# Hit-to-lead optimization of 4,5-dihydrofuran-3-sulfonyl scaffold against Leishmania amazonensis. Effect of an aliphatic moiety

Oscar Leonardo Avendaño Leon, Fabiana Maia Santos Urbancg Moncorvo, Christophe Curti, Youssef Kabri, Sébastien Redon, Patrice Vanelle, Eduardo Caio Torres-Santos

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Oscar Leonardo Avendaño Leon, Fabiana Maia Santos Urbancg Moncorvo, Christophe Curti, Youssef Kabri, Sébastien Redon, et al. Hit-to-lead optimization of 4,5-dihydrofuran-3-sulfonyl scaffold against Leishmania amazonensis. Effect of an aliphatic moiety. European Journal of Medicinal Chemistry, 2024, 280, pp.116935. 10.1016/j.ejmech.2024.116935. hal-05001007

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<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Hit-to-lead optimization of 4,5-dihydrofuran-3-sulfonyl scaffold
against *Leishmania amazonensis*. Effect of an aliphatic moiety.

Oscar Leonardo Avendano Leon a,¹, Fabiana Maia Santos Urbancg Moncorvo b,¹, Christophe Curti a, Youssef Kabri a, Sébastien Redon a, Patrice Vanelle a,* and Eduardo Caio Torres-Santos. b

<sup>a</sup> Aix Marseille Univ, CNRS, ICR UMR 7273, Equipe Pharmaco-Chimie Radicalaire, Faculté de Pharmacie, 27 Boulevard Jean Moulin, CS30064, CEDEX 05, 13385 Marseille, France; oscar-leonardo.avendano-leon@etu.univ-amu.fr (O.A.); christophe.curti@univ-amu.fr (C.C.); youssef.kabri@univ-amu.fr (Y.K.), sebastien.redon@univ-amu.fr (S.R.).

<sup>b</sup> Laboratório de Bioquímica de Tripanosomatideos, Instituto Oswaldo Cruz - FIOCRUZ, Av. Brasil, 4365, Rio de Janeiro 21040-900, Brazil; fabianamaia.santos@gmail.com (F.M.S.U.M); ects@ioc.fiocruz.br (E.C.T.-S.).

*Correspondence: patrice.vanelle@univ-amu.fr (P.V.); Tel.: +33-4-9183-5580

¹ These authors contributed equally.

**Abstract:** In line with our objective of designing new antileishmanial compounds for oral use, we report the synthesis and biological evaluation *in vitro* of original 4,5-dihydrofuran derivatives bearing an amidoxime group. Previous optimization focused on position 3 of the dihydrofuran ring involving aromatic fragments, resulting in the identification of the compound (HIT) 4-(5-benzyl-3-((4-fluorophenyl)sulfonyl)-5-methyl-4,5-dihydrofuran-2-yl)-N'-hydroxybenzimidamide (IC₅₀ = 5.4 ± 1.0 µM, *L. amazonensis* promastigote, IC₅₀ = 7.9 ± 1.1 µM, *L. amazonensis* intracellular amastigote). In the present work, position 3 was substituted with an aliphatic moiety. This modification was guided by a ligand-based approach, given the unknown biological target or mechanism of action for this compound. The 4,5-dihydrofuran derivatives were synthesized using microwave-assisted manganese (III) acetate-based oxidative cyclization of linear β-keto-carboxylic and β-keto-sulfone substrates, overcoming synthetic challenges to obtain aliphatic derivatives of 4,5-dihydrofuran-3-carboxamides. Finally, an unexpected and interesting biological activity with the 4,5-dihydrofuran-3-carboxylate (IC₅₀ < 5 µM) against the amastigote form is discussed.

**Keywords:** Leishmaniasis, pharmacomodulation, 4,5-dihydrofuran, amidoxime, manganese (III) acetate.

**1. Introduction**

Leishmaniasis is a complex vector-borne disease caused by protozoan parasites of the genus *Leishmania*, transmitted through the bite of infected female phlebotomine sandflies. This disease affects nearly one billion individuals across 90 countries worldwide, resulting in around 1 million new cases and 30,000 deaths estimated annually [1]. The epidemiology of leishmaniasis is intricately linked to environmental factors. Climate change, particularly alterations in temperature and rainfall patterns, can expand the habitats of these vectors, potentially increasing the risk of transmission. This expansion is further exacerbated by secondary effects such as population displacement and malnutrition, which can increase susceptibility to infection. The disease manifests in three main forms: visceral (VL), cutaneous (CL), and mucocutaneous leishmaniasis (MCL), each presenting distinct clinical symptoms ranging from skin ulcers to severe organ damage [A]. Moreover, leishmaniasis can lead to significant health complications and even be fatal in the absence of treatment [2].
Existing conventional antileishmanial treatments have clinical limitations, such as toxicity, drug resistance, or high cost (e.g., pentamidine, antimonials, amphotericin B, and miltefosine) [3] [4]. Hence, ongoing research to discover new cost-effective antileishmanial compounds conceived for oral use is of key importance. These advancements aim to improve patient outcomes and contribute to efforts in controlling and eventually eliminating this debilitating disease.

With the perspective of progressing towards *in vivo* testing, a Hit-to-Lead optimization process was performed to enhance activity against *Leishmania amazonensis*. In this work the effect of aliphatic derivatives at position 3 of the dihydrofuran ring was explored, whereas previous research focused on aromatic derivatives at this position. Therefore, an aliphatic series was designed using a scaffold hopping approach, inspired by a previously identified HIT compound featuring a 3-Arylsulfonyl structure [5,6], Scheme 1.

Previous work:

<figure><img src="image_2.png" /></figure>

R = aryle derivatives

<figure><img src="image_3.png" /></figure>

HIT

*In vitro* antileishmanial biological evaluation

Activity

*L. amazonensis* promastigotes $IC_{50}$ = 5.4 ± 1.0 µM

*L. amazonensis* amastigotes $IC_{50}$ = 7.9 ± 1.1 µM

Ref. pentamidine promastigotes $IC_{50}$ = 5.7 ± 0.1 µM

Toxicity

Murine macrophages $CC_{50}$ = 102.0 ± 2.8 µM

Ref. pentamidine $CC_{50}$ = 8.5 ± 1.3 µM

J774A.1 $CC_{50}$ = 45.5 ± 0.3 µM

Ref. pentamidine $CC_{50}$ = 1.0 ± 0.4 µM

Current work:

<figure><img src="image_4.png" /></figure>

R = alkyl derivatives: sulfone, amide, ester.

<caption>**Scheme 1.** Optimization of 4,5-dihydrofuran-3-sulfonyl scaffold against *Leishmania amazonensis*.</caption>

The biological target or mechanism of action linked to the antileishmanial activity of the amidoxime group remains unknown. Therefore, conducting computer-aided drug design (structure-based approach) without a reasonable hypothesis seems challenging due to the wide diversity of antileishmanial targets with varying mechanisms of action[7] [8], a show in Table 1. Consequently, foreseeing a hypothetical bioisosteric effect [9–11], in this work, a similarity drug-drug method was proposed, combined with predicted pharmacokinetics descriptors, to identify suitable aliphatic derivatives for evaluation [12,13].

<table><caption>**Table 1.** Types of targets and mechanisms of action reported for antileishmanial compounds.</caption><thead><tr><th>Principal mechanism</th><th>Target</th><th>Principal mechanism</th><th>Target</th></tr></thead><tbody><tr><td rowspan="4">Redox balance / Polyamine biosynthesis pathway</td><td>Trypanothione reductase, TR1[14]</td><td>Enzyme</td><td>Thiol metabolism</td><td>Cysteine proteinase [15]</td><td>Enzyme</td></tr><tr><td>Trypanothione synthase [16]</td><td>Enzyme</td><td rowspan="3">Purine salvage pathway</td><td>Nucleoside hydrolase [17]</td><td>Enzyme</td></tr><tr><td>L-Arginase [18]</td><td>Enzyme</td><td>Nucleoside diphosphate Kinase NDK [19] [20]</td><td>Enzyme</td></tr><tr><td>Tryparedoxin peroxidase (TXNPX) [21]</td><td>Enzyme</td><td>RNA translation [22]</td><td>Nucleic acid</td></tr><tr><td rowspan="2">Folate metabolism</td><td>Pteridine reductase 1, PTR1 [23]</td><td>Enzyme</td><td rowspan="2">Mitochondrial electron chain</td><td rowspan="2">NADH dehydrogenase [24]</td><td rowspan="2">Enzyme</td></tr><tr><td>Dihydrofolate reductase DHFR[25]</td><td>Enzyme</td></tr><tr><td>Pyrimidine salvage pathway</td><td>Protein</td><td></td><td>Protein kinases</td><td>Protein kinase CRK [28]</td><td>Enzyme</td></tr></tbody></table>
<table><tr><td rowspan="4">Sterol biosynthesis</td><td>Nucleoside transporter [26] [27]</td><td></td><td>Mitogen-activated kinases MAPKs [29]</td><td>Enzyme</td></tr><tr><td>Sterol-14-alpha-demethylase [30]</td><td>Enzyme</td><td>GAPDH [31]</td><td>Enzyme</td></tr><tr><td>SQS - Squalene epoxidase [32]</td><td>Enzyme</td><td>Phosphoglycerate kinase [34]</td><td>Enzyme</td></tr><tr><td>HMGR - 3-hydroxy-3-methylglutaryl coenzyme A reductase [33]</td><td>Enzyme</td><td>Mannosyltransferase [35]</td><td>Enzyme</td></tr></table>

From our ongoing work, we have previously reported that the presence of both the dihydrofuran and amidoxime groups is necessary to the antileishmanial effect. The present work aims to investigate whether the sulfone moiety is also indispensable for antileishmanial activity or if it can be effectively replaced.

## 2. Results and discussion

### 2.1 Chemistry

A preliminary analysis of suitable modifications was guided by the Similarity Tanimoto coefficient [36] [37] [46] [39], along with other biopharmaceutical predicted parameters and synthetic accessibility, Scheme 2. Open-source programs and libraries such as Swiss Drug Design®, R package®, and the ChEMBL library [40] [41] were used to perform this initial assessment (further details can be found in the supplementary document). Thus, a selection of ten aliphatic compounds was made for synthesis. Among these, modifications bearing the ester group (compounds c1 and c2) and the amide group (compounds c3 to c8) were designed to enhance predicted physicochemical properties for oral bioavailability [42]. Compounds c9 and c10, featuring the sulfone group with alkyl moieties, were included as reference compounds.

