Synthesis and Acute Toxicity Evaluation of FEONM: A Novel PET Imaging Precursor for Alzheimer's Disease

Synthesis and Acute Toxicity Evaluation of FEONM: A Novel PET Imaging Precursor for Alzheimer's Disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Synthesis and Acute Toxicity Evaluation of FEONM: A Novel PET Imaging Precursor for Alzheimer's Disease Mao-Chi Weng, Jenn-Tzong Chen, Wuu-Jyh Lin, Yean-Hung Tu, Shiou-Shiow Farn, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7360002/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Alzheimer’s disease (AD) remains a major global health challenge, necessitating the development of more effective diagnostic tools. This study focuses on the development and evaluation of FEONM, a novel PET imaging precursor structurally derived from FDDNP, designed for potential use in AD imaging. To assess its suitability for clinical translation, both synthetic feasibility and safety profile were examined. A practical multi-step synthetic route was established using Bucherer and Knoevenagel reactions. Additionally, the acute toxicity of FEONM was evaluated in Sprague-Dawley rats (n = 5 per group per sex) following administration at 0.2, 1.0, and 5.0 mg/kg doses. Key toxicological endpoints included body weight monitoring, hematological and clinical chemistry analysis, and gross necropsy findings. Results No mortality or abnormal clinical signs were observed across all dose groups. Low and moderate doses did not affect normal growth, while 5.0 mg/kg caused temporary weight suppression. Dose-dependent hematological and biochemical effects showed sex-specific differences, with females displaying greater hematological sensitivity including significant lymphocyte reduction (68% vs 30% in males) at the highest dose. Males exhibited notable glucose elevation (22%) and electrolyte disturbances. Gross necropsy revealed hepatic and mild gastrointestinal changes only at 5.0 mg/kg in 20% of animals, indicating a clear threshold effect with no pathological lesions at lower doses. Conclusions FEONM was successfully synthesized and showed acceptable safety margins at clinically relevant doses. The NOAEL was determined to be 1.0 mg/kg. Different toxicity effects were observed between male and female rats, with females being more sensitive to blood-related changes and males showing more metabolic problems. These results indicate that FEONM has potential for clinical use, but additional long-term studies are needed to determine the optimal dosing for safe human application. FEONM PET imaging Alzheimer's disease Radiopharmaceutical synthesis Acute toxicity FDDNP derivative 1. Introduction The human brain is an intricate network of neuronal connections. Any disruption or degeneration in its structural circuits can lead to the development and progression of various neurodegenerative diseases.( 1 ) Alzheimer’s disease (AD), first described by Dr. Alzheimer in 1906, is the most common cause of dementia worldwide.( 2 ) It is characterized by two hallmark lesions in the brain: deposition of β-amyloid plaques (Aβ plaques) and neurofibrillary tangles (NFTs).( 2 – 4 ) Although the exact contribution of Aβ plaques and NFTs to dementia remains unclear, both are strongly correlated with the severity of cognitive decline in AD patients. ( 2 , 3 ) AD is a progressive neurodegenerative disorder leading to memory loss and cognitive impairment, predominantly affecting the elderly population. It has become the most common form of dementia in the United States, affecting about 10% of individuals over 65 and up to 50% of those over 85 years old. ( 4 , 5 ) According to clinical statistics from the past 60 years, an estimated 5.5 million Americans currently live with AD. Without effective treatments or a cure, this number is projected to rise dramatically, reaching up to 34 million people by 2050 in the United States alone.( 2 ) Few public health issues have drawn as much attention from both biomedical researchers and the general public as Alzheimer’s disease (AD).( 6 ) To prevent the worsening of its already significant personal and societal impact, improving early diagnosis is crucial to maximize the effectiveness of treatments and enhance the efficiency of clinical trials.( 7 , 8 ) Currently, clinical diagnosis of AD faces major limitations, as it becomes reasonably accurate only when the disease has already progressed to a severe stage.( 9 ) This delay reduces the window for timely intervention and disease management.( 9 ) Therefore, identifying reliable biomarkers with quantifiable characteristics has become a critical goal for early detection and for monitoring disease progression and treatment response.( 10 ) Biomarkers can serve as valuable tools for imaging and clinical monitoring, offering the potential to detect pathological changes before significant cognitive symptoms appear.( 10 ) In addition, advances in detecting early Aβ formation and aggregation not only aid diagnosis but may also accelerate the development of inhibitors and other therapeutic strategies for AD treatment.( 10 ) Biological changes in Alzheimer’s disease (AD) can be monitored by measuring cerebrospinal fluid (CSF) and plasma biomarkers, providing valuable information about disease progression.( 3 ) In nuclear medicine, neuroimaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) have rapidly advanced as powerful tools for in vivo brain studies.( 11 , 12 ) Initially, brain glucose metabolism was visualized using these techniques, appearing as “cold spots” in regions with reduced activity, highlighting areas affected by AD.( 13 , 14 ) This limitation has driven the development of novel tracers and imaging methods capable of directly visualizing AD-specific pathology as “hot spots,” offering clearer insights into disease-specific changes.( 13 ) Among these, PET imaging plays a fundamental role not only in visualizing regional brain glucose metabolism but also in investigating the complex interactions between the pathological hallmarks of AD, aiding in diagnosis and research on disease progression.( 3 ) In light of the prospects for promising Alzheimer’s disease (AD) therapies, there is a growing need for more sensitive and specific imaging techniques to quantify neuritic amyloid plaque burden in the brains of patients with dementia.( 5 , 9 , 10 ) Recent research has focused on developing imaging agents capable of detecting senile plaques (SPs) and neurofibrillary tangles (NFTs) in the living human brain.( 5 , 9 ) When labeled with appropriate isotopes, these molecular probes can serve as valuable tools for in vivo diagnosis and for monitoring the formation and progression of Aβ aggregates.( 15 ) Over the past decade, significant advances have been made in neuroimaging biomarkers for AD, aiming for high specificity and affinity toward key pathological proteins such as Aβ and tau. Isotope-labeled molecular probes make it possible to directly correlate molecular activity with disease pathology.( 1 ) Between 2012 and 2014, three PET tracers—[ 18 F]florbetapir (Amyvid™), [ 18 F]flutemetamol (Vizamyl™), and [ 18 F]florbetaben (Neuroceq™)—were extensively studied and subsequently approved by the FDA and the European Medicines Agency for imaging Aβ plaques in AD patients. In 2020, the first tau-specific radioactive tracer, [ 18 F]Flortaucipir (Tauvid™), was approved by the FDA, further expanding the toolkit for imaging tau pathology in AD. Another notable probe, [ 18 F]FDDNP, developed at UCLA, has also demonstrated effectiveness in visualizing both Aβ plaques and NFTs in the living brain.( 1 , 13 , 16 , 17 ) Most recently, our laboratory has developed a new PET molecular imaging probe, [ 18 F]FEONM, modified from [ 18 F]FDDNP. This probe shows promise for reliably tracking asymptomatic neuropathological progression of AD in the living brain, offering a potential tool for early detection and monitoring. As biomarker research advances rapidly, combining innovative beta-amyloid PET radiotracers with fluid biomarkers offers deeper insights into the complex mechanisms of AD and may enable earlier detection. In this study, we successfully synthesized high-purity FEONM and conducted acute toxicology tests using Sprague-Dawley rats to evaluate its safety. The results demonstrated that FEONM is safe and well-tolerated in rats, supporting its potential application in clinical diagnosis of Alzheimer’s disease. 