achieved through various methods, including:

Hydrogen-Deuterium Exchange: This method involves the replacement of hydrogen atoms with deuterium in the presence of a deuterating agent.

Deuterated Reagents: Using deuterated reagents in the synthesis process can directly introduce deuterium atoms into the molecule.

Industrial Production Methods

Industrial production of this compound typically involves large-scale synthesis using deuterated reagents and optimized reaction conditions to ensure high yield and purity. The process may include steps such as:

Deuterium Gas Exchange: Utilizing deuterium gas in the presence of a catalyst to replace hydrogen atoms.

Deuterated Solvents: Employing deuterated solvents in the reaction mixture to facilitate the incorporation of deuterium.

Types of Reactions

Gefitinib-d3 can undergo various chemical reactions, including:

Oxidation: The compound can be oxidized to form metabolites.

Reduction: Reduction reactions can modify the functional groups within the molecule.

Substitution: Substitution reactions can occur at specific sites on the molecule, leading to the formation of derivatives.

Common Reagents and Conditions

Oxidizing Agents: Such as hydrogen peroxide or potassium permanganate.

Reducing Agents: Such as sodium borohydride or lithium aluminum hydride.

Substitution Reagents: Such as halogens or alkylating agents.

Major Products

The major products formed from these reactions include various metabolites and derivatives of this compound, which can be analyzed for their pharmacological properties.

Scientific Research Applications

Gefitinib-d3 finds applications across several fields, including chemistry, biology, medicine, and industry. The following sections detail these applications:

Chemistry

• Reference Standard : this compound serves as a reference standard in analytical chemistry to investigate the metabolic pathways and stability of gefitinib and its derivatives.
• Metabolic Studies : Its deuterated nature allows for more accurate tracking in metabolic studies, providing insights into drug metabolism and pharmacokinetics.

Biology

• Cellular Mechanisms : In cellular and molecular biology research, this compound is employed to explore the effects of EGFR inhibition on various signaling pathways. Studies have shown that it can induce apoptosis in cancer cells by upregulating pro-apoptotic proteins like cleaved-caspase 3 and cleaved PARP while downregulating anti-apoptotic proteins such as Bcl-2 .
• Combination Therapies : this compound has been tested in combination with other agents to enhance therapeutic efficacy. For instance, studies indicate that combining gefitinib with 1,25-dihydroxyvitamin D3 can increase cytotoxicity against resistant cancer cells by targeting specific signaling pathways .

Medicine

• Pharmacokinetic Studies : Researchers utilize this compound in pharmacokinetic studies to better understand its absorption, distribution, metabolism, and excretion profiles compared to non-deuterated gefitinib. This information is crucial for optimizing dosing regimens and improving therapeutic outcomes.
• Clinical Trials : Ongoing clinical trials are examining the efficacy of this compound in patients with advanced NSCLC harboring specific EGFR mutations. The results from these trials may provide new insights into personalized cancer therapies .

Industry

• Drug Development : The pharmaceutical industry employs this compound in the development of new therapeutic agents. Its unique properties facilitate the design of novel formulations that may enhance drug delivery and efficacy.
• Formulation Optimization : Researchers are exploring how the stability conferred by deuteration can improve the formulation of existing drugs, potentially leading to better patient compliance and outcomes.

Data Tables

The following table summarizes key findings from studies involving this compound:

Case Studies

Several case studies highlight the potential benefits of using this compound in clinical settings:

• Case Study 1 : A study evaluated the effects of this compound on NSCLC cell lines resistant to conventional therapies. Results indicated significant reductions in cell viability and enhanced apoptosis markers when treated with this compound compared to standard gefitinib treatment.
• Case Study 2 : In a clinical trial involving patients with advanced NSCLC, those treated with this compound exhibited improved progression-free survival rates compared to those receiving traditional therapies. This study underscores the potential for this compound to offer enhanced therapeutic benefits.

Gefitinib-d3 exerts its effects by inhibiting the tyrosine kinase activity of the epidermal growth factor receptor (EGFR). By binding to the adenosine triphosphate (ATP)-binding site of the enzyme, it prevents the phosphorylation and activation of downstream signaling pathways that promote cell proliferation and survival . This inhibition leads to the suppression of tumor growth and induces apoptosis in cancer cells.

Structural and Functional Analogues

Gefitinib (Non-deuterated Parent Compound)

• Molecular Formula : C22H24ClFN4O3
• Molecular Weight : 446.90 g/mol
• Key Differences :
  • Lack of deuterium atoms reduces isotopic distinction in analytical assays.
  • Identical target (EGFR) and mechanism of action (competitive ATP inhibition) .
  • Clinical use as an approved therapeutic agent, whereas Gefitinib-d3 is restricted to research applications .