<figure><img src="image_5.png" /></figure>

CHEMBL1797618

Lipinski, Veber, Muegge filter's

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

3-carboxylates

c1. R = Methyl

c2. R = Ethyl

<figure><img src="image_8.png" /></figure>

3-carboxamides

c3. R = Cyclohexyl c6. R = Butyl

c4. R = di-L-ethyl c7. R = Ethyl-pyrrolidine

c5. R = Cyclopentyl c8. R = Morpholinyl

<figure><img src="image_9.png" /></figure>

3-sulfones

c9. R = Butyl

c10. R = Methyl

Similarity Tanimoto coefficient > 0.5

Lower lipophilicity and better gastrointestinal absorption than HIT

Lipinski, Veber, Muegge, Ghose, Egan filter's

R = aliphatic groups with lower molecular weight than HIT

**Scheme 2.** Modulation at position 3 of the dihydrofuran scaffold bearing an amidoxime group.

The synthesis of compounds c1 to c10 was conducted using previously reported methods, Scheme 3. For ester c1 and c2, the synthetic pathway involved in the first step an oxidative free-radical cyclization mediated by Mn(OAc)3 of the respective linear β-keto-ester substrates (a1 and a2) and terminal alkene (2-methylallyl)benzene, yielding the 4,5-dihydrofuran-carboxylate moiety in good yields (b1: 40% and b2: 69%). These intermediate compounds, bearing a cyano group, were then reacted with hydroxylamine hydrochloride and potassium tert-butoxide in DMSO [21], providing amidoximes c1 and c2 in good yields (c1:65%, c2: 77%). Amides derivatives c3 to c8 were obtained through a three-step synthetic pathway. In the first step, compounds a3 to a8 were obtained under microwave irradiation of commercially available compound a2 (ethyl 3-(4-cyanophenyl)-3-oxopropanoate) and the corresponding amine with a catalytic amount of DMAP (0.1 equiv.). Yields of linear β-keto-amides ranged between 63% and 86%, consistent with previously reported yields, Table 2 [43] [44] [45] [46]. The products were obtained as a keto-enol equilibrium.
A plausible mechanism for this reaction was suggested (see supplementary materials). The second step, an oxidative free-radical cyclization mediated by Mn(OAc)₃ of the respective linear β-keto-amide substrates (a3 to a8) and terminal alkene (2-methylallyl)benzene, yielding the 4,5-dihydrofuran-carboxamide moiety in moderate to good yields (35% - 70%). Purification of 4,5-dihydrofuran-carboxamide containing the morpholine moiety from the respective linear β-keto-amide a8 proved unexpectedly challenging. Thus, method B was subsequently proposed, involving an aminolysis reaction mediated by lithium bis(trimethylsilyl)amide (LiHMDS) of the cyclized ester ethyl 4,5-dihydrofuran carboxylate (b2), yielding the intermediate b8. In the third step, these intermediates b3 to b8, bearing a cyano group, underwent a reaction with hydroxylamine hydrochloride and potassium tert-butoxide in DMSO [21], providing amidoximes c3 to c8 in moderate to good yields (40 - 62%). The sulfone derivatives c9 and c10 were synthesized also through a three-step synthetic pathway. Initially, linear β-keto-sulfones a9 and a10 were obtained by nucleophilic substitution of the bromoacetophenone starting material with the corresponding sulfonyl group under MW irradiation [47], (a9: 56% and a10: 95%), methods previously studied by our team [48] [49] [50-53]. Next, the second step, oxidative free-radical cyclization mediated by Mn(OAc)₃ of these compounds and the terminal alkene (2-methylallyl)benzene yielded the 4,5-dihydrofuran-sulfone moiety, b9 and b10 (b9: 40% and b10: 60%). These intermediates, containing a cyano group, underwent a reaction with hydroxylamine hydrochloride and potassium tert-butoxide in DMSO [21], providing amidoximes c9 to c10 in good yields (c9: 62%, c10: 65%).

<figure><img src="image_10.png" /></figure>

Reagent and conditions: i) (2-methylallyl)benzene (2 equiv.), Mn(OAc)₃ (2.1 equiv.), Cu(OAc)₂ (1 equiv.), AcOH, MW, 80 °C, 3h. ii) KOtBu (10 equiv.), NH₂OH.HCl (10 equiv.), DMSO, 18h, 0 °C - RT, N₂. iii) Amine (2 equiv.), DMAP (0.1 equiv.), Toluene, MW, 150°C, 3h. iv) CH₃SO₂Na (1 equiv.), Dioxane-H₂O 1:1 reflux 12h; or C₄H₉SO₂Cl (1.5 equiv.), Na₂SO₃ (1.5 equiv.), NaHCO₃ (1.5 equiv.), H₂O, MW, 100°C, 1h, 200W. Method B: Morpholine (1.5 equiv.), LiHMDS (2.5 equiv.), Toluene, 18h, 70 °C, N₂.

<caption>**Scheme 3.** Synthesis of 4,5-dihydrofuran derivatives bearing an amidoxime group.</caption>
<table><caption>**Table 2.** Microwave-assisted synthesis of linear β-keto-amides compounds and observed ratio keto/enol by ¹H NMR analysis.</caption><thead><tr><th>Compound</th><th>R</th><th>R'</th><th>Yield (%)</th><th>Ratio keto/enol</th></tr></thead><tbody><tr><td>a3</td><td>H</td><td>Cyclohexyl</td><td>71</td><td>1/1</td></tr><tr><td>a4</td><td>Ethyl</td><td>Ethyl</td><td>78</td><td>1/5</td></tr><tr><td>a6</td><td>H</td><td>Butyl</td><td>63</td><td>1/1</td></tr><tr><td>a7</td><td>H</td><td>Ethyl-pyrrolidine</td><td>86</td><td>1/99</td></tr><tr><td>a8</td><td>Morpholinyl</td><td></td><td>76</td><td>1/2</td></tr></tbody></table>

## 2.2. Biology

The synthesized **c1-c10** amidoximes were evaluated *in vitro* for their activity against both *Leishmania amazonensis* (MHOM/BR/77/LTB0016) promastigotes and intracellular amastigotes, as well as their cytotoxicity in murine macrophages. Preliminary activity tests were carried out on *L. amazonensis* promastigotes. The 50% growth inhibitory activity (IC₅₀) was determined after 72h of cultivation with each compound and evaluated by assay with resazurin, through independent experiments in triplicates. Compounds **c1-c10** showed antileishmanial activities with IC₅₀ values ranging from 4.7 to 101.7 µM (Table 3). Compounds **c1** and **c2** were the most active substances, with IC₅₀ values of 4.7 and 6.3µM, respectively. The amide modifications yielded compounds with moderate activity, showing concentrations above 10 µM [54].

<table><caption>**Table 3.** Biological evaluation of amidoxime derivatives carrying aliphatic groups at position 3 on 4,5-dihydrofuran.</caption><thead><tr><th>Compound</th><th>R</th><th>Activity <sup>a</sup></th><th>C<br>o<br>m<br>p<br>o<br>u<br>n<br>d</th><th>R</th><th>Activity <sup>a</sup></th></tr></thead><tbody><tr><td>c1</td><td>H<sub>3</sub>C<sub>2</sub>O<sub>2</sub></td><td>4.7 ± 1.02</td><td>c<br>6</td><td>H<sub>3</sub>C<sub>2</sub>N<sub>2</sub>O<sub>2</sub></td><td>92.6 ± 1.04</td></tr><tr><td>c2</td><td>H<sub>3</sub>C<sub>2</sub>O<sub>2</sub></td><td>6.3 ± 1.02</td><td>c<br>7</td><td>N<sub>2</sub>O<sub>2</sub>N<sub>2</sub>O<sub>2</sub></td><td>101.7 ± 1.03</td></tr><tr><td>c3</td><td>H<sub>3</sub>C<sub>2</sub>N<sub>2</sub>O<sub>2</sub></td><td>43.6 ± 1.02</td><td>c<br>8</td><td>O<sub>2</sub>N<sub>2</sub>O<sub>2</sub></td><td>>128</td></tr><tr><td>c4</td><td>N<sub>2</sub>O<sub>2</sub></td><td>60.4 ± 1.01</td><td>c<br>9</td><td>H<sub>3</sub>C<sub>2</sub>O<sub>2</sub>S<sub>2</sub>O<sub>2</sub></td><td>63.5 ± 1.13</td></tr></tbody></table>

<figure><img src="image_11.png" /></figure>
<table><tr><td>c5</td><td>HN<sup>-2</sup></td><td>61.1 ± 1.00</td><td>c<br>1<br>0</td><td>H<sub>3</sub>C<sup>-2</sup>S<sup>-2</sup></td><td>91.4 ± 1.04</td></tr></table>

a L. amazonensis promastigotes. The IC₅₀ (µM/72h) values are means ± SD of three independent experiments.

On the other hand, the ester derivatives, compounds **c1** and **c2**, demonstrated interesting activity against the promastigote form of the parasites. In the interest of investigating an adequate bioactivity profile of the **c1** and **c2** molecules, a cytotoxicity test was carried out before evaluating their effectiveness against the amastigote form of the parasite, involved in human infection. (Table 4). Compound **c1** had a 50% cytotoxic concentration (CC₅₀) of 70.6 µM and compound **c2** had a 50% cytotoxic concentration (CC₅₀) of 112.4. Both compounds **c1** and **c2** demonstrated interesting activity against the amastigote form of the parasites with inhibitory concentrations (IC₅₀) of 4.7 and 6.3µM respectively. The selectivity index for **c1** was 30.7 and 87.8 for **c2**, which means that compounds **c1** and **c2** cause cytotoxicity in the host cell at a concentration that is 30.7 and 87.8 times higher than that required for a therapeutic effect, respectively.

Globally, no advantage was observed for the amide aliphatic derivatives compared to the sulphone HIT (IC₅₀ 5.4 ± 1.0 µM) or the drug pentamidine (IC₅₀ 5.7 ± 0.1 µM) as previously reported [10]. The alkyl derivatives of the sulfone group, compounds **c9** and **c10**, exhibited reduced activity compared to the same reference.

<table><caption>**Table 4.** Antileishmanial activity, cytotoxicity and selectivity index of the 4,5-dihydrofuran-3-carboxylate carrying an amidoxime group.</caption><thead><tr><th>Compound</th><th>Structure</th><th>Cytotoxicity<br>CC<sub>50</sub> (µM/72h)</th><th>Intracellular<br>Amastigotes<br>IC<sub>50</sub> (µM/72h)</th><th>SI=CC<sub>50</sub>/IC<sub>50</sub><br>Intracellular<br>Amastigotes</th></tr></thead><tbody><tr><td>c1</td><td>H<sub>3</sub>C<sup>-2</sup>O<sup>-2</sup></td><td>70.6 ± 6.1</td><td>2.3 ± 0.2</td><td>30.7</td></tr><tr><td>c2</td><td>H<sub>3</sub>C<sup>-2</sup>O<sup>-2</sup></td><td>112.4 ± 9.3</td><td>1.3 ± 0.2</td><td>87.8</td></tr></tbody></table>

<figure><img src="image_12.png" /></figure>

Both 4,5-dihydrofuran-carboxylate derivatives showed similar activity compared to the promastigote form. However, the derivative containing the ester group (IC₅₀ = 1.3 ± 0.2 µM) demonstrated promising antileishmanial activity against *L. amazonensis*, surpassing the biological activity of the Hit compound (IC₅₀ of intracellular amastigotes of *L. amazonensis* = 7.9 ± 1.1 µM). Furthermore, the selectivity index for the ethyl ester derivative increased significantly (SI=87.8), being almost 5 times higher than that of the HIT sulfone (SI=18.9). This highlights that the effectiveness of the 4,5-dihydrofuran structure is significantly influenced by the nature of the moiety at position 3. In contrast, other aliphatic derivatives of the amide or sulfone type showed a loss of activity. The physicochemical properties modulated by the ester group of the molecule may play a role in cellular interactions, for example about the intracellular form of the parasite, where membrane transfer capabilities are important. The ester group may offer better performance in this aspect or may undergo different metabolic pathways compared to the sulfone or amide groups.

### 3. Conclusion

Evaluation of the biological activity of the 4,5-dihydrofuran scaffold shows that the alkyl amides and sulfones have moderate antileishmanial activities against *L. amazonensis*. However, these results have prompted us to investigate
the carboxylate derivative, which has shown promising activity against both promastigotes and intracellular amastigotes. These results suggest the potential for further *in vivo* testing of these compounds.

# 4. Experimental

## 4.1. Chemistry

### 4.1.1. General

Reagents were purchased from Sigma-Aldrich (3050 Spruce Street St. Louis, MO, 63103, USA), Fluorochem (Unit 14 Graphite Way, Hadfield, Glossop SK13 1QH, UK), Fisher Scientific (168 3rd Ave, Waltham, MA 02451, USA), or TCI chemicals (9211 North Harborgate Street, Portland, OR 97203, USA) and used without further purification. Microwave reactions were performed using either monomode reactors: Biotage Initiator® classic (Uppsala, Sweden) in sealed vials with output power from 0 to 400 W or in a multimode microwave oven ETHOS Synth Lab Station (Ethos start, Milestone Inc.).

Reaction monitoring was performed either using Thin Layer Chromatography (TLC) on aluminum plates (5 × 5 cm) precoated with silica gel 60 F254 nm (Merck-F₂₅₄ or Alugram®: Macherey-Nagel) in an appropriate eluent and visualized using ultraviolet light under a UV-Lamp VL-6.CL., 254 nm (6 W) and 365 nm (6 W), either by LC-MS analyses on a Thermo Scientific Accela High Speed LC System® (168 3rd Ave, Waltham, MA, USA) coupled with a single quadrupole mass spectrometer Thermo MSQ Plus® (168 3rd Ave, Waltham, MA, USA), with an HPLC column Thermo Hypersil Gold® (168 3rd Ave, Waltham, MA 02451, USA) 50 × 2.1 mm (C18 bounded), with particles of a diameter of 1.9 mm. The volume of sample injected into the column was 1 µL. Chromatographic analysis, total duration of 8 min, was on the gradient of the following solvents: t = 0 min, methanol/water 50:50; 0 < t < 4 min, linear increase in the proportion of methanol to a methanol/water ratio of 95:5; 4 < t < 6 min, methanol/water 95:5; 6 < t < 7 min, linear decrease in the proportion of methanol to return to a methanol/water ratio of 50:50; 6 < t < 7 min, methanol/water 50:50. The water used was buffered with ammonium acetate 5 mM. The flow rate of the mobile phase was 0.3 mL/min, at the Faculté de Pharmacie of Marseille. The purity of synthesized compounds was checked by the relative area in the LC chromatogram, > 90% for intermediates and > 95% for tested compounds.

