Identification of novel N-benzyloxy-amino acid hydroxamates as inhibitors of the virulence factor LasB from Pseudomonas aeruginosa†

Riccardo Di Leo^ab, Enrico Crispino^a, Doretta Cuffaro^a, Giuseppantonio Maisetta^c, Andrea Bertacca^c, Marta Bianchi^c, Giovanna Batoni^c, Imin Wushur^d, Fatema Amatur Rahman^d, Jan-Olof Winberg^d, Ingebrigt Sylte^de, Armando Rossello^a and Elisa Nuti*^a
aDepartment of Pharmacy, University of Pisa, via Bonanno 6, 56126 Pisa, Italy. E-mail: elisa.nuti@unipi.it; Tel: +39 0502219551
^bInstitute of Clinical Physiology, National Research Council (CNR), via Moruzzi 1, 56124 Pisa, Italy
^cDepartment of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Via S. Zeno 37-39, 56123 Pisa, Italy
^dDepartment of Medical Biology, Faculty of Health Sciences, UiT—The Arctic University of Norway, NO-9037 Tromsø, Norway
^eCenter for Research and Education, University Hospital of North Norway (UNN), Norway

First published on 13th August 2025

---

---

1. Introduction

Over the past decade, the healthcare system worldwide has been faced with enormous challenges due to the burden of ravaging bacterial infections. Such issues include nosocomial infections (NIs) usually caused by both Gram-negative and Gram-positive bacterial pathogens¹ which primarily affect patients with compromised immune systems. Among the major contributors to NIs are the “ESKAPE” group of pathogens, which includes Gram-positive bacteria such as Enterococcus faecium and Staphylococcus aureus, and Gram-negative bacteria, which include Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.^2,3 Due to their antibiotic resistance, ESKAPE pathogens are associated with the highest risk of mortality thereby resulting in increased health care costs.⁴ The bacterial priority pathogens list released by the World Health Organization in 2024 included P. aeruginosa in the high priority category. P. aeruginosa is generally found in patients with chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), different types of cancer, traumatic wounds, septicemia, and ventilator-associated pneumonia (VAP).^5–12 In addition to the rapid appearance of genetic mutations and adaptive resistance mechanisms to antibiotics, the difficulty of eradicating P. aeruginosa infections is due to its ability to form biofilm.¹³ Biofilm-residing bacteria exhibit significantly increased antibiotic resistance, up to 1000 times more than planktonic bacteria.¹⁴ The treatment of P. aeruginosa infections urgently requires the development of new antibacterials with novel mechanisms of action to overcome these resistance strategies.¹⁵ Considering these issues, medicinal chemistry research has focused on the development of new putative therapeutics known as “pathoblockers” which are described as antibacterial agents that specifically target key pathogenic processes and seek to reduce bacterial virulence rather than kill the bacteria. The aim is to reduce selection pressure and the likelihood of resistance development.^16–18 P. aeruginosa secretes several virulence factors, including four major proteases: elastase B (LasB), elastase A (LasA), alkaline protease (AprA), and protease IV.¹⁹ Among these, LasB plays a key role in the virulence of P. aeruginosa and therefore has become a central focus of research. LasB, also known as pseudolysin, is an extracellular zinc-dependent metalloprotease with multiple activities. It has been clearly shown to degrade several important host proteins, including elastin, collagen, fibrinogen, immunoglobulins, and complement components.²⁰ This degradation leads to extensive tissue damage and enables P. aeruginosa to evade the host immune response. Furthermore, emerging evidence indicates that LasB is implicated in respiratory infections, particularly in patients with cystic fibrosis (CF).¹⁰ In these patients, LasB compromises lung structural integrity by degrading elastin and components of the extracellular matrix. Other studies have shown that LasB can modulate immune responses through the degradation of cytokines and other immune mediators, resulting in the weakening of the host defense mechanisms.²¹ Moreover, LasB promotes biofilm formation, which increases the persistence of bacteria and their resistance to antimicrobial therapy.²² Given its central role in pathogenesis, LasB is considered a promising therapeutic target for the treatment of P. aeruginosa infections. Consequently, a combined approach using a LasB inhibitor and common antibiotics could improve therapeutic outcomes by hindering tissue degradation and immune evasion.²³

The MEROPS database classifies LasB as a member of the M4 family of metalloproteases that are characterized by the presence of a Zn²⁺ located at the catalytic site.²⁴ The ion is tetrahedrally coordinated by the two histidines of the conserved HEXXH motif and a glutamic acid located 20 amino acid C-terminal of this motif and a water molecule. The glutamic acid of the HEXXH motif is an important catalytical residue. The zinc-binding group (ZBG) is a critical feature of metalloenzyme inhibitors that represents a valuable tool for enzyme inhibition.²⁵ Commonly studied ZBGs include hydroxamate, carboxylic acids, tropolones, and 3-hydroxypyridine-4(1H)-thiones.^26–31 The development of selective inhibitors for LasB represents a significant challenge due to its strong structural similarity to human matrix metalloproteinases (MMPs) and A disintegrin and metalloproteinases (ADAMs).^32,33 This similarity hampered the development of highly specific therapeutic agents. Notwithstanding this high degree of homology, the depth of the S1′ sub-pocket represents a putative selectivity factor between LasB and MMPs.³⁴ Specifically, the Tyr114 and Trp115 residues in LasB are replaced by leucine residues in MMPs, with the side chains of these leucine residues pointing towards the binding site of MMPs, thereby limiting the accessibility of the S1′ sub-pocket. Furthermore, an arginine residue (Arg198 in LasB) is positioned between S1′ and S2′ sub-pockets in the M4 enzymes, influencing the access and size of the S1′ sub-pocket. In summary, the S1′ sub-pocket of LasB is not as deep as the S1′ sub-pocket of the MMPs and this pocket also differs in size and shape among MMPs.^35,36 Thereby, a good fitting with the S1′ sub-pocket may allow to develop selective LasB inhibitors. Previous studies have reported several LasB inhibitors with sub-micromolar activity, bearing diverse ZBGs. Among these, carboxylate derivative A and thiol B demonstrated significant activity (Fig. 1).^29,37 In 2023, Hirsch's research group developed a promising compound, the phosphonic acid C (Fig. 1), which emerged as a potent inhibitor of the elastase LasB from P. aeruginosa.²³ In vivo, compound C combined with levofloxacin significantly reduced bacterial burden and LasB protein levels in a neutropenic murine lung infection model, confirming its efficacy and target engagement. In a previous effort to discover new LasB inhibitors, we performed an enzymatic screening of an in-house chemical library of MMP inhibitors. This screening identified a N-benzyloxy-aminoacid-hydroxamate, LM-2 (Fig. 2), as the most effective LasB inhibitor, with high activity and selectivity for M4 enzymes over MMPs.³⁸ In the present study, the structure of LM-2 and its binding mode, as predicted through computational studies (Fig. 2), were exploited to synthesize a series of LM-2 analogues presenting three kinds of structural modifications, compounds 1–24 (Fig. 3). The synthesis of these analogues was guided by a structure-based design, with the aim of enhancing efficacy and selectivity against LasB. To demonstrate the potential of the new derivatives, an initial enzymatic screening was conducted on isolated enzymes to identify the most promising candidates. Subsequently, their antibacterial activity, cytotoxicity, and ability to inhibit the secreted LasB of P. aeruginosa were evaluated in vitro. Based on these findings, four compounds were selected, and their capacity to prevent P. aeruginosa biofilm formation was further assessed in the PAO1 strain.

