Exploratory scaffold hopping strategies led to identification of novel histamine H 3 and H 4 receptor ligands

1 Introduction

Scaffold hopping is a widely used strategy for designing structurally novel compounds from known chemical scaffolds, leading to novel chemotypes with the desired activity (Sun et al., 2012). Although the concept involved in scaffold hopping is not new (it was introduced by Schneider et al., in 1999), this strategy has been applied from the beginning of the drug discovery era until current days (Schneider et al., 1999). The key concept consists of hopping from existing bioactive molecules to an uncovered chemical space with distinct physicochemical and pharmacokinetic properties, while retaining the molecular features necessary to interact with the biological target, which means that key substituents involved in binding to the target must be retained to preserve the biological activity (Langdon et al., 2010). Although several methods have been applied in scaffold hopping strategies, Sun et al. categorized them into four major types–heterocycle replacement (Callis et al., 2022), ring opening/closure (Lamberth, 2023), peptidomimetics and topology-based hopping (Sun et al., 2012) – from the smaller hops to the more sophisticated. Although there are several computational approaches to suggest structural hops (Böhm et al., 2004), the knowledge about the key structural features contained in the prototypes is essential for these strategies to succeed.

In this context, it is interesting to note that classical antihistamines arose from scaffold hopping strategies such as bioisosteric replacements and modifications on molecular flexibility. This generated different antihistamines with unique properties, affording them different clinical applications besides their similar antihistamine activity (Tiligada and Ennis, 2020; Fernandes, 2022). Bioisosteric replacements and scaffold hops from ethanolamines (e.g., diphenhydramine) to phenothiazines (e.g., promethazine) led to drugs with distinct physicochemical and pharmacokinetic properties such as loratadine and other non-sedating antihistamines, or antipsychotics such as clozapine with antihistamine activity (Figure 1) (Humbert-Claude et al., 2012). This enabled the characterization of four known types of histamine receptors (H₁R-H₄R), and turned them into attractive targets for discovering drugs with different clinical applications (Tiligada and Ennis, 2020).

Histamine has nanomolar affinities for both H₃R and H₄R, denoting its importance as chemical mediator at these receptors in low concentrations (Tiligada and Ennis, 2020). The H₃R and H₄R share the highest structural similarity among histamine receptors, presenting two key conserved amino acids on their structures (Asp3.32 and Glu5.46) responsible for the recognition of the basic amine and imidazole moieties of histamine, respectively (Peng et al., 2022; Xia et al., 2024). The H₃R is the most attractive histamine receptor as drug target for CNS disorders, since it is expressed as an auto- and heteroreceptor in neurons (Panula and Nuutinen, 2013; Nieto-Alamilla et al., 2016). Consequently, the activation of H₃R by histamine regulates the production and release of histamine itself as well as other neurotransmitters. Antagonists or inverse agonists of H₃R (e.g., ABT-239, clobenpropit and its non-imidazole analog FUB 661, Figure 2) may increase the levels of these substances, making these compounds potential drug candidates for narcolepsy, Alzheimer’s and Parkinson’s diseases, drug addiction treatment, obesity and other neuropsychological disorders (Liedtke et al., 2003; Cowart et al., 2004). On the other hand, H₄R is mainly expressed in immune cells and is related to the immune-inflammatory effects of histamine (Correa and Fernandes, 2018). Therefore, H₄R antagonists/inverse agonists (e.g., JNJ-7777120, Figure 2) have noteworthy potential as anti-inflammatory drugs (Venable et al., 2005). Both H₃R and H₄R are expressed in glial cells, also enabling the potential application of H₃R/H₄R ligands (e.g., clobenpropit) in the treatment of neuroinflammatory diseases.

Ligands of these receptors usually share common structural frameworks, comprising an aromatic group and a basic moiety, separated by a variable linker group. In general, H₃R ligands present a more flexible structure due to a longer linker in between (e.g., the LINS05 compounds, clobenpropit and FUB 661) (Aranha et al., 2023; Lopes et al., 2026). Regarding H₄R ligands, the most potent compounds are frequently more rigid and compact structures (Correa and Fernandes, 2018). JNJ-7777120 is a well-studied H₄R antagonist/inverse agonist (pK _i 8.3) with high selectivity over H₃R (Venable et al., 2005). In the last years, our group reported the dihydrobenzofuran analog LINS01007, which is structurally related to JNJ-7777120. Despite its lower affinity for H₄R, this compound presents comparable affinities to both H₃R and H₄R (pK _i 6.1) (Corrêa et al., 2017a). The benzofuran analogues of JNJ-7777120 (Figure 2) possess considerable affinity for H₄R but low affinity for H₃R, reinforcing the role of the indole NH and the carbonyl group in determining the affinities for such receptors (Engelhardt et al., 2012; Corrêa et al., 2021). On the other hand, urea derivatives were designed to keep the hydrogen-bond donor in the structure (NH), but they have shown low affinity to H₄R (Latacz et al., 2016; Domínguez-Álvarez et al., 2019), while compounds containing amide linkers remain less explored. Considering this SAR background, in this work we designed two sets of compounds as H₃R/H₄R ligands using homologation, ring opening and bioisosteric replacements as structural hops, aiming to explore novel chemical spaces in the search for novel ligands. The first set presents similarities to more flexible compounds (LINS05 series), while the second set was designed from the more compact structure of the LINS01 chemotype.

2 Results and discussion

2.1 Synthesis of the compounds

The compounds were synthesized as depicted in Schemes 1, 2. Anilides 1–7 were synthesized from the corresponding anilines by reacting them with the appropriate ω-chlorocarboxylic acid, using equimolar amounts of EDC and HOBt (Scheme 1), following a previously described protocol (Varela et al., 2023). The ω-chlorinated products (A1-A4) were substituted with the corresponding piperazines to give the final desired products. Benzylamides 8 and 10 were prepared from the N-benzyl-3-chloropropanamide intermediate B by a similar synthetic protocol. The urea derivative 9 was prepared by reacting benzyl isocyanate with 2-chloro-1-propylamine with excellent yield. The obtained 2-chloroethylurea intermediate C was then substituted with 1-phenylpiperazine to give the final compound with good yield (Scheme 1).