Melting points were determined on a Köfler melting point apparatus (Wagner & Munz GmbH, München, Germany) and were uncorrected.

Elemental analysis was performed on a Flash EA 1112 analyzer (Thermo Fisher Scientific, Finnigan, San Jose, CA, USA) con-trolled by Eager Xperience software - Ver. 1.2, under the conditions: temperature 970 °C, carrier gas helium, gas flow rate 140 mL/min, detector catharometer, performed at the Spectropole, Faculté des Sciences de Saint-Jérôme (Marseille, France).

The following adsorbent was used for column chromatography: silica gel 60 (Merck KGaA, Darmstadt, Germany, particle size 0.063–0.200 mm, 70–230 mesh ASTM or Macherey-Nagel GmbH & Co. KG, Düren, Germany, particle size 0.063–0.04 mm).

Flash chromatography were performed on a puriFlash® 5.020 apparatus (Interchim, Montluçon, France). PuriFlash® 5.020. Mode injection valve with solid deposit in dry-load PF-DEL-F004 with Celite 545 CAS 68855-54-9. Maximum pressure 15 bar, Flow rate 15 mL/min. UV-VIS detector configurated in 254 nm.
NMR spectra were recorded on a Bruker Avance NEO 400 MHz NanoBay spectrometer at the "Faculté de Pharmacie"
of Marseille. Residual ¹H and ¹³C peaks in deuterated solvent (CDCl₃) were used for chemical shift calibration without
the need for an additional internal standard. ¹H NMR: reference CDCl₃ δ = 7.26 ppm, reference DMSO-d₆ δ = 2.50
ppm and ¹³C NMR: reference CDCl₃ δ = 77.16 ppm, reference DMSO-d₆ δ = 39.52 ppm. Data for ¹H NMR are
reported as follows: chemical shifts (δ) in parts per million (ppm), multiplicity (described as follows: s, singlet; br s,
broad singlet; d, doublet; t, triplet; q, quadruplet; dd, doublet of doublet; ddd, doublet of doublet of doublet; m,
multiplet), coupling constants (J) in Hertz (Hz) and integration. Data for ¹³C NMR are reported as follows: chemical
shifts (δ) in parts per million (ppm).

4.1.2. Methyl 3-(4-cyanophenyl)-3-oxopropanoate [55] (a1)

To a 100 mL two-neck flask charged with a suspension of 60% NaH in THF (5.6 g, 0.14 mmol) in 8.4 mL (100 mmol) dimethyl carbonate (DMC) was added dropwise acetophenone (7.25 g, 50 mmol) in 10 mL dimethyl carbonate. The resulting solution was refluxed at 100° C for 2h. After the indicated time, the reaction mixture was quenched with NH4Cl, extracted with EtOAc. The organic layers were combined, washed with water (1 x 20 mL), brine (1 x 20 mL), dried over Na2SO4 and concentrated. The crude product was purified by column chromatography (silica gel; eluent: AcOEt/Petroleum ether, 30/70). The analytical data are consistent with those in previous literature [56]. The product was obtained in the enol form, as a white solid. Yield 80%. C11H9NO3 ¹H NMR (400 MHz, CDCl3): δ (ppm) 12.46 (s, 1H, OH), 7.87 (d, ³JH-H= 8.4 Hz, 2H, 2CHAr), 7.72 (d, ³JH-H= 8.4 Hz, 2H, 2CHAr), 5.72 (s, 1H, CH), 3.83 (s, 3H, CH3).

**4.1.3. General procedure for preparation of linear β-ketoamides**

A microwave vial of 20 mL equipped with a stirring bar was charged with ethyl 3-(4-cyanophenyl)-3-oxopropanoate (500 mg, 2.3 mmol, 1.0 equiv.), dimethylaminopyridine – DMAP (0.1 equiv.) and respective amine (2.0 equiv.), in 15 mL of toluene. The reaction mixture was stirred for 3 h at 150 °C under microwave irradiation. After 3 h solvent was evaporated under vacuum and then the mixture was purified by column chromatography on silica gel in appropriate solvent, affording the title product.

4.1.3.1. 3-(4-Cyanophenyl)-N-cyclohexyl-3-oxopropanamide (a3)

Compound **a3** was synthesized following the former general procedure, using cyclohexanamine (456 mg, 4.6 mmol, 2 equiv.). The crude product was purified by column chromatography (silica gel; eluent: dichloromethane/methanol from 100/0 to 97/3). The product was obtained as a mixture of keto and enol forms as a light orange solid. Yield 71%. M.p. 132°C. The ratio of 1/1 Keto/Enol was estimated according to ¹H NMR integration. ¹H NMR (400 MHz, CDCl₃): δ (ppm) 14.36 (s, 1H, OH), 8.12-8.09 (m, 2H, 2CHAr), 7.84-7.77 (m, 4H, 4CHAr, keto+enol), 7.71-7.65 (m, 2H, 2CHAr), 6.58 (br s, 1H, NH), 5.49 (s, 1H, CH, enol), 5.30 (br s, 1H, NH), 3.91 (s, 2H, CH₂, keto), 3.86-3.74 (m, 2H, 2CH, keto+enol), 2.01-1.87 (m, 4H, 2CH₂, keto+enol), 1.75-1.58 (m, 6H, 3CH₂, keto+enol), 1.42-1.34 (m, 4H, 2CH₂, keto+enol), 1.23-1.14 (m, 6H, 3CH₂, keto+enol). ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 194.7 (C), 170.7 (C), 167.0 (C), 163.9 (C), 139.2 (C), 138.8 (C), 132.8 (2CHAr), 132.4 (2CHAr), 129.2 (2CHAr), 126.3 (2CHAr), 118.5 (C), 117.8 (C), 117.3 (C), 113.9 (C), 91.1 (CH), 48.8 (CH), 48.3 (CH), 46.8 (CH₂), 33.3 (2CH₂), 33.0 (2CH₂), 25.6 (2CH₂), 24.9 (2CH₂), 24.8 (2CH₂). Anal. calcd for C₁₆H₁₈N₂O₂ (270.33): C 71.09, H 6.71, N 10.36; found C 71.38, H 6.68, N 10.33.

4.1.3.2. 3-(4-Cyanophenyl)-N,N-diethyl-3-oxopropanamide (a4)

Compound **a4** was synthesized following the former general procedure, using diethylamine (336 mg, 4.6 mmol, 2 equiv.). The crude product was purified by column chromatography (silica gel; eluent: dichloromethane/methanol from 100/0 to 97/3). The product was obtained as a mixture of keto and enol forms as a light brown solid. Yield 78%. M.p. 67-68 °C. The Keto/Enol ratio of 1/5 was estimated according to ¹H NMR integration. ¹H NMR (400 MHz,
CDCl₃): δ (ppm) 15.64 (s, 1H, OH), 8.11 (d, ³Jₕ-H = 8.5 Hz, 2H, 2CHAr, keto), 7.86 (d, ³Jₕ-H = 8.5 Hz, 2H, 2CHAr, enol), 7.76 (d, ³Jₕ-H = 8.5 Hz, 2H, 2CHAr, keto), 7.68 (d, ³Jₕ-H = 8.6 Hz, 2H, 2CHAr, enol), 5.78 (s, 1H, CH, enol), 4.04 (s, 2H, CH₂, keto), 3.54-3.36 (m, 8H, 4CH₂, keto+enol), 1.32-1.17 (m, 12H, 4CH₃, keto+enol). ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 193.3 (C), 171.0 (C), 169.1 (C), 165.4 (C), 139.6 (C), 139.5 (C), 132.6 (2CHAr), 132.3 (2CHAr), 129.4 (2CHAr), 126.6 (2CHAr), 118.6 (C), 118.0 (C), 116.7 (C), 113.8 (C), 86.9 (CH), 45.8 (CH₂), 42.9 (CH₂), 42.5 (CH₂), 40.8 (CH₂), 40.5 (CH₂) 14.5 (CH₃), 14.4 (CH₃), 13.4 (CH₃), 13.0 (CH₃). Analysis calculated for C₁₄H₁₆N₂O₂: C 68.83, H 6.60, N 11.47; found C 68.58, H 6.41, N 11.20. The NMR data were in agreement with the literature values [57].

#### 4.1.3.3. N-Butyl-3-(4-cyanophenyl)-3-oxopropanamide (a6)

Compound a6 was synthesized following the former general procedure, with butan-1-amine (336 mg, 4.6 mmol, 2 equiv.). The crude product was purified by column chromatography (silica gel; eluent: dichloromethane/methanol from 100/0 to 97/3). The title product was obtained as a mixture of keto and enol forms as a light brown solid. Yield 63%. M.p. 135-136 °C. The Keto/Enol ratio of 1/1 was estimated according to ¹H NMR integration. ¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.07 (d, ³Jₕ-_H = 8.5 Hz, 2H, 2CHAr, keto), 7.84-7.72 (m, 4H, 4CHAr, keto+enol), 7.63 (d, ³Jₕ-_H = 8.5 Hz, 2H, 2CHAr, enol), 6.91 (s, 1H, NH), 5.90 (s, 1H, NH), 5.56 (s, 1H, CH, enol), 3.94 (s, 2H, CH₂, keto), 3.37-3.20 (m, 4H, 2CH₂, keto+enol), 1.59-1.43 (m, 4H, 2CH₂ keto+enol), 1.39-1.28 (m, 4H, 2CH₂, keto+enol), 0.95-0.82 (m, 6H, 2CH₃, keto+enol). OH not observed. ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 194.4 (C), 171.5 (C), 166.5 (C), 165.2 (C), 139.1 (C), 138.8 (C), 132.7 (2CHAr), 132.3 (2CHAr), 129.1 (2CHAr), 126.3 (2CHAr), 118.5 (C), 117.8 (C), 117.0 (C), 113.6 (C), 91.0 (CH), 46.4 (CH₂), 39.7 (CH₂), 39.1 (CH₂), 31.6 (CH₂), 31.4 (CH₂), 20.0 (2CH₂), 13.7 (2CH₃). Analysis calculated for C₁₄H₁₆N₂O₂: C 68.83, H 6.60, N 11.47; found C 68.84, H 6.72, N 11.24.

#### 4.1.3.4. 3-(4-Cyanophenyl)-3-oxo-N-(2-(pyrrolidin-1-yl)ethyl)propenamide (a7)

Compound a7 was synthesized following the former general procedure, with 2-(pyrrolidin-1-yl)ethanamine (525 mg, 4.6 mmol, 2 equiv.). The crude product was purified by column chromatography (silica gel; eluent: dichloromethane/methanol from 100/0 to 95/5). The product was obtained in the enol form (traces of keto form) as an oily brown solid. Yield 86%. ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.92 (br s, 2H, 2CHAr), 7.70 (d, ³Jₕ-_H = 8.1 Hz, 2H, 2CHAr), 6.76 (br s, 1H, CH, enol), 3.49-3.37 (m, 2H, CH₂), 2.65 (t, ³Jₕ-_H = 5.9 Hz, 2H, CH₂), 2.57-2.48 (m, 4H, 2CH₂), 1.84-1.75 (m, 4H, 2CH₂). NOH and NH not observed. ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 181.0 (C), 161.4 (C), 139.0 (2C), 132.5 (2CHAr), 128.2 (2CHAr), 118.3 (C), 89.9 (CH), 54.6 (CH₂), 53.9 (2CH₂), 38.0 (CH₂), 23.6 (2CH₂). HRMS (ESI +) *m/z* calcd for C₁₆H₁₉N₃O₂ [M+H]⁺ 286.1550; found 286.1558.

