# Synthesis and Anti-mycobacterial Activity of Novel Medium-chain $\beta$-Lactone Derivatives: A Multi-Target Strategy to combat Mycobacterium abscessus

Thomas Francis, Christina Dedaki, Phoebe Ananida-Dasenaki, Dimitra Bolka, Kanellos Albanis, Filippos Foteinakis, Julie Mezquida, Marie Hance, Alexandros Athanasoulis, Anna-Krinio Papagiorgou, et al.

► To cite this version:

Thomas Francis, Christina Dedaki, Phoebe Ananida-Dasenaki, Dimitra Bolka, Kanellos Albanis, et al. Synthesis and Anti-mycobacterial Activity of Novel Medium-chain $\beta$-Lactone Derivatives: A Multi-Target Strategy to combat Mycobacterium abscessus. RSC Medicinal Chemistry, In press, 10.1039/D5MD00102A. hal-05052181

HAL Id: hal-05052181

https://hal.science/hal-05052181v1

Submitted on 7 May 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

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

Distributed under a Creative Commons Attribution 4.0 International License
RSC
Medicinal Chemistry

Accepted Manuscript

This article can be cited before page numbers have been issued, to do this please use: T. Francis, C. Dedaki, P. Ananida-Dasenaki, D. Bolka, K. Albanis, F. Foteinakis, J. Mezquida, M. Hance, A. Athanasoulis, A. Papagiorgou, I. Karampoula, G. Georgitsis, C. Jardin, S. Audebert, L. Camoin, C. Crauste, S. Canaan, V. Magrioti and J. Cavalier, *RSC Med. Chem.*, 2025, DOI: 10.1039/D5MD00102A.

This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication.

Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available.

You can find more information about Accepted Manuscripts in the **Information for Authors**.

Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal's standard **Terms & Conditions** and the **Ethical guidelines** still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.

<figure><img src="image_2.png" /></figure>
<figure><img src="image_3.png" /></figure>
# Synthesis and Anti-mycobacterial Activity of Novel Medium-chain β-Lactone

View Article Online

DOI: 10.1039/D5MD00102A

## Derivatives: A Multi-Target Strategy to combat *Mycobacterium abscessus*

Thomas Francis,\*,a Christina Dedaki,\*,b Phoebe Ananida-Dasenaki, b Dimitra Bolka, b Kanellos Albanis, b Filippos Foteinakis, b Julie Mezquida, a Marie Hance, a Alexandros Athanasoulis, b Anna-Krinio Papagiorgou, b Ioanna-Foteini Karampoula, b George Georgitsis, b Celia Jardin, c Stéphane Audebert, c Luc Camoin, c Céline Crauste, d Stéphane Canaan, a Victoria Magrioti, \*,b Jean-François Cavalier\*\,a

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

RSC Medicinal Chemistry Accepted Manuscript

<footnote>a Aix-Marseille Univ., CNRS, LISM, Institut de Microbiologie de la Méditerranée FR3479, Marseille, France</footnote>

<footnote>b Department of Chemistry, National and Kapodistrian University of Athens, Panepistimiopolis, Athens, 15771, Greece</footnote>

<footnote>c Aix-Marseille Univ., INSERM, CNRS, Institut Paoli-Calmettes, CRCM, Marseille Protéomique, Marseille, France</footnote>

<footnote>d IBMM, Univ Montpellier, CNRS, ENSCM, Montpellier, France</footnote>

<footnote>\* Corresponding authors: Jean-François Cavalier (jfcavalier@imm.cnrs.fr), and Victoria Magrioti (vmagriot@chem.uoa.gr)</footnote>

<footnote>\# These authors have contributed equally to this work.</footnote>
Abstract

View Article Online

DOI: 10.1039/D5MD00102A

The constant emergence of drug-resistant mycobacteria, together with the lack of new antibiotics entering the market, has become a global public health problem that threatens the effective treatment of infectious diseases. The development of single molecules targeting different proteins should significantly reduce the emergence of resistant strains, and therefore represent a promising strategy to overcome such an issue. In this challenging context, a new series of 30 lipophilic compounds based on the β-lactone-core has been synthesized by varying the nature of the substituents on the lactone ring. The evaluation of their antibacterial activity against *M. tuberculosis* and *M. abscessus*, two major pathogenic mycobacteria, highlighted potential candidates. The **VM038**, **VM040** and **VM045** were active only against *M. tuberculosis*, while **VM025**, **VM026** and **VM043** inhibited the growth of both *M. tuberculosis* and the S and R variants of *M. abscessus*. Competitive click chemistry activity-based protein profiling revealed several potential *M. abscessus* target enzymes of **VM043**, the best extracellular growth inhibitor. Finally, when tested against intracellular bacteria, although **VM043** was found inactive, **VM025 & VM026** proved to be potent and promising inhibitors of intramacrophagic *M. abscessus* growth with minimal inhibitory concentrations (MIC<sub>50Raw</sub>) comparable to the standard antibiotic imipenem. Overall, these results strengthen the added value of our **VM β-lactone** derivatives not only in the fight against pathogenic mycobacteria, leading to the arrest of *M. abscessus* and/or *M. tuberculosis* growth through multitarget enzyme inhibition, but also as efficient probes to identify novel potential therapeutic targets using chemoproteomics approaches.

<figure><img src="image_5.png" /></figure>
**Keywords (1 to 7 keywords)**

Activity-based protein profiling; multitarget inhibitor-like affinity-based probes; drug susceptibility;
intracellular activity; proteomic analysis; mycobacteria; *M. tuberculosis*

View Article Online

DOI: 10.1039/D5MD00102A
# 1. Introduction

View Article Online

DOI: 10.1039/D5MD00102A

The *Mycobacterium* genus consists of more than 200 species, which are mainly classified according to their pathogenicity and growth rate.¹, ² In addition to *Mycobacterium tuberculosis*, the causative agent of tuberculosis (TB),³, ⁴ nontuberculous mycobacteria (NTM) are opportunistic pathogens responsible for clinical syndromes ranging from skin to pulmonary infections (e.g., *Mycobacterium abscessus*⁵⁻⁷) especially in immunocompromised individuals.⁸⁻¹⁰ Furthermore, the emergence of multidrug-resistant isolates of *M. tuberculosis* or *M. abscessus* has significantly reduced treatment success rates, leading to higher incidence of treatment failure and mortality.⁶, ¹¹⁻¹³ Known as the "antibiotic and clinical nightmare",⁶, ¹⁴ *M. abscessus* is indeed considered one of the most drug-resistant mycobacterial species.¹⁵ This mycobacterium exists as two distinct colony morphotypes: smooth (S) and rough (R), which can adapt and develop differently in response to the host immune system therefore leading to different outcomes for the mycobacteria within its host.⁷, ¹⁶, ¹⁷ The R variant is particularly associated with severe lung infections⁵, ¹² and can persist for years, especially in patients with cystic fibrosis (CF).¹⁸

A key factor limiting the treatment of mycobacterial infections is the presence of a complex, waxy, lipid-rich cell wall containing unique lipids such as mycolic acids.¹⁹, ²⁰ These lipids play a critical role in maintaining the structural integrity of the bacterial cell envelope and in modulating interactions with the host immune system.²⁰, ²¹ Moreover, this general composition and architecture is shared by all mycobacterial species contributing to their low permeability to many antibiotics thus limiting therapeutic options.¹⁹, ²⁰ Therefore, targeting the enzymes involved in mycobacterial lipid metabolism;²², ²³ which are mainly serine and cysteine enzymes (i.e., (Ser/Cys)-based enzymes);²⁴⁻²⁶ has emerged as a promising strategy to combat not only *M. tuberculosis*,²⁷⁻³¹ but also other chronic mycobacterial infections like those caused by *M. abscessus*.²⁴, ³²

β-Lactones are a class of four-membered cyclic esters characterized by a highly strained ring structure that confers significant reactivity and makes them potent inhibitors of (Ser/Cys)-based enzymes. The unique reactivity of β-lactones allows them to form a covalent, but often reversible, long-lived acyl

<figure><img src="image_6.png" /></figure>
enzyme complex with (Ser/Cys)-based enzymes as a result of nucleophilic attack of the catalytic serine (or cysteine) residue on the β-lactone ring.33, 34 This characteristic has driven interest in their development as pharmacological agents, particularly for inhibiting enzymes involved in lipid metabolism. The best example of this family is the FDA-approved anti-obesity drug **Orlistat** (Figure 1)33, 35, which is also known to inhibit microbial (Ser/Cys)-based enzymes.36 Acting as a versatile (Ser/Cys)-hydrolase inhibitor, Orlistat impairs the activity of key mycobacterial enzymes involved in critical processes related to lipid metabolism, particularly in the biosynthesis of mycolic acids which are essential for the integrity of the bacterium's cell wall.37 Among them are the enzymes belonging to the Cutinase-like family proteins including the essential *M. tuberculosis* phospholipase/thioesterase Cut6 (Rv3802c);38-40 enzymes belonging to the hormone-sensitive lipase (HSL) family member proteins (i.e., Lip-HSL);41, 42 the polyketide synthase-13 thioesterase domain (Pks13-TE) as well as the mycolyltransferase Antigen 85C.43, 44 Finally, **Orlistat** has been shown to inhibit the growth of various mycobacterial species with minimum inhibitory concentrations (MIC) of 10-60 µg/mL45 and demonstrated strong synergistic effects with vancomycin against *M. tuberculosis* H37Rv, reducing its MIC (50 µg/mL) by approximately 16-fold46. Various structural modifications of the **Orlistat** pharmacophore have been explored to enhance the specificity and antibacterial potency of newly synthesized analogs.40, 45, 47, 48 In particular, the β-lactone EZ120 (Figure 1) displayed promising antitubercular activity (MIC ~0.7 µg/mL = 1.6 µM) and was found to target the antigen 85 enzymes and Pks13-TE.47

The multi-target nature of β-lactones makes them promising candidates for the development of new derivatives that may target lipid-processing enzymes critical for mycobacterial growth and survival. A few years ago, we reported the synthesis and antimycobacterial activity of a first series of 16 long- and medium-chain mono- and disubstituted β-lactones, namely **VM001-VM016**.45 Although *M. abscessus* growth was barely affected, six β-lactones were active against *M. tuberculosis* (MIC50 ~ 20-65 µg/mL), with **VM008** [trans-(Z)-3-(hexadec-7-en-1-yl)-4-propyloxetan-2-one = trans-VM001] (Figure 1) being the best growth inhibitor (MIC50 ~ 19.7 µg/mL).45

View Article Online

DOI: 10.1039/D5MD00102A

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

RSC Medicinal Chemistry Accepted Manuscript
Given the promising antibacterial activities of this best compound,⁴⁵ **VM008** chemical structure has been used as a template to synthesize a new set of 27 β-lactone and 3 β-lactam derivatives (**VM** compounds – **Figure 1**) by varying the nature of the R¹ and R² alkyl chains to modulate their lipophilicity as a means of improving their activity. Their respective anti-mycobacterial activity was further assessed against *M. tuberculosis* and the two variants S & R of *M. abscessus*. Remarkably, and contrary to the first series of β-lactone derivatives,⁴⁵ the determined MIC revealed that some **VM** β-lactones and β-lactams were able to inhibit *M. abscessus* growth *in vitro* in culture broth medium and/or inside infected macrophages. In addition, using a competitive activity-based protein profiling approach,²⁸,²⁹ the potential target enzymes of the newly synthesized **VM043**, identified as the most active inhibitor of extracellular bacterial growth, were further identified.

<figure><img src="image_8.png" /><figcaption>**Figure 1.** Chemical structures of **Orlistat**,⁴² and **EZ120**;⁴⁷ former **VM001** and **VM008**⁴⁵ as well as new synthesized related analogues (see **Table S1** for structure details).</figcaption></figure>
## 2. Experimental section

### 2.1. Chemistry

Compounds **1a-i, 3a-c, 8a-b, 16, 21, 27, 32a-b** and **38** were commercially available. The synthetic methods and the characterization data of all synthesized new $\beta$-lactones as well as intermediates are included in the **Supporting information** file.

View Article Online

DOI: 10.1039/D5MD00102A

#### 2.1.2. General procedure for the synthesis of final $\beta$-lactone derivatives

**General procedure I. Aldol reaction for the synthesis of $\alpha,\beta$-substituted $\beta$-hydroxy acids (see Schemes 1 and 5-6, Methods A, F, G, H and I).**

To a stirring solution of diisopropylamine (3 mmol) in dry THF (2 mL), under argon at 0 °C, a solution of 1.6 M *n*-BuLi in hexane (3 mmol) was slowly added *via* syringe and the solution of LDA was stirred at 0 °C for 10 min. The carboxylic acid (1 mmol) in dry THF (3 mL) was then added and the solution was stirred at 0 °C for 1 h. Then, the appropriate aldehyde (1.3 mmol) in dry THF (2 mL) was added and the solution was stirred at 0 °C for 1 h and at room temperature overnight. The solvent was removed under reduced pressure. The reaction mixture was acidified with 1N HCl and extracted with Et₂O (3×30 mL). The organic layers were combined, washed with brine (30 mL) and dried. The solvent was removed and the product was purified by column chromatography eluting with a gradient of CHCl₃/MeOH 97:3 to 95:5 (v/v).

**General procedure II. $\beta$-Lactone cyclization using *p*-TsCl in pyridine (see Scheme 1, Method A)**

To a stirring solution of the $\beta$-hydroxy acid (1 mmol) in dry pyridine (2 mL), under argon at 0 °C, *p*-toluenesulfonyl chloride (2 mmol) in dry pyridine (1 mL) was added slowly *via* a syringe. The solution was stirred at 0 °C for 1 h and kept at 4 °C for 3 days. Then, Et₂O (30 mL) was added, and the organic layer was washed with 10% Na₂CO₃ (2×30 mL), 1N HCl (2×30 mL) and brine (30 mL). The organic layer was dried, and the solvent was removed *in vacuo*. The product was purified by column chromatography eluting with a gradient of hexane/EtOAc.
**General procedure III. β-Lactone cyclization using EDC·HCl and DMAP (see Schemes 2-3, Methods B-C).**

View Article Online

DOI: 10.1039/D5MD00102A

In a flame-dried flask under argon, a solution of the β-hydroxy acid (1 mmol) in dry CH₂Cl₂ (18 mL) was added, followed by EDC·HCl (1.6 mmol) and DMAP (0.1 mmol) and the solution was stirred at r.t. for 3 days. CH₂Cl₂ (20 mL) and H₂O (20 mL) were added and then the organic layer was washed with brine (20 mL). The organic layer was dried, and the solvent was removed *in vacuo*. The product was purified by column chromatography eluting with a gradient of hexane/EtOAc.

**General procedure IV. Late-stage fluorination of β-lactones (see Scheme 3, Method D).**

In a flame-dried flask with a solution of diisopropylamine (1.6 mmol) in dry THF (1 mL), under argon at 0 °C, a solution of *n*-BuLi 1.6 M in hexane (1.6 mmol, 1 mL) was slowly added *via* a syringe and the solution of LDA was stirred at 0 °C for 10 min and then cooled at -78 °C. At -78 °C a solution of the β-lactone (1 mmol) in dry THF (7 mL) was added *via* a syringe and the solution was left stirring for 30 min at -78 °C. Then a solution of NFSI (2 mmol) in dry THF (2 mL) was added and the reaction was left to warm up to -15 °C and left stirring for additional 1.5 h at -15 °C. Then THF was removed *in vacuo*, EtOAc (30 mL) and 10% NaHCO₃ (30 mL) were added. The organic layer was washed with brine (30 mL), dried and the solvent was removed *in vacuo*. The product was purified by column chromatography eluting with a gradient of hexane/EtOAc.

**General procedure V. Deprotection of TIPS-protected terminal alkyne β-lactones using TBAF (see Scheme 5, Methods F-H).**

In a flame-dried flask with a solution of the TIPS-protected terminal alkyne β-lactone (1 mmol) in dry THF (5 mL) under argon, TBAF (2 mmol) in dry THF (2 mL) was added and the mixture was left stirring for 3 h at r.t. The progress of the reaction was monitored by TLC. Once the starting material was consumed, saturated NH₄Cl was added (20 mL) and the crude product was extracted with Et₂O (3×20mL). The organic layer was washed with brine (20 mL), dried and the solvent was removed *in vacuo*. The product was purified by column chromatography eluting with a gradient of hexane/EtOAc or hexane/Et₂O.

<figure><img src="image_9.png" /></figure>
2.1.2. Synthesis of new β-lactone derivatives VM025, VM026, VM027, VM043, VM045 and VM046

**3-Decyl-4-propyloxetan-2-one (VM025)**

Prepared according to **General procedure II** using 2f; purified by column chromatography eluting with a gradient of hexane/EtOAc starting 97:3 to 95:5 (v/v). **VM025** gives rise to **VM026** and **VM027**.