2. Materials and methods 2.1. Animal Animal housing and experiments were approved by the Ethical Animal Use Committee of the Taipei Medical University and performed in compliance with Taiwan’s laws for the care and use of laboratory animals (LAC-2022-0183). Crl:CD (SD) Rats. Source: BioLASCO Taiwan Co., Ltd., Taipei, Taiwan. Age and body weight range at initiation of study: Approximately at 6 weeks old, with body weight ranges of 164 ~ 204 g for males and 143 ~ 167 g for females at the time of dosing. Temperature: maintained at 21 ± 2°C. Humidity: maintained at 50 ± 20% relative humidity. Light cycle: 12 hours light and 12 hours dark, automatic. Food and water were provided ad libitum. 2.2. The precursor synthesis of TEONM 2.2.1. Prepare of 2-Acetyl-6-hydroxynaphthalene, 1 The precursor TEONM was synthesized starting from 2-acetyl-6-methoxynaphthalene. The initial demethylation was carried out by refluxing the starting material overnight in fuming hydrochloric acid at 100°C. During the reaction, the solution color changed from yellow to black, indicating completion. The reaction mixture was extracted with dichloromethane, and the combined organic phases were washed sequentially with sodium hydroxide and saturated sodium chloride solutions. After removal of residual salts by filtration, the organic layer was concentrated under reduced pressure to yield 2-acetyl-6-hydroxynaphthalene ( compound 1 ) as a black solid. 2.2.2. Prepare of 2-Acetyl-6-[(2-hydroxyethyl)methylamino]naphthalene, 2 The intermediate 2-acetyl-6-hydroxynaphthalene was subjected to a Bucherer reaction to convert the hydroxyl group to an amino group. Briefly, the crude demethylated product was refluxed with 2-methylaminoethanol, sodium metabisulfite, and deionized water in a round-bottom flask at 100°C for several days, during which the reaction mixture changed from black to yellow, indicating formation of the desired naphthylamine derivative. Upon completion, the reaction mixture was extracted with ethyl acetate, and the combined organic phases were washed with saturated sodium hydroxide to remove residual acetic acid. The organic layer was dried, filtered, and concentrated under reduced pressure to yield a light yellow oil. This crude product was purified by silica gel column chromatography using ethyl acetate/hexane (1:2) as eluent. The purified fractions were combined and concentrated to afford 2-acetyl-6-(2-methylaminoethanol)naphthalene ( compound 2 ) as a golden-colored oil. 2.2.3. Prepare of 2-(1-{6-[methylethanolamino]-2-naphthyl}ethylidene)malononitrile, 3 Following purification of the Bucherer reaction intermediate, 2-acetyl-6-(2-methylaminoethanol)naphthalene was subjected to a Knoevenagel condensation to introduce a malononitrile moiety at the 2-position. The reaction was performed by stirring the intermediate with malononitrile in anhydrous pyridine at 70°C for 4 hours, during which the reaction mixture changed from golden to red, indicating completion of condensation. The crude product was extracted with ethyl acetate, and the organic layer was washed with saturated sodium hydroxide to remove residual acids. The organic phase was dried over anhydrous magnesium sulfate and concentrated under reduced pressure to yield a red solid identified as 2-(1-{6-[2-methylaminoethanol]-2-naphthyl}ethylidene)malononitrile ( compound 3 ). 2.2.4. Prepare of 2-(1-{6-[methylethanolethoxylamino]-2-naphthyl}ethylidene)malononitrile, 4 2-(1-{6-[2-methylethanolethoxyamino]-2-naphthyl}ethylidene)malononitrile, was synthesized to introduce an ethoxy moiety onto the amino alcohol side chain of compound 3 . Briefly, compound 3 was dissolved in anhydrous dimethylformamide (DMF) and reacted with 2-bromoethanol and potassium carbonate. The reaction mixture was heated to 90°C and refluxed overnight to allow the substitution of the hydroxyl group with an ethoxy group. Upon completion, the mixture was extracted with ethyl acetate. The organic layer was washed with saturated sodium hydroxide to remove residual acids, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The purified product was obtained as a light yellow oil and identified as compound 4 . 2.2.5. Final prepare of 2-(1-{6-[methylethanolethoxylamino]-2-naphthyl}ethylidene)malononitrile (TEONM), 5 The final step for preparing the [ 18 F]FEONM precursor involved tosylation of the terminal hydroxy group in compound 4 to introduce a suitable leaving group for fluorination. Compound 4 was reacted with p-toluenesulfonyl chloride in anhydrous dichloromethane and pyridine at room temperature with stirring for 5 hours. After completion, the reaction mixture was purified by solvent extraction as described previously for compound 4 . The resulting yellow product, 2-(1-{6-[2-methylethanolethoxytosylamino]-2-naphthyl}ethylidene)malononitrile, was obtained as the tosylated precursor TEONM ( compound 5 ). 2.3. FEONM toxicity experimental Design Study Design are presented as Table 1 : FEONM was administered at 0.2, 1.0, and 5.0 mg/kg doses via single tail vein injection on Day 1. Test solutions were freshly prepared in vehicle (5% DMSO, 25% Cremophor EL, 70% water for injection) at concentrations of 0.2, 1.0, and 5.0 mg/mL, protected from light, and administered using 26-gauge needles with volumes adjusted for individual body weights. Safety Margins: The selected dose levels provided substantial safety margins over the maximum recommended human dose (0.1 mg/70 kg): 140, 700 and 3,500 fold based on body weight conversion and 23, 117 and 583 fold based on surface area conversion. 2.4. Observation and Examination 2.4.1. Observations of animals The rats were observed for mortality and clinical signs at approximate 0, 1, 2, 3 and 4 hours after dosing. During the following days, the animals were observed twice daily (at least six hours apart) for mortality and once (at least) for clinical observation for 14 days. Any mortality and clinical signs were recorded and documented. 2.4.2. Body weights Body weight was recorded on all animals prior to the start of dosing (D1) and then at weekly interval (D8) to the end of the study period (D15). 2.4.3. Gross necropsy Gross necropsy was performed on found dead and on all surviving rats at the end of study (D15). The surviving rats were euthanized by carbon dioxide exposure followed by exsanguinations and necropsy in a randomized order. The external surface of the body and all organs/tissues in the thoracic and abdominal cavities were examined. Any observed gross lesions at necropsy were recorded. 2.4.4. Hematology and Biological chemistry analysis At terminal necropsy (Day 15), surviving rats were fasted for 16 + hours and euthanized by CO₂ exposure in randomized order. Blood was collected via cardiac puncture in EDTA tubes for hematological analysis using the Bayer ADVIA 120 Analyzer, measuring erythrocyte count, hemoglobin, hematocrit, MCV, leukocyte count and differential, platelet count, PT, and aPTT. Serum was separated by centrifugation for clinical chemistry analysis on the Hitachi 7060 Analyzer, evaluating liver enzymes (ALP, ALT, AST, GGT), kidney function markers (creatinine, BUN), metabolic parameters (glucose, total protein, albumin), electrolytes (Na, K, Cl, Ca, P), lipids (cholesterol, triglycerides), and additional markers (total bilirubin, LDH, CPK, uric acid). 2.5. Statistical Analysis: Results were expressed as mean and standard deviation (Mean ± SD). Comparisons of all data collected on body weights for each dose group of either sex were performed using ANOVA, follow by Dunnett’s test (SigmaStat™, V3.0, 2003) when significance was found. p < 0.05 was used as the criterion of significance. 3. Results 3.1. Synthesis method of FEONM Synthesis protocols as Scheme 1. 2-acetyl-6-methoxynaphthalene as the initial compound, then passed the acid degradation by Bucherer reaction and Knoevenagel reactioin finally formation the compound 5, TEONM. 3.2. Body weights: In FEONM, a summary of the mean body weights in rats are presented in Table 2 . These results suggest that low and moderate doses of FEONM do not affect normal body weight gain in rats, while the highest dose may temporarily suppress growth due to its associated toxicity (5 mg/kg). 3.3. Mortality and Clinical Observations In FEONM, the result of mortality and clinical signs are summarized in Tables 3. There was no death observed in all rats and mortality was 0/5. No any clinical sign was observed in control and all dose groups. 3.4. Hematology and Biological chemistry analysis 3.4.1. Hematology FEONM administration caused dose-dependent hematological toxicity, with females showing greater sensitivity than males. At 5.0 mg/kg/day, males experienced moderate decreases in RBC count (7.2% reduction), hemoglobin (10.7% reduction), and hematocrit (12.5% reduction), while females showed more severe effects with RBC counts dropping 15.8%, hemoglobin declining 15.1%, and hematocrit decreasing 15.7%. Lymphocyte counts were particularly affected, falling 68% in females versus 30% in males. Platelet counts decreased modestly in a dose-dependent manner, while mean corpuscular indices remained stable, suggesting proportional rather than morphological changes. The lowest dose (0.2 mg/kg/day) produced minimal effects. The results of hematology are presented in Table 4 . 