# Ultrasound-Assisted Synthesis of Pyrazoline Derivatives as Potential Antagonists of RAGE-Mediated Pathologies: Insights from SAR Studies and Biological Evaluations

Anca-elena Dascălu, Christophe Furman, Steve Lancel, Emmanuelle Lipka, Maxime Liberelle, Eric Boulanger, Alina Ghinet

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Anca-elena Dascălu, Christophe Furman, Steve Lancel, Emmanuelle Lipka, Maxime Liberelle, et al. Ultrasound-Assisted Synthesis of Pyrazoline Derivatives as Potential Antagonists of RAGE-Mediated Pathologies: Insights from SAR Studies and Biological Evaluations. ChemMedChem, 2024, 20 (1), pp.e202400527. 10.1002/cmdc.202400527. hal-04930504

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# Ultrasound-Assisted Synthesis of Pyrazoline Derivatives as Potential Antagonists of RAGE-Mediated Pathologies: Insights from SAR Studies and Biological Evaluations

Anca-Elena Dascălu,*[a, b, c] Christophe Furman,[b, d] Steve Lancel,[b] Emmanuelle Lipka,[b, d] Maxime Liberelle,[d] Eric Boulanger,[b] and Alina Ghinet*[a, b, c]

In the context of age-related disorders, the receptor of advanced glycation end products (RAGE), plays a pivotal role in the pathogenesis of these conditions by triggering downstream signaling pathways associated with chronic inflammation and oxidative stress. Targeting this inflamming phenomenon with RAGE antagonists holds promise for interventions with broad implications in healthy aging and the management of age-related conditions. This study explores the structure-activity relationship (SAR) of pyrazoline-based RAGE antagonists synthesized using an ultrasound-assisted green one-pot two-steps

methodology. Our investigation identifies phenylurenyl-pyrazoline 2g as a promising candidate, demonstrating superior efficiency compared to the reference antagonist Azeliragon (IC₅₀ = 13 µM). Compound 2g exhibits potent inhibition of the AGE2-BSA/sRAGE interaction (IC₅₀ = 22 µM) and favorable affinity in Microscale Thermophoresis (MST) assays (K_d = 17.1 µM), along with a favorable safety profile, with no apparent cytotoxicity observed *in vitro* in the MTS assay. These findings underscore the potential of pyrazoline-derived RAGE antagonists as therapeutic agents for addressing age-related disorders.

## Introduction

The demographic landscape is rapidly shifting, with the World Health Organization projecting a significant increase in the global population aged 60 and over by 2030, reaching 1.4 billion by 2050.[1] This demographic transformation underscores the pressing need for new strategies to address the multitude of health issues that accompany aging. Emerging research highlights the role of inflamming,[2] a chronic, asymptomatic, low-grade inflammatory state associated with aging, linked to immunosenescence, damage-associated molecular patterns (DAMPs), and the senescence associated secretory phenotype (SASP). Inflamming is linked to the activation of receptors like RAGE (Receptor for Advanced Glycation End-products), which is

a multi-ligand cell surface receptor belonging to the immunoglobulin superfamily.[3] The binding of AGEs to RAGE leads to the activation of downstream signaling pathways that sustain chronic inflammation[4] and oxidative stress.[5] These processes are implicated in various age-related disorders, including diabetes mellitus and its vascular complications,[6] neurodegenerative conditions like Alzheimer's[7] and Huntington's diseases,[8] cardiovascular diseases such as atherosclerosis,[9] autoimmune disorders like rheumatoid arthritis,[10] lupus,[11] and chronic airway diseases like asthma[12] and chronic obstructive pulmonary disease (COPD).[13] Consequently, addressing inflamming through RAGE antagonists holds promise for interventions with broad implications for healthy aging and age-related disorders.[14-16]

Our team is continuously searching for new RAGE ligands and especially potential antagonists,[17] and within this work, we investigated pyrazoline-based molecules, a class of heterocyclic compounds known for their versatile formation through various synthetic routes such as 1,3-dipolar cycloaddition reaction, reduction of pyrazoles, acid-catalyzed condensation-cyclization of α,β-unsaturated ketones with hydrazines, microwave-assisted cyclocondensation, and their potential biological activities.[18] Our initial goal was to enhance the efficiency of ureido N-acylhydrazones, originally discovered via dynamic combinatorial chemistry (DCC),[19] by employing an isostere approach. Initial investigations into chalcone replacements proved less fruitful, prompting a natural strategic shift towards pyrazoline-based molecules.