Isolated mixture of diastereomers after column chromatography (dr 1:1). Yield 70%; ¹H NMR (200 MHz, CDCl₃) δ 4.59-4.46 (m, 0.5H), 4.26-4.15 (m, 0.5H), 3.65-3.50 (m, 0.5H), 3.21-3.08 (m, 0.5H), 1.91-1.08 (m, 24H), 0.96 (t, *J* = 7 Hz, 3H), 0.86 (t, *J* = 7 Hz, 3H). ¹³C NMR (50 MHz, CDCl₃) δ 172.5, 171.8, 78.1, 75.6, 56.3, 52.8, 36.7, 32.4, 32.1, 29.8, 29.7, 29.6, 29.5, 28.1, 27.8, 27.2, 24.1, 22.8, 19.1, 18.6, 14.3, 14.0, 13.9. HRMS (ESI) [M+Na]⁺: calcd for C₁₆H₃₀NaO₂⁺ 277.2138, found 277.2135.

**(±)-trans 3-Decyl-4-propyloxetan-2-one (VM026)**

Purified by 2nd column chromatography of **VM025** eluting with a gradient of hexane/EtOAc 98:2 to 95:5 (v/v). ¹H NMR (200 MHz, CDCl₃) δ 4.26-4.15 (m, 1H), 3.21-3.08 (m, 1H), 1.91-1.56 (m, 4H), 1.55-1.08 (m, 18H), 1.00-0.65 (m, 6H). ¹³C NMR (50 MHz, CDCl₃) δ 172.2, 78.2, 56.4, 36.7, 32.1, 29.7, 29.5, 28.1, 27.2, 22.8, 18.6, 14.3, 14.0. HRMS (ESI) [M+Na]⁺: calcd for C₁₆H₃₀NaO₂⁺ 277.2138, found 277.2137.

**(±)-cis 3-Decyl-4-propyloxetan-2-one (VM027)**

Purified by 2nd column chromatography of **VM025** eluting with a gradient of hexane/EtOAc 98:2 to 95:5 (v/v). ¹H NMR (200 MHz, CDCl₃) δ 4.59-4.46 (m, 1H), 3.65-3.50 (m, 1H), 1.90-1.40 (m, 8H), 1.40-1.11 (m, 14H), 0.98 (t, *J* = 7 Hz, 3H), 0.88 (t, *J* = 7 Hz, 3H). ¹³C NMR (50 MHz, CDCl₃) δ 171.8, 75.7, 52.8, 32.4, 32.0, 29.6, 29.5, 29.4, 29.3, 27.8, 24.1, 22.8, 19.0, 14.3, 14.0. HRMS (ESI) [M+Na]⁺: calcd for C₁₆H₃₀NaO₂⁺ 277.2138, found 277.2138.

**3-Fluoro-3-octyl-4-propyloxetan-2-one (VM043)**

Prepared according to **General procedure III** using 11a; purified by column chromatography eluting with hexane/EtOAc 95:5 (v/v). Isolated mixture of diastereomers after column chromatography (dr

View Article Online

DOI: 10.1039/D5MD00102A

<figure><img src="image_10.png" /></figure>
65:35). Yield 36%; Yellowish oil; ¹H NMR (400 MHz, CDCl₃) δ 4.71-4.61 (m, 0.35H), 4.50-4.42 (m, 0.65H), 2.07-1.65 (m, 4H), 1.65-1.25 (m, 14H), 1.02 (t, *J* = 7.4 Hz, 3H), 0.90 (t, *J* = 6.6 Hz, 3H).

View Article Online

DOI: 10.1039/D5MD00102A

¹³C NMR (101 MHz, CDCl₃) δ 167.66 (d, *J* = 25.3 Hz), 166.93 (d, *J* = 24.2 Hz), 102.90 (d, *J* = 217.2 Hz), 102.00 (d, *J* = 224.2 Hz), 83.44 (d, *J* = 25.3 Hz), 82.26 (d, *J* = 22.2 Hz), 32.36 (d, *J* = 23.2 Hz), 31.80, 31.77, 31.46 (d, *J* = 3.0 Hz), 30.95 (d, *J* = 5.1 Hz), 29.65, 29.39, 29.21, 29.11, 29.08, 28.72 (d, *J* = 23.2 Hz, CH₂CF), 22.63, 22.62, 22.58, 22.54, 22.16, 22.12, 18.76, 17.95, 14.07, 13.79, 13.66. ¹⁹F NMR (376 MHz, CDCl₃) δ -159.76, -173.43. HRMS (ESI) [M+Na]⁺: calcd for C₁₄H₂₅FNaO₂⁺ 267.1731, found 267.1732.

### 3-(8-(Octyloxy)octyl)-4-propyloxetan-2-one (VM045)

Prepared according to **General procedure II** using **2j**; purified by column chromatography eluting with a gradient of hexane/EtOAc starting 97:3 to 95:5 (v/v). Isolated mixture of diastereomers after column chromatography (dr 3:7). Yield 60%; Colorless oil; ¹H NMR (200 MHz, CDCl₃) δ 4.59-4.50 (m, 0.7H), 4.26-4.20 (m, 0.3H), 3.65-3.55 (m, 0.7H), 3.32 (t, *J* = 7.0 Hz, 4H), 3.20-3.14 (m, 0.3H), 1.91-1.50 (m, 10H), 1.50-1.22 (m, 20H), 1.00 (t, *J* = 7 Hz, 3H), 0.86 (t, *J* = 7 Hz, 3H). ¹³C NMR (50 MHz, CDCl₃) δ 172.28, 171.59, 77.92, 75.45, 70.98, 70.88, 56.17, 52.67, 36.50, 32.21, 31.83, 29.78, 29.75, 29.46, 29.36, 29.33, 29.27, 29.25, 29.22, 27.87, 27.58, 26.97, 26.20, 26.15, 23.92, 22.65, 18.89, 18.43, 14.08, 13.79, 13.75. HRMS (ESI) [M+Na]⁺: calcd for C₂₂H₄₂NaO₃⁺ 377.3026, found 377.3029.

### 3-Fluoro-3-(8-(octyloxy)octyl)-4-propyloxetan-2-one (VM046)

Prepared according to **General procedure IV** using **VM045**; purified by column chromatography eluting with hexane/Et₂O 95:5 (v/v). Isolated mixture of diastereomers after column chromatography (dr 55:45). Yield 23%; ¹H NMR (400 MHz, CDCl₃) δ 4.61-4.53 (m, 0.55H), 4.40-4.34 (m, 0.45H), 3.32 (t, *J* = 6.8 Hz, 4H), 1.98-1.14 (m, 30H), 0.93 (t, *J* = 7.3 Hz, 3H), 0.81 (t, *J* = 6.5 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 166.63 (d, *J* = 25.2 Hz), 165.91 (d, *J* = 23.90 Hz), 101.91 (d, *J* = 215.5 Hz), 101.00 (d, *J* = 224.3 Hz), 82.40 (d, *J* = 25.2 Hz), 81.24 (d, *J* = 21.4 Hz), 69.98, 69.86, 69.84, 31.31 (d, *J* = 2.5 Hz), 30.82, 30.43 (d, *J* = 2.5 Hz), 29.92 (d, *J* = 3.8 Hz), 28.75, 28.71, 28.56, 28.45, 28.30, 28.27, 28.24, 28.19, 28.18, 27.67 (d, *J* = 22.7), 25.18, 25.11, 21.65, 21.52 (d, *J* = 3.8 Hz), 21.10

<figure><img src="image_11.png" /></figure>
(d, *J* = 3.8 Hz), 17.73, 16.92, 13.09, 12.78, 12.66. ¹⁹F NMR (376 MHz, CDCl₃) *δ* -159.71, -173.39.

HRMS (ESI) [M+Na]⁺: calcd for C₂₂H₄¹FNaO₃⁺ 395.2932, found 395.2935.

View Article Online

DOI: 10.1039/D5MD00102A

### 4.1.3. Synthesis of β-lactone probes VM053p and VM055p

#### (±)-trans 3-(Oct-7-yn-1-yl)-4-propyloxetan-2-one (VM053p)

Prepared according to **General procedure V** using **35b**; purified by column chromatography eluting with a gradient of hexane/EtOAc starting 97:3 to 92:8 (v/v). Only (±)-*trans* diastereomers were isolated after column chromatography. Yield 98%; Colorless oil; ¹H NMR (400 MHz, CDCl₃) *δ* 4.27-4.23 (m, 1H), 3.21-3.17 (m, 1H), 2.21 (t, *J* = 7 Hz, 2H), 1.96 (s, 1H), 1.91-1.80 (m, 2H), 1.79-1.69 (m, 2H), 1.57-1.42 (m, 10H), 1.01 (t, *J* = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) *δ* 171.57, 84.47, 77.93, 68.29, 56.14, 36.51, 28.77, 28.35, 28.26, 27.82, 26.86, 18.45, 18.33, 13.78. HRMS (ESI) [M+Na]⁺: calcd for C₁₄H₂₂NaO₂⁺ 245.1512, found 245.1510.

#### (±)-trans 3-(8-Azidooctyl)-4-propyloxetan-2-one (VM055p)

Prepared according to **General procedure I** using **39**, followed by **General procedure II**; purified by column chromatography eluting with hexane/EtOAc 95:5 (v/v). Only (±)-*trans* diastereomers were isolated after column chromatography. Yield 12%; Colorless oil; ¹H NMR (400 MHz, CDCl₃) *δ* 4.27-4.23 (m, 1H), 3.28 (t, *J* = 7 Hz, 2H), 3.22-3.17 (m, 1H), 1.92-1.80 (m, 2H), 1.78-1.69 (m, 2H), 1.52-1.34 (m, 14H), 1.01 (t, *J* = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) *δ* 171.58, 77.91, 56.16, 51.45, 36.50, 29.18, 29.15, 29.00, 28.80, 27.87, 26.93, 26.65, 18.44, 13.77. HRMS (ESI) [M+Na]⁺: calcd for C₁₄H₂₅NaN₃O₂⁺ 290.1839, found 290.1835.

### 2.2. Biological evaluation

#### 2.2.1. Chemicals

Amikacin, kanamycin, isoniazid and imipenem were purchased from Euromedex (Souffelweyersheim, France). Stock solutions of each β-lactone derivative (5 mg/mL) in which the

<figure><img src="image_12.png" /></figure>
compounds were found to be completely soluble in dimethyl sulfoxide (DMSO), were prepared and
stored at -20 °C before use.

View Article Online
DOI: 10.1039/D5MD00102A

### 2.2.2. Bacterial strains and growth conditions.

M. abscessus CIP104536T with either a smooth (S) or a rough (R) morphotype, and M. tuberculosis mc26230 (H37Rv ΔRD1 ΔpanCD49) were cultured in Middlebrook 7H9 liquid media (BD Difco) supplemented with 0.05% Tween 80 (Sigma-Aldrich, Saint-Quentin Fallavier, France), 0.2% glycerol (Euromedex, France) and 10% Oleic Albumin Dextrose Catalase (OADC enrichment, BD Difco) (7H9-SOADC). In the case of M. tuberculosis mc26230, 24 µg/mL D-pantothenate (Sigma-Aldrich) was also added in the 7H9-SOADC medium. Recombinant M. abscessus S bacterial luciferase (Lux) reporter strains was used for measurement of bacterial load inside infected macrophages. This latter M. abscessus S-Lux strain was generated by electroporation of the M. abscessus S strain with the integrating shuttle plasmid pMV306hsp+LuxG1350 (Addgene plasmid #26161) optimized for mycobacteria, and carrying the constitutive Phsp60 and PG13 promoters driving expression of luxAB and luxCDE luciferase genes.51, 52 The M. abscessus S-LuxG13 strains were grown in 7H9-SOADC with 250 µg/mL kanamycin (Euromedex, France). All cultures were incubated at 37 °C under mild agitation at 50 rpm.

### 2.2.3. Antimycobacterial susceptibility testing

Antimycobacterial susceptibility testing was performed using the Middlebrook 7H9 broth microdilution method. MICs were determined in 96-well flat-bottom Nunclon Delta Surface microplates with lid (Thermo-Fisher Scientific, Illkirch, France) using the resazurin microtiter assay (REMA).45, 53, 54 Briefly, log-phase bacteria were diluted to a cell density of 5 × 10⁶ CFU/mL and 100 µL of this inoculum was grown in a 96-well plate in the presence of serial dilutions of each β-lactone analog. After 3-5 days (M. abscessus) or 10-14 days (M. tuberculosis) incubation at 37 °C, 20 µL of a 0.025% (w/v) resazurin solution was added to each well (200 µL) and incubation was continued

<figure><img src="image_13.png" /></figure>
until the appearance of a color change (from blue to pink) in the control well (i.e., bacteria without antibiotics). Fluorescence of the resazurin metabolite resorufin ($\lambda_{excitation}$, 530 nm; $\lambda_{emission}$, 590 nm) was then measured and the lowest compound concentration leading to 50% and 90% inhibition of bacterial growth was defined as the MIC$_{50}$ and MIC$_{90}$, respectively. Amikacin (AMK) (Euromedex, France) was used as reference drugs. All experiments were performed independently at least three times. See **Supporting Information** for detailed protocol.

View Article Online

DOI: 10.1039/D5MD00102A

### 2.2.4. Determination of cytotoxicity (resazurin assay)

The cytotoxicity of the new synthesized β-lactone analogs against eukaryotic cells was measured based on the reduction of resazurin as a value of cellular viability by metabolic activity, as previously described.⁴⁵, ⁵⁴, ⁵⁵ Murine (Raw264.7) macrophages (American Type Culture Collection TIB-71) were incubated with two-fold dilution of each compound for 24 h at 37 °C and 5% CO$_2$. Then, 20 µL of a 0.025% (w/v) resazurin solution was added to each well, and fluorescence was measured following a 4 h incubation as described above. The compound concentration leading to 50% macrophage cell death was defined as the CC$_{50}$.²⁸, ²⁹, ⁵⁶ All experiments were performed as three independent biological replicates. See **Supporting Information** for detailed protocol.

### 2.2.5. Intramacrophage killing assay.

The intracellular growth of *M. abscessus* S-LuxG13⁵¹, ⁵² luminescent strain was assessed following a 24 h exposure of infected Raw264.7 murine macrophages cell line (American Type Culture Collection TIB-71) to two-fold dilutions of the selected β-lactone analogs. To avoid growth of extracellular mycobacteria, cells were extensively washed and treated with amikacin (250 µg/mL) prior to treatment with the β-lactone analogs. Imipenem (IMP; 80 µg/mL) was used as positive control for this intracellular killing assay. After incubation, luminescence measurement was used to assess intracellular bacterial viability of *M. abscessus* S-LuxG13 strain.⁵¹, ⁵² The lowest compound concentration leading to 50% of the relative luminescence unit (RLU%) was defined as the MIC$_{50Raw}$.

<figure><img src="image_14.png" /></figure>

RSC Medicinal Chemistry Accepted Manuscript
Each experiment was performed as three independent biological replicates. See **Supporting Information** for detailed protocol.

DOI: 10.1039/D5MD00102A

### 2.2.6. Copper-free click chemistry activity-based protein profiling<sup>57-59</sup>

Bacterial suspension of *M. abscessus* S in 7H9S<sup>OADC</sup> was adjusted at a final theoretical OD<sub>600 nm</sub> of 20 and then incubated with **VM043** (122 µg/mL final concentration) or DMSO (control) at 37 °C for 4 h under shaking at 200 rpm. Bacteria were then washed 3 times with PBS containing 0.05% Tween 80, resuspended in 7H9TG<sup>OADC</sup> and then re-incubated at 37 °C with **VM055<sub>p</sub>** (54 µg/mL) or DMSO for 4 h at 200 rpm. Bacteria were harvested, washed, resuspended in PBS at a 1:1 (w/v) ratio and then lysed by mechanical disruption on a BioSpec Beadbeater. Each sample (300 µL – 0.3 mg total proteins) was further subjected to copper-free azide-alkyne cycloaddition and enrichment with DBCO-agarose bead 50% slurry (Click Chemistry Tools, ref. 1034) according to the manufacturer's instructions. The beads containing bound, biotinylated proteins were resuspended in 30 µL PBS buffer pH 7.4, snap frozen in liquid nitrogen and stored at -80 °C before mass spectrometry experiments. See **Supporting Information** for detailed protocol.