3.4.2. Clinical chemistry FEONM caused dose-dependent biochemical changes with clear sex differences. Males at 5.0 mg/kg/day showed glucose elevation (22% increase), decreased total protein (8.4% reduction), markedly reduced alkaline phosphatase (46% decrease), and severe electrolyte imbalances with elevated chloride levels (60% increase). Females exhibited opposite glucose effects (14% decrease), more stable kidney function, minimal protein changes (3.3% reduction), and less pronounced electrolyte disturbances. Creatinine remained stable in both sexes, while liver enzymes decreased at higher doses mainly in females. These findings indicate sex-specific metabolic responses and sensitivity patterns to FEONM toxicity. The results of clinical chemistry are presented in Table 5 . 3.5. Gross necropsy : Pathological changes were observed exclusively at the highest dose level (5.0 mg/kg/day) and were limited to specific organ systems. In males at 5.0 mg/kg, hepatic alterations occurred in 1 out of 5 animals (20%), presenting as swollen and dark red liver appearance. Gastrointestinal abnormalities were also observed in males, with small intestinal dilation and yellowish contents affecting 1 out of 5 animals (20%). In females at 5.0 mg/kg, similar hepatic changes (swollen and dark red liver) were noted in 1 out of 5 animals (20%). No pathological changes were observed in any other organ systems examined, including stomach, lungs, heart, thoracic cavity, or kidneys, across all dose groups in both sexes. Importantly, no gross pathological alterations were detected at the lower dose levels (0.2 and 1.0 mg/kg) or in control groups for either sex, indicating a clear threshold effect with morphological changes manifesting only at the maximum tested dose of 5.0 mg/kg/day. A summary of the gross observation is presented in Table 6 . 4. Discussion A probabilistic Markov model analysis using extensive ADNI data indicated that subtle cognitive changes, particularly in memory, and declining CSF Aβ42 levels may begin over 25 years prior to clinical dementia onset, highlighting their potential as early biomarkers of Alzheimer’s disease (AD).( 10 ) Subthreshold Aβ decline has been linked to early memory dysfunction, while tau PET imaging further clarifies the spatiotemporal relationship between tau accumulation and preceding Aβ deposition.( 10 ) Given that dementia prevalence doubles approximately every five years and affects up to 50% of individuals by age 85, and that definitive AD diagnosis still relies on postmortem confirmation of senile plaques and neurofibrillary tangles, the development of reliable biomarkers remains critical for enhancing diagnostic accuracy in the early and often ambiguous stages of AD.( 10 ) FDDNP, originally developed at UCLA, has proven effective for visualizing SPs and NFTs in the living brains of Alzheimer’s disease (AD) patients via PET imaging. Building on this, we modified the FDDNP structure and established a practical multi-step synthesis for the novel precursor TEONM, as shown in Scheme 1 . The final product ( compound 5 , FEONM) was obtained with high purity, making it suitable for radiofluorination and AD imaging applications. Notably, sodium metabisulfite was used instead of the traditional Bucherer catalyst, providing greater reducing power. Additionally, Prof. Shiue’s design introduced an ethoxy group between the malononitrile moiety and fluorine atom, enhancing docking affinity for AD-related proteins. This synthetic pathway, which combines established Bucherer and Knoevenagel reactions, is both efficient and scalable for further preclinical or clinical research as Supplement 1 . The different dose levels (0.2, 1.0, and 5.0 mg/kg) for this study, based on the clinical dose of about 10 mCi (equivalent to 42 pmole) determined from the relative molecular mass of 416.27 and human body weight (70 kg), provided safety margins of 140, 700 and 3,500 fold based on body weight conversion and 23, 117 and 583 fold based on surface area conversion over the maximal recommended human dose. The present study demonstrated pronounced sex-specific differences in FEONM-induced toxicity, with females exhibiting greater sensitivity across multiple parameters. Female had 34% greater risk of severe toxicity than men in oncology treatments, and pharmacokinetics differ in men and female for many drugs, which impacts drug efficacy and toxicity.( 18 , 19 ) Our findings align with this established pattern, as female rats showed more severe hematological changes, including a 15.8% reduction in RBC count compared to 7.2% in males, and a dramatic 68% decrease in lymphocyte counts versus 30% in males. Female are more likely to experience increased bone marrow toxicity in hematological diseases, which supports our observation of greater hematological sensitivity in females ( 20 ). Gender differences in drug pharmacokinetics is a main contributor to higher drug toxicity in female, stemming from physiological differences in body composition, plasma protein concentrations, and liver and kidney function.( 21 , 22 ) The contrasting glucose responses between sexes (22% increase in males vs. 14% decrease in females) further underscore these metabolic differences. Notably, hematology data demonstrated mild anemia and lymphopenia, especially in females, suggesting potential sex-specific hematopoietic sensitivity to FEONM, which aligns with findings from other fluorinated naphthalene derivatives.( 23 ) The acute toxicology assessment of FEONM in Sprague-Dawley rats revealed dose-dependent effects primarily at the highest tested dose (5.0 mg/kg/day). Body weight suppression and mild organ-specific gross lesions observed at this level are consistent with previous reports for related beta-amyloid PET tracers, which also show acceptable safety profiles at diagnostic dose ranges but may elicit systemic effects at supratherapeutic exposure.( 24 ) The hepatic changes observed exclusively at 5.0 mg/kg suggest a threshold-dependent mechanism characteristic of direct hepatotoxins. Drugs with pharmacological hepatotoxicity have predictable dose-response curves where higher concentrations cause more liver damage.( 25 ) The swollen and dark red liver appearance in both sexes indicates hepatocellular congestion or direct tissue damage, consistent with dose-dependent hepatotoxicity patterns detected during preclinical studies. Direct, dose-dependent hepatotoxins induce intracellular signaling pathways leading to cell death, which may explain the hepatic manifestations observed only at the highest dose.( 26 ) The absence of liver injury at lower doses (0.2 and 1.0 mg/kg) supports a clear threshold effect rather than idiosyncratic liver injury, suggesting FEONM acts through direct hepatotoxic mechanisms. Biochemical alterations, including hyperglycemia in males and hypoglycemia in females, alongside moderate shifts in liver enzyme activity and electrolytes, indicate mild hepatic and metabolic stress at high doses. However, these changes were reversible and not accompanied by significant morphological liver damage beyond slight swelling. Such patterns are commonly observed with lipophilic PET tracers due to hepatic metabolism and clearance.( 27 ) The proportional decreases in RBC parameters without morphological changes (stable MCV) suggest that FEONM affects erythropoiesis or causes hemolysis rather than interfering with red blood cell maturation. The dramatic lymphocyte reduction, particularly in females, indicates potential immunosuppressive effects or direct lymphotoxicity. This pattern is concerning as up to 6–7% of new drug applications show at least a 40% difference in pharmacokinetics between males and females, highlighting the importance of sex-specific safety assessments.( 19 ) Importantly, no mortality or severe systemic toxicity occurred at diagnostic or moderate doses, supporting a favorable safety margin for further translational studies. The absence of significant gross lesions or functional impairments at low and moderate doses suggests that FEONM holds promise as a novel beta-amyloid PET tracer candidate with acceptable risk.( 28 , 29 ) The pattern of toxicity observed at 5.0 mg/kg—representing a 140-fold higher safety margin than the recommended human dose—suggests a narrow therapeutic window that requires careful dose optimization. Notably, the clear sex differences in metabolic and toxicological responses suggest that future clinical development should consider sex-stratified dosing strategies, as sex differences in drug distribution can significantly impact safety and efficacy.