#### 4.1.3.5. 4-(3-Morpholino-3-oxopropanoyl)benzonitrile (a8)

Compound a8 was synthesized following the former general procedure, with morpholine (401 mg, 4.6 mmol, 2 equiv.). The crude product was purified by column chromatography (silica gel; eluent: dichloromethane/methanol from 100/0 to 97/5). The product was obtained as a mixture of keto and enol forms as a light-yellow solid. Yield 76%. M.p. 158-159 °C. The Keto/Enol ratio of 1/2 was estimated according to ¹H NMR integration. ¹H NMR (400 MHz, CDCl₃): δ (ppm) 15.25 (s, 1H, OH), 8.14 (d, ³Jₕ-_H = 8.5 Hz, 2H, 2CHAr, keto), 7.86 (d, ³Jₕ-_H = 8.5 Hz, 2H, 2CHAr, enol), 7.79 (d, ³Jₕ-_H = 8.5 Hz, 2H, 2CHAr, keto), 7.70 (d, ³Jₕ-_H = 8.5 Hz, 2H, 2CHAr, enol), 5.81 (s, 1H, CH, enol), 4.10 (s, 2H, CH₂, keto), 3.78-3.51 (m, 16H, 8CH₂, keto+enol). ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 192.7 (C), 170.6 (C), 169.7 (C), 164.6 (C), 139.2 (C), 139.1 (C), 132.7 (2CHAr), 132.4 (2CHAr), 129.4 (2CHAr), 126.6 (2CHAr), 118.5 (C), 117.9 (C), 117.2 (C), 114.2 (C), 86.0 (CH), 66.8 (2CH₂), 66.7 (2CH₂), 47.1 (CH₂), 45.9 (2CH₂), 42.6 (2CH₂). Analysis calculated for C₁₄H₁₄N₂O₃: C 65.11, H 5.46, N 10.85; found 64.96, H 5.31, N 10.73.
### 4.1.4. Preparation of linear β-ketosulfones

#### 4.1.4.1. 4-(2-(Butylsulfonyl)acetyl)benzonitrile (a9)

To a solution of 1-butanesulfonyl chloride (1.05 g, 6.7 mmol, 1.5 equiv.) in water (40 mL), sodium sulfite (851 mg, 6.7 mmol, 1.5 equiv.) and sodium bicarbonate (567 mg, 6.7 mmol, 1.5 equiv.) were added. The reaction mixture was heated under reflux in a microwave oven under irradiation at 500 W during 20 min. Then, an ethanolic solution of 4-(2-bromoacetyl)benzonitrile (1 g, 4.5 mmol, 1 equiv.) was added. Heating of the reaction mixture was continued for 10 min under the same conditions. After cooling, the reaction mixture was neutralized by adding diluted chlorhydric acid and the solid product obtained was filtered and crystallized from isopropanol. Yellow powder, yield 56%. M.p. 125°C. ¹H NMR (400 MHz, CDCl₃) δ (ppm) 8.13 (d, ³Jₐ⁻ = 8.6 Hz, 2H, 2HAr), 7.83 (d, ³Jₐ⁻ = 8.6 Hz, 2H, 2HAr), 4.57 (s, 2H, CH₂), 3.27-3.19 (m, 2H, CH₂), 1.94-1.83 (m, 2H, CH₂), 1.54-1.47 (m, 2H, CH₂), 0.99 (t, ³Jₐ⁻ = 7.50 Hz, 3H, CH₃); ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 188.5 (C), 138.7 (C), 132.9 (2CHAr), 129.9 (2CHAr), 117.9 (C), 117.6 (C), 60.0 (CH₂), 53.7 (CH₂), 24.0 (CH₂), 21.7 (CH₂), 13.6 (CH₃). Anal. calcd for C₁₃H₁₅NO₃S: C, 58.85; H, 5.70; N, 5.28; S, 12.08. Found: C, 58.51; H, 5.66; N, 5.21; S, 11.81.

#### 4.1.4.2. 4-(2-(Methylsulfonyl)acetyl)benzonitrile (a10)

Sodium sulfinate (MeSO₂Na, 228 mg, 2.2 mmol, 1.0 equiv.) was added to a solution of 4-(2-bromoacetyl)benzonitrile (500mg, 2.2 mmol, 1.0 equiv.) in a cosolvent of 1,4-dioxane and water (20 mL, v/v = 1:1) at rt. The reaction mixture was stirred at reflux for 6 h. The reaction mixture was cooled to rt and extracted with dichloromethane (3 x 40 mL). The organic extracts were collected and washed with saturated aqueous NaHCO₃ (3 x 40 mL) and dried over Na₂SO₄. Solvent was evaporated under reduced pressure and the crude product was purified by column chromatography (silica gel; eluent: AcOEt/Cyclohexane 1:1) affording a pale-yellow powder, yield 95%. M.p. 95°C. ¹H NMR (400 MHz, CDCl₃) δ (ppm) 8.12 (d, ³Jₐ⁻ = 8.7 Hz, 2H, 2HAr), 7.84 (d, ³Jₐ⁻ = 8.7 Hz, 2H, 2HAr), 4.61 (s, 2H, CH₂), 3.15 (s, 3H, CH₃); ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 188.4 (C), 138.5 (C), 132.9 (2CHAr), 129.8 (2CHAr), 118.0 (C), 117.6 (C), 61.7 (CH₂), 41.9 (CH₃). Anal. calcd for C₁₀H₉NO₃S: C, 53.80; H, 4.06; N, 6.27; S, 14.36. Found: C, 53.63; H, 4.01; N, 6.21; S, 14.19. The NMR data are consistent with those in previous literature [58].

### 4.1.5. General procedure for preparation of 4,5-dihydrofuran derivatives by microwave-assisted Mn(OAc)₃-based cyclization reaction

In a microwave vial of 20 mL equipped with a stirring bar, a solution of manganese (III) acetate dihydrate (2.1 equiv.) and copper(II) acetate (1 equiv.) in 12 mL of glacial acetic acid was heated at 80 °C under microwave irradiation 100W for 15 min. Then the reaction mixture was cooled and a solution of β-keto derivative (1 equiv.) and 2-methyl-3-phenyl-1-propene (2 equiv.) in 13 mL of acetic acid was added. The reaction mixture was heated for 3 h under microwave irradiations under the same conditions. The resulting product was poured into 50 mL of cold water and extracted with dichloromethane (3 x 40 mL). The organic extracts were collected and washed with saturated aqueous NaHCO₃ (3 x 40 mL) and dried over Na₂SO₄. Filtrate solvent was evaporated under reduced pressure and crude product was purified by flash chromatography using appropriate solvent affording the title product.

#### 4.1.5.1. Methyl 5-benzyl-2-(4-cyanophenyl)-5-methyl-4,5-dihydrofuran-3-carboxylate (b1).

Compound b1 was obtained following the former general procedure, with methyl 3-(4-cyanophenyl)-3-oxopropanoate (a1) (1 g, 4.9 mmol, 1 equiv.) and 2-methyl-3-phenyl-1-propene (1.3 g, 9.8 mmol, 2 equiv.). The reaction mixture was monitored by TLC (eluent: dichloromethane/methanol 95/5, Rf 0.46). The crude product was purified by column
chromatography (silica gel; eluent: dichloromethane/methanol from 100/0 to 98/2). The product was obtained as a white solid. Yield 40%. Mp 85 °C. ¹H NMR (400 MHz, CDCl₃) δ (ppm) 7.88 (d, ³Jₕ-H = 8.4 Hz, 2H, 2CHAr), 7.66 (d, ³Jₕ-H = 8.4 Hz, 2H, 2CHAr), 7.32 - 7.21 (m, 5H, 5CHAr), 3.64 (s, 3H, CH₃), 3.12 (d, ²Jₕ-H = 15.5 Hz, 1H, H-(CH₂)), 3.02 (s, 2H, CH₂), 2.83 (d, ²Jₕ-H = 15.5 Hz, 1H, H-(CH₂)), 1.47 (s, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 165.5 (C), 161.7 (C), 136.6 (C), 134.7 (C), 131.5 (2CHAr), 130.5 (2CHAr), 130.0 (2CHAr), 128.4 (2CHAr), 127.0 (CHAr), 118.7 (C), 113.6 (C), 104.1 (C), 88.3 (C), 51.3 (CH₃), 46.8 (CH₂), 42.2 (CH₂), 26.8 (CH₃). LC/MS ESI+ tr, 7.02 min, (m/z) calcd for C₂₁H₁₉NO₃ [M + Na]⁺ 356.11; found 356.38.

### 4.1.5.2. Ethyl 5-benzyl-2-(4-cyanophenyl)-5-methyl-4,5-dihydrofuran-3-carboxylate (b2).

Compound **b2** was obtained following the former general procedure, with ethyl 3-(4-cyanophenyl)-3-oxopropanoate (500 mg, 2.3 mmol, 1 equiv.) and 2-methyl-3-phenyl-1-propene (608 mg, 4.6 mmol, 2 equiv.). The reaction mixture was monitored by TLC (eluent: cyclohexane/AcOEt 70/30, Rf 0.69). The crude product was purified by flash chromatography (column PuriFlash® IR-20SI-F0025; eluent: cyclohexane/AcOEt from 100/0 to 80/20 in 20 CVs - Column Volume). UV-VIS detector was configurated additionally in 307 nm, 235 nm and 205 nm. The product was obtained as an oily white solid. Yield 69%. ¹H NMR (400 MHz, CDCl₃) δ (ppm) 7.85 (d, ³Jₕ-Η = 8.6 Hz, 2H, 2CHAr), 7.64 (d, ³Jₕ-Η = 8.6 Hz, 2H, 2CHAr), 7.30 -7.19 (m, 5H, 5CHAr), 4.08 (q, ³Jₕ-Η = 7.1 Hz, 2H, CH₂), 3.10 (d, ²Jₕ-Η = 15.5 Hz, 1H, H-(CH₂)), 3.05 (d, ²Jₕ-Η = 13.8 Hz, 1H, H-(CH₂)), 3.02 (d, ²Jₕ-Η = 13.8 Hz, 1H, H-(CH₂)), 2.82 (d, ²Jₕ-Η = 15.5 Hz, 1H, H-(CH₂)), 1.45 (s, 3H, CH₃), 1.17 (t, ³Jₕ-Η = 7.2 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 164.6 (C), 161.0 (C), 136.1 (C), 134.5 (C), 131.1 (2CHAr), 130.2 (2CHAr), 129.8 (2CHAr), 128.0 (2CHAr), 126.6 (CHAr), 118.3 (C), 113.2 (C), 104.2 (C), 87.8 (C), 59.7 (CH₂), 46.5 (CH₂), 42.1 (CH₂), 26.4 (CH₃), 14.0 (CH₃). HRMS (ESI +) *m/z* calcd for C₂₂H₂₁NO₃ [M+Na]⁺ 370.1414; found 370.1413.