These molecules serve as pivotal scaffolds in various clinically available drugs, demonstrating diverse pharmacological actions (Figure 1). One notable example is Celecoxib,[20] a selective cyclooxygenase-2 (COX-2) inhibitor, prescribed for managing pain and inflammation associated with conditions

[a] A.-E. Dascălu, A. Ghinet
Health and Environment, Laboratory of Sustainable Chemistry and Health,
Junia, F-59000, Lille, France
E-mail: alina.ghinet@junia.com
anca-elena.dascalu@junia.com

[b] A.-E. Dascălu, C. Furman, S. Lancel, E. Lipka, E. Boulanger, A. Ghinet
Univ. Lille, Inserm, CHU Lille, Institut Pasteur de Lille, UMR 1167 - RID-AGE -
Risk Factors and Molecular Determinants of Aging-Related Diseases, F-
59000 Lille, France

[c] A.-E. Dascălu, A. Ghinet
Faculty of Chemistry, 'Alexandru Ioan Cuza' University of Iasi, Bd. Carol I,
Nr. 11, 700506, Iasi, Romania

[d] C. Furman, E. Lipka, M. Liberelle
Univ. Lille, UFR Pharmacie, BP 83, F-59006, Lille, France

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Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202400527

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© 2024 The Authors. ChemMedChem published by Wiley-VCH GmbH. This is
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<figure><img src="image_7.png" /><figcaption>**Figure 1.** Representative marketed molecules bearing a pyrazoline derivative ring, structure of Azeliragon and potential RAGE antagonists developed in this study.</figcaption></figure>

like osteoarthritis and rheumatoid arthritis. Another example is phenylbutazone, a non-selective COX inhibitor commonly used in equine medicine in the US, UK, and Canada.[21] Edaravone,[22] utilized in the treatment of amyotrophic lateral sclerosis, showcases antioxidant properties crucial for neuronal protection.[23] Additionally, ibipinabant, acting as a selective cannabinoid CB1 receptor antagonist, holds promise in managing metabolic disorders.[24] These pyrazoline-derived drugs have demonstrated significant clinical efficiency and continue to be valuable therapeutic options for patients suffering from diverse inflammatory conditions.

Noteworthy, among their features is the resemblance to the imidazole-based Azeliragon scaffold (Figure 1)[14] and other nitrogen heterocycles such as pyrazole or dihydropyrazole. Azeliragon, the benchmark RAGE antagonist developed by vTv Therapeutics, underwent clinical trials but failed in phase III due to insufficient efficiency in patients with mild cognitive impairment.[25,26] This similarity between the imidazole ring of Azeliragon and pyrazoline motif gained our interest. Although pyrazolines may not inherently possess superior metabolic stability compared to other heterocycles,[27] they offer an advantage over N-acylhydrazones previously investigated, which are prone to hydrolysis into an aldehyde and hydrazide under acidic conditions. Therefore, in the context of RAGE, we hoped that this pyrazoline-scaffold will influence its pharmacokinetic characteristics. Meanwhile, the versatility of this scaffold provides an opportunity to design compounds with varying properties, possible high potency, reduced toxicity, and through strategic chemical modifications, pyrazolines can be tailored to exhibit favorable physicochemical properties, enabling them to interact with specific binding sites on the RAGE receptor.

## Results and Discussion

Preliminary docking studies on targeted pyrazolines have demonstrated the ability to form multiple types of interactions with the RAGE receptor, including hydrogen bonding, hydro-

phobic interactions, and π-interactions. This diversity in binding interactions provides a broader range of options for optimizing the affinity and selectivity of RAGE antagonists, leading us to the synthetic variations presented in Scheme 1.

The synthesis of pyrazolines from chalcones was achieved using ultrasounds, highlighting a sustainable and environmentally friendly approach. Ultrasound-assisted synthesis, a green technique utilizing high-frequency sound waves, eliminates conventional heating, significantly reducing energy consumption, reaction times, and solvent usage.[28] In the first synthetic step, chalcone formation relied on a Claisen-Schmidt condensation,[29] specifically developed for synthesizing all phenylurenyl chalcones **1a–k**. This approach aimed to elaborate structure-activity relationships (SARs) concerning substituent variations (Scheme 1). In a previous study, our team demonstrated the superiority of LiOH as an optimal catalyst for the Claisen-Schmidt condensation.[30] Therefore, we chose to use LiOH for its ability to afford the best yields under ultrasounds and obtain intermediary chalcones. Building on this finding and considering that sodium methoxide and potassium carbonate led to lower yields under the same reaction conditions (Table S1, ESI), we maintained our choice of LiOH for the synthesis of phenylurenyl chalcone intermediates. In our study, we pursued a one-pot two-step synthesis to efficiently produce pyrazolines **2–3** while adhering to green chemistry principles. Unlike conventional methods, we opted not to isolate the intermediate chalcones, only a sample being taken for characterization purposes.[31] Instead, hydrazine was added directly after the completion of the chalcone formation reaction which was monitored by ¹H NMR, which then undergoes a 1,4-addition followed by rearrangement and cyclization to afford the pyrazoline scaffold.[32,33] This decision was primarily motivated by the lower interest in chalcones due to their reduced inhibition of the RAGE receptor, a crucial factor in our research focus. Also, by directly adding hydrazine after chalcone formation, we minimized the intermediate loss, maximized recovery yields of the final pyrazoline products, and minimized waste generation, contributing to a more sustainable process. Our investigation into different substitutions in the positions 3 and 5 of the pyrazoline scaffold allowed us to systematically explore the structure-activity relationship (SAR), resulting in the identification of promising derivatives with enhanced biological activities. We also employed several strategies to introduce chemical diversity at the free NH in the position 1 of the pyrazoline scaffold: again, as a one-pot two-steps reaction approach, we successfully synthesized N-substituted pyrazolines like **3a** and **3b** by adding pentanoic acid in the reaction medium, along with hydrazine. This strategy facilitated the formation of pentanoyl-substituted pyrazolines.