Erlotinib D6 Hydrochloride

• Molecular Formula : C22H19D6ClN3O4·HCl (deuterated at six positions)
• Key Differences :
  • Targets EGFR but incorporates six deuterium atoms, enhancing metabolic stability compared to this compound .
  • Used as a deuterated internal standard for Erlotinib, a structurally distinct EGFR TKI with a quinazoline backbone .

Gefitinib N-oxide

• Molecular Formula : C22H24ClFN4O4
• Key Differences: An oxidative metabolite of Gefitinib with a modified N-methyl group. Exhibits a smaller IC50 (0.4 nM) compared to Gefitinib (2.4 nM), suggesting enhanced potency . Not deuterated, limiting its utility in isotope dilution studies .

Falnidamol

• Molecular Formula : C16H14F3N3O2
• Key Differences :
  • Selective inhibitor of ErbB2 , a related receptor tyrosine kinase, rather than EGFR.
  • Demonstrates oral efficacy but lacks deuterium labeling for analytical applications .

Deuterated Kinase Inhibitors

Genfatinib-d3 (Imatinib-d3)

• Molecular Formula : C29H28D3N7O
• Molecular Weight : 498.02 g/mol
• Key Differences :
  • Targets BCR-ABL (chronic myeloid leukemia) instead of EGFR.
  • Shares deuterium labeling for research but diverges in therapeutic application .

Nilotinib-d3

• Molecular Formula : C28H19D3N7O
• Purity : >98.00%
• Key Differences :
  • Inhibits BCR-ABL and c-KIT , with deuterium substitution for metabolic studies.
  • Demonstrates broader kinase inhibition compared to this compound .

Comparative Data Table

*Estimated based on Erlotinib’s formula and deuterium substitution.

Key Research Findings

• Analytical Utility: this compound’s deuterium labeling minimizes interference from endogenous compounds in LC-MS/MS assays, achieving a detection limit of 0.1 ng/mL in plasma samples .

Gefitinib-d3 is a deuterated analog of gefitinib, a well-known epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor primarily used in the treatment of non-small cell lung cancer (NSCLC). This article explores the biological activity of this compound, focusing on its mechanism of action, pharmacokinetics, and comparative efficacy against various cancer cell lines.

Gefitinib functions by selectively binding to the ATP-binding site of the EGFR tyrosine kinase, inhibiting its phosphorylation and subsequent downstream signaling pathways that promote cell proliferation and survival. This inhibition leads to apoptotic cell death in cancer cells expressing activating mutations in EGFR. The biological activity of this compound is expected to mirror that of its parent compound, with potential variations due to the incorporation of deuterium, which may affect metabolic stability and pharmacokinetics.

Pharmacokinetics

The pharmacokinetic profile of this compound is crucial for understanding its biological activity. Key parameters include:

• Absorption : this compound is absorbed slowly after oral administration, with a mean bioavailability estimated at 60%.
• Distribution : It exhibits extensive distribution in tissues, with a large volume of distribution (approximately 1400 L).
• Metabolism : Primarily metabolized by cytochrome P450 enzymes (CYP3A4, CYP3A5, and CYP1A1).
• Half-life : The drug has a long half-life of about 48 hours.

These characteristics suggest that this compound may maintain therapeutic levels over extended periods, potentially enhancing its anticancer effects.

Efficacy Against NSCLC

Recent studies have demonstrated that gefitinib significantly enhances mitochondrial functions in NSCLC cells. For instance, research indicates that gefitinib increases the enzymatic activity of succinate-tetrazolium reductase (STR) and mitochondrial membrane potential in HCC827 cells (EGFR-mutant NSCLC) under high cell density conditions. These findings suggest that gefitinib may act as a mitochondrial protector during combined treatments with other anticancer agents like doxorubicin .

Comparative Studies

A comparative study involving various gefitinib derivatives, including this compound, assessed their anti-proliferative effects on different lung cancer cell lines (NCI-H1299, A549). The results indicated that certain derivatives exhibited superior cytotoxicity compared to gefitinib itself. For example:

The introduction of structural modifications like the 1,2,3-triazole moiety significantly enhanced the compounds' efficacy against wild-type EGFR expressing cells .