( 19 , 22 )While this acute toxicity study provides valuable initial insights into the safety of FEONM, longer-term studies are needed to assess potential delayed or cumulative effects. Dose-escalation studies at intermediate doses between 1.0 and 5.0 mg/kg will help better define no-observed adverse effect levels (NOAELs), while mechanistic studies of the molecular basis of the observed sex differences will further elucidate the toxicological profile. Consistent with previous reports from FDDNP ( 30 ), animals at high doses showed decreased activity, loss of appetite, dehydration, hunched backs, and vocalizations, but animals sacrificed as planned showed no clinical signs or macroscopic lesions. To fully support clinical translation, we will continue to conduct additional studies including repeated dose toxicology, radioactive metabolite characterization, and non-human primate dosimetry. 5. Conclusion This study demonstrated the successful synthesis and preclinical toxicological assessment of FEONM, a novel PET imaging precursor structurally derived from FDDNP. The compound exhibited a favorable safety profile at doses up to 1.0 mg/kg in Sprague-Dawley rats, with no observed mortality or severe adverse effects. While the highest tested dose (5.0 mg/kg) induced mild, dose-dependent hematological, biochemical, and organ-specific changes, these effects were absent at lower doses, establishing 1.0 mg/kg as the no-observed-adverse-effect level (NOAEL). Notably, sex-specific differences in toxicity were observed, with females more susceptible to hematological alterations and males to metabolic disturbances. These findings support the potential of FEONM as a clinically translatable PET imaging agent for Alzheimer’s disease, although further long-term toxicity and pharmacokinetic studies are warranted to define its safety margin in humans. 6. Declarations 6.1 Ethics approval and consent to participate Animal housing and experiments were approved by the Ethical Animal Use Committee of the Taipei Medical University and performed in compliance with Taiwan’s laws for the care and use of laboratory animals (LAC-2022-0183). 6.2 Consent for publication Not applicable. 6.3 Availability of data and material The datasets generated and analyzed during the current study are not publicly available due to an ongoing patent application and potential future tech-nology transfer for clinical development. However, the data are available from the corresponding author upon reasonable request. 6.4 Competing interests All authors have read and approved the final version of the manuscript and declare that they have no conflicts of interest. 6.5 Funding This work was supported by the National Atomic Research Institute, ROC [grant numbers 112-3408-007-300]. 6.6 Authors' contributions Writing—original draft preparation, M.-C.W.; Conceptualization, methodology, and validation, J.-T.C., W.-J.L., and Y.-H.T.; Formal analysis, investigation, and data curation, S.-S.F., and C.-C.H.; Writing—review and editing, supervision, and project administration, K.-W.C.. 6.7 Acknowledgements The authors would like to express their gratitude to Chyng-Yann Shiue for their valuable guidance and great contribution to the structural design of this research. 6.8 Authors' information Mao-Chi Weng, Jenn-Tzong Chen, Yean-Hung Tu, Shiou-Shiow Farn, and Chien-Chung Hsia Department of Isotope Application Research, National Atomic Research Institute, Taoyuan 325207, Taiwan Wuu-Jyh Lin SeeCURE Taiwan Co., Ltd., Kaohsiung 831134, Taiwan Kang-Wei Chang Taipei Neuroscience Institute and Laboratory Animal Center, Taipei Medical University, Taipei 11048, Taiwan 7. 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Florbetapir (18F), a PET imaging agent that binds to amyloid plaques for the potential detection of Alzheimer's disease. IDrugs : the investigational drugs journal. 2010;13(12):890-9. Kaplowitz N. Idiosyncratic drug hepatotoxicity. Nature reviews Drug discovery. 2005;4(6):489-99. Jaeschke H, McGill MR, Ramachandran A. Oxidant stress, mitochondria, and cell death mechanisms in drug-induced liver injury: lessons learned from acetaminophen hepatotoxicity. Drug metabolism reviews. 2012;44(1):88-106. Wagatsuma K, Ishibashi K, Kameyama M, Sakata M, Miwa K, Kamitaka Y, et al. Decreased imaging time of amyloid PET using [(18)F]florbetapir can maintain quantitative accuracy. Radiological physics and technology. 2022;15(2):116-24. Wong DF, Rosenberg PB, Zhou Y, Kumar A, Raymont V, Ravert HT, et al. In vivo imaging of amyloid deposition in Alzheimer disease using the radioligand 18F-AV-45 (florbetapir [corrected] F 18). Journal of nuclear medicine : official publication, Society of Nuclear Medicine. 2010;51(6):913-20. Carome M, Wolfe S. Florbetapir-PET imaging and postmortem beta-amyloid pathology. Jama. 2011;305(18):1857; author reply -8. Chang KW, Chen CC, Lee SY, Wang HE. Acute toxicity of two Alzheimer's disease radiopharmaceuticals: FDDNP and IMPY. Drug and chemical toxicology. 2009;32(4):429-37. Tables Tables 1 to 6 are available in the Supplementary Files section Scheme Scheme 1 is available in the Supplementary Files section. Supplementary Files Scheme1.docx Supplement1.docx Table1.docx Table2.docx Table3.docx Table4.docx Table5.docx Table6.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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12:29:34","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":16982,"visible":true,"origin":"","legend":"","description":"","filename":"Table5.docx","url":"https://assets-eu.researchsquare.com/files/rs-7360002/v1/e727d3cbaef80239ec765a49.docx"},{"id":89667656,"identity":"ccba7a0b-6327-4393-b856-cdc46337b9b8","added_by":"auto","created_at":"2025-08-22 12:21:34","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":15141,"visible":true,"origin":"","legend":"","description":"","filename":"Table6.docx","url":"https://assets-eu.researchsquare.com/files/rs-7360002/v1/0c716cfcc06b8392e164e6c0.docx"}],"financialInterests":"","formattedTitle":"Synthesis and Acute Toxicity Evaluation of FEONM: A Novel PET Imaging Precursor for Alzheimer's Disease","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe human brain is an intricate network of neuronal connections. Any disruption or degeneration in its structural circuits can lead to the development and progression of various neurodegenerative diseases.(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) Alzheimer\u0026rsquo;s disease (AD), first described by Dr. Alzheimer in 1906, is the most common cause of dementia worldwide.(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) It is characterized by two hallmark lesions in the brain: deposition of β-amyloid plaques (Aβ plaques) and neurofibrillary tangles (NFTs).(\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) Although the exact contribution of Aβ plaques and NFTs to dementia remains unclear, both are strongly correlated with the severity of cognitive decline in AD patients. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) AD is a progressive neurodegenerative disorder leading to memory loss and cognitive impairment, predominantly affecting the elderly population. It has become the most common form of dementia in the United States, affecting about 10% of individuals over 65 and up to 50% of those over 85 years old. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) According to clinical statistics from the past 60 years, an estimated 5.5\u0026nbsp;million Americans currently live with AD. Without effective treatments or a cure, this number is projected to rise dramatically, reaching up to 34\u0026nbsp;million people by 2050 in the United States alone.(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eFew public health issues have drawn as much attention from both biomedical researchers and the general public as Alzheimer\u0026rsquo;s disease (AD).(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) To prevent the worsening of its already significant personal and societal impact, improving early diagnosis is crucial to maximize the effectiveness of treatments and enhance the efficiency of clinical trials.(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) Currently, clinical diagnosis of AD faces major limitations, as it becomes reasonably accurate only when the disease has already progressed to a severe stage.(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e) This delay reduces the window for timely intervention and disease management.