Another modification involved the reaction of synthesized pyrazolines **2** with selected isocyanates, yielding urea molecules such as **4** and **5** with improved binding potential according to preliminary molecular docking studies, attributed to the presence of additional interactions, including alkyl and π-alkyl interactions, as well as additional hydrogen bonds with key amino acids GLN1047 and VAL1078 (Table 1). We further explored the incorporation of a different scaffold, the cyclohex-
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<figure><img src="image_9.png" /><figcaption>**Scheme 1.** Synthesis of pyrazolines 2–5, cyclohex-2-en-1-one 6 and pyrrole 7 compounds.</figcaption></figure>

2-en-1-one, leading to the synthesis of molecule 6, which showed promising preliminary docking results. Molecule 6 was synthesized via a Robinson annulation[34] using an ultrasound-assisted one-pot, two-step approach. In this synthesis, KOH was used as the base, as LiOH yielded poor results in the second step. As for pyrazoline synthesis, chalcone formation was achieved through a Claisen-Schmidt condensation, affording the chalcone as the α,β-unsaturated carbonyl compound, after ethyl acetoacetate was directly added to the reaction mixture, facilitating the Robinson annulation. The reaction conditions promoted ethyl ester saponification in situ and, consequently, compound 6 was isolated as a potassium carboxylate (Scheme 1). Compared to the formation of pyrazolines, which were obtained in 64–82% yields (Table S1, ESI), molecule 6 was obtained with a moderate yield of 48% after 25 min under ultrasounds. Additionally, aiming to introduce the acylpyrrole