2. Results and discussion

2.1. Design strategy

According to previous docking predictions in the LasB catalytic site, LM-2 placed its hydroxamate group to form a bidentate interaction with the catalytic Zn²⁺, exploiting the oxygen atoms of the CO and OH groups. The aromatic ring of LM-2 occupied the S1′ sub-site, while the n-butyl chain extended into the S2′ sub-site. Additionally, the hydroxamate OH group donated a proton to Glu141, and its NH group established a hydrogen bond with the backbone of Ala113 (Fig. 2B).³⁸ Before designing and synthesizing novel inhibitors, three previously reported N-benzyl-oxy derivatives of LM-2 from our in-house library, 1, 8, and 9 (Fig. 3, Table 1),³⁹ were tested against LasB using a fluorometric assay. In compounds 1 and 8 the n-butyl chain of LM-2 (R group in Fig. 3) was replaced by a sec-butyl and a methyl group, respectively, and 8 also presented a chlorine atom in the para position of the benzyl group (R₁). These modifications did not enhance inhibitory activity (Table 1). Instead, compound 9, which features an n-butyl chain and a chlorine atom in R₁, exhibited activity comparable to the reference compound (Table 1). This suggested that the nature of the chain in α to the hydroxamate group plays a crucial role in inhibiting LasB.

---

Based on the predicted binding mode of LM-2 (Fig. 2) and these preliminary data, the n-butyl chain of LM-2 was further replaced by longer chains to explore potential hydrophobic interactions within the S2′ sub-pocket (compounds 2–4). Additionally, the n-butyl fragment was substituted with functionalized moieties to facilitate interaction with the key Arg198 (compounds 5–7). Moreover, analogues with a chlorine atom or a trifluoromethyl group in R₁ were synthesized (compounds 10–15), and a novel derivative endowed with a ZBG different from hydroxamate was also prepared (compound 16). Given the quite hydrophobic nature of the S2′ sub-pocket,⁴⁰ the n-butyl chain was initially replaced with an n-octyl fragment (compound 2), a longer branched chain (compound 3), or a butyl chain terminating with a cyano group (compound 4). The S2′ sub-pocket, characterized by a solvent-exposed environment and hydrophobic residues lining its walls provided a favorable site for such modifications. For these reasons, compounds 3 and 4 were prepared to investigate how the extended aliphatic chains along the S2′ sub-site might influence inhibitory activity, while the cyano group was selected for its potential to form hydrogen bonds through its electron-rich nitrogen.

Subsequently, our study focused on the importance of the configuration of the chiral center in alpha position to the ZBG. Computational studies⁴¹ carried out on LM-2 enantiomers, indicated that the S-enantiomer ((S)-17, Table 2) showed better interactions than the R-enantiomer ((R)-18). To verify this prediction, (S)-17 and (R)-18 were synthesized together with compound (S)-19 (Table 2), a bioisoster of (S)-17 devoid of the oxygen atom close to the benzyl fragment. This compound and its derivatives (S)-20 and (S)-21 were prepared to elucidate the specific role of this oxygen atom in the pharmacophore of this class of inhibitors. Finally, a small series of N-benzyloxy iminoacetic derivatives (compounds 22–24, Fig. 3 and Table 3) with an oxime-ether group and a carboxylate in R was synthesized to seek for further interactions with Arg198.

---

---

2.2. Chemistry

N-Benzyloxy amino acid derivatives 2, 3, 7, 10 and 16 were prepared as reported in Scheme 1. The previously described (E)-2-((benzyloxy)imino) acetic acid 25 and (E)-2-(((4-chlorobenzyl)oxy)imino)acetic acid 26 (ref. 39) were protected as methyl esters by treatment with thionyl chloride (SOCl₂) in methanol (MeOH). This step was essential for facilitating the subsequent nucleophilic addition reaction with Grignard reagents. The appropriate α-oxime esters 27 and 28 were then converted into the proper alkyl-esters 29–31 by an alkyl addition in a mixture of zinc chloride (ZnCl₂), boron trifluoride diethyl etherate (BF₃·OEt₂), and the corresponding commercially available magnesium bromide (RMgBr, octyl magnesium bromide or (2-ethylhexyl)magnesium bromide) in DCM.⁴² Subsequently, a mild hydrolysis in basic conditions of methyl esters 29–31 afforded the carboxylates 32–34, respectively. Finally, a two-steps reaction was performed to convert carboxylates 32–34 into the corresponding hydroxamates 2, 3 and 10 by condensation with O-(tert-butyl-dimethyl-silyl)-hydroxylamine (TBDMSiONH₂) to give the silyl intermediates 35–37, followed by acid cleavage by trifluoroacetic acid (TFA) in DCM. The synthesis of 7 involved the treatment of methyl ester 27 with tert-butyl 2-iodoacetate (38), which previously had been synthesized via a reported halogen substitution reaction.⁴³ An electrophilic radical addition catalyzed by triethyl borane (Et₃B) between the oxime ether 27 and the iodide compound 38 in a MeOH/H₂O mixture resulted in the formation of the tert-butyl ester 39.⁴⁴ The orthogonally protected ester 39 was treated with TFA in controlled condition, to selectively remove the tert-butyl protection on the α-chain leading to the formation of carboxylic acid 40. Subsequently, the amino acid ester 41 was obtained by a condensation reaction between the carboxylic acid 40 and L-phenylalanine tert-butyl ester, using tetramethyl uronium hexafluorophosphate (HBTU) as carboxylic acid activator and N,N-diisopropylethylamine (DIPEA) as a base.⁴⁵ The hydroxamate 42 was obtained directly from methyl ester 41 under strongly basic reaction conditions using a methanolic solution both of potassium hydroxide (KOH) and hydroxylamine hydrochloride (NH₂OH·HCl).⁴⁶ The instability of the substrate, particularly the N-benzyloxy fragment under strongly basic conditions, might have affected the reaction yield, as confirmed by NMR analysis of the reaction byproducts, which revealed aliphatic moieties lacking the N-benzyloxy aromatic ring. Finally, the tert-butyl ester 42 was deprotected by acid hydrolysis with a solution of TFA in DCM, to give final compound 7. The sulfonamide derivative 16 was synthesized by condensing the previously described N-alkyloxy amino acid 43 (ref. 39) with trifluoromethanesulfonamide in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 4-dimethylaminopyridine (DMAP) in DCM solution.⁴⁷