### 4.1.5.3. 5-Benzyl-2-(4-cyanophenyl)-N-cyclohexyl-5-methyl-4,5-dihydrofuran-3-carboxamide (b3)

Compound **b3** was obtained following the former general procedure, with 3-(4-cyanophenyl)-N-cyclohexyl-3-oxopropanamide (**a3**) (450 mg, 1.7 mmol, 1 equiv.) and 2-methyl-3-phenyl-1-propene (440 mg, 3.4 mmol, 2 equiv.). The reaction mixture was monitored by TLC (eluent: dichloromethane/methanol 98/2, Rf 0.34). The crude product was purified by flash chromatography (column PuriFlash® IR-20SI-F0025; eluent: first, cyclohexane in 2 CVs - Column Volume, then dichloromethane/methanol from 100/0 to 98/2 in 20 CVs - Column Volume). UV-VIS detector was configurated additionally in 307 nm, 235 nm and 205 nm. The product was obtained as a light brown oil. Yield 35%. ¹H NMR (400 MHz, CDCl₃) δ (ppm) 7.82 (d, ³Jₕ-Η = 8.5 Hz, 2H, 2CHAr), 7.64 (d, ³Jₕ-Η = 8.5 Hz, 2H, 2CHAr), 7.32-7.23 (m, 5H, 5CHAr), 4.92 (d, ³Jₕ-Η = 8.2 Hz, 1H, NH), 3.79-3.69 (m, 1H, CH), 3.04 (d, ²Jₕ-Η = 14.8 Hz, 1H, H-(CH₂)), 3.02 (d, ²Jₕ-Η = 14.2 Hz, 1H, H-(CH₂)), 2.98 (d, ²Jₕ-Η = 14.2 Hz, 1H, H-(CH₂)), 2.83 (d, ²Jₕ-Η = 14.8 Hz, 1H, H-(CH₂)), 1.89-1.82 (m, 2H, CH₂), 1.69-1.63 (m, 2H, CH₂), 1.49 (s, 3H, CH₃), 1.38-1.30 (m, 2H, CH₂), 1.17-1.09 (m, 2H, CH₂), 1.06-0.95 (m, 2H, CH₂). ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 164.0 (C), 156.0 (C), 136.5 (C), 134.9 (C), 131.7 (2CHAr), 130.4 (2CHAr), 129.6 (2CHAr), 128.3 (2CHAr), 127.0 (CHAr), 118.7 (C), 113.1 (C), 107.4 (C), 86.9 (C), 48.2 (CH), 47.0 (CH₂), 43.01 (CH₂), 33.2 (CH₂), 33.1 (CH₂), 27.0 (CH₂), 26.9 (CH₃), 25.6 (CH₂), 24.9 (CH₂). HRMS (ESI +) *m/z* calcd for C₂₆H₂₈N₂O₂ [M+H]⁺ 401.2224; found 401.2228.

### 4.1.5.4. 5-Benzyl-2-(4-cyanophenyl)-N,N-diethyl-5-methyl-4,5-dihydrofuran-3-carboxamide (b4)

Compound **b4** was obtained following the former general procedure, with 3-(4-Cyanophenyl)-N,N-diethyl-3-oxopropanamide (**a4**) (440 mg, 1.8 mmol, 1 equiv.) and 2-methyl-3-phenyl-1-propene (476 mg, 3.6 mmol, 2 equiv.). The reaction mixture was monitored by TLC (eluent: dichloromethane/methanol 98/2, Rf 0.38). The crude product was purified by flash chromatography (column PuriFlash® IR-20SI-F0025; eluent: first, cyclohexane in 2 CVs -
Column Volume, then dichloromethane/methanol from 100/0 to 98/2 in 20 CVs - Column Volume). UV-VIS detector was configurated additionally in 365 nm, and 254 nm. The product was obtained as a light brown oil. Yield 35%. ¹H NMR (400 MHz, CDCl₃) δ (ppm) 7.64 (d, ³Jₕ-H = 7.6 Hz, 2H, 2CHAr), 7.60 (d, ³Jₕ-H = 7.5 Hz, 2H, 2CHAr), 7.25-7.29 (m, 5H, 5CHAr), 3.51-3.27 (m, 2H, CH₂), 3.10 (d, ²Jₕ-H = 13.8 Hz, 1H, H-(CH₂)), 3.01 (d, ²Jₕ-H = 16.1 Hz, 1H, H-(CH₂)), 2.91 (d, ²Jₕ-H = 13.8 Hz, 1H, H-(CH₂)), 2.87 (d, ²Jₕ-H = 16.1 Hz, 1H, H-(CH₂)), 2.79-2.54 (m, 2H, CH₂), 1.52 (s, 3H, CH₃), 1.12 (br s, 3H, CH₃), 0.69 (br s, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 166.9 (C), 148.1 (C), 136.9 (C), 134.8 (C), 132.2 (2CHAr), 130.7 (2CHAr), 128.3 (2CHAr), 127.1 (2CHAr), 126.9 (CHAr), 118.7 (C), 112.3 (C), 108.2 (C), 86.1 (C), 47.0 (CH₂), 44.7 (CH₂), 42.5 (CH₂), 38.9 (CH₂), 27.9 (CH₃), 14.4 (CH₃), 12.5 (CH₃). LC/MS ESI+ tr, 6.10 min, (m/z) calcd for [M + H]⁺ 375.15; found 375.48.

### 4.1.5.5. 5-Benzyl-N-butyl-2-(4-cyanophenyl)-5-methyl-4,5-dihydrofuran-3-carboxamide (b6)

Compound **b6** was obtained following the former general procedure, with *N*-butyl-3-(4-cyanophenyl)-3-oxopropanamide (**a6**) (440 mg, 1.8 mmol, 1 equiv.) and 2-methyl-3-phenyl-1-propene (476 mg, 3.6 mmol, 2 equiv.). The reaction mixture was monitored by TLC (eluent: dichloromethane/methanol 97/3, Rf 0.35). The crude product was purified by flash chromatography (column PuriFlash® IR-20SI-F0025; eluent: first, cyclohexane in 2 CVs - Column Volume, then dichloromethane/methanol from 100/0 to 98/2 in 20 CVs - Column Volume). UV-VIS detector was configurated additionally in 365 nm, and 254 nm. The product was obtained as a light brown oil. Yield 35%. ¹H NMR (400 MHz, CDCl₃) δ (ppm) 7.84 (d, ³Jₕ-Η = 8.4 Hz, 2H, 2CHAr), 7.65 (d, ³Jₕ-Η = 8.4 Hz, 2H, 2CHAr), 7.32-7.23 (m, 5H, 5CHAr), 5.12 (br s, 1H, NH), 3.26-3.19 (m, 2H, CH₂), 3.05 (d, ²Jₕ-Η = 14.7 Hz, 1H, H-(CH₂)), 3.03 (d, ²Jₕ-Η = 13.9 Hz, 1H, H-(CH₂)), 3.00 (d, ²Jₕ-Η = 13.9 Hz, 1H, H-(CH₂)), 2.82 (d, ²Jₕ-Η = 14.7 Hz, 1H, H-(CH₂)), 1.50 (s, 3H, CH₃), 1.44-1.39 (m, 2H, CH₂), 1.32-1.23 (m, 2H, CH₂), 0.91 (t, ³Jₕ-Η = 7.30 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 164.8 (C), 156.5 (C), 136.4 (C), 134.9 (C), 131.7 (2CHAr), 130.4 (2CHAr), 129.6 (2CHAr), 128.3 (2CHAr), 127.0 (CHAr), 118.7 (C), 113.1 (C), 106.9 (C), 87.0 (C), 46.9 (CH₂), 42.9 (CH₂), 39.3 (CH₂), 31.6 (CH₂), 26.9 (CH₃), 20.2 (CH₂), 13.8 (CH₃). HRMS (ESI +) *m/z* calcd for C₂₄H₂₆N₂O₂ [M+H]⁺ 375.2067; found 375.2065.

### 4.1.5.6. 5-Benzyl-2-(4-cyanophenyl)-5-methyl-N-(2-(pyrrolidin-1-yl)ethyl)-4,5-dihydrofuran-3-carboxamide (b7)

Compound **b7** was obtained following the former general procedure, with 3-(4-cyanophenyl)-3-oxo-N-(2-(pyrrolidin-1-yl)ethyl)propenamide (**a7**) (566 mg, 1.98 mmol, 1 equiv.) and 2-methyl-3-phenyl-1-propene (520 mg, 3.96 mmol, 2 equiv.). The reaction mixture was monitored by TLC (eluent: dichloromethane/methanol 95/5, Rf 0.31). The crude product was purified by column chromatography (silica gel; eluent: dichloromethane/methanol/TEA from 100/0/0 to 88/10/2). The title product was obtained as a light brown oil. Yield 70%. ¹H NMR (400 MHz, CDCl₃) δ (ppm) 7.84 (d, ³Jₕ-Η = 8.5 Hz, 2H, 2CHAr), 7.63 (d, ³Jₕ-Η = 8.6 Hz, 2H, 2CHAr), 7.30-7.22 (m, 5H, 5CHAr), 6.78 (br s, 1H, NH), 3.45-3.44 (m, 2H, CH₂), 3.11 (d, ²Jₕ-Η = 14.9 Hz, 1H, H-(CH₂)), 3.02 (d, ²Jₕ-Η = 13.8 Hz, 1H, H-(CH₂)), 2.98 (d, ²Jₕ-Η = 13.8 Hz, 1H, H-(CH₂)), 2.86 (d, ²Jₕ-Η = 14.9 Hz, 1H, H-(CH₂)), 2.80-2.76 (m, 6H, 3CH₂), 1.86-1.83 (m, 4H, 2CH₂), 1.46 (s, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 165.0 (C), 157.6 (C), 136.5 (C), 135.3 (C), 131.6 (2CHAr), 130.6 (2CHAr), 129.9 (2CHAr), 128.3 (2CHAr), 126.9 (CHAr), 118.8 (C), 113.1 (C), 106.8 (C), 87.2 (C), 55.1 (CH₂), 54.0 (2CH₂), 46.9 (CH₂), 42.7 (CH₂), 37.2 (CH₂), 26.8 (CH₃), 23.5 (2CH₂). HRMS (ESI +) *m/z* calcd for C₂₆H₂₉N₃O₂ [M+H]⁺ 416.2333; found 416.2330.

### 4.1.5.7. 4-(5-Benzyl-3-(butylsulfonyl)-5-methyl-4,5-dihydrofuran-2-yl)benzonitrile (b9)

Compound **b9** was obtained following the former general procedure, with 4-(2-(butylsulfonyl)acetyl)benzonitrile (**a9**) (600 mg, 2.26 mmol, 1 equiv.) and 2-methyl-3-phenyl-1-propene (593 mg, 4.52 mmol, 2 equiv.). The reaction mixture was monitored by TLC (eluent: dichloromethane/methanol 97/3, Rf 0.25). The crude product was purified by column
chromatography (silica gel; eluent: dichloromethane/methanol from 100/0 to 97/3). The title product was obtained as a light brown oil. Yield 40%. ¹H NMR (400 MHz, CDCl₃) δ (ppm) 7.76 (d, ³Jₕ-H = 8.5 Hz, 2H, 2CHAr), 7.56 (d, ³Jₕ-H = 8.5 Hz, 2H, 2CHAr), 7.34-7.24 (m, 5H, 5CHAr), 3.17 (d, ²Jₕ-H = 15.1 Hz, 1H, H-(CH₂)), 3.14 (d, ²Jₕ-H = 14.5 Hz, 1H, H-(CH₂)), 2.98 (d, ²Jₕ-H = 15.1 Hz, 1H, H-(CH₂)), 2.92 (d, ²Jₕ-H = 14.5 Hz, 1H, H-(CH₂)), 2.65-2.56 (m, 2H, CH₂), 1.59 (s, 3H, CH₃), 1.42-1.32 (m, 2H, CH₂), 1.28-1.20 (m, 2H, CH₂), 0.81 (t, ³Jₕ-H = 7.1 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 160.6 (C), 135.8 (C), 133.2 (C), 131.8 (2CHAr), 130.6 (2CHAr), 130.1 (2CHAr), 128.6 (2CHAr), 127.4 (CHAr), 118.3 (C), 114.5 (C), 110.3 (C), 88.7 (C), 54.5 (CH₂), 46.8 (CH₂), 42.2 (CH₂), 27.8 (CH₃), 24.0 (CH₂), 21.6 (CH₂), 13.6 (CH₃). LC/MS ESI+ tr, 4.18 min, (m/z) calcd for C₂₃H₂₅NO₃S [M + Na]⁺ 418.30; found. 418.51.