### 2.2.7. Mass spectrometry analysis for protein identification and quantification

The beads were further processed for mass spectrometry as described in Babin *et al*.⁵⁹ Briefly, proteins on beads were digested with 0.5 µg trypsin sequencing grade (Promega Inc.) in 50 mM TEAB for 16h at 37 °C. Peptides were extracted with 20% acetonitrile, dried, and further desalted on C18 Micro SpinColumns (Harvard Bioscience, Inc), dried again and diluted in 15 µL water/acetonitrile/ (98/2, v/v) containing 0.05% TFA. 20% of each sample was analyzed twice by liquid chromatography (LC)-tandem MS (MS/MS) using a Q-Exactive plus Mass Spectrometer (Thermo Fisher Scientific, San Jose, CA) online with a nanoRSLC Ultimate 3000 chromatography system (Thermo Fisher Scientific, Sunnyvale, CA). Relative intensity-based label-free quantification (LFQ) was processed using the DIA-NN 1.8 algorithm. Raw files were searched against the *M. abscessus* database (UP000007137) extracted from UniProt (date 2021-11-15; 4,940 entries).⁶⁰ The
statistical analysis was done with the Perseus program (version 1.6.15.0)⁶¹ from the MaxQuant
environment (www.maxquant.org). Differential proteins were detected using a two-sample *t*-test
using permutation-based FDR-controlled at 5 and employing 250 permutations. The *p*-value was
adjusted using a scaling factor s0 with a value of 1.⁶² See **Supporting Information** for detailed
protocol.

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium
(www.proteomexchange.org)⁶³ *via* the PRIDE partner repository⁶⁴
(https://www.ebi.ac.uk/pride/login) with the dataset identifiers PXD057836.

View Article Online

DOI: 10.1039/D5MD00102A

<figure><img src="image_15.png" /></figure>
# 3. Results and Discussion

View Article Online

DOI: 10.1039/D5MD00102A

## 3.1 Chemistry

Based on our previous results on the activity of long and medium chain substituted β-lactones,⁴⁵ we decided to synthesize a new set of β-lactone derivatives bearing saturated aliphatic chains located at the α-position such as C₆, C₈, C₁₀, C₁₂, and an ether analogue of our most potent β-lactone **VM001**; while at the β-position a propyl chain that had proved to be the optimal substituent⁴⁵ was used. We also synthesized β-lactone **VM036**, which has an oleyl chain at the β-position and a propyl chain at the α-position, *i.e.* in opposite positions compared to β-lactone **VM001**. Finally, we resynthesized β-lactones **VM009**, **VM010**, **VM011** and **VM012** to separate the racemic mixtures of *cis*- and *trans*-β-lactones to be tested independently. For the synthesis of these β-lactones, Method A was followed (**Scheme 1**). The first step is an aldol reaction between the appropriate carboxylic acid and aldehyde using LDA as the base which deprotonates the proton in the α-position of the acid function and yields the corresponding β-hydroxy acids **2** after treatment with aqueous HCl. The β-hydroxy acids **2** were then cyclized using *para*-toluenesulfonyl chloride (*p*-TsCl) in dry pyridine, and the resulting β-lactones were further purified by column chromatography (**Scheme 1**). In all cases, the mixture of all 4 diastereomers was obtained. In the cases of **VM019** and **VM045** the *cis*- and *trans*-diastereomeric mixture were inseparable, while in the cases of **VM009-VM012**, **VM025**, **VM036**, **VM038** and **VM040** both *cis*- and *trans*- pure racemic mixtures were obtained after a second chromatography of the initial mixture of all 4 diastereomers. The *cis*- and *trans*-isomers were assigned by comparison of the corresponding ¹H NMR and ¹³C NMR chemical shifts of α-CH and β-CH to our previous work for both β-hydroxy acids and β-lactones.⁴⁵

<figure><img src="image_16.png" /></figure>
**Method A**

<figure><img src="image_17.png" /></figure>

1a-j

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

2a-j

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

VM lactones

View Article Online
DOI: 10.1039/D5MD00102A

<table><thead><tr><th></th><th>R<sup>1</sup></th><th>R<sup>2</sup></th></tr></thead><tbody><tr><td>1-2a, VM009, VM013, VM020</td><td>cis-CH<sub>3</sub>(CH<sub>2</sub>)<sub>7</sub>CH=CH(CH<sub>2</sub>)<sub>6</sub>-</td><td>CH<sub>3</sub>-</td></tr><tr><td>1-2b, VM010, VM017, VM018</td><td>cis-CH<sub>3</sub>(CH<sub>2</sub>)<sub>7</sub>CH=CH(CH<sub>2</sub>)<sub>6</sub>-</td><td>CH<sub>3</sub>CH<sub>2</sub>-</td></tr><tr><td>1-2c, VM011, VM022, VM024</td><td>cis-CH<sub>3</sub>(CH<sub>2</sub>)<sub>7</sub>CH=CH(CH<sub>2</sub>)<sub>6</sub>-</td><td>CH<sub>3</sub>(CH<sub>2</sub>)<sub>5</sub>-</td></tr><tr><td>1-2d, VM012, VM021, VM023</td><td>cis-CH<sub>3</sub>(CH<sub>2</sub>)<sub>7</sub>CH=CH(CH<sub>2</sub>)<sub>6</sub>-</td><td>CH<sub>3</sub>(CH<sub>2</sub>)<sub>8</sub>-</td></tr><tr><td>1-2e, VM019</td><td>CH<sub>3</sub>(CH<sub>2</sub>)<sub>5</sub>-</td><td>CH<sub>3</sub>(CH<sub>2</sub>)<sub>2</sub>-</td></tr><tr><td>1-2f, VM025, VM026, VM027</td><td>CH<sub>3</sub>(CH<sub>2</sub>)<sub>9</sub>-</td><td>CH<sub>3</sub>(CH<sub>2</sub>)<sub>2</sub>-</td></tr><tr><td>1-2g, VM036, VM037</td><td>CH<sub>3</sub>(CH<sub>2</sub>)<sub>2</sub>-</td><td>cis-CH<sub>3</sub>(CH<sub>2</sub>)<sub>7</sub>CH=CH(CH<sub>2</sub>)<sub>7</sub>-</td></tr><tr><td>1-2h, VM038, VM039</td><td>CH<sub>3</sub>(CH<sub>2</sub>)<sub>7</sub>-</td><td>CH<sub>3</sub>(CH<sub>2</sub>)<sub>2</sub>-</td></tr><tr><td>1-2i, VM040, VM041</td><td>CH<sub>3</sub>(CH<sub>2</sub>)<sub>11</sub>-</td><td>CH<sub>3</sub>(CH<sub>2</sub>)<sub>2</sub>-</td></tr><tr><td>1-2j, VM045</td><td>CH<sub>3</sub>(CH<sub>2</sub>)<sub>7</sub>O(CH<sub>2</sub>)<sub>8</sub>-</td><td>CH<sub>3</sub>(CH<sub>2</sub>)<sub>2</sub>-</td></tr></tbody></table>

**Scheme 1.** Synthesis of β-lactones form an aldol reaction (**Method A**). Reagents and conditions: (a) i) LDA (prepared *in situ* from (i-Pr)<sub>2</sub>NH and *n*-BuLi), dry THF, 0 °C, 1 h; ii) R<sup>2</sup>CHO, dry THF, 0 °C, 1 h, then r.t. 16 h; iii) 1N HCl, 33-93%; (b) i) *p*-TsCl, dry pyridine, 0 °C, 1 h, then 4 °C, 3 days, 36-70%; ii) chromatographic separation of the racemic mixtures of *cis*- and *trans*-β-lactones.

For the synthesis of β-lactones **VM028**, **VM047** and **VM048**, a different synthetic route (**Method B** – **Scheme 2**) was chosen either to insert the desired substituent from a commercially available starting material or to avoid any side reactions that would be caused by the use of LDA in the aldol reaction mentioned above. In this procedure, butyric acid **3** reacted with 1,1'-carbonyl diimidazole (CDI) and monoethyl malonic acid magnesium salt using Masamune's method<sup>65</sup> to yield the corresponding β-keto esters **4**. Deprotonation of the more acidic proton in α-position of the β-keto ester carbonyl function using less reactive K<sub>2</sub>CO<sub>3</sub> base, followed by substitution reaction with an alkyl iodide bearing the desired R<sup>1</sup> chain as substituent yielded the substituted β-keto esters **5** which were subsequently reduced using NaBH<sub>4</sub>. Hydrolysis of the resulting β-hydroxy esters **6** to the corresponding β-hydroxy acids **7** and a final cyclization reaction as described above using *p*-TsCl/pyridine afforded the final β-lactone **VM028**. For β-lactones **VM047** and **VM048**, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) with a catalytic amount of 4-dimethylaminopyridine (DMAP) was used. In the case of **VM028** both racemic mixtures of *cis*- and

<figure><img src="image_22.png" /></figure>
trans-β-lactones were isolated by column chromatography, whereas the diastereomers of VM047 and VM048 were inseparable by column chromatography.

View Article Online
DOI: 10.1039/D5MD00102A

### Method B

<figure><img src="image_23.png" /></figure>

<table><thead><tr><th></th><th>R<sup>1</sup></th><th>R<sup>2</sup></th></tr></thead><tbody><tr><td>5-7a, VM028, VM029, VM030</td><td>cis-CH<sub>3</sub>(CH<sub>2</sub>)<sub>7</sub>CH=CH(CH<sub>2</sub>)<sub>8</sub>-</td><td>CH<sub>3</sub>(CH<sub>2</sub>)<sub>2</sub>-</td></tr><tr><td>5-7b, VM047</td><td>CH<sub>3</sub>(CH<sub>2</sub>)<sub>5</sub>NHCO(CH<sub>2</sub>)<sub>7</sub>-</td><td>CH<sub>3</sub>(CH<sub>2</sub>)<sub>2</sub>-</td></tr><tr><td>5-7c, VM048</td><td>CH<sub>3</sub>(CH<sub>2</sub>)<sub>6</sub>CONH(CH<sub>2</sub>)<sub>6</sub>-</td><td>CH<sub>3</sub>(CH<sub>2</sub>)<sub>2</sub>-</td></tr></tbody></table>

**Scheme 2.** Synthesis of β-lactones from appropriately substituted β-keto ester (Method B). Reagents and conditions: (a) i) CDI, dry THF, r.t., 6 h; ii) MgCl₂, EtOCOCH₂COOK, r.t., 18 h; iii) aq. HCl 1N, 95%; (b) K₂CO₃, R¹I, acetone/DMF, reflux, 18 h, 50-78%; (c) NaBH₄, EtOH, 0 °C 30 min, then r.t. 3 h, 70-86%; (d) NaOH 1N, 1,4-dioxane, r.t., 16 h, 54-85%; (e) For VM028: i) p-TsCl, dry pyridine, 0 °C, 1 h, then 4 °C, 3 days, 60%; ii) chromatographic separation of the racemic mixtures of cis- and trans-β-lactones; or for VM047 and VM048: EDC·HCl, DMAP, dry DCM, r.t., 72 h, 39-45%.

We also decided to incorporate a fluorine atom as second substituent at the α-position of the β-lactone ring. Fluorine atoms are widely used in compounds of medicinal interest as they enhance physicochemical properties such as solubility and lipophilicity, but also often increase the affinity of bioactive compounds for enzymes and receptors. To that end, two different synthetic procedures, Methods C & D, were established as depicted in **Scheme 3**. Method C is similar to the previously described Method B (**Scheme 2**) with the addition of an extra synthetic fluorination step (**Scheme 3** – Method C, step c) after the synthesis of the monosubstituted β-keto esters **8** and before the cyclisation step. The fluorination step that we introduced here for the first time included the use of sodium hydride for the deprotonation of the monosubstituted β-keto ester **8**, followed by the use of Selectfluor to insert the fluorine atom. The β-lactones VM043 and VM044 were successfully prepared according to this procedure.

<figure><img src="image_24.png" /></figure>
Furthermore, in order to incorporate the fluorine atom directly on the β-lactone ring, we further developed a more efficient fluorination method where the fluorination step would be performed at the last step of the synthesis, after the cyclisation. This proposed late-stage fluorination method would be much more efficient since it could be applied to already synthesized β-lactone molecules, or to new β-lactones such as **VM045**. Indeed, this new Method D (**Scheme 3**) includes the use of LDA on the β-lactone and treatment with *N*-fluorobenzenesulfonimide (NFSI) as fluorinating electrophile. **VM042** and **VM046** which are the fluorinated analogues of **VM001** and **VM045**, respectively, have been prepared *via* this late-stage fluorination Method D.

View Article Online
DOI: 10.1039/D5MD00102A

### Method C

<figure><img src="image_25.png" /></figure>

3

4

8a-b

9a-b

<figure><img src="image_26.png" /></figure>

10a-b

e

<figure><img src="image_27.png" /></figure>

11a-b

f

<figure><img src="image_28.png" /></figure>

VM lactones

<table><thead><tr><th></th><th>R<sup>1</sup></th><th>R<sup>2</sup></th></tr></thead><tbody><tr><td>8-11a, VM043</td><td>CH<sub>3</sub>(CH<sub>2</sub>)<sub>7</sub>-</td><td>CH<sub>3</sub>(CH<sub>2</sub>)<sub>2</sub>-</td></tr><tr><td>8-11b, VM044</td><td>CH<sub>3</sub>(CH<sub>2</sub>)<sub>11</sub>-</td><td>CH<sub>3</sub>(CH<sub>2</sub>)<sub>2</sub>-</td></tr></tbody></table>

### Method D

<figure><img src="image_29.png" /></figure>

VM001
VM045

<figure><img src="image_30.png" /></figure>

VM042
VM046

<table><thead><tr><th></th><th>R<sup>1</sup></th><th>R<sup>2</sup></th></tr></thead><tbody><tr><td>VM001, VM042</td><td rowspan="2">cis-CH<sub>3</sub>(CH<sub>2</sub>)<sub>7</sub>CH=CH(CH<sub>2</sub>)<sub>8</sub>-</td><td>CH<sub>3</sub>(CH<sub>2</sub>)<sub>2</sub>-</td></tr><tr><td>VM045, VM046</td><td>CH<sub>3</sub>(CH<sub>2</sub>)<sub>7</sub>O(CH<sub>2</sub>)<sub>8</sub>-</td><td>CH<sub>3</sub>(CH<sub>2</sub>)<sub>2</sub>-</td></tr></tbody></table>

**Scheme 3.** Synthesis of fluorinated β-lactones (**Methods C & D**). Reagents and conditions: (a) i) CDI, dry THF, r.t., 6 h; ii) MgCl₂, EtOCOCH₂COOK, r.t., 18 h; iii) aq. HCl 1N, 95%; (b) K₂CO₃, R¹I, acetone/DMF, reflux, 18 h, 67-70%; (c) i) NaH, dry THF, -20 °C, 1 h, ii) Selectfluor, dry MeCN, -20 °C, 2.5 h, 78-82%; (d) NaBH₄, EtOH, 0 °C 30 min, then r.t. 3 h, 61-82%; (e) NaOH 1N or LiOH 1N, THF or 1,4-dioxane, r.t., 16 h, 51-75%; (f) EDC·HCl, DMAP, dry DCM, r.t., 72 h, 29-36%; (g) i) LDA (prepared *in situ* from (i-Pr)₂NH and *n*-BuLi), dry THF, -78 °C, 30 min, ii) NFSI, dry THF, -78 °C to r.t., 2.5 h, 23-39%.

Finally, three β-lactam analogues were synthesized. For this purpose, a synthetic method (**Scheme 4** - Method E) starting from the corresponding β-lactone was established. After opening the β-lactone ring by the nucleophilic attack of NaN₃ to give the corresponding β-azido acids **12**, the azide group
was reduced by catalytic hydrogenation using 10% Pd/C, and finally the β-lactam ring of **13** was closed using Mukaiyama's reagent⁶⁶ in a very dilute solution to avoid the intermolecular reaction (Scheme 4).

View Article Online
DOI: 10.1039/D5MD00102A

### Method E

<figure><img src="image_31.png" /></figure>

**Scheme 4.** Synthesis of β-lactams **VM056** and **VM057** (Method E). Reagents and conditions: (a) NaN₃, DMF, 60 °C, 16 h, 65-77%; (b) H₂, 10% Pd/C, MeOH, r.t., 2h, 36-44%; (c) i) Mukaiyama's reagent, Et₃N, CH₃CN, reflux, 4 h, then r.t., 16 h, 65-71%; ii) chromatographic separation of the racemic mixture of the *trans*-β-lactone **VM058**.