The benzofuran derivatives were prepared using the same synthetic protocol previously described (Corrêa et al., 2016; 2017b; 2021). The 2,3-dihydrobenzofurans (11, 12 and 15) were prepared by reacting the substituted 2-iodo-2,3-dihydrobenzofuran intermediate with the corresponding amines in moderate to good yields (Scheme 2). To prepare the compounds 13 and 16, benzofuran-2-carbaldehyde was reacted with the appropriate amines using a reductive amination protocol with STAB (Varela et al., 2019). Finally, the amides were prepared from the aminolysis of benzofuran-2-carbonyl chloride in high yields (Varela et al., 2023).

2.2 Binding affinities of the compounds at H₃R and H₄R

The summary of the results from the binding assays at H₃R and H₄R are presented in Tables 1, 2. The results showed that only two compounds (13 and 14) presented reasonable affinity for H₄R, while nearly all compounds (except 5) displayed preference toward H₃R, with affinity values lower than 10 µM (pK _i > 5.0). This indicates that the scaffold modifications generated compounds with higher affinity for the H₃R, displaying considerable selectivity over the H₄R (>10-fold). Among the compounds from LINS05 series (Table 1), compound 7 displayed submicromolar affinity for H₃R, (pK _i 6.27), while most of the compounds from LINS01 series (Table 2) showed nanomolar affinities for H₃R, with exception of compound 17 (pK _i 5.11).

TABLE 1. Results of binding assays at histamine H₃R and H₄R for the LINS05 series. ^a

Competition binding assay at HEK293-SP-FLAG-hH₃R, or HEK293-SP-FLAG-hH₄R, cells.

Displacement of 4 nM [3H]UR-PI294 (K _d = 4.0 nM) (Igel et al., 2009).

Displacement of 4 nM [3H]UR-PI294 (K _d = 3.6 nM) (Igel et al., 2009). Data shown are mean values ±SEM, of at least three independent experiments, each performed in triplicate. Data were analyzed by nonlinear regression and were best fitted to sigmoidal concentration-response curves.

TABLE 2. Results of binding assays at histamine H₃R and H₄R for the LINS01 series. ^a

Competition binding assay at HEK293-SP-FLAG-hH₃R, or HEK293-SP-FLAG-hH₄R, cells.

Displacement of 4 nM [3H]UR-PI294 (K _d = 4.0 nM) (Igel et al., 2009).

Displacement of 4 nM [3H]UR-PI294 (K _d = 3.6 nM) (Igel et al., 2009). Data shown are mean values ±SEM, of at least three independent experiments, each performed in triplicate. Data were analyzed by nonlinear regression and were best fitted to sigmoidal concentration-response curves.

Efficiency values for H₄R affinity.

The structural modifications performed on LINS05 chemotype led to interesting changes in the binding affinity profile of the compounds. Bioisosteric replacement of the piperazine with a homopiperazine led to equipotent compound 2, although the ligand efficiency (LE) of this compound is lower in comparison to the parent compound. The introduction of one additional methylene unit penalizes its LE, and thus maintaining the piperazine on the structure seems advantageous. Removal of the chlorine substituent also produced a compound with similar affinity (3) but higher LE and lipophilic efficiency (LLE), indicating that this atom provided no specific contribution to binding affinity. The summary of the SAR findings for the LINS05 compounds is depicted in Figure 3.

Although literature data suggested that higher homologation in the linker region could increase the H₃R affinity (Kuder et al., 2016), the insertion of a methylene unit in the linker of compound 1 considerably decreased the affinity and efficiency values of the compound 4, even though this compound has the same number of heavy atoms as compound 2. This clearly shows that homologation was better tolerated in the basic region than in the linker. However, the insertion of a phenyl substituent in the piperazine nitrogen produced comparable affinity to compound 5, and the removal of the chlorine substituent increased the affinity of compound 6, as expected. The higher homologation found in compound 7 also increased the affinity, achieving binding at submicromolar concentrations.

The role of linker length and substitution variations in the basic moiety is frequently investigated in H₃R ligands (Łażewska et al., 2009; 2016; Kuder et al., 2016). Long linkers between the aromatic and basic regions tend to be well tolerated by the H₃R but not as much by the H₄R, while bulkier substituents in the basic region are only tolerated by H₃R. Compounds such as pitolisant and DL-80 clearly illustrate these roles (Kuder et al., 2014; Lamb, 2020). However, compounds with two basic nitrogens (such as the piperazine in compound 1) may exhibit two protonation states that can bind on different modes at the receptor, while the phenylpiperazine group prevent the protonation of the piperazine N4 at physiological pH, allowing only one protonation state for compound 7 (Tagiuri et al., 2016; Szczepańska et al., 2022). The distances between the anilide region and the protonated N-methyl group of 1 versus and the basic nitrogen of 7 are similar (Figure 3), suggesting that the N-phenyl group can perform efficient interactions with H₃R. This was previously hypothesized for DL-80 (Kuder et al., 2014) and some previously reported LINS01 and LINS05 compounds (Corrêa et al., 2019; Aranha et al., 2023).

Variations on the arrangement of the amide in the linker were also assessed, since urea and benzylamidine derivatives of FUB 661 and clobenpropit are described in literature. Rearrangement of the anilide group of 7 to the benzylamide analog 8 decreased the affinity by 10-fold. The urea bioisostere 9 showed increased affinity but the efficiency values were comparable to those of the more potent analog 7. Finally, the bioisosteric replacement of the phenylpiperazine with a 4-pyridylpiperazine slightly increased the affinity of 10 in relation to 8 as well, but with higher efficiency values, especially with regard to LLE. The role of the 4-pyridyl group on the affinity of H₃R ligands is noteworthy; Szczepanska et al. first verified that the introduction of a 4-pyridyl substituent in the basic region represented by both piperazine and piperidine provided high affinity at H₃R (Szczepańska et al., 2018; 2023). This role was also observed in compounds reported by our group (Aranha et al., 2023; Lopes et al., 2026). It was suggested that 4-pyridyl group interacts with Glu5.46 of H₃R along with the protonated piperazine performing a salt bridge with Asp3.32 (Lopes et al., 2026). These key interactions may explain the high affinity of compounds containing this structural motif.

The modifications in the LINS01007 structure showed a different activity profile (Figure 4). Bioisosteric replacement of piperazine with its higher homologue in compound 11 increased the affinity toward H₃R and produced a sharp reduction in affinity for H₄R. Moreover, removal of the chlorine from the dihydrobenzofuran core (12) and its aromatization to benzofuran (13) reduced the affinity in relation to 11, leading to similar pK _i values to those observed for the prototype LINS01007. This strongly suggests that the homopiperazine plays a role in the selectivity profile, increasing the affinity for H₃R and reducing the affinity for H₄R. In addition, the higher efficiency values for compound 11 in relation to 12 denote a specific effect of the chlorine on the binding to H₃R.