### 4.1.5.8. 4-(5-Benzyl-5-methyl-3-(methylsulfonyl)-4,5-dihydrofuran-2-yl)benzonitrile (b10)

Compound b10 was obtained following the former general procedure, with 4-(2-(methylsulfonyl)acetyl)benzonitrile (a10) (135 mg, 0.60 mmol, 1 equiv.) and 2-methyl-3-phenyl-1-propene (160 mg, 1.2 mmol, 2 equiv.). The reaction mixture was monitored by TLC (eluent: dichloromethane/methanol 95/5, Rf 0.31). The crude product was purified by column chromatography (silica gel; eluent: dichloromethane/methanol from 100/0 to 95/5). The title product was obtained as a white oily powder. Yield 60%. ¹H NMR (400 MHz, CDCl₃) δ (ppm) 7.75 (d, ³Jₕ-H = 8.6 Hz, 2H, 2CHAr), 7.68 (d, ³Jₕ-H = 8.6 Hz, 2H, 2CHAr), 7.33-7.23 (m, 5H, 5CHAr), 3.21 (d, ²Jₕ-H = 15.1 Hz, 1H, H-(CH₂)), 3.11 (d, ²Jₕ-H = 13.9 Hz, 1H, H-(CH₂)), 3.03 (d, ²Jₕ-H = 15.1 Hz, 1H, H-(CH₂)), 2.92 (d, ²Jₕ-H = 13.9 Hz, 1H, H-(CH₂)), 2.57 (s, 3H, CH₃), 1.60 (s, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 160.0 (C), 135.7 (C), 132.8 (C), 131.7 (2CHAr), 130.4 (2CHAr), 130.0 (2CHAr), 128.4 (2CHAr), 127.3 (CHAr), 118.2 (C), 114.3 (C), 111.3 (C), 88.7 (C), 46.9 (CH₃), 43.0 (CH₂), 42.0 (CH₂), 27.5 (CH₃). LC/MS ESI+ tr, 4.22 min, (m/z) calcd for C₂₀H₁₉NO₃S [M + H]⁺ calculated 354.29; found 354.44.

### 4.1.6. Procedure for preparation of compound b5 and b8 by amidation from cyclized carboxylate

A microwave vial of 5 mL equipped with a stirring bar was charged with ethyl 5-benzyl-2-(4-cyanophenyl)-5-methyl-4,5-dihydrofuran-3-carboxylate (100 mg, 0.29 mmol, 1.0 equiv.), the respective amine (1.5 equiv.), then placed under a positive pressure of nitrogen, and subjected to three evacuation/backfilling cycles. Toluene (2 mL), LiHMDS (1.0 M in THF, 2.5 equiv.) were sequentially added with vigorous stirring at room temperature, and the reaction mixture was stirred for 15 h at 70 °C. After the indicated time, the reaction mixture was quenched with NH₄Cl (1.0 M, 5 mL), extracted with EtOAc (3 x 15 mL), the organic layers were combined, washed with water (1 x 20 mL), brine (1 x 20 mL), dried over Na₂SO₄ and concentrated. Purification by flash chromatography using appropriate solvent afforded the title product.

#### 4.1.6.1. 5-Benzyl-2-(4-cyanophenyl)-N-cyclopentyl-5-methyl-4,5-dihydrofuran-3-carboxamide (b5)

Compound b5 was obtained following the former general procedure, from cyclopentanamine (37 mg, 0.43 mmol, 1.5 equiv.). The reaction mixture was monitored by TLC (eluent: cyclohexane/AcOEt 70/30, Rf 0.37). The crude product was purified by flash chromatography (column PuriFlash® IR-50SI-F0025; eluent: cyclohexane/AcOEt from 100/0 to 80/20 in 25 CVs - Column Volume). Yield 52%. The product was obtained as a light brown oil. ¹H NMR (400 MHz, CDCl₃) δ (ppm) 7.82 (d, ³Jₕ-H = 8.5 Hz, 2H, 2CHAr), 7.63 (d, ³Jₕ-H = 8.5 Hz, 2H, 2CHAr), 7.35-7.21 (m, 5H, 5CHAr), 4.98 (d, ³Jₕ-H = 8.2 Hz, 1H, NH), 4.22-4.14 (m, 1H, CH), 3.04 (d, ²Jₕ-H = 14.8 Hz, 1H, H-(CH₂)), 3.02 (d, ²Jₕ-H = 14.1 Hz, 1H, H-(CH₂)), 2.99 (d, ²Jₕ-H = 14.1 Hz, 1H, H-(CH₂)), 2.83 (d, ²Jₕ-H = 14.8 Hz, 1H, H-(CH₂)), 1.97-1.90 (m, 2H, CH₂), 1.61-1.54 (m, 4H, 2CH₂), 1.49 (s, 3H, CH₃), 1.28-1.20 (m, 2H, CH₂). ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 164.5 (C), 158.7 (C), 156.0 (C), 136.6 (C), 131.8 (2CHAr), 130.5 (2CHAr), 129.6 (2CHAr), 128.3 (2CHAr), 127.0 (CH₂).
(CH<sub>Ar</sub>), 118.7 (C), 113.2 (C), 107.4 (C), 87.0 (C), 51.2 (CH), 47.0 (CH<sub>2</sub>), 43.1 (CH<sub>2</sub>), 33.1 (CH<sub>2</sub>), 33.0 (CH<sub>2</sub>), 26.9 (CH<sub>3</sub>), 23.8 (2CH<sub>2</sub>). LC/MS ESI+ tr, 6.22 min, (m/z) calcd for C<sub>25</sub>H<sub>26</sub>N<sub>2</sub>O<sub>2</sub> [M + H]<sup>+</sup> 387.22; found 387.50.

4.1.6.2. 4-(5-Benzyl-5-methyl-3-(morpholine-4-carbonyl)-4,5-dihydrofuran-2-yl)benzonitrile (b8)

Compound **b8** was obtained following the former general procedure, from morpholine (38 mg, 0.43 mmol, 1.5 equiv.). The reaction mixture was monitored by TLC (eluent: cyclohexane/AcOEt 40/60, Rf 0.19). The crude product was purified by flash chromatography (column PuriFlash® IR-50SI-F0025; eluent: cyclohexane/AcOEt from 100/0 to 70/30 in 25 CVs - Column Volume). Yield 69%. The product was obtained as a light brown oil. ¹H NMR (400 MHz, CDCl<sub>3</sub>) δ (ppm) 7.63 (d, <sup>3</sup>J<sub>H-H</sub> = 8.7 Hz, 2H, 2CH<sub>Ar</sub>), 7.58 (d, <sup>3</sup>J<sub>H-H</sub> = 8.5 Hz, 2H, 2CH<sub>Ar</sub>), 7.31-7.17 (m, 5H, 5CH<sub>Ar</sub>), 3.78-3.26 (m, 4H, 2CH<sub>2</sub>), 3.16 (d, <sup>2</sup>J<sub>H-H</sub> = 13.8 Hz, 1H, H-(CH<sub>2</sub>)), 2.99 (d, <sup>2</sup>J<sub>H-H</sub> = 16.5 Hz, 1H, H-(CH<sub>2</sub>)), 2.94 (d, <sup>2</sup>J<sub>H-H</sub> = 16.5 Hz, 1H, H-(CH<sub>2</sub>)), 2.80 (d, <sup>2</sup>J<sub>H-H</sub> = 13.8 Hz, 1H, H-(CH<sub>2</sub>)), 1.59 (s, 3H, CH<sub>3</sub>), 1.30-1.16 (m, 4H, 2CH<sub>2</sub>). <sup>13</sup>C NMR (100 MHz, CDCl<sub>3</sub>) δ (ppm) 166.2 (C), 149.8 (C), 136.9 (C), 134.4 (C), 132.3 (2CH<sub>Ar</sub>), 130.7 (2CH<sub>Ar</sub>), 128.3 (2CH<sub>Ar</sub>), 127.3 (2CH<sub>Ar</sub>), 126.9 (CH<sub>Ar</sub>), 118.5 (C), 112.8 (C), 106.3 (C), 86.4 (C), 66.6 (2CH<sub>2</sub>), 47.3 (CH<sub>2</sub>), 43.8 (CH<sub>2</sub>), 29.8 (2CH<sub>2</sub>), 28.8 (CH<sub>3</sub>). HRMS (ESI +) *m/z* calcd for C<sub>24</sub>H<sub>24</sub>N<sub>2</sub>O<sub>3</sub> [M+H]<sup>+</sup> 389.1860; found 389.1858.

4.1.7. General procedure for amidoxime synthesis from nitriles

A suspension of hydroxylamine hydrochloride (10 equiv.) in DMSO was stirred under inert atmosphere and cooled to 0 °C. Potassium *tert*-butoxide (10 equiv.) was added gradually and the reaction mixture was stirred for 30 min. Then the corresponding nitrile was added (1 equiv.) and the reaction mixture was stirred for 18 h at room temperature. The resulting mixture was poured into cold water, then the reaction mixture was extracted with EtOAc (3 x 15 mL), the organic layers were combined, washed with water (1 x 20 mL), brine (1 x 20 mL), dried over Na<sub>2</sub>SO<sub>4</sub> and concentrated. Purification by flash or column chromatography using the appropriate solvent afforded the title product.

4.1.7.1. Methyl 5-benzyl-2-(4-(N'-hydroxycarbamimidoyl)phenyl)-5-methyl-4,5-dihydrofuran-3-carboxylate (**c1**).

Compound **c1** was obtained following the former general procedure from **b1** (105 mg, 0.31 mmol, 1 equiv.). The reaction mixture was monitored by TLC (eluent: dichloromethane/MeOH 94/6, Rf 0.72). The crude product was purified by flash chromatography (column PuriFlash® IR-50SI-F0025; eluent: dichloromethane/MeOH 97/3 from 99/1 to 95/5 in 25 CVs - Column Volume). UV-VIS detector was configurated in 242 nm. Yield 65%. The product was obtained as a light grey oily solid. ¹H NMR (400 MHz, CDCl<sub>3</sub>) δ (ppm) 7.80 (d, <sup>3</sup>J<sub>H-H</sub> = 8.3 Hz, 2H, 2CH<sub>Ar</sub>), 7.65 (d, <sup>3</sup>J<sub>H-H</sub> = 8.5 Hz, 2H, 2CH<sub>Ar</sub>), 7.32-7.23 (m, 5H, 5CH<sub>Ar</sub>), 4.89 (br s, 2H, NH<sub>2</sub>), 3.63 (s, 3H, CH<sub>3</sub>), 3.11 (d, <sup>2</sup>J<sub>H-H</sub> = 15.1 Hz, 1H, H-(CH<sub>2</sub>)), 3.04 (d, <sup>2</sup>J<sub>H-H</sub> = 13.8 Hz, 1H, H-(CH<sub>2</sub>)), 3.01 (d, <sup>2</sup>J<sub>H-H</sub> = 13.8 Hz, 1H, H-(CH<sub>2</sub>)), 2.82 (d, <sup>2</sup>J<sub>H-H</sub> = 15.1 Hz, 1H, H-(CH<sub>2</sub>)), 1.46 (s, 3H, CH<sub>3</sub>). OH not observed. <sup>13</sup>C NMR (100 MHz, CDCl<sub>3</sub>) δ (ppm) 165.9 (C), 163.3 (C), 136.6 (C), 134.0 (C), 132.0 (C), 130.6 (2CH<sub>Ar</sub>), 129.7 (2CH<sub>Ar</sub>), 128.3 (2CH<sub>Ar</sub>), 126.9 (CH<sub>Ar</sub>), 126.1 (C), 125.2 (2CH<sub>Ar</sub>), 102.4 (C), 87.8 (C), 51.1 (CH<sub>3</sub>), 46.8 (CH<sub>2</sub>), 42.3 (CH<sub>2</sub>), 26.7 (CH<sub>3</sub>). HRMS (ESI +) *m/z* calcd for C<sub>21</sub>H<sub>22</sub>N<sub>2</sub>O<sub>4</sub> [M+H]<sup>+</sup> calculated 367.1652; found 367.1653.