## 3.2 Antimycobacterial Activity of the new β-lactone Compounds

Drug susceptibility testing of the 27 β-lactone and 3 β-lactam derivatives was assessed against two pathogenic mycobacterial species: *M. tuberculosis* mc²6230,⁴⁹ and the two S & R variants of *M. abscessus*. The corresponding MIC₅₀/MIC₉₀ values for each molecule, as determined by the resazurin microtiter assay (REMA),³⁰, ⁴⁵, ⁵³, ⁵⁴ are reported in **Table 1**. Amikacin (AMK) which is a core drug used in the treatment of *M. abscessus*, with MIC₅₀ / MIC₉₀ of 4 / 32 and 8 / 32 µg/mL against the S & R variant,⁶⁷ respectively; as well as a second-line injectable drug for the treatment of multidrug resistant TB, with MIC₅₀ / MIC₉₀ ~1 µg/mL against *M. tuberculosis* H37Rv,⁶⁸ was used as reference drug.

Nearly all β-lactone derivatives were active against *M. tuberculosis* mc²6230 (i.e., 18 compounds) growth (**Table 1**). With *M. tuberculosis*, MIC₅₀ values were indicative of either a poor (MIC₅₀ > 100 µg/mL for **VM020** to **VM024**, **VM027** to **VM030**, **VM036**, **VM037**, and **VM045**), a weak (MIC₅₀ ~45-66 µg/mL for **VM017** to **VM019**, **VM025**, **VM026**, **VM047**, and **VM048**), a moderate (MIC₅₀ ~20-35 µg/mL for **VM039**, **VM043**, **VM044**, **VM046**, **VM057**, and **VM058**), or a quite good

<figure><img src="image_32.png" /></figure>
(~9.6-17.7 µg/mL for VM038, VM040 to VM042, and VM056) antibacterial activity comparable to that of Orlistat (11.0 µg/mL) (Table 1). Of note, 15 of the 18 active β-lactone derivatives exhibited interesting MIC<sub>90</sub> values in the range of 21-67 µg/mL. Remarkably, most of these latter β-lactones exhibited MIC<sub>50</sub> and MIC<sub>90</sub> values in the same range. Collectively, the best growth inhibitors of *M. tuberculosis* mc²6230 were VM040, VM044 and VM056 with MIC<sub>50</sub> / MIC<sub>90</sub> in the range 9.6-20.8 / 21.8-29.1 µg/mL, as compared to 0.24 / 0.37 µg/mL for amikacin (Table 1).

View Article Online

DOI: 10.1039/D5MD00102A

In the case of *M. abscessus*, only 6 β-lactones were found to block the growth of the S variant. These included VM025 (MIC<sub>50</sub> = 49.9±1.3 µg/mL / MIC<sub>90</sub> = 52.4±2.5 µg/mL), VM026 (MIC<sub>50</sub> = 47.2±1.5 µg/mL / MIC<sub>90</sub> = 56.7±2.2 µg/mL), VM043 (MIC<sub>50</sub> = 33.8±1.2 µg/mL / MIC<sub>90</sub> = 49.2±3.4 µg/mL), VM056 (MIC<sub>50</sub> = 47.3±6.0 µg/mL / MIC<sub>90</sub> = 74.1±3.9 µg/mL), VM057 (MIC<sub>50</sub> = 41.9±7.4 µg/mL / MIC<sub>90</sub> = 44.4±6.2 µg/mL), and VM058 (MIC<sub>50</sub> = 41.8±6.3 µg/mL / MIC<sub>90</sub> = 46.6±4.0 µg/mL) for which both MIC<sub>50</sub> & MIC<sub>90</sub> values were in the same order of magnitude (Table 1). Among these, three molecules were also active against *M. abscessus* R: VM025 (MIC<sub>50</sub> = 27.1±0.80 µg/mL) and VM026 (MIC<sub>50</sub> = 49.9±1.3 µg/mL) but with MIC<sub>90</sub> values higher than 100 µg/mL; and VM043 (MIC<sub>50</sub> = 59.8±5.6 µg/mL and a MIC<sub>90</sub> = 88.9±0.3 µg/mL). Interestingly, although VM022 and VM024 were inactive against *M. abscessus* S (MIC > 100 µg/mL), these two molecules were however found to impair the growth of the R morphotype with interesting, albeit moderate, MIC<sub>50</sub> / MIC<sub>90</sub> values (MIC<sub>50</sub> = 59.9±2.1 µg/mL / MIC<sub>90</sub> = 68.9±2.7 µg/mL, and MIC<sub>50</sub> = 41.9±2.9 µg/mL / MIC<sub>90</sub> = 64.7±0.66 µg/mL, respectively). In addition, although VM023, which is also inactive against the S variant, has an interesting MIC<sub>50</sub> value (31.0±2.8 µg/mL), its MIC<sub>90</sub> was however higher than 100 µg/mL. Overall, the best inhibitors of *M. abscessus* R growth were VM022, VM024, and VM043 which exhibited similar MIC<sub>50</sub> / MIC<sub>90</sub> values, respectively (Table 1).

<table><caption>**Table 1.** Antibacterial activities of Orlistat and β-lactone derivatives against *M. tuberculosis* mc²6230 and *M. abscessus* S and R variants. a</caption><thead><tr><td>Compounds</td><td>MIC<sub>50</sub> / MIC<sub>90</sub> (µg/mL) <sup>a</sup></td></tr></thead></table>
<table><thead><tr><th rowspan="2"></th><th colspan="2">M. abscessus CIP 104536<sup>T</sup></th><th rowspan="2">M. tuberculosis<br/>mc<sup>26320</sup><br/>DOI: 10.1039/D5MD00102A</th></tr><tr><th>Smooth</th><th>Rough</th></tr></thead><tbody><tr><td>Orlistat<sup>b</sup></td><td>44.4±0.68 / 45.6±0.56</td><td>57.0±0.11 / 73.5±0.50</td><td>11.0±0.50 / 13.5±1.0</td></tr><tr><td>VM001 (C16:1 ω9)<sup>b</sup></td><td>>100</td><td>>100</td><td>31.8±1.5 / >100</td></tr><tr><td>VM008 = trans-(R,R)-VM001<sup>b</sup></td><td>>100</td><td>>100</td><td>19.7±1.3 / 31.9±1.4</td></tr><tr><td>VM009<sup>b</sup></td><td>>100</td><td>>100</td><td>>100</td></tr><tr><td>VM013 = trans-VM009<sup>b</sup></td><td>>100</td><td>>100</td><td>33.6±0.21 / 42.9±1.3</td></tr><tr><td>VM020 = cis-VM009</td><td>>100</td><td>>100</td><td>>100</td></tr><tr><td>VM010<sup>b</sup></td><td>>100</td><td>>100</td><td>33.5±0.26 / 69.9±0.94</td></tr><tr><td>VM017 = trans-VM010</td><td>>100</td><td>>100</td><td>45.8±1.0 / 47.8±1.8</td></tr><tr><td>VM018 = cis-VM010</td><td>>100</td><td>>100</td><td>47.2±1.2 / 53.1±2.0</td></tr><tr><td>VM011<sup>b</sup></td><td>>100</td><td>>100</td><td>>100</td></tr><tr><td>VM022 = trans-VM011</td><td>>100</td><td>59.9±2.1 / 68.9±2.7</td><td>>100</td></tr><tr><td>VM024 = cis-VM011</td><td>>100</td><td>41.9±2.9 / 64.7±0.66</td><td>>100</td></tr><tr><td>VM012<sup>b</sup></td><td>>100</td><td>>100</td><td>>100</td></tr><tr><td>VM021 = trans-VM012</td><td>>100</td><td>>100</td><td>>100</td></tr><tr><td>VM023 = cis-VM012</td><td>>100</td><td>31.0±2.8 / >100</td><td>>100</td></tr><tr><td>VM019 (trans)</td><td>>100</td><td>>100</td><td>65.2±0.93 / 67.2±0.61</td></tr><tr><td>VM025</td><td>49.9±1.3 / 52.4±2.5</td><td>27.1±0.80 / >100</td><td>63.9±1.6 / 65.1±1.5</td></tr><tr><td>VM026 = trans-VM025</td><td>47.2±1.5 / 56.7±2.2</td><td>25.6±1.4 / >100</td><td>59.2±2.8 / >100</td></tr><tr><td>VM027 = cis-VM025</td><td>>100</td><td>>100</td><td>>100</td></tr><tr><td>VM028 (C18:1 ω9)</td><td>>100</td><td>>100</td><td>>100</td></tr><tr><td>VM029 = trans-VM028</td><td>>100</td><td>>100</td><td>>100</td></tr><tr><td>VM030 = cis-VM028</td><td>>100</td><td>>100</td><td>>100</td></tr><tr><td>VM037</td><td>>100</td><td>>100</td><td>>100</td></tr><tr><td>VM036 = trans-VM037</td><td>>100</td><td>>100</td><td>>100</td></tr><tr><td>VM039</td><td>>100</td><td>>100</td><td>24.7±1.7 / 51.3±1.3</td></tr><tr><td>VM038 = trans-VM039</td><td>>100</td><td>>100</td><td>17.7±0.22 / 46.5±1.6</td></tr><tr><td>VM040 (trans)</td><td>>100</td><td>>100</td><td>9.6±0.45 / 29.1±1.6</td></tr><tr><td>VM041 (cis)</td><td>>100</td><td>>100</td><td>17.4±1.6 / 44.2±1.7</td></tr><tr><td>VM042</td><td>>100</td><td>>100</td><td>10.2±3.6 / 40.1±7.5</td></tr><tr><td>VM043 = fluorinated VM038-039</td><td>33.8±1.2 / 49.2±3.4</td><td>59.8±5.6 / 88.9±0.3</td><td>30.2±1.3 / 45.9±2.5</td></tr><tr><td>VM044 = fluorinated VM040-041</td><td>>100</td><td>>100</td><td>20.8±0.2 / 24.1±0.1</td></tr><tr><td>VM045</td><td>>100</td><td>>100</td><td>>100</td></tr><tr><td>VM046 = fluorinated VM045</td><td>>100</td><td>>100</td><td>32.2±4.2 / 45.0±3.3</td></tr><tr><td>VM047</td><td>>100</td><td>>100</td><td>46.2±4.0 / >100</td></tr><tr><td>VM048</td><td>>100</td><td>>100</td><td>46.8±8.7 / >100</td></tr><tr><td>VM056 = VM040 β-lactam</td><td>47.3±6.0 / 74.1±3.9</td><td>>100</td><td>15.2±0.6 / 21.8±0.7</td></tr><tr><td>VM057 = trans-VM058 β-lactam</td><td>41.9±7.4 / 44.4±6.2</td><td>>100</td><td>35.5±1.2 / 44.2±1.1</td></tr><tr><td>VM058 = β-lactam</td><td>41.8±6.3 / 46.6±4.0</td><td>>100</td><td>23.7±0.2 / 44.2±2.4</td></tr><tr><td>AMK</td><td>2.3±0.12 / 3.4±0.11</td><td>4.3±0.15 / 5.9±0.26</td><td>0.24±0.01 / 0.37±0.02</td></tr></tbody></table>

<footnote><sup>a</sup> Minimum inhibitory concentration leading to 50% or 90% growth inhibition (MIC<sub>50</sub> / MIC<sub>90</sub>) as determined by the resazurin microtiter assay (REMA). <sup>b</sup> Data from our previous study.<sup>45</sup> AMK: amikacin. All reported values are expressed as mean ± SD of three independent assays.</footnote>
From all these results, some structure-activity relationships (SAR) tendencies can however be set up.

First, and as mentioned in our previous report,⁴⁵ *trans*-β-lactones were almost >2 times more active than the corresponding *cis* isomers, and nearly as active as the *cis/trans* isomeric mixtures. This is best illustrated by the antibacterial activity of **VM026** (*trans*-**VM025**) on *M. tuberculosis* mc²6230 and *M. abscessus* compared to the *cis* isomer **VM027** (*cis*-**VM025**) and the *cis/trans* isomeric mixture **VM025** (Table 1).

Remarkably, the three β-lactones **VM025**, **VM026** (*trans*-**VM025**), and **VM043** which were able to efficiently inhibit the growth of all mycobacteria were bearing a medium R¹ octyl or decyl chain, and a short R² propyl chain (Figure 1). In terms of lipophilicity this translates into a narrow range of calculated Log P_o/w, determined using the iLOGP⁶⁹ method from the SwissADME web tool,⁷⁰ between 4.3 and 4.8 for these latter three compounds (Table S1). Indeed, the insertion of a fluorine atom on the β-lactone ring of **VM038** and **VM039** (Log P_o/w ~ 4.12) slightly increased the lipophilicity of the fluorinated derivative **VM043** (Log P_o/w ~ 4.31). This fluorine will also affect the electrophilic nature of the carbonyl, making it even more reactive and sensitive to nucleophilic attack by enzymes with nucleophilic residues, thus resulting in a significantly improved antibacterial activity for **VM043** as compared to the parent molecules. Finally, switching from a β-lactone core (**VM038** and **VM039**, and **VM040** and **VM041**) to a β-lactam core (**VM057** and **VM058**, and **VM056**, respectively) led to a partial improvement in antibacterial properties against *M. abscessus* S.

In summary, while **VM025**, **VM026** and **VM043** are active against all three mycobacterial strains, **VM043** is the best growth inhibitor with MIC₅₀ / MIC₉₀ values very similar to those of **Orlistat**.⁴⁵,⁴⁶
As already discussed in the case of others families of mycobacterial growth inhibitors,²⁸,⁵⁴ the reason for such differences in β-lactones activity against *M. tuberculosis* compared to *M. abscessus* S and R variants still remains unclear and would require further study. However, differences in membrane composition and permeability between these three mycobacterial species are likely to play a key role in this phenotype.

View Article Online

DOI: 10.1039/D5MD00102A

<figure><img src="image_33.png" /></figure>

RSC Medicinal Chemistry Accepted Manuscript
### 3.3 Synthesis of β-lactone Activity-Based Probes

View Article Online

DOI: 10.1039/D5MD00102A

Having established the promising antibacterial activity of **VM025**, **VM026** and **VM043** against *M. tuberculosis* mc²6230 and more especially *M. abscessus*, we further aimed at identifying their target enzymes using click-chemistry activity-based protein profiling (CC-ABPP) approach.²⁶, ⁷¹⁻⁷⁴ Considering the covalent mechanism of action attributed to **Orlistat**³⁴, ⁴² and related β-lactone derivatives,⁷² a series of eight new molecules were synthesized by substituting the R¹ or R² alkyl chain with a terminal alkyne or azide group, to provide a panel of new activity-based probes (ABP), i.e., **VM035*<sub>p</sub>**, and **VM049*<sub>p</sub>** to **VM055*<sub>p</sub>** probes, that would be capable of retaining the antibacterial activity of their respective parent molecules (**Schemes 5-6** and **Table 2**).

For the synthesis of the β-lactones ABP that would be tested in CC-ABPP, several different synthetic routes were established (Scheme 5). The previous Method B was first applied on 4-pentynoic acid **14** as starting material following the steps previously presented in Scheme 2. Reaction of **14** with CDI and monoethyl malonic acid magnesium salt afforded the β-keto ester **15**. Then, substitution with K₂CO₃ as base and the appropriate alkyl iodide followed by NaBH₄ reduction and hydrolysis, led to the β-hydroxy acids **18**. Finally, β-lactone ring formation using *p*-TsCl in dry pyridine produced the final **VM035*<sub>p</sub>** and **VM054*<sub>p</sub>** probes that bear a terminal alkyne at the β-position of the ring. Methods F-H were further designed to synthesize β-lactones that bear the terminal alkyne at the α-position of the ring. Method F utilizes commercially available 6-heptynoic acid **19** that was protected as a *tert*-butyl ester at the carboxyl end using DCC, catalytic DMAP and *tert*-butanol. Then, a TIPS protecting group was added with *n*-BuLi and TIPS-Cl at the terminal alkyne for protection, followed by TFA deprotection of the ester to yield the free acid **22**. With the terminal alkyne group protected, the acid undergoes an aldol reaction using LDA and butyraldehyde, as previously described in Scheme 1, followed by cyclization with *p*-TsCl in dry pyridine. The final step of this synthesis is the deprotection of the terminal alkyne using TBAF in dry THF for the production of the diastereomeric mixture of β-lactone **VM049*<sub>p</sub>** and the racemic *trans*-mixture of **VM050*<sub>p</sub>**.