The known SAR for JNJ-7777120 suggests that indole NH, carbonyl group and the basic nitrogen from piperazine are involved in key interactions with H₄R, and that the chlorine substituent increases its affinity over other substituents (Venable et al., 2005; Engelhardt et al., 2012). Interestingly, the insertion of the carbonyl into the LINS01 chemotype increased the affinity for H₄R (compound 14), reinforcing the previous observation on LINS01018 that the carboxamide motif seems to be a more critical determinant for high affinity at H₄R than the chlorine. Previous studies from our group identified compound LINS01018 as nanomolar affinity H₄R ligand (Corrêa et al., 2021). Curiously, compound 14 exhibited nanomolar affinity for H₃R, while LINS01018 lacks affinity for this receptor. This also reinforces the role of homopiperazine in increasing affinity at H₃R in this chemotype.

The ring opening of the piperazine moiety in compounds 15 and 16 led to comparable affinity profiles at the H₃R, suggesting that the cyclic structure of piperazine is not required for H₃R binding, but seems important for the H₄R. Nonetheless, the ethylenediamine compounds 15 and 16 showed much higher ligand efficiency than the rigid homopiperazine counterparts 12 and 13, suggesting that these compounds can adapt better to the receptor topology due to their more flexible structure. However, the carboxamide derivative 17 showed 10-fold reduction in affinity in relation to 14, denoting that the hydrogen-bond donor NH group can negatively affect the interaction with the H₃R.

Finally, it is interesting to note that the 4-pyridyl substituted analogue 18 had higher affinity for the H₃R than its methyl and phenylpiperazine counterparts (LINS01018 and LINS01019, respectively) (Corrêa et al., 2021), but showed comparable affinity to the remaining compounds from the current set. The lower ligand efficiency values for compound 18 in comparison to the other analogues reinforce the poor contribution of the 4-pyridyl substitution on this chemotype, which adds heavy atoms in the molecule but does not efficiently increase the pK _i value. The result sheds light on the role of the 4-pyridyl group on H₃R affinity, and strongly suggests that at least one ionizable nitrogen on piperazine at physiological pH is important to achieve high affinity toward H₃R (possibly to perform an ionic interaction with Asp3.32). In addition, the protonated N-methylpiperazine group seems important for binding at H₄R with high affinity.

2.3 Docking experiments

To have a better picture of the role of these modifications on the binding interactions at the receptors, docking experiments were carried out with selected compounds using the structures of human H₃R (PDB 7F61) and H₄R (PDB 8JXX). The experimental structures of the ligands PF-03654746 (H₃R) and clobenpropit (H₄R) (Peng et al., 2022; Xia et al., 2024) were used as reference to validate the experiments by redocking procedure and to evaluate the obtained binding modes of the test ligands. The symmetric distribution of the conserved acidic amino acids Asp3.32 and Glu5.46 on both receptors frequently provides two opposite but plausible binding modes (Kiss et al., 2008; Corrêa et al., 2019; Lopes et al., 2026). With regard to H₃R, a third residue, Glu7.36, interacts with amide carbonyl from the experimental ligand, also enabling additional interactions with ligands containing longer linkers between their aromatic and basic moieties. Considering the data from experimental structures and availability of mutagenesis studies (Lim et al., 2010; Istyastono et al., 2011; Nijmeijer et al., 2013), a salt bridge between Asp3.32 and the protonated amine from the basic moiety, along with the overlap between the regions defined in Figure 2 with the reference experimental structures was used as primary criterion for choosing the more plausible binding modes from the docking results.

The compounds 1, 7 and 10 were selected from LINS05 series for docking studies at H₃R. The most potent ligand 7 exhibited a binding pose that aligns with the reference compound PF-03654746, which is consistent to its nanomolar affinity. The presence of two aromatic regions on this compound allows obtaining two plausible but opposite binding modes at the receptor; the first mode shows a salt bridge between the protonated N1 atom of the 4-phenylpiperazine and Asp3.32 (Figure 5A), while in the second binding mode, the salt bridge is performed with the Glu7.36 residue. Similar behavior was also observed for other benzamides recently reported by our group (Lopes et al., 2026). The first binding mode was selected as the most plausible according to our predefined criteria, and therefore it was used to discuss the results presented herein. The binding mode for compound 7 demonstrated a key hydrogen bond between the anilide NH group and Glu7.36, and complementary hydrophobic or π-contacts were observed between the aromatic anilide and phenylpiperazine groups of the 7 with the Phe and Tyr residues from ECL2 and TM2 domains (Figure 5A).

Shorter compounds (such as 1) may also present two binding modes: one with the anilide motif directed to the N-terminal Arg residue and another where this motif is directed to the hydrophobic pocket on TM2. The phenyl ring linked to piperazine prevents the protonation of the N4 nitrogen under physiological conditions, allowing only one protonation state for compound 7 at pH 7.4, whereas two possible protonation states for compound 1 are plausible. In this case, the first binding mode was selected based on a better overlap of the anilide regions of both 1 and 7, as well as the alignment of the protonated N4 from compound 1 with the protonated N1 from compound 7 (Figure 3). With regard to compound 10, the 4-pyridyl group was positioned toward the hydrophobic pocket of TM2 and may perform interactions with both Asp3.32 and Glu5.46, which may suggest why this motif increases the experimental binding affinity of (4-pyridyl)piperazine-containing compounds to H₃R.

With respect to compounds from the LINS01 series, docking experiments were carried out with compounds LINS01007, 11 and 12 at H₃R in order to investigate the role of the chlorine and homopiperazine modifications. It is worth mentioning that docking experiments were carried out with both R and S-enantiomers of the ligands, but no significant differences were observed among them, possibly to highly similar alignments of the aromatic and basic moieties in the binding site; This was also previously demonstrated in our previous publication regarding LINS01007 (Corrêa et al., 2021). The selected binding poses for all the compounds displayed the expected salt bridge interaction with Asp3.32, and their aromatic regions were positioned to perform hydrophobic contacts with Tyr residues from ECL2 (Figure 5B). As previously identified in other literature studies (Corrêa et al., 2021), the chlorine atom may interact with these Tyr residues, suggesting that this substituent may be involved in interactions that increase the affinity of 11 in comparison to 12. Furthermore, the higher affinity of 11 compared to LINS01007 may be attributed to the higher lipophilicity of the homopiperazine relative to piperazine, which enables additional hydrophobic contacts with Tyr6.51, Tyr3.33 and Phe7.39 residues. Similar results were observed for other H₃R ligands containing lipophilic substituents close to the basic moiety (Peng et al., 2022).