motif, we reacted the chalcone **1k** with toluenesulfonylmethyl isocyanide (TosMIC) in DMSO at 90 °C,[35] and obtained pyrrole derivative **7**. While this reaction initially seemed promising due to its simplicity, our investigation was restricted to a single example because the synthesized molecule **7** exhibited low solubility, limiting its practical application. Subsequent computational analyses via docking studies (Figure S17, ESI) revealed limited binding interactions with the target receptor, further tempering our expectations. Aiming to verify whether the synthesis of pyrazolines follows green chemistry principles,[36] we chose derivative **2g**, to calculate the green metrics. Analyzing its synthesis revealed an Atom Economy (AE) of 87.79%, signifying efficient atom utilization. Carbon Efficiency (CE) reached 100%, demonstrating optimal carbon utilization, while Reaction Mass Efficiency (RME) approached 64.5%, indicating efficient conversion of reactants. The Environmental
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<table><caption>Table 1. The molecular docking studies of the selected compounds in the active site of the V domain of RAGE.</caption><thead><tr><th rowspan="2">Entry</th><th rowspan="2">Compound</th><th rowspan="2">Docking score (kcal/mol)</th><th colspan="2">Interactions</th></tr><tr><th>H-bonds</th><th>Non-covalent interactions</th></tr></thead><tbody><tr><td>1</td><td><strong>Azeliragon</strong></td><td>-6.9</td><td>LEU1049</td><td>Attractive charge (GLU1050), π-alkyl (LYS1044, PRO1045, PRO1066, VAL1078)</td></tr><tr><td>2</td><td><strong>2a</strong></td><td>-8.4</td><td>GLN1047, LEU1049, LEU1079</td><td>π-alkyl (PRO1080, PRO1066), Van der Waals (PRO1045, GLU1050, TRP1051, VAL1063, LEU1064, GLN1067, SER1065)</td></tr><tr><td>3</td><td><strong>2c</strong></td><td>-8.0</td><td>LEU1049</td><td>π-alkyl (VAL1078, PRO1066, PRO1045, PRO1080), Van der Waals (GLN1047, ARG1048, GLU1050, TRP1051, LEU1064, SER1065, GLY1082)</td></tr><tr><td>4</td><td><strong>1g</strong></td><td>-8.4</td><td>LEU1049, LEU1069, GLN1067, VAL1078</td><td>Amide-π-staking (SER1065), π-alkyl (PRO1080), π-anion (GLU1050), Van der Waals (PRO1045, GLN1047, ARG1048, TRP1051, VAL1063, LEU1079)</td></tr><tr><td>5</td><td><strong>2g</strong></td><td>-9.2</td><td>GLN1047, LEU1049, LEU1079</td><td>Amide-π-staking (SER1065), π-anion (GLU1050), π-alkyl (PRO1045, LYS1044, PRO1066, VAL1078), Van der Waals (ARG1048, TRP1051, VAL1063, LEU1079, PRO1080)</td></tr><tr><td>6</td><td><strong>2h</strong></td><td>-8.4</td><td>GLN1047, LEU1079</td><td>π-alkyl (PRO1045, PRO1066, PRO1080), Van der Waals (ARG1048, TRP1051, LEU1064, SER1065, GLN1047, GLU1050, VAL1078, GLY1082)</td></tr><tr><td>7</td><td><strong>2j</strong></td><td>-9.0</td><td>GLN1047, ARG1048</td><td>halogen (LEU1049), π-alkyl (ARG1104, PRO1042, PRO1045, PRO1046, PRO1066, VAL1078), Van der Waals (LEU1079, PRO1080, GLY1082, MET1102, ASN1103)</td></tr><tr><td>8</td><td><strong>3a</strong></td><td>-8.0</td><td>VAL1078</td><td>π-alkyl (PRO1045, VAL1063, VAL1068, PRO1066), Van der Waals (GLN1047, LEU1049, GLU1050, TRP1051, LEU1064, SER1065, GLN1067, LEU1079, PRO1080)</td></tr><tr><td>9</td><td><strong>3c</strong></td><td>-7.4</td><td>LEU1049</td><td>π-alkyl (PRO1066, VAL1078, PRO1080), Van der Waals (PRO1045, GLU1050, TRP1051, VAL1063, LEU1064, SER1065, GLN1067)</td></tr><tr><td>10</td><td><strong>4a</strong></td><td>-7.3</td><td>GLN1047, VAL1078</td><td>π-alkyl (PRO1066, PRO1080, VAL1078), Van der Waals (LEU1049, LEU1064, SER1065, PRO1046, PRO1045, LEU1079, GLN1067)</td></tr><tr><td>11</td><td><strong>4c</strong></td><td>-7.1</td><td>LEU1049, LEU1064, TRP1051</td><td>Amide-π-staking (SER1065), π-alkyl (PRO1045, PRO1066, PRO1080, VAL1078), Van der Waals (PRO1046, GLN1047, GLU1050, SER1065, GLN1067, VAL1063)</td></tr><tr><td>12</td><td><strong>5a</strong></td><td>-8.9</td><td>VAL1078</td><td>π-alkyl (VAL1063), Amide-π-staking (SER1065, PRO1066), Van der Waals (PRO1045, GLN1048, LEU1049, GLU1050, TRP1051, LEU1064, SER1065, LEU1079, PRO1080, PRO1086)</td></tr><tr><td>13</td><td><strong>5b</strong></td><td>-9.1</td><td>GLN1047, GLN1067</td><td>π-alkyl (VAL1063, VAL1078, PRO1045, PRO1046, PRO1066), Van der Waals (LEU1049, GLU1050, TRP1051, LEU1064, SER1065, LEU1079, PRO1080)</td></tr><tr><td>14</td><td><strong>6</strong></td><td>-8.6</td><td>GLN1047</td><td>Amide-π-staking (SER1065), π-alkyl (LYS1044, PRO1045, LEU1049, PRO1066, PRO1080, VAL1078), Van der Waals (GLU1050, GLN1067, LEU1079, LEU1064, GLY1082)</td></tr></tbody></table>

Factor (E-Factor) of approximately 0.55 when the solvent is recovered at the end of the reaction and 31.5 if the solvent becomes waste, respectively. This showcased minimal waste generation relative to product mass when recovering the solvent, highlighting the effectiveness of our synthesis method (Table S2, ESI). These findings underscore the sustainability and efficiency of our ultrasound-assisted stepwise synthesis approach in producing pyrazoline compounds, exemplified by compound **2g**.