<figure><img src="image_34.png" /></figure>
**Method B**

<figure><img src="image_35.png" /></figure>

14

a

<figure><img src="image_36.png" /></figure>

15

b

<figure><img src="image_37.png" /></figure>

c

View Article Online
DOI: 10.1039/D5MD00102A

16a-b

<figure><img src="image_38.png" /></figure>

17a-b

d

<figure><img src="image_39.png" /></figure>

18a-b

e

<figure><img src="image_40.png" /></figure>

VM probes

16-18a, VM035<sub>p</sub>, R<sup>1</sup>: cis-CH<sub>3</sub>(CH<sub>2</sub>)<sub>7</sub>CH=CH(CH<sub>2</sub>)<sub>8</sub>-

16-18b, VM054<sub>p</sub>, R<sup>1</sup>: CH<sub>3</sub>(CH<sub>2</sub>)<sub>11</sub>-

**Method F**

<figure><img src="image_41.png" /></figure>

19

<figure><img src="image_42.png" /></figure>

<figure><img src="image_43.png" /></figure>

<figure><img src="image_44.png" /></figure>

<figure><img src="image_45.png" /></figure>

<figure><img src="image_46.png" /></figure>

<figure><img src="image_47.png" /></figure>

22

20

21

<figure><img src="image_48.png" /></figure>

23

<figure><img src="image_49.png" /></figure>

24

<figure><img src="image_50.png" /></figure>

<figure><img src="image_51.png" /></figure>

VM049<sub>p</sub>, VM050<sub>p</sub>

**Method G**

<figure><img src="image_52.png" /></figure>

25

<figure><img src="image_53.png" /></figure>

<figure><img src="image_54.png" /></figure>

<figure><img src="image_55.png" /></figure>

<figure><img src="image_56.png" /></figure>

<figure><img src="image_57.png" /></figure>

<figure><img src="image_58.png" /></figure>

28

26

27

<figure><img src="image_59.png" /></figure>

29

<figure><img src="image_60.png" /></figure>

<figure><img src="image_61.png" /></figure>

VM051<sub>p</sub>

**Method H**

<figure><img src="image_62.png" /></figure>

30a-b

<figure><img src="image_63.png" /></figure>

31a-b

<figure><img src="image_64.png" /></figure>

<figure><img src="image_65.png" /></figure>

32a-b

<figure><img src="image_66.png" /></figure>

<figure><img src="image_67.png" /></figure>

33a-b

<figure><img src="image_68.png" /></figure>

34a-b

<figure><img src="image_69.png" /></figure>

35a-b

<figure><img src="image_70.png" /></figure>

<figure><img src="image_71.png" /></figure>

VM lactones

30-35a, VM052<sub>p</sub>, n=6

30-35b, VM053<sub>p</sub>, n=5

**Scheme 5.** Synthesis of β-lactone probes bearing a terminal alkyne. Reagents and conditions: **Method B.** (a) i) CDI, dry THF, r.t., 6 h; ii) MgCl₂, EtOCOCH₂COOK, r.t., 18 h; iii) aq. HCl 1N, 82%; (b) K₂CO₃, R¹I, acetone/DMF, reflux, 18 h, 76-83%; (c) NaBH₄, EtOH, 0 °C 30 min, then r.t. 3 h, 69-75%; (d) NaOH 1N, 1,4-dioxane, r.t., 16 h, 30-87%; (e) p-TsCl, dry pyridine, 0 °C, 1 h, then 4 °C, 3 days, 40-71%. **Method F.** (f) t-BuOH, DMAP, dry DCM, DCC, 75%; (g) i) n-BuLi, dry THF; ii) TIPS-Cl, 77%; (h) 15% TFA in DCM, 75%; (i) i) LDA (prepared *in situ* from (i-Pr)₂NH and n-BuLi), dry THF; ii) butyraldehyde, dry THF, 0 °C to
r.t., 66-67%; (j) TBAF, dry THF, 65-100%. **Method G.** (k) NaI, acetone, 95%; (l) *n*-BuLi, TIPS-acetylene, dry THF, -78 °C, 62%. **Method H.** (m) propargyl alcohol, *n*-BuLi, THF/HMPA, -78 °C to r.t., 83-84%; (n) *n*-BuLi, 1,2-ethylenediamine, 0 °C to 70 °C, 65-66%; (o) Jones reagent, acetone, 0 °C, 81%; (p) *i*) *n*-BuLi, dry THF; ii) TIPS-Cl, iii) K₂CO₃, THF/MeOH/H₂O 3:1:1 (v/v/v), 38-64%.

View Article Online
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.

In method G, an ω-bromo carboxylic acid may be used. Here, 11-bromoundecanoic acid **25** is transformed into the corresponding 11-iodoundecanoic acid **26** using NaI in acetone, that is then used to a substitution reaction to TIPS-acetylene using *n*-BuLi in dry THF and HMPA, to yield the corresponding TIPS protected tridec-12-ynoic acid **27**. This acid is then used in an aldol reaction, cyclization and final deprotection, as described in the above-mentioned Method B to yield β-lactone **VM051p**.

All of the above-mentioned methods used commercially ω-alkynoic acids or ω-bromo acids as starting materials that are expensive and only few are commercially available. To that end, we designed a synthetic procedure (Method H) starting from any bromoalkane and propargyl alcohol that would lead to any ω-alkynoic acid using inexpensive starting materials. Indeed, starting from 1-bromooctane or 1-bromomonane **30** using propargyl alcohol and *n*-BuLi in dry THF and HMPA, the corresponding internal alkynyl alcohols **31** were prepared. A Zipper reaction⁷⁵ using NaH and ethylenediamine moved the triple bond to the ω-end of the alcohol **32** and subsequent oxidation using Jones reagent in acetone yielded the desired terminal alkyne carboxylic acids **33**. Protection of the alkyne with TIPS group, aldol reaction with butyraldehyde, cyclization and finally TIPS deprotection produced the β-lactones **VM052p** and **VM053p**.

**Method I**

<figure><img src="image_72.png" /><figcaption>**Scheme 6.** Synthesis of β-lactone probes bearing a terminal azide (**Method I**). Reagents and conditions: (a) conc. HBr, toluene, reflux, 68%; (b) TBAB, NaN₃, benzene, reflux, 95%; (c) Jones reagent, acetone, 0 °C,</figcaption></figure>

RSC Medicinal Chemistry Accepted Manuscript
71%; (d) i) LDA (prepared in situ from (i-Pr)₂NH and *n*-BuLi), dry THF ; ii) butyraldehyde, dry THF, 0 °C to r.t.; iii) column chromatography ; iv) *p*-TsCl, dry pyridine, 0 °C to 4 °C, 12%.

View Article Online

DOI: 10.1039/D5MD00102A

For the terminal azide β-lactone probes, we started from 1,10-decanediol **36** which was monobrominated using HBr in toluene under reflux (**Scheme 6**, Method I). Then, the bromine was substituted by the azide group using NaN₃ and TBAB in benzene, followed by Jones oxidation, aldol reaction and cyclization to yield the terminal azide β-lactone **VM055p**.

### 3.4 Activity-Based Protein Profiling (ABPP) Approach for Targets Identification

The antimicrobial potency of the synthesized β-lactone probes [VM035p, VM049p to VM055p] was first evaluated against *M. tuberculosis* mc²6230 and *M. abscessus* S and R variants (**Table 2**). Partial loss of activity was observed for VM049p and VM050p (i.e., pentynyl version of VM019), and even no activity for VM035p (i.e., butynyl version of VM028) as compared to unmarked parent molecules (**Table 2**). On the other hand, VM054p (i.e., pentynyl version of VM040 and VM041) bearing the alkyne on the alkyl chain in β-position, and VM051p (i.e., undecynyl version of VM025) retained comparable activity to their parent molecules against *M. tuberculosis* mc²6230. The same trend was observed with VM052p and VM053p, the nonyl and octyl derivatives, respectively, of VM038 and VM039, which gained some antibacterial activity against *M. abscessus* S while displaying similar activity to the parent β-lactones against *M. tuberculosis* mc²6230. Remarkably, the azide derivative VM055p not only retained good antibacterial activities as the unmarked parent VM038 and VM039 molecules against *M. tuberculosis* mc²6230, but also showed same moderate antibacterial activity as VM043 against *M. abscessus* S and R variants despite the absence of the fluorine atom, with fold changes in MIC₅₀ / MIC₉₀ of around ×0.9-1.2 / >2.0.

Overall, these results indicate that the choice of the alkyl chain length for the incorporation of a terminal alkyne bond, as well as its location in either the R¹ or R² position of the lactone cycle, is of great importance to avoid major changes in the antibacterial activity of the resulting β-lactone ABPs.

<figure><img src="image_73.png" /></figure>
<figure><img src="image_74.png" /></figure>

<table><caption>**Table 2.** Antibacterial activities of β-lactone probes against *M. tuberculosis* mc²6230 and *M. abscessus* S and R variants. <sup>a</sup></caption><thead><tr><th rowspan="3">Compounds</th><th colspan="3">MIC<sub>50</sub> / MIC<sub>90</sub> (µg/mL) <sup>a</sup></th></tr><tr><th colspan="2">M. abscessus CIP 104536<sup>T</sup></th><th>M. tuberculosis<br/>mc<sup>2</sup>6230</th></tr><tr><th>Smooth</th><th>Rough</th><th></th></tr></thead><tbody><tr><td>VM035<sub>p</sub><br/> (= VM028 probe)</td><td></td><td>>100<br/>(>100)</td><td>>100<br/>(>100)</td></tr><tr><td>VM049<sub>p</sub><br/> (= VM019 probe)</td><td></td><td>>100<br/>(>100)</td><td>>100<br/>(>100)</td></tr><tr><td>VM050<sub>p</sub><br/> (= VM019 probe)</td><td></td><td>>100<br/>(>100)</td><td>>100<br/>(>100)</td></tr><tr><td>VM051<sub>p</sub><br/> (= VM025 probe)</td><td></td><td>35.0±7.1 / >100<br/>(49.9±1.3 / 52.4±2.5)</td><td>64.6±0.1 / >100<br/>(27.1±0.80 / >100)</td></tr><tr><td>VM052<sub>p</sub><br/> (= VM038-VM039 probe)<br/> (= VM043 probe)</td><td></td><td>52.1±5.5 / >100<br/>(>100)<br/>(33.8±1.2 / 49.2±3.4)</td><td>>100<br/>(>100)<br/>(59.8±5.6 / 88.9±0.3)</td></tr><tr><td>VM053<sub>p</sub><br/> (= VM038-VM039 probe)<br/> (= VM043 probe)</td><td></td><td>63.4±1.3 / >100<br/>(>100)<br/>(33.8±1.2 / 49.2±3.4)</td><td>>100<br/>(>100)<br/>(59.8±5.6 / 88.9±0.3)</td></tr><tr><td>VM054<sub>p</sub><br/> (= VM040-VM041 probe)</td><td></td><td>>100<br/>(>100)</td><td>>100<br/>(>100)</td></tr><tr><td>VM055<sub>p</sub><br/> (= VM038-VM039 probe)<br/> (= VM043 probe)</td><td></td><td>39.0±4.5 / >100<br/>(>100)<br/>(33.8±1.2 / 49.2±3.4)</td><td>55.7±3.3 / >100<br/>(>100)<br/>(59.8±5.6 / 88.9±0.3)</td></tr></tbody></table>

<footnote><sup>a</sup> Minimum inhibitory concentration leading to 50% or 90% growth inhibition (MIC<sub>50</sub> / MIC<sub>90</sub>) as determined by the resazurin microtiter assay (REMA). All values are expressed as mean ± SD of three independent assays. The MIC values of the parent molecules derived from **Table 1** are shown in parentheses.</footnote>
According to these results, **VM055<sub>p</sub>** proved to be the best inhibitor of *M. abscessus* S & R and *M. tuberculosis* mc²6230 growth among the eight β-lactone probes tested, with MICs very similar to those of its parent molecules. This probe was therefore a logical choice for target enzyme identification through CC-ABPP workflow.<sup>26</sup>

View Article Online

DOI: 10.1039/D5MD00102A

To do so, exponential growth phase *M. abscessus* S cells were incubated with dimethyl sulfoxide (DMSO) and then treated with **VM055<sub>p</sub>** ABP (**Figure 2A**). Bacteria were lysed, and the probe-labeled proteins was subjected to strain-promoted azide-alkyne cycloaddition with DBCO-agarose beads.<sup>58</sup> Indeed, such strained cyclooctynes can exclusively react with azide-tagged biomolecules to form a stable 1,2,3-triazole linkage without the need for copper catalyst.<sup>76</sup> Here, the use of crosslinked agarose resin activated with DBCO functional groups, allows affinity enrichment of **VM055<sub>p</sub>**-labeled proteins. Following on-bead tryptic digestion, the resulting peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and quantified by label-free quantitative analysis<sup>59</sup> (**Figure 2**). The comparative proteomic analysis between a control sample (i.e. DMSO-treated *M. abscessus* cells for non-specific binding to DBCO-agarose beads) and **VM055<sub>p</sub>**-treated samples led to the identification of 12 target enzymes, when applying *p*-value ≤ 0.05 and Fold change ≥ 1.0 thresholds on the proteomics analysis results (**Figure 2A, Table 3 & Table S2**).

Even though at this stage no genes have been reported to be essential for *M. abscessus* growth by saturated transposon mutagenesis;<sup>77</sup> their corresponding orthologs in *M. tuberculosis* H37Rv were nevertheless reported using the KEGG database<sup>78, 79</sup> and then cross-referenced with the Mycob browser database<sup>80</sup> to provide further information on their essentiality, activity and predicted location.<sup>81</sup> Although three of the captured proteins are only conserved hypotheticals, the remaining nine ranged in their functional category from intermediary metabolism/respiration (2 proteins), lipid metabolism (1 protein), regulatory pathways (1 protein), cell wall/cell processes (3 proteins), and PE/PPE family proteins (2 proteins). Interestingly, these include the probable non-specific serine/threonine protein kinase MAB_3436 whose ortholog Rv0014c is annotated as essential for the *in vitro* growth of *M. tuberculosis*.<sup>82, 83</sup>
<figure><img src="image_75.png" /><figcaption>**Figure 2.** Click chemistry activity-based protein profiling on living *M. abscessus* S culture. CC-ABPP typical workflow for the identification of proteins covalently bound to (A) VM055<sub>p</sub> or (B) significantly outcompeted by VM043. A mid-log phase culture of *M. abscessus* S was pre-treated with either DMSO or VM043, and then labelled with VM055<sub>p</sub> probe. After cell lysis and strain-promoted azide-alkyne cycloaddition with DBCO-agarose beads, enriched samples were subjected to tryptic digestion. Tandem mass spectrometry analyses and subsequent differential peptides analysis allowed the identification of the target enzymes captured by VM055<sub>p</sub> and those outcompeted by addition of VM043. (C) Volcano plot of the differential analysis of the labeled proteome of samples pre-incubated with VM043 inhibitor prior to VM055<sub>p</sub> probe labeling vs. VM055<sub>p</sub> probe-labeled samples only, and showing the significance of two-sample t-test (–Log(p-value)) versus Fold change (Log<sub>2</sub>(LFQ normalized intensity in [VM043+VM055<sub>p</sub>] vs. VM055<sub>p</sub>)) on the y and x axes, respectively (n = 3 biological independent experiments per group). The dashed lines indicate the threshold of p-value ≤ 0.1 and a Fold change ≥ 1.0. Blue dots indicate VM055<sub>p</sub> targets proteins that are outcompeted by addition of VM043; while red dots represent VM055<sub>p</sub> targets proteins that were not inhibited by VM043. See also **Supplementary Tables S2-S6**.</figcaption></figure>