The synthesis of N-benzyloxy amino acids 4–6 and 11–15 is described in Scheme 2. The carboxylic acids 47–52 were obtained through a Et₃B-catalyzed intermolecular radical addition reaction in water between the oxime ethers 25, 26 and 44 and the corresponding iodo-derivatives 38, 45 and 46, which were previously synthesized.^44,48 Carboxylic acid 52, was obtained as by-product of the reaction in a mixture with 51. Subsequently, carboxylic acids 47 and 49–52 were reacted with TBDMSiONH₂ and EDC as a condensing agent in DCM. The selective hydrolysis of the silyl hydroxylamine functionality was achieved by washing the crude with HCl during the reaction work-up, which resulted in the formation of the hydroxamic derivatives 6 and 12–15. The same reaction conducted with carboxylic acids 47–49 in the absence of HCl treatment resulted in the isolation of the O-silylated hydroxamates 53–55, which gave the hydroxamates 4, 5 and 11 by treatment with TFA in DCM.

The synthesis of optically active N-benzyloxy amino acid derivatives 17–21 is described in Scheme 3. The corresponding α-hydroxy amino acids 59 and 60 were obtained by a substitution reaction via diazonium salt formation from commercially available enantiomerically pure D-norleucine 56 and L-norleucine 57, respectively.⁴⁹ Methyl esters 61 and 62 were synthesized by reaction of carboxylic acids 59 and 60 with SOCl₂ in MeOH. Then, the N-benzyloxy intermediates 63, 64 were prepared by an S_N2 reaction from the corresponding hydroxyl derivatives, 61 and 62, using triflic anhydride (Tf₂O), 2,6-lutidine as base and O-benzylhydroxylamine as nucleophile in DCM. Subsequently, the final enantiomers (S)-17 and (R)-18 were obtained as described above. For the synthesis of (S)-19, L-norleucine was treated with SOCl₂ in MeOH to give methyl ester 69. The latter was used as a nucleophile to react with the (2-bromoethyl)benzene to give the alkylated compound 70. Due to the low nucleophilicity of the methyl ester amino acid 69, this alkylation reaction proceeded with low yield. Finally, the hydroxamate 19 was prepared by a MW-assisted reaction in strongly basic conditions in the presence of KOH, using directly the methyl ester 70 with NH₂OH·HCl in a MeOH solution. The synthesis of (S)-N-benzyl amino acid derivatives 20 and 21 was conducted from the corresponding (S)-amino acids 57 and 58, as illustrated in Scheme 3. The esters 69 and 71 were reacted with benzyl bromide to afford the corresponding N-benzyl derivatives 72 and 73 via a nucleophilic substitution reaction (S_N2). Finally, (S)-N-benzyl amino acids 20 and 21 were achieved by treating the corresponding methyl esters 72 and 73 with a methanolic solution of hydroxylamine hydrochloride and KOH in methanol.

Scheme 4 shows the synthesis of N-benzyloxy iminoacetic acids 22–24. The carboxylic acid 22 was prepared by a condensation reaction between commercial O-benzylhydroxylamine hydrochloride (74) and commercial α-ketoglutaric acid in a solution of THF and H₂O. To achieve the hydroxamate 23, the carboxylic acid groups of 22 were protected as O-(tetrahydropyranyl) derivatives to obtain 75 by condensation with O-(tetrahydropyranyl)hydroxylamine (THPONH₂), using EDC as a condensing agent, N-methyl morpholine (NMM) and 1-hydroxybenzotriazole (HOBt) in dry DMF. The protecting groups were then removed by acid hydrolysis (HCl 4 N) in a mixture MeOH/dioxane. The use of MeOH as solvent promoted the replacement of THPONH– on the side chain by a methyl ester, giving the final compound 23. Finally, carboxylic acid 26 was converted into the corresponding hydroxamate 24 according to the synthetic procedure described above.

2.3. LasB inhibitory activity and SAR study

All newly synthesized compounds were initially tested by a fluorometric assay measuring hydrolysis of a fluorogenic substrate McaRPPGFSAFK(Dnp)-OH by purified LasB and K_i values were determined (Tables 1–3). The previously reported LM-2 was used as reference compound, while its derivatives 1, 8 and 9, already published as MMPs inhibitors,³⁹ were also evaluated against LasB.

Among non-R₁ substituted hydroxamates (1–7, Table 1), the cyanoalkyl derivative 4 showed an inhibitory activity comparable to that of LM-2 (K_i = 4.5 μM) while compound 2 with a n-octyl group in R resulted 2-fold more active than LM-2 (K_i = 1.7 μM). Otherwise, derivative 3 with a long and branched chain in R was unable to reduce the enzymatic activity by 50% even at a concentration of 100 μM (Table 1). Remarkably, these results found support in molecular docking studies. As shown in Fig. 4, the long and linear side chain of 2 was correctly positioned in the S2′ sub-pocket by forming hydrophobic interactions, the aromatic ring was retained in the S1′ sub-pocket, while the hydroxamic acid coordinated in a bidentate manner the Zn²⁺. On the other hand, the analysis of compound 3 within the LasB binding site revealed two potential scenarios. In the first one, the branched side chain induced important steric clashes whereas the hydroxamate chelated the Zn²⁺ and the aromatic ring occupied the S1′ sub-site. Alternatively, when the longer portion of the side chain was located within the groove of the S2′ sub-pocket and its shorter branch was oriented similarly to the n-butyl fragment of LM-2, the carbonyl group of the hydroxamate moiety was too distant to effectively coordinate the zinc ion in a bidentate manner (Fig. 4). Since chelation is an essential feature of this class of compounds, the lack of proper zinc chelation is most likely the reason for the inactivity of compound 3.