Several compounds demonstrated highly favorable docking scores, indicating strong binding affinity to the RAGE receptor. The initial choice of the chalcone bridge as the bioisosteric replacement for the N-acylhydrazone (NAH) link of the HIT molecule previously found by DCC,[18] revealed limitations during the docking analysis, exhibiting less favorable interactions with the target, compared to NAHs. Therefore, we were inclined towards pyrazoline as the preferred alternative due to its superior interaction profile observed during the docking analysis. For instance, compounds **2a**, **2g**, **2h**, **2j**, and **5a** exhibited notably high docking scores between -8.4 and -9.2 kcal/mol, respectively, suggesting robust binding interactions with the target protein (Table 1). Azeliragon, with its established role as a RAGE antagonist, sets the benchmark for our comparative analysis. Among the docked pyrazolines, we

identified **2g** having an enhanced binding affinity, firstly reflected by the docking score of -9.2 kcal/mol, indicating a stronger interaction with the RAGE receptor compared to Azeliragon, for which we found -6.9 kcal/mol. Moreover, in our comparative analysis, we observed common interactions between Azeliragon and compound **2g**, suggesting similar binding patterns within the RAGE binding pocket (Figure 2). Both compounds engaged in hydrogen bonding interactions with GLN1047 and LEU1049, indicating a shared binding interface. Additionally, they exhibited π-alkyl interactions with PRO1045 and PRO1066, suggesting a conserved binding mode targeting specific amino acid residues. These shared interactions imply that Azeliragon and compound **2g** likely occupy overlapping regions within the binding pocket. Furthermore, the presence of the attractive charge interaction with GLU1050, observed exclusively in the cases of Azeliragon and **2g**, emphasizes a distinct interaction characteristic to these ligands within the binding pocket of the RAGE receptor. Given that both Azeliragon and **2g** were found antagonists of the AGE2-BSA/sRAGE interaction in our ELISA assays, the π-anion interaction with GLU1050 plays an efficient role in their inhibition actions. Crosslinking our docking analyses and MST ligand binding results, we have identified critical amino acid residues within the V-domain pocket of RAGE. Notably, LEU1049, GLN1047,
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<figure><img src="image_12.png" /><figcaption>**Figure 2.** Molecular docking analysis depicting the binding modes and interactions of Azeliragon and pyrazoline 2g with the target protein RAGE.</figcaption></figure>

LEU1079, VAL1078, and potentially ARG1048 and TRP1051 are involved in various types of interactions, strengthening the anchoring of the ligand within the binding pocket, thereby enhancing its overall binding affinity. This pocket may play a crucial role in RAGE inhibition, potentially disrupting signaling pathways and could help to identify possible antagonists. Several pyrazoline derivatives, including **2c**, **2g**, **2i**, **4a**, **4c**, and **5a**, showed superior binding affinities compared to Azeliragon in the Microscale Thermophoresis (MST) experiments. Confirming our docking predictions, pyrazolines outperformed their chalcone precursors, respectively corresponding N-acylhydrazones, proving the effectiveness of the pyrazoline ring as a bioisosteric replacement. Chalcone **1g** displayed decreased affinity ($K_d$=90.2 µM) compared to compound **2g** ($K_d$=17.1 µM), where the introduction of the pyrazoline scaffold improved the RAGE affinity through enhanced structural complementarity and additional binding interactions (Table 2, entries 4 and 5). In comparison to **2g**, the difluorinated compound **2i**, presents a $K_d$ of 10.7 µM (Table 2, entry 6). As demonstrated from its docking, this difference may originate from its distinct binding orientations and interaction profiles within the RAGE receptor binding site, elucidated in the docking analysis (Figure S9, ESI). Regarding **2a** and **2c**, we can observe a variance in affinity we assume to be due to the positioning of the phenyl-urea moiety on the pyrazoline scaffold. Even slight alterations in this placement can significantly impact the binding affinity to RAGE, highlighting the significance of precise structural adjustments in the development of pyrazoline derivatives as RAGE antagonists. This is

demonstrated by the shift in binding affinity from 59.3 µM for derivative **2a** to 15.1 µM for derivative **2c** (Table 2, entries 2 and 3). Among the molecules containing a single urea fragment, pyrazolines **2a**–**f**, chlorophenyl substituted demonstrates good affinity for RAGE, presenting a $K_d$ value of 15.1 µM. Surprisingly, substituting the NH-group with a pentanoyl chain in compound **3c** (Table 2, entry 7), similar to the butyl group in Azeliragon, intended to enhance solvent accessibility and interactions, conserved the affinity to RAGE, resulting in a $K_d$ of 22.6 µM. Regarding compounds **4a** and **4c**, we observed a consistency in activity despite the placement of phenyl-urea fragment on the pyrazoline scaffold (Table 2, Entries 8 and 9).