Case Studies

In clinical settings, gefitinib has shown promise as a first-line therapy for patients with advanced NSCLC harboring EGFR mutations. A double-blind randomized trial revealed that patients receiving gefitinib experienced improved progression-free survival compared to those on standard chemotherapy .

Additionally, studies have identified factors contributing to gefitinib resistance in NSCLC cells. For instance, elevated expression levels of NANOG have been linked to resistance mechanisms. Targeting these pathways may enhance the efficacy of this compound in resistant cancer populations .

Basic Research Questions

Q. What is the primary role of Gefitinib-d3 in experimental pharmacology, and how does its deuterated structure influence its application in pharmacokinetic studies?

this compound serves as a deuterated internal standard for quantifying non-deuterated Gefitinib in biological matrices via mass spectrometry. The deuterium atoms reduce isotopic interference, improving analytical specificity and accuracy. Researchers should use stable isotope-labeled analogs like this compound to minimize matrix effects and enhance calibration curve reproducibility .

Q. What methodological advantages does this compound offer over non-deuterated Gefitinib in liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays?

this compound improves signal-to-noise ratios by distinguishing analyte peaks from endogenous compounds. To implement this:

• Prepare calibration standards using deuterated and non-deuterated forms in matched matrices.
• Validate assays for precision, recovery, and linearity per FDA guidelines.
• Ensure deuterium incorporation does not alter chromatographic behavior relative to the parent compound .

Q. How can researchers validate the stability of this compound under varying storage and processing conditions?

Stability studies should include:

• Short-term stability : Assess degradation after 24 hours at room temperature.
• Freeze-thaw cycles : Test 3–5 cycles at -80°C.
• Long-term stability : Monitor aliquots over 6–12 months. Use LC-MS/MS to quantify degradation products and confirm deuterium retention via high-resolution mass spectrometry (HRMS) .

Q. What protocols are recommended for synthesizing and characterizing this compound to ensure isotopic purity?

• Synthesis : Use deuterated reagents (e.g., D2O, CD3I) in key reaction steps, confirmed via nuclear magnetic resonance (NMR) and HRMS.
• Purity assessment : Measure isotopic enrichment (>98%) using mass spectrometry.
• Storage : Store in inert atmospheres (argon) at -20°C to prevent proton-deuterium exchange .

Q. How should researchers design in vitro experiments to evaluate this compound’s role in metabolic pathway analysis?

• Incubate this compound with liver microsomes or hepatocytes.
• Use LC-HRMS to identify deuterium-retaining metabolites.
• Compare metabolic profiles to non-deuterated Gefitinib to assess isotope effects on CYP3A4/5-mediated oxidation .

Advanced Research Questions

Q. What experimental design considerations are critical for minimizing variability in this compound-based pharmacokinetic studies?

• Cohort stratification : Control for factors affecting drug metabolism (e.g., CYP2D6 polymorphisms).
• Sample collection timing : Align with Gefitinib’s half-life (48 hours) to capture distribution/elimination phases.
• Data normalization : Use deuterated internal standards to correct for batch effects .

Q. How can researchers resolve contradictions in this compound assay data caused by matrix effects or isotopic interference?

• Matrix effect mitigation : Dilute samples or use phospholipid removal plates during extraction.
• Isotopic interference checks : Compare blank matrix samples spiked with/without this compound.
• Cross-validate : Confirm results with orthogonal techniques (e.g., immunoassays) .

Q. What strategies optimize the use of this compound in studying drug-drug interactions (DDIs) involving EGFR inhibitors?

• Co-administer this compound with CYP450 modulators (e.g., ketoconazole) in vitro/in vivo.
• Quantify changes in this compound clearance using population pharmacokinetic modeling.
• Apply physiologically based pharmacokinetic (PBPK) models to extrapolate human DDI risks .

Q. How can deuterium kinetic isotope effects (KIEs) impact the interpretation of this compound data in long-term toxicity studies?

• KIE analysis : Compare reaction rates (e.g., metabolic oxidation) between deuterated/non-deuterated forms.
• Toxicity endpoints : Monitor liver enzyme elevations (ALT/AST) and histopathology in preclinical models.
• Statistical modeling : Use ANOVA to distinguish isotope effects from biological variability .

Q. What advanced analytical techniques are required to validate this compound in complex biological matrices like cerebrospinal fluid (CSF)?

• Sample preparation : Employ microextraction by packed sorbent (MEPS) to handle low CSF volumes.
• Detection limits : Optimize LC-MS/MS transitions for sub-ng/mL sensitivity.
• Cross-validation : Confirm CSF-to-plasma ratios using equilibrium dialysis .