Subsequently, the substitution of the alkyl chain in R with a functionalized fragment was explored in derivatives 5–7 (Table 1). The rationale was to establish an interaction with Arg198 by introducing a carboxylic group on the α chain, keeping the N-benzyloxy moiety and the hydroxamate as the ZBG. As mentioned above, Arg198 is strategically located between S1′ and S2′ and influences the dimensions and depth of S1′ sub-pocket. Unfortunately, the hydroxamate 5 did not show even 50% enzyme inhibition at a concentration of 100 μM. Computational models suggested that the observed lack of inhibition may result from compound 5 coordinating the catalytic Zn²⁺ via its carboxyl group, while the hydroxamate formed hydrogen bonds with the side chain of the crucial Arg198 (Fig. S1†). It is well known that hydroxamates are stronger chelating groups than carboxylates, which are more prone to forming monodentate interactions with the zinc ion.⁵⁰ To prevent this undesirable shift in chelation, the carboxyl group in compound 5 was then substituted with a tert-butyl ester in compound 6, or the carboxylic acid was condensed with L-phenylalanine as for compound 7. Esterification failed to preserve inhibitory activity (K_i > 100 μM), likely because the carbonyl group of the tert-butyl ester in compound 6 is positioned too far to effectively accept a hydrogen bond from the Arg198 side chain. This could be due to a deeper positioning of the benzyloxy fragment within the S1′ sub-pocket. In contrast, the condensation with L-phenylalanine preserved some affinity, although it was 8-fold lower than that of LM-2 (K_i = 35 μM). It is possible that the amino acid fragment in compound 7 is too hydrophobic and bulky to remain solvent-exposed and properly position itself within the S2′ sub-pocket, with the amino acid size potentially impeding optimal alignment and reducing the inhibitory effect. Based on these findings, the next step was targeting the interactions within the S1′ sub-pocket to improve activity. To leverage the hydrophobic nature of this sub-pocket (e.g. Leu132, Val137), a chlorine atom was introduced in R₁ position of the benzyl group in compound 2, to give hydroxamate 10 (Table 1). Similarly, a chlorine atom was added in compound 6 to try to preserve the correct orientation of the benzyl group within the S1′ sub-pocket. This modification was expected to be essential for maintaining the optimal positioning of the hydroxamate group and ensuring effective bidentate chelation of the zinc ion, leading to the development of tert-butyl ester 12. As a matter of fact, while the p-Cl derivative 10 exhibited an activity comparable to LM-2 and 2 (K_i = 4.3 μM), 12 was found to be 5-fold more potent than LM-2, with a K_i = 0.92 μM. As displayed in Fig. 5, the hydroxamate group formed a bidentate coordination with the catalytic Zn²⁺, as observed for LM-2. Additionally, the incorporation of the p-chlorine atom on the benzyl group slightly forced out the latter from the S1′ sub-pocket, enabling the carbonyl group of the tert-butyl ester to form a hydrogen bond with Arg198. Consequently, derivative 12 was firmly anchored within the LasB binding site through a synergistic network of interactions: the bidentate coordination of the hydroxamate with Zn²⁺, the hydrophobic interaction of the p-chlorine benzyl moiety, and two critical hydrogen bonds, one involving the NH group of the –NH–O-benzyl functionality with Asn112 and the other, as previously mentioned, between the carbonyl group of the tert-butyl ester and Arg198. Notably, the N–O-benzyl moiety—typically considered chemically labile—participates in a key hydrogen bond within the catalytic pocket, suggesting a stabilizing effect from its engagement in the binding site. To further support this observation, we evaluated the integrity of this functionality under physiologically and chemically relevant conditions. Compound 12 was incubated at 37 °C in PBS at pH 1, 7.4, and 10, and no significant degradation of the N–O bond was observed, indicating good chemical stability across a broad pH range (ESI†).

To verify if the introduction of the p-Cl substituent could induce a similar increase in potency also in compound 5 with a carboxylate in R, its analogue 11 was synthesized and tested. On the contrary, this derivative showed no activity (K_i > 100 μM), suggesting that the chlorine atom alone did not facilitate hydrogen bond formation between Arg198 and the carboxyl group of 11, thereby preventing the switch of the ZBG. To enlarge the SAR study, the p-CF₃ substituted analogues of LM-2 and 6 were synthesized, to give compounds 14 and 15 respectively (compound 13 was obtained as a by-product). While the introduction of a CF₃ in R₁ led to a slight decrease of activity in 14 with respect to LM-2, the same substituent in 15 caused a strong increase in potency with respect to 6, with a K_i = 1.1 μM. As aforementioned for the p-Cl substituted 12, this enhanced activity could be due to the positioning of a substituent in the para position of the benzyl group, which slightly moved the aromatic group out of the S1′ sub-site thus facilitating the interaction of the carbonyl group of the tert-butyl ester with the key Arg198.

The last N-benzyloxy amino acid LM-2 analogue to be synthesized was compound 16, bearing an N-acyl(trifluoromethane)sulfonamide as ZBG. However, the introduction of this “soft binder”⁴⁷ for the catalytic Zn²⁺ led to complete loss of affinity (K_i > 100 μM).

As regards LM-2 enantiomers, the enzymatic assays confirmed a stronger binding of (S)-17 than of (R)-18 (Table 2). Particularly, (S)-17 had 10-fold stronger affinity than (R)-18 on LasB, but the improvement of affinity with respect to racemic LM-2 was negligible (Table 2). Considering the complex synthesis to obtain optically pure N-benzyloxy derivatives (as described in Scheme 3), and the small difference of activity between the (S) enantiomer and the respective racemate, the idea to obtain (S) enantiomers of inhibitors was abandoned.

At this point, with the aim to prove the role of the oxygen atom in the N–O benzyl moiety, a bioisoster of (S)-17 devoid of the oxygen atom, compound (S)-19, was synthesized followed by two N-benzyl derivatives, (S)-20 and (S)-21. These last two compounds were designed to optimize the selectivity for M4 enzymes over human MMPs. The preference for the N-benzyl fragment was based on its shorter length compared to the N-benzyloxy counterpart thus reducing the probability of penetrating deeply into the S1′ sub-pocket of MMPs, which is a feature to obtain M4 inhibitors selective over MMPs. N-Benzyl derivatives (S)-20 and (S)-21 bore a hydroxamate moiety as ZBG and an n-butyl and n-octyl chain in R, respectively. As a result of this isosteric substitution, compound (S)-19 showed approximately a 26-fold weaker binding against LasB compared to (S)-17, and both N-benzyl derivatives, (S)-20 and (S)-21, were inactive, with a K_i > 100 μM (Table 2). These findings clearly pointed out that the N–O– fragment is crucial for a correct orientation of the aromatic system within the S1′ sub-pocket of the enzyme.

As a final step, the structure of the inactive compound 5 was further modified to synthesize unsaturated oxime-ether derivatives (–CN–O–R, 22–24, Fig. 3) to obtain compounds potentially able to interact with Arg198 in LasB. Compared to 5, its analogue 22 had a carboxylate group as ZBG, and its side chain was extended by one carbon atom. On the other hand, both 23 and 24 contained a hydroxamate as ZBG but differed from 5 for the aliphatic chain in R. Specifically, 23 had an additional carbon atom like 22 and a methyl ester group in R, while 24 lacked the aliphatic side chain in R and contained a chlorine atom in R₁. Unfortunately, all these compounds were ineffective (Table 3) probably due to the geometric constraints introduced by the oxime double bond (–CN–O–), which did not allow a proper orientation of the ZBG. Thus, the design and synthesis of other unsaturated compounds was interrupted due to these unsuccessful results.