Regarding H₄R, docking studies were performed with compounds JNJ-7777120, LINS01007, LINS01018 and 14. Given that the experimental co-crystallized ligand of H₄R, clobenpropit, presents a structure considerably different from the LINS01 compounds, we used the binding pose of JNJ-7777120 as reference to compare with the remaining ligands, due to the abundance of published experimental data available for this compound. Again, more than a single binding orientation was identified, but literature data for JNJ-7777120 support that the interaction of the basic nitrogen of piperazine with Asp3.32 and hydrogen bonds between the indole NH and carbonyl groups with Glu5.46 and Gln7.42, respectively, may occur (Lim et al., 2010; Nijmeijer et al., 2013; Corrêa et al., 2021); hence, this orientation of JNJ-7777120 was used as reference. Additionally, hydrophobic contacts of the N-methyl group with the aromatic residues from TM7 were also observed. The remaining compounds showed similar binding modes that align with the JNJ-7777120 structure, as depicted in Figure 5C.

The obtained binding orientation suggested that the aromatic moiety of the compounds may perform hydrophobic contacts with Phe, Leu and Thr residues on TM5, and the chlorine may increase the affinity by performing additional contacts in this pocket. The absence of the carbonyl group on LINS01007 and 11 prevent the interaction with Gln7.42, which may decrease the affinity of these compounds at H₄R (Corrêa et al., 2021). Nonetheless, the lack of the indole NH on compound 14 and other LINS01 compounds may also be the reason behind their lower affinity toward H₄R. Moreover, the additional methylene unit of the homopiperazine from compound 14 promotes a distorted positioning of the basic nitrogen and the carbonyl groups, influencing their contacts with Asp3.32 and Gln7.42 and thereby reducing its affinity in relation to piperazine analogue LINS01018. This also may play a role on increasing the selectivity of compound 11 toward H₃R.

In conclusion, the scaffold hopping strategies employed in this work enabled the identification of novel ligands, especially for targeting the H₃R. Compound 11 (LINS01040) is highlighted due to its high affinity for H₃R and its selectivity over H₄R, while compound 7 stood out in the LINS05 series, displaying submicromolar affinity for the H₃R. These findings provide important novel SAR data to the field of H₃R/H₄R ligands and shed light on the design of future potent compounds.

3 Experimental

3.1 Chemicals and reagents

The starting materials and chemicals were purchased from LabSynth (Diadema, SP, Brazil) and Sigma-Aldrich (Saint Louis, MO, United States of America) with adequate purity level for the procedures and used without any further purification. The starting materials containing the 2-iodomethyl-2,3-dihydrobenzofuran nucleus were prepared as previously reported (Corrêa et al., 2016; 2017a; 2017b; 2021). For the binding assays, histamine dihydrochloride was purchased from Alfa Aesar GmbH and Co. KG (Karlsruhe, Germany). [³H]N ^α-methylhistamine (specific activity: 85.3 Ci/mmol) and [³H]histamine (specific activity: 25.0 Ci/mmol) were acquired from Hartmann analytic (Braunschweig, Germany).

¹H and ¹³C NMR spectra were recorded on a Bruker Ultrashield 300 spectrometer, operating at 300 and 75 MHz, respectively, using CDCl₃ or DMSO-d ₆ as solvents and tetramethylsilane (TMS) as internal standard. Chemical shifts (δ) were determined from TMS and are reported in parts per million (ppm). Coupling constants (J) are reported in units of Hertz (Hz), if applicable. All NMR data were obtained using the compounds as free bases. HRMS analysis was performed using a micrOTOF-QII LC-ESI-HRMS mass spectrometer (Bruker Daltonics, Bremen, Germany) or a QTOF LCMS-9030 mass spectrometer (Shimadzu, Kyoto, Japan). HPLC-UV analyses were performed in a LC-20AT chromatograph with a RF20A UV-Vis detector (Shimadzu Corp., Kyoto, Japan) for the hydrogenmaleate salts, using a C18 column (ACE 5) in the conditions described in the Supplementary Material. The accepted purity level for the final compounds was >95%. The analytical data (spectra and chromatograms) are presented in the Supplementary Material.

3.2 Synthesis of amides A1-A4 and B

In a 50 mL reaction flask, equimolar quantities (3 mmol) of the appropriate ω-chlorocarboxylic acid (2-chloroacetic, 3-chloropropionic or 4-chlorobutyric acids), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC.HCl) and 1-hydroxybenzotriazole hydrate (HOBt.xH₂O) were dissolved into 20 mL of dichloromethane (DCM). The reaction mixture was stirred at room temperature for 1 h, and then 3.3 mmol of the appropriate aniline (aniline or 4-chloroaniline) or benzylamine was added. The reaction mixture was kept under stirring for 18–22 h, and afterwards washed with 2x15 mL of aqueous saturated NaHCO₃ solution and purified water. The organic layer was dried with anhydrous Na₂SO₄ and the solvent evaporated. The raw material was purified through silica gel column chromatography, using hexane:AcOEt as eluent.

2-chloro-N-(4-chlorophenyl)acetamide (A1): Yield 32%. Yellowish oil. ¹H NMR (300 MHz, CDCl₃) δ 8.24 (s, 1H), 7.51 (d, J = 8.8 Hz, 2H), 7.33 (d, J = 8.8 Hz, 2H), 4.19 (s, 2H). NMR data is in accordance with literature (Braga et al., 2017).

3-chloro-N-phenylpropanamide (A2): Yield 56%. Off-white solid, m.p. 92 °C–94 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.51 (d, J = 8.1 Hz, 2H), 7.46–7.15 (m, 3H), 3.95 (t, J = 6.7 Hz, 2H), 2.81 (t, J = 6.7 Hz, 2H). NMR data is in accordance with literature (Igoe et al., 2017).