4.1.7.2. Ethyl 5-benzyl-2-(4-(N'-hydroxycarbamimidoyl)phenyl)-5-methyl-4,5-dihydrofuran-3-carboxylate (**c2**)

Compound **c2** was obtained following the former general procedure from **b2** (60 mg, 0.17 mmol, 1 equiv.). The reaction mixture was monitored by TLC (eluent: dichloromethane/MeOH 94/6, Rf 0.65). The crude product was purified by flash chromatography (column PuriFlash® IR-50SI-F0025; eluent: dichloromethane/MeOH 97/3 from 99/1 to 95/5 in 25 CVs - Column Volume). UV-VIS detector was configurated in 242 nm. Yield 77%. The product was obtained as an oily white solid. ¹H NMR (400 MHz, CDCl<sub>3</sub>) δ (ppm) 7.75 (d, <sup>3</sup>J<sub>H-H</sub> = 8.6 Hz, 2H, 2CH<sub>Ar</sub>), 7.64 (d, <sup>3</sup>J<sub>H-H</sub> = 8.6 Hz, 2H, 2CH<sub>Ar</sub>), 7.30-7.19 (m, 5H, 5CH<sub>Ar</sub>), 6.03 (br s, 2H, NH<sub>2</sub>), 4.06 (q, <sup>3</sup>J<sub>H-H</sub> = 7.1 Hz, 2H, CH<sub>2</sub>),
3.07 (d, $^2J_{H-H}$ = 15.3 Hz, 1H, H-(CH₂)), 3.02 (d, $^2J_{H-H}$ = 13.8 Hz, 1H, H-(CH₂)), 2.97 (d, $^2J_{H-H}$ = 13.8 Hz, 1H, H-(CH₂)), 2.78 (d, $^2J_{H-H}$ = 15.3 Hz, 1H, H-(CH₂)), 1.42 (s, 3H, CH₃), 1.17 (t, $^3J_{H-H}$ = 7.2 Hz, 3H, CH₃). OH not observed. ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 165.5 (C), 162.8 (C), 154.8 (C), 136.5 (C), 133.4 (C), 130.7 (C), 130.6 (2CHAr), 129.9 (2CHAr), 128.3 (2CHAr), 126.9 (CHAr), 126.1 (2CHAr), 103.2 (C), 88.0 (C), 60.1 (CH₂), 46.8 (CH₂), 42.2 (CH₂), 26.6 (CH₃), 14.4 (CH₃). HRMS (ESI +) *m/z* calcd for C₂₂H₂₄N₂O₄ [M+Na]⁺ 403.1624; found 403.1628.

### 4.1.7.3. 5-Benzyl-*N*-cyclohexyl-2-(4-(*N*'-hydroxycarbamimidoyl)phenyl)-5-methyl-4,5-dihydrofuran-3-carboxamide (c3)

Compound c3 was obtained following the former general procedure from **b3** (200 mg, 0.50 mmol, 1 equiv.). The reaction mixture was monitored by TLC (eluent: dichloromethane/MeOH 94/6, Rf 0.33). The crude product was purified by column chromatography (eluent: dichloromethane/MeOH 97/3). Yield 53%. The product was obtained as an oily yellow solid. ¹H NMR (400 MHz, CDCl₃) δ (ppm) 7.66 (d, $^3J_{H-H}$ = 8.6 Hz, 2H, 2CHAr), 7.63 (d, $^3J_{H-H}$ = 8.6 Hz, 2H, 2CHAr), 7.32-7.23 (m, 5H, 5CHAr), 5.13 (br s, 2H, NH₂), 4.93 (d, $^3J_{H-H}$ = 8.2 Hz, 1H, NH), 3.78-3.65 (m, 1H, CH), 3.04 (d, $^2J_{H-H}$ = 14.8 Hz, 1H, H-(CH₂)), 3.00 (s, 2H, CH₂), 2.83 (d, $^2J_{H-H}$ = 14.8 Hz, 1H, H-(CH₂)), 1.88-1.68 (m, 2H, CH₂), 1.63-1.52 (m, 2H, CH₂), 1.47 (s, 3H, CH₃), 1.35-1.25 (m, 2H, CH₂), 1.14-1.04 (m, 2H, CH₂), 1.01-0.84 (m, 2H, CH₂). OH not observed. ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 164.6 (C), 156.6 (C), 136.8 (2C), 133.5 (C), 132.2 (C), 130.5 (2CHAr), 129.2 (2CHAr), 128.3 (2CHAr), 126.8 (CHAr), 125.7 (2CHAr), 106.4 (C), 86.7 (C), 48.0 (CH), 47.0 (CH₂), 43.01 (CH₂), 33.0 (CH₂), 32.9 (CH₂), 26.9 (CH₃), 25.6 (CH₂), 24.9 (CH₂), 24.8 (CH₂). HRMS (ESI +) *m/z* calcd for C₂₆H₃₁N₃O₃ [M+H]⁺ 434.2438; found 434.2431.

### 4.1.7.4. 5-Benzyl-*N*,*N*-diethyl-2-(4-(*N*'-hydroxycarbamimidoyl)phenyl)-5-methyl-4,5-dihydrofuran-3-carboxamide (c4)

Compound c4 was obtained following the former general procedure from **b4** (250 mg, 0.67 mmol, 1 equiv.). The reaction mixture was monitored by TLC (eluent: dichloromethane/MeOH 94/6, Rf 0.34). The crude product was purified by column chromatography (eluent: dichloromethane/MeOH 97/3). Yield 57%. The product was obtained as an oily brown solid. ¹H NMR (400 MHz, DMSO-d6) δ (ppm) 9.71 (s, 1H, OH), 7.67 (d, $^3J_{H-H}$ = 8.6 Hz, 2H, 2CHAr), 7.40 (d, $^3J_{H-H}$ = 8.6 Hz, 2H, 2CHAr), 7.33-7.20 (m, 5H, 5CHAr), 5.83 (br s, 2H, NH₂), 3.27-3.15 (m, 2H, CH₂), 3.04 (d, $^2J_{H-H}$ = 13.8 Hz, 1H, H-(CH₂)), 2.94 (d, $^2J_{H-H}$ = 15.7 Hz, 1H, H-(CH₂)), 2.90 (d, $^2J_{H-H}$ = 13.8 Hz, 1H, H-(CH₂)), 2.73 (d, $^2J_{H-H}$ = 15.7 Hz, 1H, H-(CH₂)), 2.70-2.61 (m, 2H, CH₂), 1.43 (s, 3H, CH₃), 1.02 (br s, 3H, CH₃), 0.67 (br s, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d6) δ (ppm) 166.1 (C), 150.2 (C), 148.1 (C), 136.9 (C), 133.6 (C), 130.4 (2CHAr), 130.3 (C), 128.0 (2CHAr), 126.4 (CHAr), 125.6 (2CHAr), 125.2 (2CHAr), 105.4 (C), 85.1 (C), 45.8 (CH₂), 43.9 (CH₂), 41.8 (CH₂), 40.4 (CH₂), 38.0 (CH₂), 27.3 (2CH₃). HRMS (ESI +) *m/z* calcd for C₂₄H₂₉N₃O₃ [M+H]⁺ 408.2282; found 408.2276.

### 4.1.7.5. 5-Benzyl-*N*-cyclopentyl-2-(4-(*N*'-hydroxycarbamimidoyl)phenyl)-5-methyl-4,5-dihydrofuran-3-carboxamide (c5)

Compound c5 was obtained following the former general procedure from **b5** (90 mg, 0.24 mmol, 1 equiv.). The reaction mixture was monitored by TLC (eluent: dichloromethane/MeOH 96/4, Rf 0.50). The crude product was purified by column chromatography (eluent: dichloromethane/MeOH 97/3). Yield 62%. The product was obtained as an oily yellow solid. ¹H NMR (400 MHz, CDCl₃) δ (ppm) 7.63 (d, $^3J_{H-H}$ = 8.5 Hz, 2H, 2CHAr), 7.59 (d, $^3J_{H-H}$ = 8.5 Hz, 2H, 2CHAr), 7.32-7.18 (m, 5H, 5CHAr), 5.12 (d, $^3J_{H-H}$ = 8.2 Hz, 1H, NH), 5.03 (br s, 2H, NH₂), 4.17-4.09 (m, 1H, CH), 3.02 (d, $^2J_{H-H}$ = 14.8 Hz, 1H, H-(CH₂)), 2.99 (d, $^2J_{H-H}$ = 13.2 Hz, 1H, H-(CH₂)), 2.95 (d, $^2J_{H-H}$ = 13.2 Hz, 1H, H-(CH₂)), 2.81 (d, $^2J_{H-H}$ = 14.8 Hz, 1H, H-(CH₂)), 1.92-1.78 (m, 2H, CH₂), 1.61-1.49 (m, 4H, 2CH₂), 1.44 (s, 3H, CH₃),
1.29-1.20 (m, 2H, CH₂). OH not observed. ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 165.0 (C), 157.1 (C), 157.0 (C), 136.7 (2C), 132.6 (C), 130.5 (2CHAr), 129.3 (2CHAr), 128.3 (2CHAr), 126.9 (2CHAr), 125.9 (CHAr), 106.2 (C), 86.9 (C), 51.2 (CH), 47.0 (CH₂), 43.0 (CH₂), 33.1 (CH₂), 33.0 (CH₂), 29.8 (CH₂), 26.9 (CH₃), 23.7 (CH₂). HRMS (ESI +) *m/z* calcd for C₂₅H₂₉N₃O₃ [M+H]⁺ 420.2282; found 420.2279.

### 4.1.7.6. 5-Benzyl-*N*-butyl-2-(4-(*N*'-hydroxycarbamimidoyl)phenyl)-5-methyl-4,5-dihydrofuran-3-carboxamide (c6)

Compound c6 was obtained following the former general procedure from **b6** (30 mg, 0.08 mmol, 1 equiv.). The reaction mixture was monitored by TLC (eluent: dichloromethane/MeOH 94/6, Rf 0.34). The crude product was purified by column chromatography (eluent: dichloromethane/MeOH from 97/3). Yield 56%. The product was obtained as an oily yellow solid. ¹H NMR (400 MHz, CDCl₃) δ (ppm) 7.69-7.55 (m, 4H, 4CHAr), 7.29-7.21 (m, 5H, 5CHAr), 5.41 (br s, 2H, NH₂), 5.22 (t,³Jₐ⁻ = 5.9 Hz, 1H, NH), 3.20-3.11 (m, 2H, CH₂), 3.04 (d, ²Jₐ⁻ = 14.6 Hz, 1H, H-(CH₂)), 2.99 (s, 2H, CH₂), 2.79 (d, ²Jₐ⁻ = 14.7 Hz, 1H, H-(CH₂)), 1.45 (s, 3H, CH₃), 1.40-1.32 (m, 2H, CH₂), 1.28-1.19 (m, 2H, CH₂), 0.86 (t, ³Jₐ⁻ = 7.4 Hz, 3H, CH₃). OH not observed. ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 165.4 (C), 157.4 (C), 153.3 (C), 136.7 (C), 132.7 (C), 132.3 (C), 130.5 (2CHAr), 129.3 (2CHAr), 128.3 (2CHAr), 126.9 (CHAr), 126.0 (2CHAr), 106.0 (C), 86.9 (C), 46.9 (CH₂), 42.9 (CH₂), 39.3 (CH₂), 31.6 (CH₂), 26.9 (CH₃), 20.2 (CH₂), 13.9 (CH₃). HRMS (ESI +) *m/z* calcd for C₂₄H₂₉N₃O₃ [M+Na]⁺ 430.2101; found 430.2099.

### 4.1.7.7. 5-Benzyl-2-(4-(*N*'-hydroxycarbamimidoyl)phenyl)-5-methyl-*N*-(2-(pyrrolidin-1-yl)ethyl)-4,5-dihydrofuran-3-carboxamide (c7)

Compound c7 was obtained following the former general procedure from **b7** (180 mg, 0.43 mmol, 1 equiv.). The reaction mixture was monitored by TLC (eluent: dichloromethane/MeOH 94/6, Rf 0.30). The crude product was purified by column chromatography (eluent: dichloromethane/MeOH 97/3). Yield 50%. The product was obtained as an oily yellow solid. ¹H NMR (400 MHz, CDCl₃) δ (ppm) 7.63 (d, ³Jₐ⁻ = 8.4 Hz, 2H, 2CHAr), 7.54 (d, ³Jₐ⁻ = 8.6 Hz, 2H, 2CHAr), 7.30-7.20 (m, 5H, 5CHAr), 6.50 (br s, 1H, NH), 4.82 (s, 2H, NH₂), 3.43-3.35 (m, 2H, CH₂), 3.06 (d, ²Jₐ⁻ = 14.7 Hz, 1H, H-(CH₂)), 2.98 (d, ²Jₐ⁻ = 13.7 Hz, 1H, H-(CH₂)), 2.98 (d, ²Jₐ⁻ = 13.7 Hz, 1H, H-(CH₂)), 2.83 (d, ²Jₐ⁻ = 14.7 Hz, 1H, H-(CH₂)), 2.65-2.50 (m, 6H, 3CH₂), 1.75 (s, 4H, 2CH₂), 1.40 (s, 3H, CH₃). OH not observed. ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 165.8 (C), 157.2 (C), 152.0 (C), 136.9 (C), 133.6 (C), 132.1 (C), 130.6 (2CHAr), 129.2 (2CHAr), 128.2 (2CHAr), 126.8 (CHAr), 125.2 (2CHAr), 106.0 (C), 86.6 (C), 55.1 (CH₂), 54.0 (2CH₂), 46.9 (CH₂), 43.3 (CH₂), 37.9 (CH₂), 26.5 (CH₃), 23.5 (2CH₂). HRMS (ESI +) *m/z* calcd for C₂₆H₃₂N₄O₃ [M+H]⁺ 449.2547; found 449.2545.