<figure><img src="image_76.png" /></figure>
<table><caption>Table 3. VM055<sub>p</sub> target proteins identified in *M. abscessus* S culture by LC-ESI-MS/MS analysis <sup>a</sup></caption><thead><tr><th rowspan="2">Gene name</th><th rowspan="2">Protein name</th><th colspan="4">M. tuberculosis orthologs</th></tr><tr><th>Rv number</th><th>Essentiality <sup>b</sup></th><th>Location <sup>c</sup></th><th>Activity / Function</th><th>Functional category <sup>d</sup></th></tr></thead><tbody><tr><td>MAB_3436</td><td>Non-specific serine/threonine protein kinase</td><td>Rv0014c</td><td>in vitro growth</td><td>CF; WCL</td><td>Serine/threonine-protein kinase PknB</td><td>RP</td></tr><tr><td>MAB_4551c</td><td>Possible acylglycerol lipase</td><td>Rv0183</td><td></td><td>CM; CW; WCL</td><td>Monoacylglycerol lipase</td><td>LM</td></tr><tr><td>MAB_1026c</td><td>Hypothetical protein</td><td>Rv1926c</td><td></td><td>CF; CM; WCL</td><td>Immunogenic protein Mpt63</td><td>CW/CP</td></tr><tr><td>MAB_0302</td><td>Putative quinolone synthase</td><td>Rv1260</td><td></td><td>WCL</td><td>Probable oxidoreductase</td><td>IM/R</td></tr><tr><td>MAB_4284c</td><td>Hypothetical protein</td><td>Rv3514</td><td></td><td></td><td>PE-PGRS family protein PE_PGRS57</td><td>PE/PPE</td></tr><tr><td>MAB_1895c</td><td>Hypothetical protein</td><td>Rv0278c</td><td></td><td></td><td>PE-PGRS family protein PE_PGRS3</td><td>PE/PPE</td></tr><tr><td>MAB_0974</td><td>Hypothetical protein</td><td>Rv3876</td><td></td><td>CW; M; WCL</td><td>ESX-1 secretion-associated protein EspI</td><td>CW/CP</td></tr><tr><td>MAB_3501</td><td>Putative hydrolase</td><td>Rv3195</td><td></td><td></td><td>Conserved hypotheticals</td><td>-</td></tr><tr><td>MAB_3566c</td><td>Putative cyclase</td><td>-</td><td></td><td></td><td></td><td>-</td></tr><tr><td>MAB_4924</td><td>Hypothetical protein</td><td>Rv0040c</td><td></td><td>CL; WCL</td><td>Secreted proline rich protein Mtc28</td><td>CW/CP</td></tr><tr><td>MAB_2394</td><td>Hypothetical protein</td><td>Rv0910</td><td></td><td></td><td>Conserved hypotheticals</td><td>-</td></tr><tr><td>MAB_2912c</td><td>Probable aldolase</td><td>Rv0727c</td><td></td><td>M</td><td>Possible L-fuculose phosphate aldolase FucA</td><td>IM/R</td></tr></tbody></table>

<figure><img src="image_77.png" /></figure>

<footnote><sup>a</sup> See also **Supplementary Tables S2 & S4**. <sup>b</sup> From references<sup>82, 83</sup>. <sup>c</sup> CF: Culture filtrate; CW: Cell wall; M: Membrane fraction; WCL: Whole cell lysate. <sup>d</sup> CW/CP: cell wall/cell processes; IM/R: intermediary metabolism/respiration; IP: information pathways; LM: lipid metabolism; Pe/PPE: PE-PGRS family protein; RP: regulatory protein.</footnote>
Interestingly, several hydrolases were detected, including a putative hydrolase (MAB_3501 / Rv3195), a putative cyclase (MAB_3566c), a probable aldolase (MAB_2912c / Rv0727c), a putative quinolone synthase (MAB_0302 / Rv1260), and the monoacylglycerol lipase (MAB_4551c / Rv0183) which has previously been reported to be inhibited by **Orlistat**.⁸⁴

View Article Online

DOI: 10.1039/D5MD00102A

Since all β-lactones share a similar mechanism of enzyme inhibition, resulting from the formation of a covalent bond between the β-lactone ring and the catalytic site of the enzyme,³³, ³⁴ and since **VM055<sub>p</sub>** showed comparable antibacterial activities to **VM043** against both *M. abscessus* and *M. tuberculosis* mc²6230, we decided to also use this latter probe in an *in situ* competitive ABPP approach.²⁶ The *M. abscessus* S culture was then pre-incubated with **VM043** prior to treatment with the **VM055<sub>p</sub>** probe and subsequent CC-ABPP experiment (**Figure 2B**). To gain in statistically relevant results, a differential comparative proteomic analysis between the [*VM043+VM055<sub>p</sub>*) captured proteome and that of the **VM055<sub>p</sub>** probe alone was then performed. As shown in the resulting volcano plot (**Figure 2C**), pre-incubation with **VM043** significantly and selectively impaired (≥2-fold inhibition) the capture of four proteins by **VM055<sub>p</sub>** (see blue dots in **Figure 2C**), thereby suggesting that these latter enzymes are preferential targets of **VM043** vs. **VM055<sub>p</sub>** (**Table S3**). These included the probable aldolase MAB_2912c (Rv0727c), the putative quinolone synthase MAB_0302 (Rv1260), the uncharacterized protein MAB_2394 (Rv0910), and the non-specific serine/threonine protein kinase MAB_3436 (Rv0014c) (**Table 4** and **Table S3**).

<figure><img src="image_78.png" /></figure>

RSC Medicinal Chemistry Accepted Manuscript
<caption>**Table 4.** VM055<sub>p</sub> captured proteins that are either significantly outcompeted (blue list) or not inhibited (red list) by VM043.<sup>a</sup></caption>

View Article Online
DOI: 10.1039/D5MD00102A

<table><thead><tr><th rowspan="2">#</th><th rowspan="2">Protein IDs</th><th rowspan="2">Protein names</th><th rowspan="2">Gene names</th><th colspan="2">[VM043+VM055<sub>p</sub>] vs. VM055<sub>p</sub></th></tr><tr><th>-LOG(p-value)</th><th>Fold Change (Log<sub>2</sub>)</th></tr></thead><tbody><tr><td>1</td><td>B1MCL9</td><td>Probable aldolase</td><td>MAB_2912c</td><td>1.319</td><td>-1.810</td></tr><tr><td>2</td><td>B1MFK1</td><td>2-heptyl-3-hydroxy-4(1H)-quinolone synthase</td><td>MAB_0302</td><td>1.411</td><td>-1.189</td></tr><tr><td>3</td><td>B1MEQ6</td><td>non-specific serine/threonine protein kinase</td><td>MAB_3436</td><td>1.169</td><td>-1.080</td></tr><tr><td>4</td><td>B1MB54</td><td>Uncharacterized protein</td><td>MAB_2394</td><td>1.201</td><td>-1.073</td></tr><tr><td>5</td><td>B1MF33</td><td>Hypothetical dipeptidyl aminopeptidase</td><td>MAB_3564c</td><td>3.543</td><td>2.606</td></tr><tr><td>6</td><td>B1MDI7</td><td>SnoaL-like domain-containing protein</td><td>MAB_3230c</td><td>1.098</td><td>1.712</td></tr><tr><td>7</td><td>B1MBL9</td><td>Peptidyl-prolyl cis-trans isomerase</td><td>MAB_2559c</td><td>1.897</td><td>1.431</td></tr><tr><td>8</td><td>B1MEW6</td><td>Uncharacterized protein</td><td>MAB_3496</td><td>3.839</td><td>1.030</td></tr></tbody></table>

<sup>a</sup> See also **Supplementary Tables S2-S6**. The two lists of proteins are deduced from the differential volcano plot depicted in Figure 2C.

Remarkably, this pre-incubation with VM043 also resulted in a statistically significant number of four enzymes being captured by the VM055<sub>p</sub> probe (see red dots in **Figure 2C**); namely a hypothetical dipeptidyl aminopeptidase MAB_3564c, a snoaL-like domain-containing protein MAB_3230c (Rv2910c), an uncharacterized protein MAB_3496, and the peptidyl-prolyl cis-trans isomerase MAB_2559c whose ortholog Rv3909 is annotated as essential for *M. tuberculosis* H37Rv *in vitro* growth (**Figure 2C, Table 4 and Table S3**). Of particular interest, although these latter four enzymes were also captured by the VM055<sub>p</sub> probe alone, they were however found to be below our *p*-value ≤ 0.05 and Fold change ≥ 1.0 thresholds when compared to DMSO non-specific conditions (**Table S3**). These later findings thus suggest that the antibacterial activity of VM043 and VM055<sub>p</sub> against *M. abscessus* growth should be due to the inhibition of a comparable spectrum of target enzymes by these two molecules, resulting in a similar anti-mycobacterial activity. Therefore, *a posteriori*, it is not surprising that pre-incubation of *M. abscessus* S cells with VM043 modifies the availability/accessibility/selectivity of these target enzymes with respect to VM055<sub>p</sub>, since proteins that were covalently inhibited by VM043 can no longer react and be captured by VM055<sub>p</sub>.

Moreover, these proteomic data also suggest that, as previously reported for two other families of multi-target inhibitors,<sup>24, 28, 29, 54, 85</sup> VM β-lactones would impair the growth of *M. abscessus* and *M. tuberculosis* by inhibiting the activity of several enzymes involved in various important

<figure><img src="image_79.png" /></figure>
physiological processes. As a direct consequence, the likelihood of a strain developing resistance to such inhibitors would be very low, because resistant mutants should acquire multiple mutations in the same bacterial genome; making it difficult or impossible for the bacteria to adapt and survive.

View Article Online

DOI: 10.1039/D5MD00102A

## 3.5 Cell toxicity of beta-lactone derivatives

One of the keys to the success of *M. tuberculosis*<sup>86, 87</sup> as well as *M. abscessus*<sup>7, 16, 88</sup> as lung pathogens is their ability to survive and replicate inside infected macrophages. As a result, new drugs must be able to inhibit only the intracellular growth of the pathogen and not be cytotoxic to host cells. In this regard, we determined for each β-lactone analog the concentration required to induce a 50% decrease in Raw264.7 murine macrophage cells viability,<sup>89</sup> i.e. $CC_{50}$<sup>55</sup> (**Table S7**). Among the 38 tested derivatives, **VM049<sub>p</sub>** and **VM050<sub>p</sub>** bearing a short pentynyl $R^2$ chain and the three β-lactam analogs **VM056** to **VM058** exhibited high toxicity towards Raw264.7 cells with $CC_{50}$ values in the range of 30-70 µg/mL, close to their MIC<sub>90</sub>. Otherwise, except **VM039** which is slightly toxic ($CC_{50}$ = 119±4 µg/mL), the other molecules had no cytotoxic effect against Raw264.7 macrophages at the highest concentration tested (125 µg/mL).

## 3.6. Intramacrophagic susceptibility of *M. abscessus* to selected β-lactone derivatives

Given these latter findings, we further investigated the ability of the β-lactones to inhibit the intra-macrophagic growth of *M. abscessus* S.<sup>30, 54, 85, 90, 91</sup> To achieve this goal, Raw264.7 cells were infected with *M. abscessus* S at a multiplicity of infection (MOI) of 10, and then incubated for 24 h with selected β-lactone compounds or with imipenem (IMP; 80 µg/mL ~ 9.5×MIC<sub>50Raw</sub>) used as positive drug control for this intracellular killing assay.<sup>54, 85</sup> The selected inhibitors tested were **VM025**, **VM026** (= *trans*-**VM025**), and **VM043** which are the best inhibitors of *M. abscessus* S & R growth in broth medium. The inactive growth inhibitor **VM027** (= *cis*-**VM025**), as well as the two probes of **VM043**, **VM053<sub>p</sub>** and **VM055<sub>p</sub>** which differ in their “clickable” group (alkyne vs. azide), were also included (**Table 5** and **Figure 3**).

<figure><img src="image_80.png" /></figure>
Among the compounds tested, **VM025** and its *trans*-isomer **VM026** exhibited promising antibacterial activity against intracellular *M. abscessus* growth, with an approximated MIC<sub>50Raw</sub> of around 13 µg/mL which is only 1.5 times higher than that of IMP (MIC<sub>50Raw</sub> = 8.5 µg/mL) (**Figure 3** and **Table 5**). Interestingly, the *cis*-isomer **VM027** which was not active against extracellular mycobacteria (**Table 1**), was however able to significantly decrease the intramacrophagic *M. abscessus* present 24 h post-infection, with a MIC<sub>50Raw</sub> of around 28 µg/mL. On the other hand, **VM043** bearing a fluorine atom at the C-3 position of the β-lactone ring had no activity against the intracellular growth of *M. abscessus* (**Figure 3**, **Table 5** and **Figure S1**). The potential “negative” effect of the fluorine atom was further confirmed by testing **VM046** and its non-fluorinated analog, **VM045** (**Table 5** and **Figure S1**). Indeed, although **VM046** was inactive, **VM045** was able to significantly inhibit the intracellular growth of *M. abscessus* with a MIC<sub>50Raw</sub> of around 19 µg/mL. Finally, the two non-fluorinated probes displayed quite good (MIC<sub>50Raw</sub> = 24.9 µg/mL for **VM055<sub>p</sub>**) to moderate (MIC<sub>50Raw</sub> = 48.6 µg/mL for **VM053<sub>p</sub>**) antibacterial activities against intracellular *M. abscessus* S growth (**Table 5** and **Figure S1**).

View Article Online

DOI: 10.1039/D5MD00102A

<figure><img src="image_81.png" /></figure>

RSC Medicinal Chemistry Accepted Manuscript
<table><caption>**Table 5.** Intracellular antibacterial activities of the selected β-lactone derivatives against *M. abscessus* S-LuxG13 infected macrophages <sup>a</sup></caption><thead><tr><th>Compounds</th><th></th><th>CC<sub>50</sub><br>(µg/mL)</th><th>MIC<sub>50Raw</sub><br>(µg/mL)</th><th>SI</th></tr></thead><tbody><tr><td>VM025</td><td></td><td>&gt;125</td><td>13.6±1.1</td><td>&gt;9.2</td></tr><tr><td>VM026<br>= trans-VM025</td><td></td><td>&gt;125</td><td>12.9±1.2</td><td>&gt;9.7</td></tr><tr><td>VM027<br>= cis-VM025</td><td></td><td>&gt;125</td><td>27.9±1.4</td><td>&gt;4.5</td></tr><tr><td>VM043</td><td></td><td>&gt;125</td><td>&gt;100</td><td>-</td></tr><tr><td>VM045</td><td></td><td>&gt;125</td><td>18.9±1.3</td><td>&gt;6.5</td></tr><tr><td>VM046<br>= fluorinated-VM045</td><td></td><td>&gt;125</td><td>&gt;100</td><td>-</td></tr><tr><td>VM053<sub>p</sub><br>= non-fluorinated VM043 probe</td><td></td><td>&gt;125</td><td>48.6±3.8</td><td>&gt;2.6</td></tr><tr><td>VM055<sub>p</sub><br>= non-fluorinated VM043 probe</td><td></td><td>&gt;125</td><td>24.9±3.3</td><td>&gt;5.0</td></tr><tr><td>IMP<sup>b</sup></td><td></td><td></td><td>8.5±1.4</td><td>-</td></tr></tbody></table>

<figure><img src="image_82.png" /></figure>

<footnote><sup>a</sup> Experiments were performed as described in the **Experimental Section**. CC<sub>50</sub>: compound concentration leading to 50% Raw264.7 macrophages toxicity. IC<sub>50Raw</sub>: minimal compound concentration leading to a 50% decrease in RLU count as compared to untreated cells. Raw264.7 macrophages were infected by *M. abscessus* S-LuxG13 at a MOI of 10, and further treated with each β-lactone or IMP for 24 h. The viable mycobacteria were quantified by measurement of luminescence from luciferase-expressing *M. abscessus* S-LuxG13 within Raw264.7 macrophages. Untreated infected macrophages were used as control representing 100% of bacterial viability. MIC<sub>50Raw</sub> were calculated from curve fitting of RLU% as a function of the inhibitor concentration and are expressed as mean values of three independent assays. <sup>b</sup> Data from<sup>54</sup>. IMP, imipenem. ND: not determined. SI, selectivity index, SI = CC<sub>50</sub>/MIC<sub>50Raw</sub>.</footnote>
<figure><img src="image_83.png" /></figure>

View Article Online

DOI: 10.1039/D5MD00102A

<caption>Figure 3. Intracellular activity of VM025, VM026, VM027, and VM043 as compared to imipenem (IMP).</caption>

The activity of selected β-lactones on intracellular *M. abscessus* growth was tested in Raw264.7 murine macrophages. Cells were infected at a multiplicity of infection (MOI) of 10 with *M. abscessus* S-LuxG13 and treated with various concentrations of each inhibitor or IMP for 24 h. The viable mycobacteria were quantified by measurement of luminescence from luciferase-expressing *M. abscessus* S-LuxG13 within Raw264.7 macrophages. Untreated infected macrophages were used as control representing 100% of bacterial viability. Untreated infected macrophages were used as control representing 100% of bacterial viability. Results are shown as mean ± standard error of the mean (SEM) of three independent assays performed in triplicate. SC, solvent control (DMSO). ***, *p*-value <0.001. *, *p*-value <0.05. Statistical analysis was done using a Student's *t*-test.