3-chloro-N-(4-chlorophenyl)propanamide (A3): Yield 38%. Off-white solid, m.p. 106 °C–108 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.49 (d, J = 8.8 Hz, 2H), 7.26 (d, J = 8.8 Hz, 2H), 3.95 (t, J = 6.7 Hz, 2H), 2.81 (t, J = 6.7 Hz, 2H). NMR data is in accordance with literature (Grimm et al., 2020).

4-chloro-N-phenylbutanamide (A4): Yield 50%. Off-white solid, m.p. 66 °C–68 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.54 (br.s, 1H), 7.50 (d, J = 7.9 Hz, 2H), 7.31 (t, J = 7.8 Hz, 2H), 7.10 (t, J = 7.4 Hz, 1H), 3.64 (t, J = 6.1 Hz, 2H), 2.54 (t, J = 7.1 Hz, 2H), 2.28–2.10 (m, 2H). NMR data is in accordance with literature (Gagné-Boulet et al., 2021).

N-benzyl-3-chloropropanamide ( *B* ): Yield 48%. Off-white solid, m.p. 82 °C–84 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.38–7.19 (m, 5H), 6.47 (s, 1H), 4.40 (d, J = 5.7 Hz, 2H), 3.76 (t, J = 6.4 Hz, 2H), 2.61 (t, J = 6.4 Hz, 2H). NMR data is in accordance with literature (Ang et al., 2022).

3.3 Synthesis of 1-benzyl-3-(2-chloroethyl)urea C

In a 25 mL reaction flask, equimolar quantities (3 mmol) of benzyl isocyanate and 1-(2-chloro)ethylamine hydrochloride were dissolved into 10 mL of DCM. Afterwards, 3 mmol of triethylamine (TEA) was added and the reaction mixture was stirred at room temperature for 2 h. The mixture was washed with 3x10 mL of purified water, and the organic layer was dried with anhydrous Na₂SO₄ and the solvent evaporated to furnish the product C1 with 90% yield. White solid, m.p. 96 °C–98 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.32–7.25 (m, 5H), 5.04 (s, 1H), 4.45–4.35 (m, 3H), 4.28 (t, J = 8.5 Hz, 2H), 3.79 (t, J = 8.5 Hz, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 158.00, 138.98, 128.66, 127.40, 127.37, 44.97, 44.49, 42.13.

3.4 General method for the synthesis of compounds 1–10

To 15 mL of acetonitrile in a 25 mL reaction flask, 1 mmol of chloroalkyl intermediates (A1-A4, B or C), 1 mmol of K₂CO₃, 1 mmol of KI and the appropriate piperazine (2 mmol of 1-phenylpiperazine or 1-(4-pyridyl)piperazine, or 3 mmol of 1-methylpiperazine) were added. The reaction mixture was stirred at 80 °C for 12–24 h, when the solvent was evaporated. The residue was taken up in 15 mL of AcOEt and washed with 3x15 mL of purified water. The organic layer was dried with anhydrous Na₂SO₄ and the solvent evaporated. The crude material was purified by silica gel column chromatography, using DCM:MeOH as eluent.

N-(4-chlorophenyl)-2-(4-methylpiperazin-1-yl)acetamide (1). Yield 50%. Yellowish oil. ¹H NMR (300 MHz, CDCl₃) δ 9.15 (s, 1H), 7.58–7.46 (m, 2H), 7.29 (d, J = 8.8 Hz, 2H), 3.14 (s, 2H), 2.66 (s, 4H), 2.51 (s, 4H), 2.33 (s, 3H). NMR data is in accordance with literature (Braga et al., 2017). HRMS (ESI+) m/z [M + H]⁺ calcd.: 268.1211; [M + H]⁺ found: 268.1221.

N-(4-chlorophenyl)-2-(4-methyl-1,4-diazepan-1-yl)acetamide (2). Yield 41%. Yellowish oil. ¹H NMR (300 MHz, CDCl₃) δ 9.41 (s, 1H), 7.56 (d, J = 8.8 Hz, 2H), 7.29 (d, J = 8.8 Hz, 2H), 3.27 (s, 2H), 2.85 (dd, J = 7.9, 4.0 Hz, 4H), 2.67 (dd, J = 11.2, 6.7 Hz, 4H), 2.40 (s, 3H), 1.93–1.76 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 169.38, 136.39, 129.05, 129.01, 120.65, 62.72, 58.14, 57.09, 55.93, 55.08, 47.27, 28.05. HRMS (ESI+) m/z [M + H]⁺ calcd.: 282.1367; [M + H]⁺ found: 282.1378.

2-(4-methylpiperazin-1-yl)-N-phenyl-acetamide (3). Yield 52%. Yellowish oil. ¹H NMR (300 MHz, CDCl₃) δ 9.11 (s, 1H), 7.63–7.49 (m, 2H), 7.34 (dd, J = 10.7, 5.1 Hz, 2H), 7.11 (t, J = 7.4 Hz, 1H), 3.14 (s, 2H), 2.66 (d, J = 4.2 Hz, 4H), 2.52 (s, 4H), 2.33 (s, 3H). NMR data is in accordance with literature (Braga et al., 2017). HRMS (ESI+) m/z [M + H]⁺ calcd.: 234.1601; [M + H]⁺ found: 234.1610.

N-(4-chlorophenyl)-3-(4-methylpiperazin-1-yl)propanamide (4). Yield 66%. Yellowish oil. ¹H NMR (300 MHz, CDCl₃) δ 11.09 (s, 1H), 7.49 (d, J = 7.7 Hz, 2H), 7.29–7.24 (m, 2H), 2.72 (t, J = 6.2 Hz, 2H), 2.63 (br.s, 8H), 2.51 (t, J = 6.2 Hz, 2H), 2.38 (s, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 170.54, 137.36, 128.94, 128.45, 120.68, 55.34, 53.55, 52.25, 45.98, 32.45. HRMS (ESI+) m/z [M + H]⁺ calcd.: 282.1367; [M + H]⁺ found: 282.1369.

N-(4-chlorophenyl)-3-(4-phenylpiperazin-1-yl)propanamide (5). Yield 46%. Yellowish oil. ¹H NMR (300 MHz, CDCl₃) δ 10.96 (s, 1H), 7.55–7.41 (m, 2H), 7.36–7.18 (m, 5H), 6.97 (d, J = 7.9 Hz, 2H), 6.92 (t, J = 7.3 Hz, 1H), 3.38–3.24 (m, 4H), 2.89–2.71 (m, 6H), 2.65–2.49 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 170,36, 150,78, 137,27, 129,30, 121,01, 128,54, 120,62, 120,44, 116,23, 53,68, 52,43, 49,47, 32,50. HRMS (ESI+) m/z [M + H]⁺ calcd.: 344.1524; [M + H]⁺ found: 344.1536.