2.4. Molecular dynamics simulations

To investigate the stability and dynamic behavior of compound 12, the most promising inhibitor in this series, a 100 ns molecular dynamics (MD) simulation was performed in explicit solvent (TIP3P) at 300 K. The root mean square deviation (RMSD) of the protein heavy atoms (yellow) and the ligand (aligned to the protein, blue), as shown in Fig. S2,† was analyzed to assess the conformational stability of the complex. The protein RMSD remained stable, indicating that LasB retains its structural integrity, while the ligand RMSD exhibited initial fluctuations (10–15 ns), followed by stabilization, suggesting conformational adaptation within the binding pocket (Fig. S3†). A transient RMSD increase in the final phase suggests localized intra-pocket reorganization, likely due to dynamic interactions with active-site residues. This aligns with the K_i value of 0.92 μM, suggesting that while compound 12 remains stably engaged within the catalytic pocket, its residual flexibility, particularly due to the solvent-exposed tert-butyl fragment, may limit its potency. Overall, these MD results confirm the stability of compound 12 within LasB, supporting its potential as an effective inhibitor while highlighting opportunities for structural refinement to enhance affinity.

2.5. Selectivity against MMPs

The most promising LasB inhibitors were selected for further characterization, including selectivity studies against human recombinant MMP-2, -9 and -14. These enzymes were chosen among the 23 human MMPs because represent the most well-studied MMPs, due to their involvement in cancer-induced angiogenesis and invasion.⁵² MMP-2 and -9 (gelatinase A and B) are secreted MMPs, while MMP-14 is a collagenase membrane-type MMP (MT1-MMP), so they could represent two different sub-types of human MMPs.⁵³ Compounds 2, 10, 12, 15 and (S)-17 were tested on MMPs by a fluorometric assay which uses a fluorogenic peptide⁵⁴ as the substrate, in comparison with the parent compound LM-2.

As can be seen from data in Table 4, all compounds were highly selective over MMP-14, which is anchored to the cell membrane and has important roles in cell migration implicated in many physiological and pathological events.⁵⁵ A good selectivity for LasB over MMP-2 and MMP-9 was also found, even if to a lower extent. In particular, hydroxamate 12 displayed a 90-fold selectivity for LasB over MMP-14 and a 24-fold selectivity over MMP-2. The same selectivity profile was found for the CF₃-analogue of 12, compound 15. Overall, compounds 12 and 15 presented an improved activity and selectivity for LasB with respect to the parent compound LM-2.

---

Based on the results of enzymatic assays, the LasB inhibitors LM-2, 2, 10, 12, 15, and (S)-17 were selected for further investigation in vitro against Pseudomonas aeruginosa PAO1 strain.

2.6. Antibacterial activity

None of the N-benzyloxy amino acid derivatives tested against the PAO1 strain showed any bacteriostatic effect when tested up to a concentration of 200 μM (minimum inhibitory concentration, MIC > 200 μM), confirming their potential use as pathoblockers.

2.7. Cytotoxicity

To further explore the translational potential of LM-2, 2, 10, 12, 15, and (S)-17, the cytotoxic effect of these inhibitors was tested on the human lung epithelial cell line NCI H441. After incubation for 24 h, cell viability was evaluated using the WST-1 assay. All compounds did not exhibit any evident effect on cell viability at the tested concentrations, except 2 and 10, which markedly reduced viability at the concentration of 200 μM (Fig. 6).

2.8. Inhibition of secreted LasB activity of PAO1 strain

The ability of LM-2, 2, 10, 12, 15, and (S)-17 to inhibit the enzymatic activity of native LasB produced by P. aeruginosa was evaluated (Fig. 7). For this purpose, PAO1-CFS (cell free supernatant) was pre-incubated for 30 min with each derivative, and then the mixture was incubated for 18 h with LasB substrate (elastin Congo-red). The elastase activity was determined by measuring the absorbance at OD₄₉₀ and expressed as percent of LasB activity compared to the untreated controls. Among the tested compounds, 12 exhibited the highest efficacy, reducing LasB activity by 50% at 10.7 μM (Fig. 7) while LM-2, 2, 10, 15, and (S)-17 showed similar efficacy in inhibiting LasB activity (IC₅₀ from 21.2 to 29.7 μM) (Fig. 7). Notably, these data were in accordance with the inhibitory activity determined on the isolated enzyme LasB (Table 1), since compound 12 resulted the most active in both assays.

2.9. Antibiofilm activity

Although the biofilm formation is a multifactorial process, it has been found that the increased expression of LasB elastase leads to higher rhamnolipid production, which in turn has a role in the biofilm formation in P. aeruginosa.⁵⁶ Thus, to evaluate the potential antibiofilm activity of N-benzyloxy amino acids, we tested whether selected compounds, LM-2, 2, 12, and (S)-17, at sub-MIC concentrations (25, 50, 100, 200 μM) inhibited P. aeruginosa PAO1 biofilm after 24 h of treatment. The biofilm biomass was evaluated by crystal-violet staining. Compounds 2 and (S)-17 significantly reduced the biofilm biomass in a dose-dependent manner (Fig. 8). Both reduced the biofilm biomass by approximately 50% at concentrations as low as 50 μM. In sharp contrast, a similar effect was observed with LM-2 and compound 12 but only when tested at 200 μM (Fig. 8). This apparent discrepancy between LasB inhibition and antibiofilm activity of compound 12 could be explained considering that there may be other factors which contribute to biofilm formation, in addition to LasB activity. The importance of quorum sensing (QS) system in P. aeruginosa biofilm has been largely documented.⁵⁷

3. Conclusions

This study presents the design and synthesis of novel N-benzyloxy-amino acid derivatives as potential pathoblockers targeting the LasB enzyme secreted by Pseudomonas aeruginosa. Through structure-based design, we identified key modifications that could enhance selectivity and inhibitory activity, particularly focusing on interactions within the S1′ and S2′ sub-pockets of the catalytic site. Hydroxamate 12, bearing a p-Cl benzyl group and a tert-butyl ester moiety in R, demonstrated the highest efficacy with significant inhibition of LasB enzymatic activity and promising antibiofilm properties. Importantly, the selectivity of these compounds against LasB over human matrix metalloproteinases underscores their therapeutic potential while minimizing off-target effects. The inhibitors were effective in attenuating LasB-mediated virulence without exhibiting antibacterial effects, confirming their potential use as pathoblockers. Cytotoxicity evaluations on human lung epithelial cells revealed a favorable safety profile for most compounds at relevant concentrations, further supporting their translational viability. This work highlights the importance of targeting bacterial virulence mechanisms to combat multidrug-resistant pathogens such as P. aeruginosa.