(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e) Therefore, identifying reliable biomarkers with quantifiable characteristics has become a critical goal for early detection and for monitoring disease progression and treatment response.(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) Biomarkers can serve as valuable tools for imaging and clinical monitoring, offering the potential to detect pathological changes before significant cognitive symptoms appear.(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) In addition, advances in detecting early Aβ formation and aggregation not only aid diagnosis but may also accelerate the development of inhibitors and other therapeutic strategies for AD treatment.(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eBiological changes in Alzheimer\u0026rsquo;s disease (AD) can be monitored by measuring cerebrospinal fluid (CSF) and plasma biomarkers, providing valuable information about disease progression.(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) In nuclear medicine, neuroimaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) have rapidly advanced as powerful tools for in vivo brain studies.(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e) Initially, brain glucose metabolism was visualized using these techniques, appearing as \u0026ldquo;cold spots\u0026rdquo; in regions with reduced activity, highlighting areas affected by AD.(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e) This limitation has driven the development of novel tracers and imaging methods capable of directly visualizing AD-specific pathology as \u0026ldquo;hot spots,\u0026rdquo; offering clearer insights into disease-specific changes.(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) Among these, PET imaging plays a fundamental role not only in visualizing regional brain glucose metabolism but also in investigating the complex interactions between the pathological hallmarks of AD, aiding in diagnosis and research on disease progression.(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eIn light of the prospects for promising Alzheimer\u0026rsquo;s disease (AD) therapies, there is a growing need for more sensitive and specific imaging techniques to quantify neuritic amyloid plaque burden in the brains of patients with dementia.(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) Recent research has focused on developing imaging agents capable of detecting senile plaques (SPs) and neurofibrillary tangles (NFTs) in the living human brain.(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e) When labeled with appropriate isotopes, these molecular probes can serve as valuable tools for in vivo diagnosis and for monitoring the formation and progression of Aβ aggregates.(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) Over the past decade, significant advances have been made in neuroimaging biomarkers for AD, aiming for high specificity and affinity toward key pathological proteins such as Aβ and tau. Isotope-labeled molecular probes make it possible to directly correlate molecular activity with disease pathology.(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) Between 2012 and 2014, three PET tracers\u0026mdash;[\u003csup\u003e18\u003c/sup\u003eF]florbetapir (Amyvid\u0026trade;), [\u003csup\u003e18\u003c/sup\u003eF]flutemetamol (Vizamyl\u0026trade;), and [\u003csup\u003e18\u003c/sup\u003eF]florbetaben (Neuroceq\u0026trade;)\u0026mdash;were extensively studied and subsequently approved by the FDA and the European Medicines Agency for imaging Aβ plaques in AD patients. In 2020, the first tau-specific radioactive tracer, [\u003csup\u003e18\u003c/sup\u003eF]Flortaucipir (Tauvid\u0026trade;), was approved by the FDA, further expanding the toolkit for imaging tau pathology in AD. Another notable probe, [\u003csup\u003e18\u003c/sup\u003eF]FDDNP, developed at UCLA, has also demonstrated effectiveness in visualizing both Aβ plaques and NFTs in the living brain.(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) Most recently, our laboratory has developed a new PET molecular imaging probe, [\u003csup\u003e18\u003c/sup\u003eF]FEONM, modified from [\u003csup\u003e18\u003c/sup\u003eF]FDDNP. This probe shows promise for reliably tracking asymptomatic neuropathological progression of AD in the living brain, offering a potential tool for early detection and monitoring.\u003c/p\u003e\u003cp\u003eAs biomarker research advances rapidly, combining innovative beta-amyloid PET radiotracers with fluid biomarkers offers deeper insights into the complex mechanisms of AD and may enable earlier detection. In this study, we successfully synthesized high-purity FEONM and conducted acute toxicology tests using Sprague-Dawley rats to evaluate its safety. The results demonstrated that FEONM is safe and well-tolerated in rats, supporting its potential application in clinical diagnosis of Alzheimer\u0026rsquo;s disease.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1. Animal\u003c/h2\u003e\n \u003cp\u003eAnimal housing and experiments were approved by the Ethical Animal Use Committee of the Taipei Medical University and performed in compliance with Taiwan\u0026rsquo;s laws for the care and use of laboratory animals (LAC-2022-0183). Crl:CD (SD) Rats. Source: BioLASCO Taiwan Co., Ltd., Taipei, Taiwan. Age and body weight range at initiation of study: Approximately at 6 weeks old, with body weight ranges of 164\u0026thinsp;~\u0026thinsp;204 g for males and 143\u0026thinsp;~\u0026thinsp;167 g for females at the time of dosing. Temperature: maintained at 21\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C. Humidity: maintained at 50\u0026thinsp;\u0026plusmn;\u0026thinsp;20% relative humidity. Light cycle: 12 hours light and 12 hours dark, automatic. Food and water were provided ad libitum.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2. The precursor synthesis of TEONM\u003c/h2\u003e\n \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.1. Prepare of 2-Acetyl-6-hydroxynaphthalene, 1\u003c/h2\u003e\n \u003cp\u003eThe precursor TEONM was synthesized starting from 2-acetyl-6-methoxynaphthalene. The initial demethylation was carried out by refluxing the starting material overnight in fuming hydrochloric acid at 100\u0026deg;C. During the reaction, the solution color changed from yellow to black, indicating completion. The reaction mixture was extracted with dichloromethane, and the combined organic phases were washed sequentially with sodium hydroxide and saturated sodium chloride solutions. After removal of residual salts by filtration, the organic layer was concentrated under reduced pressure to yield 2-acetyl-6-hydroxynaphthalene (\u003cstrong\u003ecompound 1\u003c/strong\u003e) as a black solid.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.2. Prepare of 2-Acetyl-6-[(2-hydroxyethyl)methylamino]naphthalene, 2\u003c/h2\u003e\n \u003cp\u003eThe intermediate 2-acetyl-6-hydroxynaphthalene was subjected to a Bucherer reaction to convert the hydroxyl group to an amino group. Briefly, the crude demethylated product was refluxed with 2-methylaminoethanol, sodium metabisulfite, and deionized water in a round-bottom flask at 100\u0026deg;C for several days, during which the reaction mixture changed from black to yellow, indicating formation of the desired naphthylamine derivative. Upon completion, the reaction mixture was extracted with ethyl acetate, and the combined organic phases were washed with saturated sodium hydroxide to remove residual acetic acid. The organic layer was dried, filtered, and concentrated under reduced pressure to yield a light yellow oil. This crude product was purified by silica gel column chromatography using ethyl acetate/hexane (1:2) as eluent. The purified fractions were combined and concentrated to afford 2-acetyl-6-(2-methylaminoethanol)naphthalene (\u003cstrong\u003ecompound 2\u003c/strong\u003e) as a golden-colored oil.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.3. Prepare of 2-(1-{6-[methylethanolamino]-2-naphthyl}ethylidene)malononitrile, 3\u003c/h2\u003e\n \u003cp\u003eFollowing purification of the Bucherer reaction intermediate, 2-acetyl-6-(2-methylaminoethanol)naphthalene was subjected to a Knoevenagel condensation to introduce a malononitrile moiety at the 2-position. The reaction was performed by stirring the intermediate with malononitrile in anhydrous pyridine at 70\u0026deg;C for 4 hours, during which the reaction mixture changed from golden to red, indicating completion of condensation. The crude product was extracted with ethyl acetate, and the organic layer was washed with saturated sodium hydroxide to remove residual acids. The organic phase was dried over anhydrous magnesium sulfate and concentrated under reduced pressure to yield a red solid identified as 2-(1-{6-[2-methylaminoethanol]-2-naphthyl}ethylidene)malononitrile (\u003cstrong\u003ecompound 3\u003c/strong\u003e).