N-phenyl-3-(4-phenylpiperazin-1-yl)propanamide (6). Yield 43%. White solid, m.p. 134 °C–136 °C. ¹H NMR (300 MHz, CDCl₃) δ 10.88 (s, 1H), 7.52 (d, J = 7.7 Hz, 2H), 7.36–7.26 (m, 4H), 7.06 (t, J = 7.4 Hz, 1H), 6.98 (d, J = 8.0 Hz, 2H), 6.92 (t, J = 7.3 Hz, 1H), 3.41–3.22 (m, 4H), 2.90–2.68 (m, 6H), 2.68–2.46 (m, 2H). ¹³C NMR (75 MHz, CDCl₃, ppm): δ 170.34, 150.86, 138.68, 129.28, 129.03, 123.75, 120.34, 119.45, 116.22, 53.79, 52.45, 49.44, 32.62. HRMS (ESI+) m/z [M + H]⁺ calcd.: 310.1913; [M + H]⁺ found: 310.1925.

N-phenyl-4-(4-phenylpiperazin-1-yl)butanamide (7). Yield 43%. Yellowish oil. ¹H NMR (300 MHz, CDCl₃) δ 8.28 (s, 1H), 7.51 (d, J = 7.9 Hz, 2H), 7.35–7.23 (m, 4H), 7.08 (t, J = 7.4 Hz, 1H), 6.94 (d, J = 8.8 Hz, 2H), 6.87 (t, J = 6.8 Hz, 1H), 3.28–3.20 (m, 4H), 2.70–2.62 (m, 4H), 2.53 (t, J = 6.7 Hz, 2H), 2.47 (t, J = 7.0 Hz, 2H), 1.96 (quint, J = 6.7 Hz, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 171.18, 151.14, 138.19, 129.16, 129.02, 124.05, 119.97, 119.66, 116.16, 56.83, 53.02, 49.15, 35.55, 30.93. HRMS (ESI+) m/z [M + H]⁺ calcd.: 324.2070; [M + H]⁺ found: 324.2058.

N-benzyl-3-(4-phenylpiperazin-1-yl)propanamide (8). Yield 50%. Off-white solid; m.p. 110 °C–112 °C. ¹H NMR (300 MHz, CDCl₃) δ 8.59 (s, 1H), 7.35–7.15 (m, 7H), 6.90–6.82 (m, 3H), 4.43 (d, J = 5.3 Hz, 2H), 3.10–2.98 (m, 4H), 2.69 (t, J = 5.9 Hz, 2H), 2.65–2.57 (m, 4H), 2.47 (t, J = 5.9 Hz, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 172.13, 151.01, 138.54, 129.15, 128.70, 127.68, 127.41, 120.10, 116.24, 53.88, 52.45, 49.16, 43.36, 32.08. HRMS (ESI+) m/z [M + H]⁺ calcd.: 324.2070; [M + H]⁺ found: 324.2065.

1-benzyl-3-(2-(4-phenylpiperazin-1-yl)ethyl)urea (9). Yield 67%. White solid; m.p. 148 °C–150 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.28–7.21 (m, 7H), 6.88–6.83 (m, 3H), 5.69 (br.s, 1H), 5.10 (br.s, 1H), 4.33 (d, J = 5.7 Hz, 2H), 3.27 (q, J = 5.6 Hz, 2H), 3.09–2.94 (m, 4H), 2.61–2.51 (m, 4H), 2.49 (t, J = 5.6 Hz, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 158.82, 151.16, 139.39, 129.10, 128.63, 127.58, 127.30, 119.82, 116.08, 57.99, 53.01, 48.99, 44.59, 37.55. HRMS (ESI+) m/z [M + H]⁺ calcd.: 325.2022; [M + H]⁺ found: 325.2013.

N-benzyl-3-[4-(pyridin-4-yl)piperazin-1-yl]propanamide (10). Yield 35%. Yellowish oil. ¹H NMR (300 MHz, CDCl₃) δ 8.26 (d, J = 6.2 Hz, 2H), 8.16 (s, 1H), 7.35–7.19 (m, 5H), 6.63 (d, J = 6.3 Hz, 2H), 4.43 (d, J = 5.3 Hz, 2H), 3.23–3.17 (m, 4H), 2.71 (t, J = 6.1 Hz, 2H), 2.62–2.54 (m, 4H), 2.47 (t, J = 6.1 Hz, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 171.75, 154.97, 149.30, 138.48, 128.72, 127.70, 127.52, 108.35, 53.84, 51.90, 45.82, 43.44, 32.32. HRMS (ESI+) m/z [M + H]⁺ calcd.: 339.2179; [M + H]⁺ found: 339.2191.

3.5 General method for the synthesis of 2,3-dihydrobenzofurans 11, 12 and 15

To a solution 1 mmol of the appropriate 2-iodomethyl-2,3-dihydrobenzofuran derivative in 15 mL of tetrahydrofuran (THF), 4 mmol of 1-methyl-homopiperazine or N′,N′-dimethylethane-1,2-diamine and 1 mmol of K₂CO₃ were added. The mixture was stirred at 80 °C for 12–24 h and then the reactional mixture was filtered and evaporated. The residue was taken up into 15 mL of aqueous 1M HCl solution and washed with 2x15 mL of AcOEt. The aqueous layer was alkalinized with NaOH (pH 10) and extracted with 3x15 mL of AcOEt. The organic layer was dried with anhydrous Na₂SO₄ and the solvent evaporated. The crude material was purified by silica gel column chromatography, using DCM:MeOH as eluent.

1-[(5-chloro-2,3-dihydro-1-benzofuran-2-yl)methyl]-4-methyl-1,4-diazepane (11). Yield 45%. Yellowish oil. ¹H NMR (300 MHz, CDCl₃) δ 7.10 (s, 1H), 7.04 (d, J = 8.5 Hz, 1H), 6.67 (d, J = 8.5 Hz, 1H), 4.99–4.81 (m, 1H), 3.23 (dd, J = 15.8, 9.1 Hz, 1H), 3.02–2.94 (m, 1H), 2.94–2.89 (m, 1H), 2.89–2.78 (m, 4H), 2.72 (dd, J = 13.5, 4.9 Hz, 1H), 2.65–2.52 (m, 4H), 2.36 (s, 3H), 1.87–1.74 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 158.27, 128.65, 127.72, 124.93, 110.31, 82.29, 62.14, 57.82, 57.05, 55.20, 54.82, 47.00, 33.76, 27.34. HRMS (ESI+) m/z [M + H]⁺ calcd.: 281.1415; [M + H]⁺ found: 281.1425.