4. Experimental section

4.1. Chemistry

Air sensitive reactions were carried out under an inert atmosphere (N₂ or Ar) and monitored by thin layer chromatography (TLC) on Merck aluminum silica gel sheets (60 F254). TLCs were visualized with a UV lamp at 254 or 365 nm and, for non-chromophore substances, stained with phosphomolybdic acid solution and then heated. In addition, an aqueous solution of FeCl₃ was used to detect hydroxamic acids. Evaporation was performed in vacuo (rotating evaporator). Na₂SO₄ was used as the drying agent for organic solutions. Reaction mixtures were purified by chromatographic separations on silica gel using an automated system or prepacked ISOLUTE Flash Si II cartridges (Biotage). The automated system was the Isolera Prime from Biotage (Uppsala, Sweden) equipped with a UV detector with a range of 200–400 wavelengths (λ) and using SFÄR HC Duo silica cartridges (Biotage, Uppsala, Sweden). The microwave-assisted reactions were carried out in a Biotage Initiator⁺ Microwave Synthesizer. The purified compounds were characterized by ¹H, ¹³C, and ¹⁹F NMR spectra using a Bruker Avance III HD 400 MHz spectrometer. Chemical shifts (δ) are given in ppm, J in Hz, and various abbreviations have been used for the multiplicities (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = double doublet, dt = double triplet, br s = broad singlet). Melting points were determined on a Leica Galen III microscope (Leica/Cambridge Instruments) and were not corrected. The ESI-MS spectra were recorded by direct injection at 5 (positive) and 7 (negative) μL min⁻¹ flow rate in an Orbitrap high-resolution mass spectrometer (Thermo, San Jose, CA, USA), equipped with HESI source. Optical rotations were measured at 20 °C using an ATAGO AP-300 polarimeter equipped with a continuous sodium lamp (λ = 589 nm). The final compounds 2–7 and 10–24 were synthesized with a purity of at least 95%, as confirmed by combustion analysis and by NMR analysis reported in the ESI.† Analytical results are within ±0.4% of the theoretical values. Commercially available chemicals were purchased from Sigma-Aldrich (Merck) or ABCR. Compounds 1, 8 and 9 were synthesized as previously described.³⁹

4.2. Biology assays

4.3. Computational methods

The crystal structure used to perform non-covalent docking was downloaded from the Protein Data Bank (PDB). PDB ID of LasB is 1U4G. Modelling studies were performed with Maestro, release 2023-01, version 13.1, with the tool named Glide.

Data availability

The data supporting this article have been included as part of the ESI.†

Author contributions

Conceptualization, E. N., A. R., I. S., and J.-O. W.; investigation, R. D. L., E. C., A. B., M. B., I. W., and F. A. R.; formal analysis, J.-O. W., G. M., and R. D. L.; data curation, E. N., D. C., and R. D. L.; writing – original draft, R. D. L., E. C., and E. N.; writing – review and editing, E. N., D. C., I. S., G. M., and J.-O. W.; supervision, E. N., A. R., I. S., J.-O. W., and G. B.; funding acquisition, A. R., I. S., E. N., G. B., and G. M. All authors have read and approved the final version of the manuscript.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by the Northern Norway Health Authorities (Helse Nord) (grant number HNF 1514-20) to I. S. and A. R., by University of Pisa (PRA_2022_19) to E. N., by the European Union – Next Generation EU, PNRR MUR M4 C2 Inv. 1.5 CUP I53C22000780001 to G. B. and by Telethon Foundation “Seed Grant Spring 2024 PCD” ref. number 5737 to G. M. We thank CISUP – Centre for Instrumentation Sharing – University of Pisa for the acquisition and elaboration of the HRMS spectra. We are grateful to the Cheminformatics & Nutrition research group from the Universitat Rovira i Virgili (Tarragona, Spain) for allowing us to use the Schrödinger Drug Discovery suite in their computers.