<table><caption>**Table 2.** Microscale thermophoresis screening results.</caption><thead><tr><td>Entry</td><td>Compound</td><td>MST K<sub>d</sub>±SD (µM)</td></tr></thead><tbody><tr><td>1</td><td>1g</td><td>90.2 ± 7.4</td></tr><tr><td>2</td><td>2a</td><td>59.3 ± 7.6</td></tr><tr><td>3</td><td>2c</td><td>15.1 ± 2.4</td></tr><tr><td>4</td><td>2g</td><td>17.1 ± 2.0</td></tr><tr><td>5</td><td>2h</td><td>40.3 ± 5.1</td></tr><tr><td>6</td><td>2i</td><td>10.7 ± 4.0</td></tr><tr><td>7</td><td>3c</td><td>22.6 ± 1.1</td></tr><tr><td>8</td><td>4a</td><td>13.8 ± 3.1</td></tr><tr><td>9</td><td>4c</td><td>13.0 ± 3.2</td></tr><tr><td>10</td><td>5a</td><td>10.5 ± 1.7</td></tr><tr><td>11</td><td>Azeliragon</td><td>45.0 ± 3.0</td></tr></tbody></table>
This suggests a remarkable tolerance within the RAGE receptor binding site. This could imply that the introduced urea in the position 5 of the pyrazoline scaffold, along with the attached alkyl chain, maintains similar steric and chemical properties. As a result, crucial binding interactions within the receptor binding site are preserved despite the substitution, as seen in docking result as well (Figure S11 and S12, ESI). **5a** exhibits the highest affinity for the RAGE receptor among the tested compounds (Table 2, entry 10), with its low K<sub>d</sub> value correlating with a favorable docking score (Table 1, entry 12). The slightly higher affinity of **5a** compared to **4a**, can be due to its phenyl urea substitution, that enhanced binding affinity through additional hydrophobic interactions, increased structural rigidity, and potentially greater specificity for the receptor binding site.

Compared to the MST assay, where we evaluate the binding affinity of compounds to the RAGE receptor, without considering the presence of AGE2-BSA (glyceraldehyde-modified AGE), RAGE ligand, in the ELISA inhibition assay we specifically examine the effect of experimental drugs on the inhibition of the AGE2-BSA/sRAGE (soluble RAGE) interaction, providing insights into the compound's ability to disrupt this specific protein-ligand interaction. Compound **2a** features a urea group adjacent to the position 5 of the pyrazoline ring, while compound **2c** incorporates a urea group adjacent to the position 3 of the pyrazoline. This difference in the positioning of the urea group could lead to variations in the spatial orientation and electronic properties of the molecules, affecting their inhibition of the AGE2-BSA/sRAGE interaction. The discrepancy between MST K<sub>d</sub> values and ELISA inhibition percentages for these two compounds exemplifies the complexity of molecular interactions involved in modulating inhibitory activity against this interaction. However, several compounds demonstrate significant inhibitory activity against the AGE2-BSA/sRAGE interaction, as evidenced by substantial reductions in inhibition percentages. A recurring trend suggests that the inclusion of a rigid pyrazoline scaffold is important for achieving optimal inhibitory activity against RAGE. For instance, chalcone derivative **1g** (Table 3, entry 1) shows moderate inhibitory effects, with an ELISA inhibition of 40%, compared to the corresponding pyrazoline **2g** (94% inhibition), at 100 µM concentration. Pyrazolines bearing a bis phenyl urea substitution, such as compounds **2g**, **2h**, **2i**, and **2j** consistently exhibit potent inhibitory activity against the sRAGE/AGE2-BSA interaction (Table 3, entries 9–12). For instance, compound **2h** demonstrates a high inhibition of 89%, indicative of its efficiency in disrupting the studied interaction. Compounds incorporating alkyl chains or linkers, exhibit intriguing trends in inhibitory activity against the AGE2-BSA/sRAGE interaction. Despite the structural modification, compound **3a** induces a comparable inhibitory potency, with a 30% inhibition, compared to 35% for **2a**. This suggests that the introduction of the carboxyalkyl chain in **3a** does not significantly impact its inhibitory activity. Considering other structural aspects, when compared, the difference between compounds **4a** and **4b** lies in the substitution of chlorine in **4a** and fluorine in **4b**. Compound **4a**, exhibits robust inhibitory potency, having an IC<sub>50</sub> of 25 µM (Table 3, entry 15). Conversely, compound **4b**, fluorine substi-

tuted, shows moderate inhibition of 34%, at 100 µM. This highlights that the nature of halogen substitution can have an important impact on the inhibitory efficiency. Comparatively to pyrazolines, different scaffold containing molecules such as **6**, exhibited a more promising inhibition of 55% and a slightly better binding energy of -8.7 kcal/mol. The enhanced inhibitory activity and binding affinity of molecule **6** can be attributed to its different scaffold, featuring a six-membered ring, the cyclohex-2-en-1-one, which may confer favorable spatial orientation and electronic properties for disrupting the AGE2-BSA/sRAGE interaction. Additionally, from ELISA assays, we observed that molecule **7**, containing the pyrrole motif, exhibited little to no activity, as seen from the preliminary docking studies as well. After the selection of the best inhibitors at 100 µM in ELISA assays, we further proceeded with IC<sub>50</sub> determinations, in triplicate. Among these compounds, **2g**, characterized by its bis-phenyl urea structure, emerged as the most potent inhibitor with an IC<sub>50</sub> of 22 µM.