The fact that MIC values determined in broth medium do not always correlate with the activity of the compounds against intracellular bacteria, is not new. In 2015, Vanderven *et al*. demonstrated that among 1,359 hits tested in a screening assay against *M. tuberculosis*, only 141 compounds were able to inhibit both the extracellular and intracellular growth of the bacillus, and 132 inhibited bacterial replication inside macrophages with little or no inhibitory activity in broth medium.⁹² Another best example is the first-line anti-TB drug pyrazinamide⁹³ which is inactive *in vitro* at neutral pH in conventional culture media, but displays potent activity against *M. tuberculosis* in an acidic environment (pH 5.5 or below) mimicking the endolysosomal pH of macrophages. Regarding

<figure><img src="image_84.png" /></figure>

RSC Medicinal Chemistry Accepted Manuscript
multi-target inhibitors, such a discrepancy between extracellular and intracellular activities has also been reported and discussed in previous works with two other families of growth inhibitors of *M. tuberculosis* and *M. abscessus*; namely the Oxadiazolone (OX) derivatives²⁸, ⁸⁵ and the Cyclipostins & Cyclophostin (CyC) analogs.²⁹, ³⁰, ³², ⁵⁴ In particular, we have recently demonstrated that the CyCs which were active against intracellular *M. abscessus* growth accumulated in acidic compartments inside macrophage cells, and that this accumulation was essential for their delivery to mycobacteria-containing phagosomes and thus for their antimicrobial activity against intramacrophagic *M. abscessus*.³⁰

Here, the fact that fluorine derivatives were fully inactive against intramacrophagic *M. abscessus*, as in the case of the best extracellular growth inhibitor **VM043**, may suggest a specific behavior or mode of action of these molecules inside infected macrophages. On the other hand, **VM025** and **VM026** which had a promising extracellular activity (MIC$_{90}$ ~54 µg/mL) were 4 times more active against *M. abscessus* intracellular growth within macrophages (MIC$_{50Raw}$ ~13 µg/mL). One hypothesis to explain this clear preference for intracellularly replicating mycobacteria could be that the corresponding target enzyme(s) of these inhibitors would be more accessible and/or vulnerable during the intracellular lifestyle of *M. abscessus* compared to its extracellular replication. However, a specific response of the infected macrophages resulting from the stress effect of these compounds and leading to bacterial clearance cannot be excluded.

Given the previously determined very low toxicity of the β-lactones toward Raw264.7 cells with CC$_{50}$ > 125 µg/mL, the selectivity index (SI = CC$_{50}$/MIC$_{50Raw}$) of the intracellular inhibitors on *M. abscessus* vs. Raw264.7 cells was calculated and found to be in a promising range from >2.6 and up to >9.0 (Table 4).

From these findings, it can be assumed that the observed inhibitory potency of the β-lactone derivatives *i*) might result from the inhibition of specific but most likely distinct mycobacterial target enzymes between intramacrophagic- vs. extracellularly-replicating mycobacteria; or *ii*) might reflect differences in the uptake and accumulation of the different compounds inside the macrophage.

<figure><img src="image_85.png" /></figure>
Overall, these results suggest that the non-fluorinated derivatives would be able to enter the macrophages and arrest bacterial replication without exhibiting significant toxicity to the host cell, with comparable antibacterial activity to imipenem.

DOI: 10.1039/D5MD00102A

### 3. Conclusion

In the present work, a new series of lipophilic compounds based on β-lactone-core were synthesized by varying the nature of the substituents on the lactone ring. Evaluation of their antibacterial activity first highlighted **VM038**, **VM040**, **VM043** and **VM045** as potential candidates against *M. tuberculosis*. With respect to *M. abscessus*, the MIC determination of this set of 30 derivatives provided **VM025** and **VM026**, in addition to the latter **VM043**, as efficient inhibitors of both S and R variants. A competitive click-chemistry ABPP approach and comparative chemical proteomics with a newly synthesized customized activity-based probe **VM055<sub>p</sub>** revealed several *M. abscessus* target enzymes of **VM043**, the best inhibitor of extracellular growth, compared to **VM055<sub>p</sub>**, thus confirming the multi-target nature of this family of molecules. When tested against intracellular bacteria, while **VM043** was found inactive, **VM025 & VM026** emerged as potent and promising inhibitors of intramacrophagic *M. abscessus* growth with MIC<sub>50Raw</sub> values comparable to the standard antibiotic imipenem. This dual activity is of major importance as it may affect the different stages of the infection process.

Hence, thanks to their multitargeted covalent mechanism of action, our results underscore the added value of β-lactone probes. In particular, we anticipate that these probes would represent attractive tools against mycobacterial infections, and provide interesting insights into the different stages of the infection process that may lead to the arrest of two main mycobacterial pathogens, *M. tuberculosis* and/or *M. abscessus*. Identifying the proteins inactivated by our antibacterial activity-based probes would indeed reveal new potential targets for treating mycobacterial-related diseases, and contribute to background information for the development of new therapeutic strategies for elimination of either actively replicating or latent mycobacteria from infected individuals.

<figure><img src="image_86.png" /></figure>

RSC Medicinal Chemistry Accepted Manuscript
Further work to better understand the behavior of our β-lactone derivatives inside macrophage cells and consequently to elucidate their mode of action against intracellular *M. abscessus* is currently in progress.

DOI: 10.1039/D5MD00102A

### CRediT authorship contribution statement

**Thomas Francis:** Formal analysis - Investigation – Visualization - Writing-review & editing.

**Christina Dedaki:** Formal analysis - Investigations - Resources - Writing-review & editing. **Phoebe**

**Ananida-Dasenaki:** Investigations – Resources. **Dimitra Bolka:** Investigations – Resources.

**Kanellos Albanis:** Investigations – Resources. **Filippos Foteinakis:** Investigations – Resources.

**Julie Mezquida:** Formal analysis – Investigation. **Marie Hance:** Formal analysis – Investigation.

**Alexandros Athanasoulis:** Investigations – Resources. **Anna-Krinio Papagiorgou:** Investigations – Resources. **Ioanna-Foteini Karampoula:** Investigations – Resources. **George Georgitsis:**

Investigations – Resources. **Celia Jardin:** Data curation - Formal analysis – Investigation. **Stéphane**

**Audebert:** Data curation - Formal analysis – Investigation - Writing-review & editing. **Luc Camoin:**

Data curation - Writing-review & editing. **Céline Crauste:** Writing-review & editing. **Stéphane**

**Canaan:** Writing-review & editing. **Victoria Magrioti:** Conceptualization - Formal analysis – Methodology - Project administration - Resources - Supervision - Validation - Visualization - Writing-original draft - Writing-review & editing. **Jean-François Cavalier:** Conceptualization - Data curation - Formal analysis – Investigation - Methodology - Project administration - Supervision - Validation - Visualization - Writing-original draft - Writing-review & editing.

### Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

<figure><img src="image_87.png" /></figure>
## Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files. The mass spectrometry proteomics data are available online through the ProteomeXchange Consortium (www.proteomexchange.org) with the dataset identifiers PXD057836.

Supplementary information for this article is available online.

**Additional file 1: Detailed protocols** related to biological evaluation and to the synthesis of intermediates as well as new β-lactone derivatives; **Table S1**, Code numbers, structures and Log P<sub>O/W</sub> of all β-lactone compounds; **Figure S1**, Intracellular activity of **VM045**, **VM046**, as well as the two probes, **VM053<sub>p</sub>** and **VM055<sub>p</sub>**, against *M. abscessus* S infected macrophages; NMR spectra of the new compounds synthesized (PDF).

**Additional file 2: Tables S2-S6**, Target proteins identified from *M. abscessus* S culture, through CC-ABPP by LC-ESI-MS/MS analysis, using either **VM055<sub>p</sub>** probe alone, or following **VM043** pre-incubation and labeling with **VM055<sub>p</sub>** probe; **Table S7**, Cytotoxic activities of the β-lactone derivatives towards Raw264.7 murine macrophage cells (pdf).

## Acknowledgements

This work was supported by the Centre National de la Recherche Scientifique (CNRS) and Aix-Marseille Université (AMU) and by the Special Account for Research Grants of the National and Kapodistrian University of Athens (SARG/NKUA). Proteomics analyses were done using the mass spectrometry facility of Marseille Proteomics (marseille-proteomique.univ-amu.fr) supported by IBISA, the Cancéropôle PACA, the Provence-Alpes-Côte d'Azur Region, the Institut Paoli-Calmettes, and Fonds Européen de Développement Regional (FEDER). T.F. PhD fellowship was supported by the Ministère de l'Enseignement Supérieur et de la Recherche Française.

## Abbreviations

<figure><img src="image_88.png" /></figure>

View Article Online
DOI: 10.1039/D5MD00102A
ABP, activity-based probe; ABPP, activity-based protein profiling; AMK, amikacin; $CC_{50}$, compound concentration leading to 50% of cell cytotoxicity; CC-ABPP, click-chemistry activity-based protein profiling; CF, cystic fibrosis; IMP, imipenem; $MIC_{50}/MIC_{90}$, minimal inhibitory concentration leading to 50% and 90% of bacterial growth inhibition, respectively; $MIC_{50\text{Raw}}$, minimal inhibitory concentration leading to 50% of bacterial growth inhibition inside infected macrophages NTM, nontuberculous mycobacterial; REMA, resazurin microtiter assay; RLU, relative luminescence unit; SI, selectivity index.

View Article Online

DOI: 10.1039/D5MD00102A

<figure><img src="image_89.png" /></figure>

RSC Medicinal Chemistry Accepted Manuscript
References

View Article Online

DOI: 10.1039/D5MD00102A

1. E. Tortoli, Microbiological features and clinical relevance of new species of the genus *Mycobacterium*, *Clin Microbiol Rev*, 2014, **27**, 727-752.

2. E. Tortoli, T. Fedrizzi, C. J. Meehan, A. Trovato, A. Grottola, E. Giacobazzi, G. F. Serpini, S. Tagliazucchi, A. Fabio, C. Bettua, R. Bertorelli, F. Frascaro, V. De Sanctis, M. Pecorari, O. Jousson, N. Segata and D. M. Cirillo, The new phylogeny of the genus *Mycobacterium*: The old and the news, *Infect Genet Evol*, 2017, **56**, 19-25.

3. WHO, Global tuberculosis report, https://www.who.int/teams/global-tuberculosis-programme/data, (accessed 20 November 2024).

4. D. G. Russell, *Mycobacterium tuberculosis*: here today, and here tomorrow, *Nat Rev Mol Cell Biol.*, 2001, **2**, 569-577.

5. E. Catherinot, J. Clarissou, G. Etienne, F. Ripoll, J. F. Emile, M. Daffe, C. Perronne, C. Soudais, J. L. Gaillard and M. Rottman, Hypervirulence of a rough variant of the *Mycobacterium abscessus* type strain, *Infect Immun*, 2007, **75**, 1055-1058.

6. R. Nessar, E. Cambau, J. M. Reyrat, A. Murray and B. Gicquel, *Mycobacterium abscessus*: a new antibiotic nightmare, *J Antimicrob Chemother*, 2012, **67**, 810-818.

7. M. D. Johansen, J. L. Herrmann and L. Kremer, Non-tuberculous mycobacteria and the rise of *Mycobacterium abscessus*, *Nat Rev Microbiol*, 2020, **18**, 392-407.

8. J. E. Stout, W. J. Koh and W. W. Yew, Update on pulmonary disease due to non-tuberculous mycobacteria, *Int J Infect Dis*, 2016, **45**, 123-134.

9. J. M. Leung and K. N. Olivier, Nontuberculous mycobacteria in patients with cystic fibrosis, *Semin Respir Crit Care Med*, 2013, **34**, 124-134.

10. A. L. Roux, E. Catherinot, F. Ripoll, N. Soismier, E. Macheras, S. Ravilly, G. Bellis, M. A. Vibet, E. Le Roux, L. Lemonnier, C. Gutierrez, V. Vincent, B. Fauroux, M. Rottman, D. Guillemot, J. L. Gaillard and O. M. A. G. Jean-Louis Herrmann for the, Multicenter study of prevalence of nontuberculous mycobacteria in patients with cystic fibrosis in france, *J Clin Microbiol.*, 2009, **47**, 4124-4128.

11. P. H. Candido, S. Nunes Lde, E. A. Marques, T. W. Folescu, F. S. Coelho, V. C. de Moura, M. G. da Silva, K. M. Gomes, M. C. Lourenco, F. S. Aguiar, F. Chitolina, D. T. Armstrong, S. C. Leao, F. P. Neves, F. C. Mello and R. S. Duarte, Multidrug-resistant nontuberculous mycobacteria isolated from cystic fibrosis patients, *J Clin Microbiol.*, 2014, **52**, 2990-2997.

12. E. Catherinot, A. L. Roux, E. Macheras, D. Hubert, M. Matmar, L. Dannhoffer, T. Chinet, P. Morand, C. Poyart, B. Heym, M. Rottman, J. L. Gaillard and J. L. Herrmann, Acute respiratory

<figure><img src="image_90.png" /></figure>
failure involving an R variant of *Mycobacterium abscessu*, *J Clin Microbiol.*, 2009, **47**, 271-274.

View Article Online

DOI: 10.1039/D5MD00102A

13. K. Dheda, T. Gumbo, G. Maartens, K. E. Dooley, R. McNerney, M. Murray, J. Furin, E. A. Nardell, L. London, E. Lessem, G. Theron, P. van Helden, S. Niemann, M. Merker, D. Dowdy, A. Van Rie, G. K. H. Siu, J. G. Pasipanodya, C. Rodrigues, T. G. Clark, F. A. Sirgel, A. Esmail, H.-H. Lin, S. R. Atre, H. S. Schaaf, K. C. Chang, C. Lange, P. Nahid, Z. F. Udwadia, C. R. Horsburgh, G. J. Churchyard, D. Menzies, A. C. Hesseling, E. Nuermberger, H. McIlleron, K. P. Fennelly, E. Goemaere, E. Jaramillo, M. Low, C. M. Jara, N. Padayatchi and R. M. Warren, *The epidemiology, pathogenesis, transmission, diagnosis, and management of multidrug-resistant, extensively drug-resistant, and incurable tuberculosis*, *The Lancet. Respiratory medicine*, 2017, **5**, 291-360

14. R. C. Lopeman, J. Harrison, M. Desai and J. A. G. Cox, *Mycobacterium abscessus*: Environmental Bacterium Turned Clinical Nightmare, *Microorganisms*, 2019, **7**.

15. B. J. Seaworth and D. E. Griffith, Therapy of Multidrug-Resistant and Extensively Drug-Resistant Tuberculosis, *Microbiology spectrum*, 2017, **5**.

16. A. L. Roux, A. Viljoen, A. Bah, R. Simeone, A. Bernut, L. Laencina, T. Deramaudt, M. Rottman, J. L. Gaillard, L. Majlessi, R. Brosch, F. Girard-Misguich, I. Vergne, C. de Chastellier, L. Kremer and J. L. Herrmann, The distinct fate of smooth and rough *Mycobacterium abscessus* variants inside macrophages, *Open biology*, 2016, **6**, 160185.

17. Y. M. Boudehen and L. Kremer, *Mycobacterium abscessus*, *Trends Microbiol*, 2021, **29**, 951-952.

18. B. E. Jönsson, M. Gilljam, A. Lindblad, M. Ridell, A. E. Wold and C. Welinder-Olsson, Molecular epidemiology of *Mycobacterium abscessus*, with focus on cystic fibrosis, *Journal of Clinical Microbiology*, 2007, **45**, 1497-1504.

19. P. J. Brennan and H. Nikaido, The envelope of mycobacteria, *Annual review of biochemistry*, 1995, **64**, 29-63.

20. M. Jackson, The mycobacterial cell envelope-lipids, *Cold Spring Harb Perspect Med*, 2014, **4**.

21. S. M. Batt, D. E. Minnikin and G. S. Besra, The thick waxy coat of mycobacteria, a protective layer against antibiotics and the host's immune system, *Biochemical Journal*, 2020, **477**, 1983-2006.

22. G. Gago, L. Diacovich and H. Gramajo, Lipid metabolism and its implication in mycobacteria-host interaction, *Curr Opin Microbiol*, 2018, **41**, 36-42.

<figure><img src="image_91.png" /></figure>

RSC Medicinal Chemistry Accepted Manuscript
23. T. van der Klugt, R. van den Biggelaar and A. Saris, Host and bacterial lipid metabolism during tuberculosis infections: possibilities to synergise host- and bacteria-directed therapies, *Curr Re视频·Microbiol*, 2024, DOI: https://doi.org/10.1080/1040841X.2024.2370979, 1-21.

24. J. F. Cavalier, C. D. Spilling, T. Durand, L. Camoin and S. Canaan, Lipolytic enzymes inhibitors: A new way for antibacterial drugs discovery, *Eur J Med Chem.*, 2021, **209**, 112908.

25. G. Johnson, The alpha/beta Hydrolase Fold Proteins of *Mycobacterium tuberculosis*, With Reference to their Contribution to Virulence, *Curr Protein Pept Sci*, 2017, **18**, 190-210.