References

1. M. Haque, M. Sartelli, J. McKimm and M. B. Abu Bakar, Infect. Drug Resist., 2018, 11, 2321–2333 CrossRef PubMed .
2. M. Rodríguez-Alvarado, R. Russo, N. D. Connell and S. E. Brenner-Moyer, Org. Biomol. Chem., 2020, 18, 5867–5878 RSC .
3. J.-L. Vincent, JAMA, J. Am. Med. Assoc., 2009, 302, 2323 CrossRef CAS PubMed .
4. U. Theuretzbacher, R. P. Jumde, A. Hennessy, J. Cohn and L. J. V. Piddock, Nat. Rev. Microbiol., 2025, 23, 474–490 CrossRef CAS PubMed .
5. N. Safdar, C. J. Crnich and D. G. Maki, Respir. Care, 2005, 50, 725–739 Search PubMed  ; discussion 739–741.
6. N. A. M. Cobben, M. Drent, M. Jonkers, E. F. M. Wouters, M. Vaneechoutte and E. E. Stobberingh, J. Hosp. Infect., 1996, 33, 63–70 CrossRef CAS PubMed .
7. S. Schelenz and G. French, J. Hosp. Infect., 2000, 46, 23–30 CrossRef CAS PubMed .
8. H. A. Khan, F. K. Baig and R. Mehboob, Asian Pac. J. Trop. Biomed., 2017, 7, 478–482 CrossRef .
9. S. Qin, W. Xiao, C. Zhou, Q. Pu, X. Deng, L. Lan, H. Liang, X. Song and M. Wu, Signal Transduction Targeted Ther., 2022, 7, 199 CrossRef CAS PubMed .
10. S. Malhotra, D. Hayes and D. J. Wozniak, Clin. Microbiol. Rev., 2019, 32, e00138-18 CrossRef PubMed .
11. T. E. Woo, J. Duong, N. M. Jervis, H. R. Rabin, M. D. Parkins and D. G. Storey, Microbiology, 2016, 162, 2126–2135 CrossRef CAS PubMed .
12. J. L. L. Ferreiro, J. Á. Otero, L. G. González, L. N. Lamazares, A. A. Blanco, J. R. B. Sanjurjo, I. R. Conde, M. F. Soneira and J. De La Fuente Aguado, PLoS One, 2017, 12, e0178178 CrossRef PubMed .
13. A. David, A. Tahrioui, A.-S. Tareau, A. Forge, M. Gonzalez, E. Bouffartigues, O. Lesouhaitier and S. Chevalier, Antibiotics, 2024, 13, 688 CrossRef CAS PubMed .
14. N. Høiby, C. Moser and O. Ciofu, Microorganisms, 2024, 12, 2115 CrossRef PubMed .
15. Z. Pang, R. Raudonis, B. R. Glick, T.-J. Lin and Z. Cheng, Biotechnol. Adv., 2019, 37, 177–192 CrossRef CAS PubMed .
16. S. Wagner, R. Sommer, S. Hinsberger, C. Lu, R. W. Hartmann, M. Empting and A. Titz, J. Med. Chem., 2016, 59, 5929–5969 CrossRef CAS PubMed .
17. M. B. Calvert, V. R. Jumde and A. Titz, Beilstein J. Org. Chem., 2018, 14, 2607–2617 CrossRef CAS PubMed .
18. A. B. H. Khalifa, D. Moissenet, H. Vu Thien and M. Khedher, Ann. Biol. Clin., 2011, 69, 393–403 Search PubMed .
19. Y. Kida, Y. Higashimoto, H. Inoue, T. Shimizu and K. Kuwano, Cell. Microbiol., 2008, 10, 1491–1504 CrossRef CAS PubMed .
20. E. Kessler and D. E. Ohman, in Handbook of Proteolytic Enzymes, Elsevier, 2013, pp. 582–592 Search PubMed .
21. B. Wretlind and O. R. Pavlovskis, Clin. Infect. Dis., 1983, 5, S998–S1004 CrossRef CAS PubMed .
22. S. Sharma, J. Mohler, S. D. Mahajan, S. A. Schwartz, L. Bruggemann and R. Aalinkeel, Microorganisms, 2023, 11, 1614 CrossRef CAS PubMed .
23. J. Konstantinović, A. M. Kany, A. Alhayek, A. S. Abdelsamie, A. Sikandar, K. Voos, Y. Yao, A. Andreas, R. Shafiei, B. Loretz, E. Schönauer, R. Bals, H. Brandstetter, R. W. Hartmann, C. Ducho, C.-M. Lehr, C. Beisswenger, R. Müller, K. Rox, J. Haupenthal and A. K. H. Hirsch, ACS Cent. Sci., 2023, 9, 2205–2215 CrossRef PubMed .
24. N. D. Rawlings, A. J. Barrett, P. D. Thomas, X. Huang, A. Bateman and R. D. Finn, Nucleic Acids Res., 2018, 46, D624–D632 CrossRef CAS PubMed .
25. O. A. Adekoya and I. Sylte, Chem. Biol. Drug Des., 2009, 73, 7–16 CrossRef CAS PubMed .
26. G. R. A. Cathcart, D. Quinn, B. Greer, P. Harriott, J. F. Lynas, B. F. Gilmore and B. Walker, Antimicrob. Agents Chemother., 2011, 55, 2670–2678 CrossRef CAS PubMed .
27. J. Zhu, X. Cai, T. L. Harris, M. Gooyit, M. Wood, M. Lardy and K. D. Janda, Chem. Biol., 2015, 22, 483–491 CrossRef CAS PubMed .
28. D. Grobelny, L. Poncz and R. E. Galardy, Biochemistry, 1992, 31, 7152–7154 CrossRef CAS PubMed .
29. M. J. Everett, D. T. Davies, S. Leiris, N. Sprynski, A. Llanos, J. M. Castandet, C. Lozano, C. N. LaRock, D. L. LaRock, G. Corsica, J.-D. Docquier, T. D. Pallin, A. Cridland, T. Blench, M. Zalacain and M. Lemonnier, ACS Infect. Dis., 2023, 9, 270–282 CrossRef CAS PubMed .
30. J. L. Fullagar, A. L. Garner, A. K. Struss, J. A. Day, D. P. Martin, J. Yu, X. Cai, K. D. Janda and S. M. Cohen, Chem. Commun., 2013, 49, 3197 RSC .
31. A. L. Garner, A. K. Struss, J. L. Fullagar, A. Agrawal, A. Y. Moreno, S. M. Cohen and K. D. Janda, ACS Med. Chem. Lett., 2012, 3, 668–672 CrossRef CAS PubMed .
32. C. Camodeca, D. Cuffaro, E. Nuti and A. Rossello, Curr. Med. Chem., 2019, 26, 2661–2689 CrossRef CAS PubMed .
33. L. G. N. De Almeida, H. Thode, Y. Eslambolchi, S. Chopra, D. Young, S. Gill, L. Devel and A. Dufour, Pharmacol. Rev., 2022, 74, 714–770 CrossRef CAS PubMed .
34. D. Cuffaro, E. Nuti, F. D'Andrea and A. Rossello, Pharmaceuticals, 2020, 13, 376 CrossRef CAS PubMed .
35. R. Di Leo, D. Cuffaro, A. Rossello and E. Nuti, Molecules, 2023, 28, 4378 CrossRef CAS PubMed .
36. B. Fabre, A. Ramos and B. De Pascual-Teresa, J. Med. Chem., 2014, 57, 10205–10219 CrossRef CAS PubMed .
37. C. Kaya, I. Walter, A. Alhayek, R. Shafiei, G. Jézéquel, A. Andreas, J. Konstantinović, E. Schönauer, A. Sikandar, J. Haupenthal, R. Müller, H. Brandstetter, R. W. Hartmann and A. K. H. Hirsch, ACS Infect. Dis., 2022, 8, 1010–1021 CrossRef CAS PubMed .
38. S. Sjøli, E. Nuti, C. Camodeca, I. Bilto, A. Rossello, J.-O. Winberg, I. Sylte and O. A. Adekoya, Eur. J. Med. Chem., 2016, 108, 141–153 CrossRef PubMed .
39. E. Nuti, E. Orlandini, S. Nencetti, A. Rossello, A. Innocenti, A. Scozzafava and C. T. Supuran, Bioorg. Med. Chem., 2007, 15, 2298–2311 CrossRef CAS PubMed .
40. O. A. Adekoya, S. Sjøli, Y. Wuxiuer, I. Bilto, S. M. Marques, M. A. Santos, E. Nuti, G. Cercignani, A. Rossello, J.-O. Winberg and I. Sylte, Eur. J. Med. Chem., 2015, 89, 340–348 CrossRef CAS PubMed .