1-[(2,3-dihydro-1-benzofuran-2-yl)methyl]-4-methyl-1,4-diazepane (12). Yield 48%. Yellowish oil. ¹H NMR (300 MHz, CDCl₃) δ 7.15 (d, J = 7.3 Hz, 1H), 7.09 (t, J = 7.8 Hz, 1H), 6.82 (t, J = 7.4 Hz, 1H), 6.77 (d, J = 8.0 Hz, 1H), 4.97–4.82 (m, 1H), 3.26 (dd, J = 15.6, 9.2 Hz, 1H), 3.03–2.79 (m, 6H), 2.72 (dd, J = 13.4, 4.9 Hz, 1H), 2.62 (dd, J = 10.7, 5.2 Hz, 4H), 2.36 (s, 3H), 1.82 (quint, J = 6.0 Hz, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 159.56, 127.88, 126.61, 124.92, 120.21, 109.49, 81.49, 62.41, 57.80, 57.13, 55.24, 54.81, 47.03, 33.94, 27.40. HRMS (ESI+) m/z [M + H]⁺ calcd.: 247.1804; [M + H]⁺ found: 247.1814.

[(2,3-dihydro-1-benzofuran-2-yl)methyl][2-(dimethylamino)ethyl]amine (15). Yield 38%. Yellowish oil. ¹H NMR (300 MHz, CDCl₃) δ 7.18–7.05 (m, 2H), 6.86–6.74 (m, 2H), 4.92 (ddd, J = 16.8, 7.7, 4.1 Hz, 1H), 3.28 (dd, J = 15.5, 9.2 Hz, 1H), 2.99–2.81 (m, 3H), 2.76 (t, J = 6.5 Hz, 2H), 2.44 (t, J = 6.0 Hz, 2H), 2.34 (s, 1H), 2.23 (s, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 159.38, 127.95, 126.64, 124.97, 120.29, 109.50, 82.27, 59.15, 54.61, 47.50, 45.58, 33.52. HRMS (ESI+) m/z [M + H]⁺ calcd.: 221.1648; [M + H]⁺ found: 221.1656.

3.6 Synthesis of 1-[(1-benzofuran-2-yl)methyl]-4-methylamines 13 and 16

In a 50 mL reaction flask, 1 mmol of benzofuran-2-carbaldehyde and 2 mmol of appropriate amine (1-methyl-homopiperazine for 13 and N′,N′-dimethylethane-1,2-diamine for 16) were dissolved into 25 mL of MeOH. The mixture was stirred at room temperature for 1 h and then 3 mmol of sodium triacetoxyborohydride (STAB, for 13) or NaBH₄ (for 16) were added. The reaction was kept under stirring for 2–18 h, and the solvent was evaporated. The residue was taken up in 15 mL of DCM and washed with 3x15 mL of saturated NaHCO₃ solution. The organic solution was dried with anhydrous Na₂SO₄ and the solvent evaporated. The crude material was purified by silica gel column chromatography, using DCM:MeOH as eluent.

1-[(1-benzofuran-2-yl)methyl]-4-methyl-1,4-diazepane (13). Yield 51%. Yellowish oil. ¹H NMR (300 MHz, CDCl₃) δ 7.52 (d, J = 6.7 Hz, 1H), 7.46 (d, J = 7.9 Hz, 1H), 7.29–7.16 (m, 2H), 6.58 (s, 1H), 3.83 (s, 2H), 2.82 (m, 8H), 2.44 (s, 3H), 1.91 (quint, J = 6.0 Hz, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 155.48, 155.03, 155.27, 128.32, 123.87, 122.65, 120.68, 111.22, 105.22, 57.60, 56.54, 55.20, 53.90, 53.36, 46.49, 26.56. HRMS (ESI+) m/z [M + H]⁺ calcd.: 245.1648; [M + H]⁺ found: 245.1653.

1-[(1-benzofuran-2-yl)methyl][2-(dimethylamino)ethyl]amine (16). Yield 55%. Yellowish oil. ¹H NMR (300 MHz, CDCl₃) δ 7.51 (d, J = 6.9 Hz, 1H), 7.44 (d, J = 7.9 Hz, 1H), 7.25–7.16 (m, 2H), 6.57 (s, 1H), 3.95 (s, 2H), 2.74 (t, J = 6.0 Hz, 2H), 2.44 (t, J = 6.0 Hz, 2H), 2.21 (s, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 156.91, 154.95, 128.48, 123.71, 122.57, 120.65, 111.07, 103.71, 58.97, 46.73, 46.45, 45.48. HRMS (ESI+) m/z [M + H]⁺ calcd.: 219.1491; [M + H]⁺ found: 219.1499.

3.7 Synthesis of 1-[(1-benzofuran-2-yl)methyl]carboxamides 14, 17 and 18

In a 25 mL reaction flask, 1 mmol of the appropriate amine (1-methyl-homopiperazine for 14, N′,N′-dimethylethane-1,2-diamine for 17 or 1-(4-pyridyl)piperazine for 18) was dissolved in 10 mL of DCM and then a solution of 1 mmol of benzofuran-2-carbonyl chloride in 5 mL of DCM was added dropwise. The solution was stirred for 1–2 h until completion (TLC monitored). Afterwards the organic mixture was washed with 3x15 mL of aqueous NaHCO₃ and 15 mL of purified water, dried with anhydrous Na₂SO₄ evaporated. The crude material was purified by silica gel column chromatography, using DCM:MeOH as eluent.

1-(1-benzofuran-2-carbonyl)-4-methyl-1,4-diazepane (14). Yield 90%. Yellowish oil. ¹H NMR (300 MHz, CDCl₃) δ 7.65 (d, J = 7.7 Hz, 1H), 7.52 (d, J = 8.3 Hz, 1H), 7.39 (t, J = 7.7 Hz, 1H), 7.33–7.22 (m, 2H), 3.91–3.87 (m, 2H), 3.96–3.71 (m, 2H), 2.85–2.70 (m, 2H), 2.64–2.59 (m, 2H), 2.40 (s, 3H), 2.04 (quint, J = 6.0 Hz, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 161.01, 154.63, 149.56, 149.48, 127.03, 126.32, 123.51, 122.21, 111.83, 111.69, 111.64, 59.61, 57.55, 57.50, 56.73, 48,27, 48,08, 46,69, 46,63, 46,27, 46,18, 28,55, 26,78. HRMS (ESI+) m/z [M + H]⁺ calcd.: 259.1441; [M + H]⁺ found: 259.1451.