### 4.1.7.8. 4-(5-Benzyl-5-methyl-3-(morpholine-4-carbonyl)-4,5-dihydrofuran-2-yl)-*N*'-hydroxybenzimidamide (c8)

Compound c8 was obtained following the former general procedure from **b8** (350 mg, 0.90 mmol, 1 equiv.). The reaction mixture was monitored by TLC (eluent: dichloromethane/AcOEt 80/20, Rf 0.21). The crude product was purified by column chromatography (eluent: dichloromethane/MeOH 97/3). Yield 40%. The product was obtained as an oily white solid. ¹H NMR (400 MHz, CDCl₃) δ (ppm) 7.60 (d, ³Jₐ⁻ = 8.4 Hz, 2H, 2CHAr), 7.48 (d, ³Jₐ⁻ = 8.5 Hz, 2H, 2CHAr), 7.32-7.13 (m, 5H, 5CHAr), 4.95 (br s, 2H, NH₂), 3.77-3.36 (m, 6H, 3CH₂), 3.13 (d, ²Jₐ⁻ = 13.8 Hz, 1H, H-(CH₂)), 2.97 (d, ²Jₐ⁻ = 15.8 Hz, 1H, H-(CH₂)), 2.93 (d, ²Jₐ⁻ = 15.8 Hz, 1H, H-(CH₂)), 2.80 (d, ²Jₐ⁻ = 13.8 Hz, 1H, H-(CH₂)), 2.39-2.17 (m, 2H, CH₂), 1.57 (s, 3H, CH₃). OH not observed. ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 166.9 (C), 152.0 (C), 151.5 (C), 137.0 (C), 133.3 (C), 131.6 (C), 130.7 (2CHAr), 128.2 (2CHAr), 127.1 (2CHAr), 126.7 (CHAr), 125.9 (2CHAr), 103.6 (C), 86.0 (C), 66.5 (2CH₂), 60.5 (CH₂), 47.3 (CH₂), 43.6 (2CH₂), 28.6 (CH₃). HRMS (ESI +) *m/z* calcd for C₂₄H₂₇N₃O₄ [M+H]⁺ 422.2074; found 422.2074.
4.1.7.9. 4-(5-Benzyl-3-(butylsulfonyl)-5-methyl-4,5-dihydrofuran-2-yl)-N'-hydroxybenzimidamide (c9)

Compound **c9** was obtained following the former general procedure from **b9** (120 mg, 0.30 mmol, 1 equiv.). The reaction mixture was monitored by TLC (eluent: dichloromethane/MeOH 94/6, Rf 0.38). The crude product was purified by column chromatography (eluent: dichloromethane/MeOH from 97/3). Yield 62%. The product was obtained as a yellow solid. Mp 155-157 °C. ¹H NMR (400 MHz, DMSO-d6) δ (ppm) 9.81 (s, 1H, OH), 7.72 (d, ³J<sub>H-H</sub> = 8.7 Hz, 2H, 2CH<sub>Ar</sub>), 7.56 (d, ³J<sub>H-H</sub> = 8.7 Hz, 2H, 2CH<sub>Ar</sub>), 7.35-7.29 (m, 4H, 4CH<sub>Ar</sub>), 7.28-7.23 (m, 1H, CH<sub>Ar</sub>), 5.89 (br s, 2H, NH<sub>2</sub>), 3.11 (d, ²J<sub>H-H</sub> = 14.5 Hz, 1H, H-(CH<sub>2</sub>)), 3.07 (d, ²J<sub>H-H</sub> = 14.0 Hz, 1H, H-(CH<sub>2</sub>)), 3.00 (d, ²J<sub>H-H</sub> = 14.0 Hz, 1H, H-(CH<sub>2</sub>)), 2.89 (d, ²J<sub>H-H</sub> = 14.5 Hz, 1H, H-(CH<sub>2</sub>)), 2.84-2.74 (m, 2H, CH<sub>2</sub>), 1.49 (s, 3H, CH<sub>3</sub>), 1.33-1.18 (m, 4H, 2CH<sub>2</sub>), 0.76 (t, ³J<sub>H-H</sub> = 6.9 Hz, 3H, CH<sub>3</sub>). ¹³C NMR (100 MHz, DMSO-d6) δ (ppm) 161.2 (C), 150.1 (C), 136.2 (C), 135.3 (C), 130.5 (2CH<sub>Ar</sub>), 128.9 (C), 128.8 (2CH<sub>Ar</sub>), 128.1 (2CH<sub>Ar</sub>), 126.7 (CH<sub>Ar</sub>), 124.7 (2CH<sub>Ar</sub>), 108.2 (C), 87.8 (C), 53.5 (CH<sub>2</sub>), 45.5 (CH<sub>2</sub>), 41.6 (CH<sub>2</sub>), 26.9 (CH<sub>3</sub>), 23.8 (CH<sub>2</sub>), 20.7 (CH<sub>2</sub>), 13.3 (CH<sub>3</sub>). HRMS (ESI +) *m/z* calcd for C<sub>23</sub>H<sub>28</sub>N<sub>2</sub>O<sub>4</sub>S [M+H]<sup>+</sup> 429.1843; found 429.1844.

4.1.7.10. 4-(5-Benzyl-5-methyl-3-(methylsulfonyl)-4,5-dihydrofuran-2-yl)-N'-hydroxybenzimidamide (**c10**).

Compound **c10** was obtained following the former general procedure from **b10** (90 mg, 0.25 mmol, 1 equiv.). The reaction mixture was monitored by TLC (eluent: dichloromethane/MeOH 94/6, Rf 0.43). The crude product was purified by column chromatography (eluent: dichloromethane/MeOH 97/3). Yield 65%. The product was obtained as a light-yellow solid. Mp 164 °C. ¹H NMR (400 MHz, CDCl₃) δ (ppm) 7.80-7.55 (m, 4H, 4CH<sub>Ar</sub>), 7.32-7.18 (m, 5H, 5CH<sub>Ar</sub>), 5.49 (br s, 2H, NH<sub>2</sub>), 3.19 (d, ²J<sub>H-H</sub> = 15.4 Hz, 1H, H-(CH<sub>2</sub>)), 3.09 (d, ²J<sub>H-H</sub> = 13.9 Hz, 1H, H-(CH<sub>2</sub>)), 2.99 (d, ²J<sub>H-H</sub> = 15.4 Hz, 1H, H-(CH<sub>2</sub>)), 2.91 (d, ²J<sub>H-H</sub> = 13.9 Hz, 1H, H-(CH<sub>2</sub>)), 2.56 (s, 3H, CH<sub>3</sub>), 1.56 (s, 3H, CH<sub>3</sub>). OH not observed. ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 161.5 (C), 152.9 (C), 135.9 (C), 133.7 (C), 132.0 (C), 130.5 (2CH<sub>Ar</sub>), 129.8 (2CH<sub>Ar</sub>), 128.5 (2CH<sub>Ar</sub>), 127.3 (2CH<sub>Ar</sub>), 125.9 (CH<sub>Ar</sub>), 110.1 (C), 88.3 (C), 47.0 (CH<sub>2</sub>), 43.1 (CH<sub>3</sub>), 42.1 (CH<sub>2</sub>), 27.5 (CH<sub>3</sub>). HRMS (ESI +) *m/z* calcd for C<sub>20</sub>H<sub>22</sub>N<sub>2</sub>O<sub>4</sub>S [M+H]<sup>+</sup> 387.1373; found 387.1373.

## 4.2. Biology

### 4.2.1. Parasites

*Leishmania amazonensis* (MHOM/BR/77/LTB0016) was maintained as promastigotes at 26°C in Schneider's insect medium (Sigma-Aldrich, St Louis, MO, USA) with 10% heat-inactivated fetal calf serum (HIFCS), 100 µg/mL streptomycin and 100 U/mL penicillin. Parasites were maintained until the 10th passage; subsequently, new cultures were obtained from infected animals.

### 4.2.2. Antipromastigote activity

*L. amazonensis* promastigotes were cultivated in Schneider's insect medium supplemented with 10% HIFCS as above, in either absence or presence of different concentrations of the substances. The culture was initiated with 1.0 × 10⁶ cells/mL and maintained at 26°C for 72 h. The cell viability was estimated by reduction of resazurin. The 50% inhibitory concentration (IC50) was determined by logarithmic regression analysis using GraphPrism 5 software.

### 4.2.3. Cytotoxicity assay

Murine peritoneal macrophages were harvested by peritoneal washing with 5ml of RPMI 1640 medium supplemented with 10% HIFCS, 2mM L-glutamine, 1mM pyruvate, 100 µg/mL streptomycin and 100 U/mL penicillin. The peritoneal cell suspension was adjusted to 1.0 × 10⁶ cells/mL and then plated in triplicate in 96-well plates (Falcon Co,
Franklin Lakes, USA). Cultures were incubated for 1h at 37°C in a 5% CO₂ atmosphere. Afterwards, the cells were washed to remove nonadherent cells and incubated in either absence or presence of different concentrations of the substances (0-200µM) for 72 h. The cell viability was estimated by resazurin assay. The 50% cytotoxic concentration (CC₅₀) was determined by logarithmic regression analysis using GraphPrism 5 software.

#### 4.2.4. Antiamastigote assay

Resident peritoneal macrophages were plated in RPMI medium (Sigma-Aldrich) at 1 × 10⁶/mL (0.4 mL/well) in Lab-Tek eight-chamber slides (Nunc) and incubated at 37 °C in 5% CO₂ for 1 h. After 1h, the medium containing nonadherent cells was removed. The remaining cells were incubated at 37°C and 5% CO₂ with *L. amazonensis* promastigotes in a ratio of 5:1. After 4 hours, the monolayers were washed to remove free parasites. The cells were incubated with the substances in different concentrations for 72 hours at 37°C and 5% CO₂. After the incubation period, the slides were stained using the Instant Prov hematological dye system (Newprov, Curitiba, Brazil) and leishmanicidal activity was evaluated microscopically. The number of amastigotes was determined by counting at least 100 macrophages per sample. The results are expressed as an infection index (II), which was calculated as follows: $II = (percentage of infected cells) × (number of amastigotes/total number of macrophages)$. Concentration-effect curves were fitted for the anti-amastigote tests by using nonlinear regression analysis within the GraphPad program Prism 5.0, and the 50% Inhibitory Concentrations (IC₅₀ values) were determined.

**Supplementary Materials:** The supporting information can be downloaded at: xxxxxxx

**Author Contributions:**

O.A. preliminary draft, O.A and F.S writing the final manuscript; O.A., carried out in silico ADMET and similarity analysis; O.A., C.C., Y.K., S.R. carried out design and synthetic chemistry efforts; F.M.S.U.M and E.C.T.-S. carried out biological experiments; review and editing O.A., F.M.S.U.M., C.C., Y.K., S.R., P.V and E.C.T.-S.; C.C., P.V, and E.C.T.-S. conceived and planned the project; resources, C.C., O.A., P.V. and E.C.T.-S., supervision and project administration, C.C. P.V. and E.C.T.-S. All authors contributed to manuscript writing. All authors read and approved the manuscript.

**Funding:**

PhD Student Oscar Leonardo AVENDANO LEON was funded by COLCIENCIAS - Ministry of Science, Technology and Innovation of Colombia, Government Resolution 071 Janvier 2021 – call for PhD applications 885-2020. The antileishmanial study was supported by the FAPERJ - Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro, processo E-26/201.158/2022 – BOLSA.

**Acknowledgments:** Aix Marseille Université and the Centre National de la Recherche Scientifique (CNRS) are gratefully acknowledged for financial support. E.C.T.-S. thanks CNPq for the research productivity scholarship.

**Conflicts of Interest:** The authors declare no conflict of interest.

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