41. H. Sabishiro, Y. Nagura, N. Chimura, M. Yuguchi, C. Nakatani, S. Takenaka, H. Sugizaki and N. Kurita, J. Mol. Graphics Modell., 2025, 137, 108977 CrossRef CAS PubMed .
42. M. Mitani, Y. Tanaka, A. Sawada, A. Misu and Y. Matsumoto, Eur. J. Org. Chem., 2008, 2008, 1383–1391 CrossRef .
43. L. Gu, P. G. Luo, H. Wang, M. J. Meziani, Y. Lin, L. M. Veca, L. Cao, F. Lu, X. Wang, R. A. Quinn, W. Wang, P. Zhang, S. Lacher and Y.-P. Sun, Biomacromolecules, 2008, 9, 2408–2418 CrossRef CAS PubMed .
44. S. B. McNabb, M. Ueda and T. Naito, Org. Lett., 2004, 6, 1911–1914 CrossRef CAS PubMed .
45. N. Venugopal, J. Moser, M. Vojtíčková, I. Císařová, B. König and U. Jahn, Adv. Synth. Catal., 2022, 364, 405–412 CrossRef CAS .
46. L. Baldini, E. Lenci, F. Bianchini and A. Trabocchi, Molecules, 2022, 27, 1249 CrossRef CAS PubMed .
47. E. Nuti, D. Cuffaro, E. Bernardini, C. Camodeca, L. Panelli, S. Chaves, L. Ciccone, L. Tepshi, L. Vera, E. Orlandini, S. Nencetti, E. A. Stura, M. A. Santos, V. Dive and A. Rossello, J. Med. Chem., 2018, 61, 4421–4435 CrossRef CAS PubMed .
48. T. Naito, Chem. Pharm. Bull., 2008, 56, 1367–1383 CrossRef CAS PubMed .
49. J. Müller, S. C. Feifel, T. Schmiederer, R. Zocher and R. D. Süssmuth, ChemBioChem, 2009, 10, 323–328 CrossRef PubMed .
50. K. Kawai and N. Nagata, Eur. J. Med. Chem., 2012, 51, 271–276 CrossRef CAS PubMed .
51. J. Kyte and R. F. Doolittle, J. Mol. Biol., 1982, 157, 105–132 CrossRef CAS PubMed .
52. E. Nuti, A. R. Cantelmo, C. Gallo, A. Bruno, B. Bassani, C. Camodeca, T. Tuccinardi, L. Vera, E. Orlandini, S. Nencetti, E. A. Stura, A. Martinelli, V. Dive, A. Albini and A. Rossello, J. Med. Chem., 2015, 58, 7224–7240 CrossRef CAS PubMed .
53. M. Raeeszadeh-Sarmazdeh, L. Do and B. Hritz, Cells, 2020, 9, 1313 CrossRef CAS PubMed .
54. U. Neumann, H. Kubota, K. Frei, V. Ganu and D. Leppert, Anal. Biochem., 2004, 328, 166–173 CrossRef CAS PubMed .
55. V. Gifford and Y. Itoh, Biochem. Soc. Trans., 2019, 47, 811–826 CrossRef CAS PubMed .
56. H. Yu, X. He, W. Xie, J. Xiong, H. Sheng, S. Guo, C. Huang, D. Zhang and K. Zhang, Can. J. Microbiol., 2014, 60, 227–235 CrossRef CAS PubMed .
57. F. Soukarieh, P. Williams, M. J. Stocks and M. Cámara, J. Med. Chem., 2018, 61, 10385–10402 CrossRef CAS PubMed .
58. Z. Lin, J. Li, Q. Huang, Q. Huang, Q. Wang, L. Tang, D. Gong, J. Yang, J. Zhu and J. Deng, J. Org. Chem., 2015, 80, 4419–4429 CrossRef CAS PubMed .
59. Y. Ji, P. Xue, D.-D. Ma, X.-Q. Li, P. Gu and R. Li, Tetrahedron Lett., 2015, 56, 192–194 CrossRef CAS .
60. C. R. Ganellin, P. B. Bishop, R. B. Bambal, S. M. T. Chan, J. K. Law, B. Marabout, P. M. Luthra, A. N. J. Moore, O. Peschard, P. Bourgeat, C. Rose, F. Vargas and J.-C. Schwartz, J. Med. Chem., 2000, 43, 664–674 CrossRef CAS PubMed .
61. W. A. Gibbons, R. A. Hughes, M. Charalambous, M. Christodoulou, A. Szeto, A. E. Aulabaugh, P. Mascagni and I. Toth, Liebigs Ann. Chem., 1990, 1990, 1175–1183 CrossRef .
62. R. K. Bowman and J. S. Johnson, Org. Lett., 2006, 8, 573–576 CrossRef CAS PubMed .
63. E. Busto, N. Richter, B. Grischek and W. Kroutil, Chem. – Eur. J., 2014, 20, 11225–11228 CrossRef CAS PubMed .
64. F. Rahman, T.-M. Nguyen, O. A. Adekoya, C. Campestre, P. Tortorella, I. Sylte and J.-O. Winberg, J. Enzyme Inhib. Med. Chem., 2021, 36, 819–830 CrossRef CAS PubMed .
65. Schrödinger Release 2016-3: LigPrep, Schrödinger, LLC, New York, NY, USA, 2016 Search PubMed .
66. Schrödinger Release 2016-3: Epik, Schrödinger, LLC, New York, NY, USA, 2016 Search PubMed .
67. J. A. Bell, Y. Cao, J. R. Gunn, T. Day, E. Gallicchio, Z. Zhou, R. Levy and R. Farid, International Tables for Crystallography, Wiley, 2012, ch. 18.10, vol. F, pp. 534–538 Search PubMed .
68. Schrödinger Release 2016-3: Schrödinger Suite 2016-3 Protein Preparation Wizard; Epik, Schrödinger, LLC: New York, NY, USA; and Impact, Schrödinger, LLC: New York, NY, USA; Prime, Schrödinger, LLC: New York, NY, USA, 2016 Search PubMed.
69. J. D. Westbrook, C. Shao, Z. Feng, M. Zhuravleva, S. Velankar and J. Young, Bioinformatics, 2015, 31, 1274–1278 CrossRef PubMed .
70. M. H. M. Olsson, C. R. Søndergaard, M. Rostkowski and J. H. Jensen, J. Chem. Theory Comput., 2011, 7, 525–537 CrossRef CAS PubMed .
71. C. R. Søndergaard, M. H. M. Olsson, M. Rostkowski and J. H. Jensen, J. Chem. Theory Comput., 2011, 7, 2284–2295 CrossRef PubMed .
72. Schrödinger Release 2016-3: Maestro, Schrödinger, LLC, New York, NY, USA, 2016 Search PubMed .
73. Schrödinger Release 2016-3: Glide, Schrödinger, LLC, New York, NY, USA, 2016 Search PubMed .
74. D. A. Anil, M. F. Polat, R. Saglamtas, A. H. Tarikogullari, M. A. Alagoz, I. Gulcin, O. Algul and S. Burmaoglu, Comput. Biol. Chem., 2022, 100, 107748 CrossRef CAS PubMed .
75. O. Ozten, B. Zengin Kurt, F. Sonmez, B. Dogan and S. Durdagi, Bioorg. Chem., 2021, 115, 105225 CrossRef CAS PubMed .
76. G. J. Martyna, D. J. Tobias and M. L. Klein, J. Chem. Phys., 1994, 101, 4177–4189 CrossRef CAS .

---

Footnote

† Electronic supplementary information (ESI) available: 1H-NMR spectra of final compounds 2–7 and 10–24; 13C-NMR spectra of final compounds 2, 4–6, 10–24; 19F-NMR spectra of final compounds 13–16; HRMS spectra of final compounds 2–4, 7, 10, 12, 14, 15 and 17; Fig. S1: binding of compound 5 to LasB; Fig. S2: RMSD plot of compound 12 and protein heavy atoms in the LasB complex during MD simulations; Fig. S3: LasB residues interacting with compound 12 and their interaction frequency during the MD simulation; chemical stability of compound 12. See DOI: https://doi.org/10.1039/d5md00393h

This journal is © The Royal Society of Chemistry 2025