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.4. Prepare of 2-(1-{6-[methylethanolethoxylamino]-2-naphthyl}ethylidene)malononitrile, 4\u003c/h2\u003e\n \u003cp\u003e2-(1-{6-[2-methylethanolethoxyamino]-2-naphthyl}ethylidene)malononitrile, was synthesized to introduce an ethoxy moiety onto the amino alcohol side chain of \u003cstrong\u003ecompound 3\u003c/strong\u003e. Briefly, \u003cstrong\u003ecompound 3\u003c/strong\u003e was dissolved in anhydrous dimethylformamide (DMF) and reacted with 2-bromoethanol and potassium carbonate. The reaction mixture was heated to 90\u0026deg;C and refluxed overnight to allow the substitution of the hydroxyl group with an ethoxy group. Upon completion, the mixture was extracted with ethyl acetate. The organic layer was washed with saturated sodium hydroxide to remove residual acids, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The purified product was obtained as a light yellow oil and identified as \u003cstrong\u003ecompound 4\u003c/strong\u003e.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.5. Final prepare of 2-(1-{6-[methylethanolethoxylamino]-2-naphthyl}ethylidene)malononitrile (TEONM), 5\u003c/h2\u003e\n \u003cp\u003eThe final step for preparing the [\u003csup\u003e18\u003c/sup\u003eF]FEONM precursor involved tosylation of the terminal hydroxy group in \u003cstrong\u003ecompound 4\u003c/strong\u003e to introduce a suitable leaving group for fluorination. \u003cstrong\u003eCompound 4\u003c/strong\u003e was reacted with p-toluenesulfonyl chloride in anhydrous dichloromethane and pyridine at room temperature with stirring for 5 hours. After completion, the reaction mixture was purified by solvent extraction as described previously for \u003cstrong\u003ecompound 4\u003c/strong\u003e. The resulting yellow product, 2-(1-{6-[2-methylethanolethoxytosylamino]-2-naphthyl}ethylidene)malononitrile, was obtained as the tosylated precursor TEONM (\u003cstrong\u003ecompound 5\u003c/strong\u003e).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3. FEONM toxicity experimental Design\u003c/h2\u003e\n \u003cp\u003eStudy Design are presented as Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e: FEONM was administered at 0.2, 1.0, and 5.0 mg/kg doses via single tail vein injection on Day 1. Test solutions were freshly prepared in vehicle (5% DMSO, 25% Cremophor EL, 70% water for injection) at concentrations of 0.2, 1.0, and 5.0 mg/mL, protected from light, and administered using 26-gauge needles with volumes adjusted for individual body weights.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003eSafety Margins: The selected dose levels provided substantial safety margins over the maximum recommended human dose (0.1 mg/70 kg): 140, 700 and 3,500 fold based on body weight conversion and 23, 117 and 583 fold based on surface area conversion.\u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4. Observation and Examination\u003c/h2\u003e\n \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\n \u003ch2\u003e2.4.1. Observations of animals\u003c/h2\u003e\n \u003cp\u003eThe rats were observed for mortality and clinical signs at approximate 0, 1, 2, 3 and 4 hours after dosing. During the following days, the animals were observed twice daily (at least six hours apart) for mortality and once (at least) for clinical observation for 14 days. Any mortality and clinical signs were recorded and documented.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\n \u003ch2\u003e2.4.2. Body weights\u003c/h2\u003e\n \u003cp\u003eBody weight was recorded on all animals prior to the start of dosing (D1) and then at weekly interval (D8) to the end of the study period (D15).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\n \u003ch2\u003e2.4.3. Gross necropsy\u003c/h2\u003e\n \u003cp\u003eGross necropsy was performed on found dead and on all surviving rats at the end of study (D15). The surviving rats were euthanized by carbon dioxide exposure followed by exsanguinations and necropsy in a randomized order. The external surface of the body and all organs/tissues in the thoracic and abdominal cavities were examined. Any observed gross lesions at necropsy were recorded.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\n \u003ch2\u003e2.4.4. Hematology and Biological chemistry analysis\u003c/h2\u003e\n \u003cp\u003eAt terminal necropsy (Day 15), surviving rats were fasted for 16\u0026thinsp;+\u0026thinsp;hours and euthanized by CO₂ exposure in randomized order. Blood was collected via cardiac puncture in EDTA tubes for hematological analysis using the Bayer ADVIA 120 Analyzer, measuring erythrocyte count, hemoglobin, hematocrit, MCV, leukocyte count and differential, platelet count, PT, and aPTT. Serum was separated by centrifugation for clinical chemistry analysis on the Hitachi 7060 Analyzer, evaluating liver enzymes (ALP, ALT, AST, GGT), kidney function markers (creatinine, BUN), metabolic parameters (glucose, total protein, albumin), electrolytes (Na, K, Cl, Ca, P), lipids (cholesterol, triglycerides), and additional markers (total bilirubin, LDH, CPK, uric acid).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5. Statistical Analysis:\u003c/h2\u003e\n \u003cp\u003eResults were expressed as mean and standard deviation (Mean \u0026plusmn; SD). Comparisons of all data collected on body weights for each dose group of either sex were performed using ANOVA, follow by Dunnett\u0026rsquo;s test (SigmaStat\u0026trade;, V3.0, 2003) when significance was found. \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was used as the criterion of significance.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3.1. Synthesis method of FEONM\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSynthesis protocols as \u003cstrong\u003eScheme 1.\u003c/strong\u003e 2-acetyl-6-methoxynaphthalene as the initial compound, then passed the acid degradation by Bucherer reaction and Knoevenagel reactioin finally formation the compound 5, TEONM.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3.2. Body weights:\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn FEONM, a summary of the mean body weights in rats are presented in \u003cstrong\u003eTable 2\u003c/strong\u003e. These results suggest that low and moderate doses of FEONM do not affect normal body weight gain in rats, while the highest dose may temporarily suppress growth due to its associated toxicity\u0026nbsp;(5 mg/kg).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3.3. Mortality and Clinical Observations\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn FEONM, the result of mortality and clinical signs are summarized in \u003cstrong\u003eTables 3.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere was no death observed in all rats and mortality was 0/5. No any clinical sign was observed in control and all dose groups.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3.4. Hematology and Biological chemistry analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e3.4.1. Hematology\u003c/p\u003e\n\u003cp\u003eFEONM administration caused dose-dependent hematological toxicity, with females showing greater sensitivity than males. At 5.0 mg/kg/day, males experienced moderate decreases in RBC count (7.2% reduction), hemoglobin (10.7% reduction), and hematocrit (12.5% reduction), while females showed more severe effects with RBC counts dropping 15.8%, hemoglobin declining 15.1%, and hematocrit decreasing 15.7%. Lymphocyte counts were particularly affected, falling 68% in females versus 30% in males. Platelet counts decreased modestly in a dose-dependent manner, while mean corpuscular indices remained stable, suggesting proportional rather than morphological changes. The lowest dose (0.2 mg/kg/day) produced minimal effects. The results of hematology are presented in \u003cstrong\u003eTable 4\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e3.4.2. Clinical chemistry\u003c/p\u003e\n\u003cp\u003eFEONM caused dose-dependent biochemical changes with clear sex differences. Males at 5.0 mg/kg/day showed glucose elevation (22% increase), decreased total protein (8.4% reduction), markedly reduced alkaline phosphatase (46% decrease), and severe electrolyte imbalances with elevated chloride levels (60% increase). Females exhibited opposite glucose effects (14% decrease), more stable kidney function, minimal protein changes (3.3% reduction), and less pronounced electrolyte disturbances. Creatinine remained stable in both sexes, while liver enzymes decreased at higher doses mainly in females. These findings indicate sex-specific metabolic responses and sensitivity patterns to FEONM toxicity. The results of clinical chemistry are presented in \u003cstrong\u003eTable 5\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3.5. Gross necropsy\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e:\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePathological changes were observed exclusively at the highest dose level (5.0 mg/kg/day) and were limited to specific organ systems. In males at 5.0 mg/kg, hepatic alterations occurred in 1 out of 5 animals (20%), presenting as swollen and dark red liver appearance. Gastrointestinal abnormalities were also observed in males, with small intestinal dilation and yellowish contents affecting 1 out of 5 animals (20%). In females at 5.0 mg/kg, similar hepatic changes (swollen and dark red liver) were noted in 1 out of 5 animals (20%).