It was followed by **4a** with an IC<sub>50</sub> of 25 µM, **2i** with an IC<sub>50</sub> of 33 µM and **2a** with an IC<sub>50</sub> of 72 µM. Notably, **2g** demonstrates comparable or slightly superior inhibitory potency against the sRAGE/AGE2-BSA interaction compared to Azeliragon, underscoring the effectiveness of its binding mechanism. This observation aligns with the enhanced binding

<table><caption>Table 3. ELISA AGE2-BSA/sRAGE inhibitory screening results.</caption><thead><tr><th>Entry</th><th>Compound</th><th>ELISA inhibition % at 100 µM</th><th>IC<sub>50</sub> (AGE2-BSA/sRAGE) (µM)</th></tr></thead><tbody><tr><td>1</td><td>1g</td><td>40</td><td>N.D.</td></tr><tr><td>2</td><td>1a</td><td>15</td><td>N.D.</td></tr><tr><td>3</td><td>2a</td><td>81</td><td>72</td></tr><tr><td>4</td><td>2b</td><td>35</td><td>N.D.</td></tr><tr><td>5</td><td>2c</td><td>41</td><td>N.D.</td></tr><tr><td>6</td><td>2d</td><td>18</td><td>N.D.</td></tr><tr><td>7</td><td>2e</td><td>1</td><td>N.D.</td></tr><tr><td>8</td><td>2f</td><td>12</td><td>N.D.</td></tr><tr><td>9</td><td>2g</td><td>94</td><td>22</td></tr><tr><td>10</td><td>2h</td><td>89</td><td>&gt; 100</td></tr><tr><td>11</td><td>2i</td><td>91</td><td>33</td></tr><tr><td>12</td><td>2j</td><td>90</td><td>&gt; 100</td></tr><tr><td>13</td><td>3a</td><td>30</td><td>N.D.</td></tr><tr><td>14</td><td>3c</td><td>41</td><td>N.D.</td></tr><tr><td>15</td><td>4a</td><td>89</td><td>25</td></tr><tr><td>16</td><td>4b</td><td>34</td><td>N.D.</td></tr><tr><td>17</td><td>4c</td><td>19</td><td>N.D.</td></tr><tr><td>18</td><td>4d</td><td>32</td><td>N.D.</td></tr><tr><td>19</td><td>5a</td><td>38</td><td>N.D.</td></tr><tr><td>20</td><td>5b</td><td>32</td><td>N.D.</td></tr><tr><td>21</td><td>5c</td><td>34</td><td>N.D.</td></tr><tr><td>22</td><td>6</td><td>55</td><td>N.D.</td></tr><tr><td>23</td><td>7</td><td>5</td><td>N.D.</td></tr><tr><td>24</td><td>Azeliragon</td><td>93</td><td>13</td></tr></tbody></table>
<figure><img src="image_13.png" /></figure>

affinity seen in MST and docking studies, highlighting the potential of 2g as a lead compound for further development.

In our investigation, the cytotoxicity of the synthesized and reference compound Azeliragon, were verified via an MTS assay using the HEK293 cell line (Figure 3). Notably, the experimental results revealed no discernible toxicity for the pyrazoline compounds, experimentally, presenting a safe profile on the HEK293 cell line.

However, the reference compound Azeliragon, tested at 50 µM, exhibited cytotoxicity, resulting in only 20% of cell viability. These findings underline a lower cytotoxicity profile for the pyrazolines when compared to Azeliragon, highlighting their potential as safer alternatives in drug development. While experimental testing provides valuable insights into the cytotoxicity of compounds, it is often not feasible to cover all potential toxicity grounds due to resource constraints and ethical considerations for the in vivo evaluation. As a result, using computer predictions alongside experiments is crucial for understanding how compounds might be toxic.[37] In our study, we integrated in silico toxicity assessment using ProTox 3.0 online software[38] to complement the experimental results, which provided valuable insights into their safety profile.

Pyrazoline derivatives **2a–j** (Table 4, Entries 3–7) indicated an absence of mutagenicity, while the starting chalcones, **1g** and **1i**, could potentially present immunotoxicity (Table 4, Entries 1 and 2). Furthermore, the absence of toxicity towards Nrf2-ARE and GABAR, crucial pathways for cellular stress response and neurological function, respectively, was highlighted through in silico predictions. These findings, coupled with the experimental results, provide a comprehensive understanding of the potential toxicities associated with the pyrazoline compounds, despite not covering all experimental testing grounds.