N-[2-(dimethylamino)ethyl]-1-benzofuran-2-carboxamide (17). Yield 94%. Yellowish oil. ¹H NMR (300 MHz, CDCl₃) δ 7.65 (d, J = 7.8 Hz, 1H), 7.52 (d, J = 8.3 Hz, 1H), 7.45 (s, 1H), 7.40 (t, J = 7.1 Hz, 1H), 7.28 (t, J = 7.5 Hz, 1H), 3.56 (q, J = 6.0 Hz, 2H), 2.54 (t, J = 6.0 Hz, 2H), 2.30 (s, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 158.96, 154.78, 148.99, 127.66, 126.68, 123.57, 122.61, 111.82, 110.12, 57.87, 45.28, 36.78. HRMS (ESI+) m/z [M + H]⁺ calcd.: 233.1284; [M + H]⁺ found: 233.1294.

1-(1-benzofuran-2-carbonyl)-4-(pyridin-4-yl)piperazine (18). Yield 75%. White solid. ¹H NMR (300 MHz, CDCl₃) δ 8.33 (d, J = 6.5 Hz, 2H), 7.68 (d, J = 8.1 Hz, 1H), 7.54 (d, J = 8.4 Hz, 1H), 7.45 (dd, J = 7.2, 1.2 Hz, 1H), 7.41 (s, 1H), 7.32 (t, J = 7.5 Hz, 1H), 6.70 (d, J = 6.5 Hz, 2H), 4.04 (s, 4H), 3.55–3.38 (m, 4H). ¹³C NMR (75 MHz, CDCl₃) δ 159.82, 154.73, 154.58, 150.52, 148.76, 126.89, 126.81, 123.80, 122.42, 112.91, 111.89, 108.55, 46.12. HRMS (ESI+) m/z [M + H]⁺ calcd.: 308.1394; [M + H]⁺ found: 308.1406.

3.8 Pharmacological assays

Radioligand competition binding experiments were performed as previously described by Rosier et al. on stable HEK293-SP-FLAG-hH₃R, and HEK293-SP-FLAG-hH₄R cell lines (Rosier et al., 2021). The tested compounds were initially screened at a single concentration of 100 µM. For those that showed a considerable (>60%) displacement of the radioligand at this concentration, full concentration-response curves were carried out. In this case, ligand dilutions were prepared 10-fold concentrated in Leibovitz’ L-15 medium with 1% BSA and 10 µL/well were transferred to a flat-bottom polypropylene 96-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), as well as 10 µL/well of the respective radioligand. The cells were adjusted to a density of 1.25 x 10⁶ cells/mL and 80 µL of the cell suspension were added to each well (total volume of 100 µL). All data were analyzed using GraphPad Prism8 software (San Diego, CA, United States of America). The normalized competition binding curves were then fitted with a four-parameter logistic fit yielding pIC₅₀-values. These were transformed into pK _i-values using the Cheng-Prusoff equation (Cheng and Prusoff, 1973).

3.9 Ligand efficiency

The ligand efficiency (LE) and lipophilic ligand efficiency (LLE) values were calculated using the previously reported formulae from literature (Hopkins et al., 2014). where HA is the number of heavy (non-hydrogen) atoms in the molecule. The logP values were calculated in the SwissADME on-line platform (Daina et al., 2017) and used as the consensus value, which is an average of five logP values calculated through different methods.

LE = 1.37 × p K i HA

L L E = p K i − logP

3.10 Molecular modeling and docking experiments

The three-dimensional experimental structures of the human targets H₃R (PDB 7F61) and H₄R (PDB 8JXX) were downloaded from Protein Data Bank (PDB) in a.pdb file format. The complexed ligands and water molecules were removed; however, both receptors have no water molecules co-crystalized in the binding site. The clean protein structure was used in the molecular docking experiments (the receptor protein chain was used in each receptor; chain A in H₃R and chain F in H₄R) using the DockThor Web server (Guedes et al., 2024), which is a freely available web server (https://dockthor.lncc.br) and well-established in literature. DockThor uses genetic algorithm to predict multiple solutions and the scoring function of the Merck Molecular Force Field 94S (MMFF94S) to estimate the partial atomic charges of both receptor and ligand molecules and predict the binding poses.

The size of docking search’s grid boxes (X, Y, Z and respective coordinates) was defined as follows: H₃R - 20 × 24 × 20 (−20.46, 48, 1.08); H₄R - 22 × 23 × 22 (140.33, 146.96, 108.34). These search boxes included the key amino acid residues from each target. The default setup conditions were maintained (24 docking runs; 1,000,000 iterations per run; population of 750 and 20 maximum cluster leaders per run). For methodology validation, redocking procedures were executed by extracting the ligands within the PDB crystal structure from each target. The experimental ligands were redocked on each respective target using the same molecular docking methods, which returned low RMSD values for heavy atoms from the selected pose, which showed the expected interactions observed for the crystalized ligand (0.458 Å for H₃R ligand; 1.337 for H₄R ligand). The analysis of the results was conducted using the Discovery Studio Visualizer (Dassault Systemes Biovia Corp, version 24.1.0.23298).

The three-dimensional structures of the tested ligands were built using the Marvin Sketch software (Chemaxon Inc., version 23.17.0) on their main ionized forms as predicted by the pKa add-on implemented in the software and saved as.mol2 files. The top binding modes (ten at maximum) were ranked by the implemented scores in DockThor and visually assessed on Discovery Studio Visualizer for reasonable binding modes, guided by the identification of contacts between the basic nitrogen of the compounds and Asp3.32. The ligand−target interactions were identified using the same software.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fddsv.2026.1944403/full#supplementary-material

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Footnotes

Edited by: José L Medina-Franco, National Autonomous University of Mexico, Mexico

Reviewed by: Abel Suárez-Castro, Universidad Vasco de Quiroga, Mexico

Anaid Flores, Metropolitan Autonomous University, Mexico