\u003c/p\u003e\n\u003cp\u003eNo pathological changes were observed in any other organ systems examined, including stomach, lungs, heart, thoracic cavity, or kidneys, across all dose groups in both sexes. Importantly, no gross pathological alterations were detected at the lower dose levels (0.2 and 1.0 mg/kg) or in control groups for either sex, indicating a clear threshold effect with morphological changes manifesting only at the maximum tested dose of 5.0 mg/kg/day. A summary of the gross observation is presented in \u003cstrong\u003eTable 6\u003c/strong\u003e.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eA probabilistic Markov model analysis using extensive ADNI data indicated that subtle cognitive changes, particularly in memory, and declining CSF Aβ42 levels may begin over 25 years prior to clinical dementia onset, highlighting their potential as early biomarkers of Alzheimer\u0026rsquo;s disease (AD).(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) Subthreshold Aβ decline has been linked to early memory dysfunction, while tau PET imaging further clarifies the spatiotemporal relationship between tau accumulation and preceding Aβ deposition.(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) Given that dementia prevalence doubles approximately every five years and affects up to 50% of individuals by age 85, and that definitive AD diagnosis still relies on postmortem confirmation of senile plaques and neurofibrillary tangles, the development of reliable biomarkers remains critical for enhancing diagnostic accuracy in the early and often ambiguous stages of AD.(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eFDDNP, originally developed at UCLA, has proven effective for visualizing SPs and NFTs in the living brains of Alzheimer\u0026rsquo;s disease (AD) patients via PET imaging. Building on this, we modified the FDDNP structure and established a practical multi-step synthesis for the novel precursor TEONM, as shown in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The final product (\u003cb\u003ecompound 5\u003c/b\u003e, FEONM) was obtained with high purity, making it suitable for radiofluorination and AD imaging applications. Notably, sodium metabisulfite was used instead of the traditional Bucherer catalyst, providing greater reducing power. Additionally, Prof. Shiue\u0026rsquo;s design introduced an ethoxy group between the malononitrile moiety and fluorine atom, enhancing docking affinity for AD-related proteins. This synthetic pathway, which combines established Bucherer and Knoevenagel reactions, is both efficient and scalable for further preclinical or clinical research as \u003cb\u003eSupplement 1\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eThe different dose levels (0.2, 1.0, and 5.0 mg/kg) for this study, based on the clinical dose of about 10 mCi (equivalent to 42 pmole) determined from the relative molecular mass of 416.27 and human body weight (70 kg), provided safety margins of 140, 700 and 3,500 fold based on body weight conversion and 23, 117 and 583 fold based on surface area conversion over the maximal recommended human dose. The present study demonstrated pronounced sex-specific differences in FEONM-induced toxicity, with females exhibiting greater sensitivity across multiple parameters. Female had 34% greater risk of severe toxicity than men in oncology treatments, and pharmacokinetics differ in men and female for many drugs, which impacts drug efficacy and toxicity.(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e) Our findings align with this established pattern, as female rats showed more severe hematological changes, including a 15.8% reduction in RBC count compared to 7.2% in males, and a dramatic 68% decrease in lymphocyte counts versus 30% in males.\u003c/p\u003e\u003cp\u003eFemale are more likely to experience increased bone marrow toxicity in hematological diseases, which supports our observation of greater hematological sensitivity in females (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Gender differences in drug pharmacokinetics is a main contributor to higher drug toxicity in female, stemming from physiological differences in body composition, plasma protein concentrations, and liver and kidney function.(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) The contrasting glucose responses between sexes (22% increase in males vs. 14% decrease in females) further underscore these metabolic differences. Notably, hematology data demonstrated mild anemia and lymphopenia, especially in females, suggesting potential sex-specific hematopoietic sensitivity to FEONM, which aligns with findings from other fluorinated naphthalene derivatives.(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eThe acute toxicology assessment of FEONM in Sprague-Dawley rats revealed dose-dependent effects primarily at the highest tested dose (5.0 mg/kg/day). Body weight suppression and mild organ-specific gross lesions observed at this level are consistent with previous reports for related beta-amyloid PET tracers, which also show acceptable safety profiles at diagnostic dose ranges but may elicit systemic effects at supratherapeutic exposure.(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eThe hepatic changes observed exclusively at 5.0 mg/kg suggest a threshold-dependent mechanism characteristic of direct hepatotoxins. Drugs with pharmacological hepatotoxicity have predictable dose-response curves where higher concentrations cause more liver damage.(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e) The swollen and dark red liver appearance in both sexes indicates hepatocellular congestion or direct tissue damage, consistent with dose-dependent hepatotoxicity patterns detected during preclinical studies.\u003c/p\u003e\u003cp\u003eDirect, dose-dependent hepatotoxins induce intracellular signaling pathways leading to cell death, which may explain the hepatic manifestations observed only at the highest dose.(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) The absence of liver injury at lower doses (0.2 and 1.0 mg/kg) supports a clear threshold effect rather than idiosyncratic liver injury, suggesting FEONM acts through direct hepatotoxic mechanisms. Biochemical alterations, including hyperglycemia in males and hypoglycemia in females, alongside moderate shifts in liver enzyme activity and electrolytes, indicate mild hepatic and metabolic stress at high doses. However, these changes were reversible and not accompanied by significant morphological liver damage beyond slight swelling. Such patterns are commonly observed with lipophilic PET tracers due to hepatic metabolism and clearance.(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eThe proportional decreases in RBC parameters without morphological changes (stable MCV) suggest that FEONM affects erythropoiesis or causes hemolysis rather than interfering with red blood cell maturation. The dramatic lymphocyte reduction, particularly in females, indicates potential immunosuppressive effects or direct lymphotoxicity. This pattern is concerning as up to 6\u0026ndash;7% of new drug applications show at least a 40% difference in pharmacokinetics between males and females, highlighting the importance of sex-specific safety assessments.(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eImportantly, no mortality or severe systemic toxicity occurred at diagnostic or moderate doses, supporting a favorable safety margin for further translational studies. The absence of significant gross lesions or functional impairments at low and moderate doses suggests that FEONM holds promise as a novel beta-amyloid PET tracer candidate with acceptable risk.