## Conclusions

Our investigation rigorously explored the SAR of pyrazoline-based RAGE antagonists synthesized via an ultrasound-assisted green one-pot two-steps methodology. While NH-substituted pyrazoline **5a** displayed a notable affinity in MST ($K_d$ = 10.5 µM), bis-phenyl urea derivative **2g** emerged as the most potent inhibitor of the AGE2-BSA/sRAGE interaction, exhibiting an $IC_{50}$ of 22 µM in ELISA assays. This activity, coupled with the very good affinity found in MST of 17.1 µM, supported by the

<figure><img src="image_14.png" /><figcaption>Figure 3. MTS HEK-293 viability screening results after treatment with experimental pyrazoline derivatives **2a**, **2c**, **2g**, **2i**, **4a**, **4c**, **5a** and reference Azeliragon.</figcaption></figure>

<table><caption>Table 4. Predicted toxicity results.</caption><thead><tr><td>Entry</td><td>Compound</td><td>Hepatotoxicity</td><td>Carcinogenicity</td><td>Immunotoxicity</td><td>Mutagenicity</td><td>Cytotoxicity</td></tr></thead><tbody><tr><td>1</td><td>1g</td><td>+</td><td>—</td><td>+</td><td>—</td><td>—</td></tr><tr><td>2</td><td>1i</td><td>+</td><td>—</td><td>+</td><td>—</td><td>—</td></tr><tr><td>3</td><td>2a</td><td>+</td><td>+</td><td>—</td><td>—</td><td>—</td></tr><tr><td>4</td><td>2c</td><td>+</td><td>+</td><td>—</td><td>—</td><td>—</td></tr><tr><td>5</td><td>2g</td><td>+</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>6</td><td>2i</td><td>+</td><td>+</td><td>—</td><td>—</td><td>—</td></tr><tr><td>7</td><td>2j</td><td>+</td><td>+</td><td>—</td><td>—</td><td>—</td></tr><tr><td>8</td><td>3c</td><td>—</td><td>+</td><td>—</td><td>—</td><td>—</td></tr><tr><td>9</td><td>4a</td><td>+</td><td>+</td><td>—</td><td>—</td><td>—</td></tr><tr><td>10</td><td>5a</td><td>+</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>11</td><td>Azeliragon</td><td>—</td><td>—</td><td>+</td><td>—</td><td>—</td></tr></tbody></table>
<figure><img src="image_15.png" /></figure>

docking studies (–9.2 kcal/mol), highlights 2g's superior efficiency compared to the reference RAGE antagonist Azeliragon. Moreover, 2g demonstrated a favorable safety profile, evidenced by the absence of cytotoxicity. This is an asset for an anti-inflammating drug, providing a more comfortable therapeutic window. Comparing the ADME parameters predictions,[39] 2g exhibits several promising features and is a promising starting point for further investigation of more potent RAGE antagonists (Table S3, ESI). One notable aspect is its moderate lipophilicity, as indicated by its Log Po/w values, which suggests a potential for favorable absorption and distribution characteristics. These pharmacokinetic parameters will be evaluated in due future. Unlike Azeliragon, molecule 2g adheres to Veber[40] and Egan's rules[41] for drug-likeness, possesses favorable lipophilicity, and shares an equivalent bioavailability score with the reference compound, highlighting its potential as a promising therapeutic candidate. This needs further experimental validation. Overall findings underscore the significance of rational design and systematic evaluation in drug discovery, positioning 2g as a promising lead compound, while underlining the potential of pyrazoline derivatives as effective inhibitors in targeting RAGE-mediated pathologies. With few drugs available to address the challenge of controlling physiological and accelerated inflammingaging during chronic disease, each step forward in this direction requires careful consideration and validation. Therefore, ongoing studies aim to build upon existing research and explore alternative molecular pathways.

## Supporting Information

The Supporting Information associated with this article (¹H and ¹³C NMR spectra of newly synthesized compounds, HPLC chromatograms of pure compounds, material and methods for both chemical synthesis and biological evaluation, green metrics calculation for the synthesis of compound 2g, molecular docking and ADME predictions) is available free of charge on the publication website. The authors have cited additional references within the Supporting Information.[42-47]

## Acknowledgements

The authors thank the Agence Nationale de la Recherche (ANR), France, for financial support of this work (grant number ANR-23-CE14-0063 (project POSSAR: Preserve Our Strength when Sepsis Ages us: Let's block our RAGE)).

## Conflict of Interests

The authors declare no conflict of interest.

## Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

**Keywords:** Inflammaging • RAGE • Antagonist • Phenylurenyl Pyrazoline • Sonication • Green Synthesis

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Manuscript received: July 11, 2024

Revised manuscript received: September 17, 2024

Accepted manuscript online: September 17, 2024

Version of record online: November 5, 2024