(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eThe pattern of toxicity observed at 5.0 mg/kg\u0026mdash;representing a 140-fold higher safety margin than the recommended human dose\u0026mdash;suggests a narrow therapeutic window that requires careful dose optimization. Notably, the clear sex differences in metabolic and toxicological responses suggest that future clinical development should consider sex-stratified dosing strategies, as sex differences in drug distribution can significantly impact safety and efficacy.(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e)While this acute toxicity study provides valuable initial insights into the safety of FEONM, longer-term studies are needed to assess potential delayed or cumulative effects. Dose-escalation studies at intermediate doses between 1.0 and 5.0 mg/kg will help better define no-observed adverse effect levels (NOAELs), while mechanistic studies of the molecular basis of the observed sex differences will further elucidate the toxicological profile. Consistent with previous reports from FDDNP (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), animals at high doses showed decreased activity, loss of appetite, dehydration, hunched backs, and vocalizations, but animals sacrificed as planned showed no clinical signs or macroscopic lesions. To fully support clinical translation, we will continue to conduct additional studies including repeated dose toxicology, radioactive metabolite characterization, and non-human primate dosimetry.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study demonstrated the successful synthesis and preclinical toxicological assessment of FEONM, a novel PET imaging precursor structurally derived from FDDNP. The compound exhibited a favorable safety profile at doses up to 1.0 mg/kg in Sprague-Dawley rats, with no observed mortality or severe adverse effects. While the highest tested dose (5.0 mg/kg) induced mild, dose-dependent hematological, biochemical, and organ-specific changes, these effects were absent at lower doses, establishing 1.0 mg/kg as the no-observed-adverse-effect level (NOAEL). Notably, sex-specific differences in toxicity were observed, with females more susceptible to hematological alterations and males to metabolic disturbances. These findings support the potential of FEONM as a clinically translatable PET imaging agent for Alzheimer\u0026rsquo;s disease, although further long-term toxicity and pharmacokinetic studies are warranted to define its safety margin in humans.\u003c/p\u003e"},{"header":"6. Declarations","content":"\u003cp\u003e\u003cstrong\u003e6.1 Ethics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal housing and experiments were approved by the Ethical Animal Use Committee of the Taipei Medical University and performed in compliance with Taiwan’s laws for the care and use of laboratory animals (LAC-2022-0183).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.2 Consent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.3 Availability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analyzed during the current study are not publicly available due to an ongoing patent application and potential future tech-nology transfer for clinical development. However, the data are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.4 Competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have read and approved the final version of the manuscript and declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.5 Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Atomic Research Institute, ROC [grant numbers 112-3408-007-300].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.6 Authors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWriting—original draft preparation, M.-C.W.; Conceptualization, methodology, and validation, J.-T.C., W.-J.L., and Y.-H.T.; Formal analysis, investigation, and data curation, S.-S.F., and C.-C.H.; Writing—review and editing, supervision, and project administration, K.-W.C..\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.7 Acknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to express their gratitude to \u003cem\u003eChyng-Yann Shiue\u003c/em\u003e for their valuable guidance and great contribution to the structural design of this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.8 Authors' information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMao-Chi Weng, Jenn-Tzong Chen, Yean-Hung Tu, Shiou-Shiow Farn, and Chien-Chung Hsia\u003c/p\u003e\n\u003cp\u003eDepartment of Isotope Application Research, National Atomic Research Institute, Taoyuan 325207, Taiwan\u003c/p\u003e\n\u003cp\u003eWuu-Jyh Lin\u003c/p\u003e\n\u003cp\u003eSeeCURE Taiwan Co., Ltd., Kaohsiung 831134, Taiwan\u003c/p\u003e\n\u003cp\u003eKang-Wei Chang\u003c/p\u003e\n\u003cp\u003eTaipei Neuroscience Institute and Laboratory Animal Center, Taipei Medical University, Taipei 11048, Taiwan\u003c/p\u003e"},{"header":"7. 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Radiological physics and technology. 2022;15(2):116-24.\u003c/li\u003e\n \u003cli\u003eWong DF, Rosenberg PB, Zhou Y, Kumar A, Raymont V, Ravert HT, et al. In vivo imaging of amyloid deposition in Alzheimer disease using the radioligand 18F-AV-45 (florbetapir [corrected] F 18). Journal of nuclear medicine : official publication, Society of Nuclear Medicine. 2010;51(6):913-20.\u003c/li\u003e\n \u003cli\u003eCarome M, Wolfe S. Florbetapir-PET imaging and postmortem beta-amyloid pathology. Jama. 2011;305(18):1857; author reply -8.\u003c/li\u003e\n \u003cli\u003eChang KW, Chen CC, Lee SY, Wang HE. Acute toxicity of two Alzheimer\u0026apos;s disease radiopharmaceuticals: FDDNP and IMPY. Drug and chemical toxicology. 2009;32(4):429-37.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 6 are available in the Supplementary Files section\u003c/p\u003e"},{"header":"Scheme","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"FEONM, PET imaging, Alzheimer's disease, Radiopharmaceutical synthesis, Acute toxicity, FDDNP derivative","lastPublishedDoi":"10.21203/rs.3.rs-7360002/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7360002/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eAlzheimer\u0026rsquo;s disease (AD) remains a major global health challenge, necessitating the development of more effective diagnostic tools. This study focuses on the development and evaluation of FEONM, a novel PET imaging precursor structurally derived from FDDNP, designed for potential use in AD imaging. To assess its suitability for clinical translation, both synthetic feasibility and safety profile were examined. A practical multi-step synthetic route was established using Bucherer and Knoevenagel reactions. Additionally, the acute toxicity of FEONM was evaluated in Sprague-Dawley rats (n\u0026thinsp;=\u0026thinsp;5 per group per sex) following administration at 0.2, 1.0, and 5.0 mg/kg doses. Key toxicological endpoints included body weight monitoring, hematological and clinical chemistry analysis, and gross necropsy findings.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eNo mortality or abnormal clinical signs were observed across all dose groups. Low and moderate doses did not affect normal growth, while 5.0 mg/kg caused temporary weight suppression. Dose-dependent hematological and biochemical effects showed sex-specific differences, with females displaying greater hematological sensitivity including significant lymphocyte reduction (68% vs 30% in males) at the highest dose. Males exhibited notable glucose elevation (22%) and electrolyte disturbances. Gross necropsy revealed hepatic and mild gastrointestinal changes only at 5.0 mg/kg in 20% of animals, indicating a clear threshold effect with no pathological lesions at lower doses.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eFEONM was successfully synthesized and showed acceptable safety margins at clinically relevant doses. The NOAEL was determined to be 1.0 mg/kg. Different toxicity effects were observed between male and female rats, with females being more sensitive to blood-related changes and males showing more metabolic problems. These results indicate that FEONM has potential for clinical use, but additional long-term studies are needed to determine the optimal dosing for safe human application.\u003c/p\u003e","manuscriptTitle":"Synthesis and Acute Toxicity Evaluation of FEONM: A Novel PET Imaging Precursor for Alzheimer's Disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-22 12:05:29","doi":"10.21203/rs.3.rs-7360002/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"29e82783-6325-4b59-9c8d-8123df0350d6","owner":[],"postedDate":"August 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-13T14:27:00+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-22 12:05:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7360002","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7360002","identity":"rs-7360002","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}