26. R. Avellan, M. Sarrazin, C. D. Spilling, C. Crauste, S. Canaan and J.-F. Cavalier, Chapter 11 - Deciphering the physiological role of serine enzymes involved in mycobacterial lipid metabolism using activity-based protein profiling, in *Biology of Mycobacterial Lipids*, eds. Z. Fatima and S. Canaan, Academic Press, 2022, pp. 235-251. https://doi.org/10.1016/B978-0-323-91948-7.00001-4

27. M. Li, H. V. Patel, A. B. Cognetta, 3rd, T. C. Smith, 2nd, I. Mallick, J. F. Cavalier, M. L. Previti, S. Canaan, B. B. Aldridge, B. F. Cravatt and J. C. Seeliger, Identification of cell wall synthesis inhibitors active against *Mycobacterium tuberculosis* by competitive activity-based protein profiling, *Cell Chem Biol*, 2022, **29**, 883-896 e885.

28. P. C. Nguyen, V. Delorme, A. Benarouche, A. Guy, V. Landry, S. Audebert, M. Pophillat, L. Camoin, C. Crauste, J. M. Galano, T. Durand, P. Brodin, S. Canaan and J.-F. Cavalier, Oxadiazolone derivatives, new promising multi-target inhibitors against *M. tuberculosis*, *Bioorg Chem*, 2018, **81**, 414-424.

29. P. C. Nguyen, V. Delorme, A. Benarouche, B. P. Martin, R. Paudel, G. R. Gnawali, A. Madani, R. Puppo, V. Landry, L. Kremer, P. Brodin, C. D. Spilling, J.-F. Cavalier and S. Canaan, Cyclipostins and Cyclophostin analogs as promising compounds in the fight against tuberculosis, *Scientific Reports*, 2017, **7**, 11751.

30. M. Sarrazin, B. P. Martin, R. Avellan, G. R. Gnawali, I. Poncin, H. Le Guenno, C. D. Spilling, J. F. Cavalier and S. Canaan, Synthesis and Biological Characterization of Fluorescent Cyclipostins and Cyclophostin Analogues: New Insights for the Diagnosis of Mycobacterial-Related Diseases, *ACS Infect Dis*, 2022, **8**, 2564-2578.

31. K. R. Tallman, S. R. Levine and K. E. Beatty, Small Molecule Probes Reveal Esterases with Persistent Activity in Dormant and Reactivating *Mycobacterium tuberculosis*, *ACS Infect. Dis.*, 2016, **2**, 936-944.

32. P. C. Nguyen, A. Madani, P. Santucci, B. P. Martin, R. R. Paudel, S. Delattre, J. L. Herrmann, C. D. Spilling, L. Kremer, S. Canaan and J.-F. Cavalier, Cyclophostin and Cyclipostins

<figure><img src="image_92.png" /></figure>

RSC Medicinal Chemistry Accepted Manuscript
analogues, new promising molecules to treat mycobacterial-related diseases, *International Journal of Antimicrobial Agents*, 2018, **51**, 651-654.

DOI: 10.1039/D5MD00102A

View Article Online

DOI: 10.1039/D5MD00102A

33. P. Hadvary, W. Sidler, W. Meister, W. Vetter and H. Wolfer, The lipase inhibitor tetrahydrolipstatin binds covalently to the putative active site serine of pancreatic lipase, *J Biol Chem*, 1991, **266**, 2021-2027.

34. A. Benarouche, V. Point, F. Carriere and J. F. Cavalier, Using the reversible inhibition of gastric lipase by Orlistat for investigating simultaneously lipase adsorption and substrate hydrolysis at the lipid-water interface, *Biochimie*, 2014, **101**, 221-231.

35. A. M. Heck, J. A. Yanovski and K. A. Calis, Orlistat, a new lipase inhibitor for the management of obesity, *Pharmacotherapy*, 2000, **20**, 270-279.

36. L. Haalck and F. Spener, On the inhibition of microbial lipases by tetrahydrolipstatin, *Methods Enzymol*, 1997, **286**, 252-263.

37. L. Kremer, C. De Chastellier, G. Dobson, K. J. C. Gibson, P. Bifani, S. Balor, J.-P. Gorvel, C. Locht, D. E. Minnikin and G. S. Besra, Identification and structural characterization of an unusual mycobacterial monomeromycolyl-diacylglycerol, *Mol Microbiol*, 2005, **57**, 1113-1126.

38. S. K. Parker, R. M. Barkley, J. G. Rino and M. L. Vasil, *Mycobacterium tuberculosis* Rv3802c encodes a phospholipase/thioesterase and is inhibited by the antimycobacterial agent tetrahydrolipstatin, *PLoS One*, 2009, **4**, e4281.

39. P. K. Crellin, J. P. Vivian, J. Scoble, F. M. Chow, N. P. West, R. Brammananth, N. I. Proellocks, A. Shahine, J. Le Nours, M. C. J. Wilce, W. J. Britton, R. L. Coppel, J. Rossjohn and T. Beddoe, Tetrahydrolipstatin inhibition, functional analyses, and three-dimensional structure of a lipase essential for mycobacterial viability, *J Biol Chem*, 2010, **285**, 30050-30060.

40. N. P. West, K. M. Cergol, M. Xue, E. J. Randall, W. J. Britton and R. J. Payne, Inhibitors of an essential mycobacterial cell wall lipase (Rv3802c) as tuberculosis drug leads, *Chem Commun* (*Camb*), 2011, **47**, 5166-5168.

41. V. Delorme, S. V. Diomande, L. Dedieu, J.-F. Cavalier, F. Carriere, L. Kremer, J. Leclaire, F. Fotiadu and S. Canaan, MmPPOX inhibits *Mycobacterium tuberculosis* lipolytic enzymes belonging to the hormone-sensitive lipase family and alters mycobacterial growth, *PLoS ONE*, 2012, **7**, e46493.

42. M. S. Ravindran, S. P. Rao, X. Cheng, A. Shukla, A. Cazenave-Gassiot, S. Q. Yao and M. R. Wenk, Targeting lipid esterases in mycobacteria grown under different physiological conditions using activity-based profiling with tetrahydrolipstatin (THL), *Molecular & cellular proteomics*, 2014, **13**, 435-448.

<figure><img src="image_93.png" /></figure>

RSC Medicinal Chemistry Accepted Manuscript
43. C. M. Goins, T. D. Sudasinghe, X. Liu, Y. Wang, G. A. O'Doherty and D. R. Ronning, Characterization of Tetrahydrolipstatin and Stereoderivatives on the Inhibition of Mycobacterium tuberculosis Lipid Esterases, *Biochemistry*, 2018, **57**, 2383-2393.

View Article Online

DOI: 10.1039/D7EX002A

44. C. M. Goins, S. Dajnowicz, M. D. Smith, J. M. Parks and D. R. Ronning, Mycolyltransferase from *Mycobacterium tuberculosis* in covalent complex with tetrahydrolipstatin provides insights into antigen 85 catalysis, *J Biol Chem*, 2018, **293**, 3651-3662.

45. P. Santucci, C. Dedaki, A. Athanasoulis, L. Gallorini, A. Munoz, S. Canaan, J.-F. Cavalier and V. Magrioti, Synthesis of long chain β-lactones and their antibacterial activities against pathogenic mycobacteria, *ChemMedChem*, 2019, **14**, 349-358.

46. C. Rens, F. Laval, M. Daffe, O. Denis, R. Frita, A. Baulard, R. Wattiez, P. Lefevre and V. Fontaine, Effects of lipid-lowering drugs on vancomycin susceptibility of mycobacteria, *Antimicrob Agents Chemother*, 2016, **60**, 6193-6199.

47. J. Lehmann, T. Y. Cheng, A. Aggarwal, A. S. Park, E. Zeiler, R. M. Raju, T. Akopian, O. Kandror, J. C. Sacchettini, D. B. Moody, E. J. Rubin and S. A. Sieber, An Antibacterial beta-Lactone Kills *Mycobacterium tuberculosis* by Disrupting Mycolic Acid Biosynthesis, *Angew Chem Int Ed Engl*, 2018, **57**, 348-353.

48. S. S. Khan, T. D. Sudasinghe, A. D. Landgraf, D. R. Ronning and S. J. Scheck, Total Synthesis of Tetrahydrolipstatin, Its Derivatives, and Evaluation of Their Ability to Potentiate Multiple Antibiotic Classes against Mycobacterium Species, *ACS Infect Dis*, 2021, **7**, 2876-2888.

49. V. K. Sambandamurthy, S. C. Derrick, T. Hsu, B. Chen, M. H. Larsen, K. V. Jalapathy, M. Chen, J. Kim, S. A. Porcelli, J. Chan, S. L. Morris and W. R. Jacobs, Jr., *Mycobacterium tuberculosis* DeltaRD1 DeltapanCD: a safe and limited replicating mutant strain that protects immunocompetent and immunocompromised mice against experimental tuberculosis, *Vaccine*, 2006, **24**, 6309-6320.

50. N. Andreu, A. Zelmer, T. Fletcher, P. T. Elkington, T. H. Ward, J. Ripoll, T. Parish, G. J. Bancroft, U. Schaible, B. D. Robertson and S. Wiles, Optimisation of bioluminescent reporters for use with mycobacteria, *PLoS One*, 2010, **5**, e10777.

51. B. S. Simcox, B. R. Tomlinson, L. N. Shaw and K. H. Rohde, *Mycobacterium abscessus* DosRS two-component system controls a species-specific regulon required for adaptation to hypoxia, *Front Cell Infect Microbiol*, 2023, **13**, 1144210.

52. D. G. Lee, Y. H. Hwang, E. J. Park, J. H. Kim and S. W. Ryoo, Clomiphene Citrate Shows Effective and Sustained Antimicrobial Activity against *Mycobacterium abscessus*, *Int J Mol Sci*, 2021, **22**.

<figure><img src="image_94.png" /></figure>

RSC Medicinal Chemistry Accepted Manuscript
53. J. C. Palomino, A. Martin, M. Camacho, H. Guerra, J. Swings and F. Portaels, Resazurin microtiter assay plate: simple and inexpensive method for detection of drug resistance in *Mycobacterium tuberculosis*, *Antimicrob Agents Chemother*, 2002, **46**, 2720-2722.

*View Article Online*
*DOI: 10.1021/nc020242g*

54. A. Madani, J. N. Ridenour, B. P. Martin, R. R. Paudel, A. Abdul Basir, V. Le Moigne, J. L. Herrmann, S. Audebert, L. Camoin, L. Kremer, C. D. Spilling, S. Canaan and J.-F. Cavalier, Cyclipostins and Cyclophostin Analogues as Multitarget Inhibitors That Impair Growth of *Mycobacterium abscessus*, *ACS Infect Dis*, 2019, **5**, 1597-1608.

55. C. Rodrigues Felix, R. Gupta, S. Geden, J. Roberts, P. Winder, S. A. Pomponi, M. C. Diaz, J. K. Reed, A. E. Wright and K. H. Rohde, Selective Killing of Dormant *Mycobacterium tuberculosis* by Marine Natural Products, *Antimicrob Agents Chemother*, 2017, **61**, e00743-00717.

56. T. Christophe, M. Jackson, H. K. Jeon, D. Fenistein, M. Contreras-Dominguez, J. Kim, A. Genovesio, J. P. Carralot, F. Ewann, E. H. Kim, S. Y. Lee, S. Kang, M. J. Seo, E. J. Park, H. Skovierova, H. Pham, G. Riccardi, J. Y. Nam, L. Marsollier, M. Kempf, M. L. Joly-Guillou, T. Oh, W. K. Shin, Z. No, U. Nehrbass, R. Brosch, S. T. Cole and P. Brodin, High content screening identifies decaprenyl-phosphoribose 2' epimerase as a target for intracellular antimycobacterial inhibitors, *PLoS Pathog*, 2009, **5**, e1000645.

57. J. C. Jewett and C. R. Bertozzi, Cu-free click cycloaddition reactions in chemical biology, *Chem Soc Rev*, 2010, **39**, 1272-1279.

58. J. C. Jewett, E. M. Sletten and C. R. Bertozzi, Rapid Cu-free click chemistry with readily synthesized biarylazacyclooctynones, *J Am Chem Soc*, 2010, **132**, 3688-3690.

59. B. M. Babin, L. Atangcho, M. B. van Eldijk, M. J. Sweredoski, A. Moradian, S. Hess, T. Tolker-Nielsen, D. K. Newman and D. A. Tirrell, Selective Proteomic Analysis of Antibiotic-Tolerant Cellular Subpopulations in *Pseudomonas aeruginosa* Biofilms, *mBio*, 2017, **8**.

60. A. M. Frankenfield, J. Ni, M. Ahmed and L. Hao, Protein Contaminants Matter: Building Universal Protein Contaminant Libraries for DDA and DIA Proteomics, *J Proteome Res*, 2022, **21**, 2104-2113.

61. S. Tyanova and J. Cox, Perseus: A Bioinformatics Platform for Integrative Analysis of Proteomics Data in Cancer Research, in *Cancer Systems Biology: Methods and Protocols*, ed. L. von Stechow, Springer New York, New York, NY, 2018, pp. 133-148.

[https://doi.org/10.1007/978-1-4939-7493-1_7](https://doi.org/10.1007/978-1-4939-7493-1_7)

62. V. G. Tusher, R. Tibshirani and G. Chu, Significance analysis of microarrays applied to the ionizing radiation response, *Proc Natl Acad Sci U S A*, 2001, **98**, 5116-5121.

<figure><img src="image_95.png" /></figure>
63. E. W. Deutsch, N. Bandeira, V. Sharma, Y. Perez-Riverol, J. J. Carver, D. J. Kundu, D. Garcia-Seisdedos, A. F. Jarnuczak, S. Hewapathirana, B. S. Pullman, J. Wertz, Z. Sun, S. Kawano, S. Okuda, Y. Watanabe, H. Hermjakob, B. MacLean, M. J. MacCoss, Y. Zhu, Y. Ishihama and J. A. Vizcaino, The ProteomeXchange consortium in 2020: enabling 'big data' approaches in proteomics, *Nucleic Acids Res*, 2020, **48**, D1145-D1152.

64. Y. Perez-Riverol, J. Bai, C. Bandla, D. Garcia-Seisdedos, S. Hewapathirana, S. Kamatchinathan, D. J. Kundu, A. Prakash, A. Frericks-Zipper, M. Eisenacher, M. Walzer, S. Wang, A. Brazma and J. A. Vizcaino, The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences, *Nucleic Acids Res*, 2022, **50**, D543-D552.

65. D. W. Brooks, L. D. L. Lu and S. Masamune, C-Acylation under Virtually Neutral Conditions, *Angew Chem Int Ed Engl.*, 2003, **18**, 72-74.

66. T. Mukaiyama, Reactions based on the onium salts of azaaromatics, *Pure and Applied Chemistry*, 1979, **51**, 1337-1346.

67. S. Singh, N. Bouzinbi, V. Chaturvedi, S. Godreuil and L. Kremer, In vitro evaluation of a new drug combination against clinical isolates belonging to the *Mycobacterium abscessus* complex, *Clin Microbiol Infect*, 2014, **20**, O1124-1127.

68. Y. I. Ho, C. Y. Chan and A. F. Cheng, *In-vitro* activities of aminoglycoside-aminocyclitols against mycobacteria, *J Antimicrob Chemother*, 1997, **40**, 27-32.

69. A. Daina, O. Michielin and V. Zoete, iLOGP: a simple, robust, and efficient description of n-octanol/water partition coefficient for drug design using the GB/SA approach, *J Chem Inf Model*, 2014, **54**, 3284-3301.

70. A. Daina, O. Michielin and V. Zoete, SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules, *Scientific Reports*, 2017, **7**, 42717.

71. B. F. Cravatt, A. T. Wright and J. W. Kozarich, Activity-based protein profiling: from enzyme chemistry to proteomic chemistry, *Annu Rev Biochem*, 2008, **77**, 383-414.

72. T. Bottcher and S. A. Sieber, Beta-lactones as privileged structures for the active-site labeling of versatile bacterial enzyme classes, *Angew Chem Int Ed Engl*, 2008, **47**, 4600-4603.

73. G. M. Simon and B. F. Cravatt, Activity-based proteomics of enzyme superfamilies: serine hydrolases as a case study, *J Biol Chem*, 2010, **285**, 11051-11055.

74. H. K. Lembke and E. E. Carlson, Activity-based probes in pathogenic bacteria: Investigating drug targets and molecule specificity, *Curr Opin Chem Biol*, 2023, **76**, 102359.

<figure><img src="image_96.png" /></figure>
75. C. A. Brown and A. Yamashita, Saline hydrides and superbases in organic reactions. IX. Acetylene zipper. Exceptionally facile contrathermodynamic multipositional isomerization of alkynes with potassium 3-aminopropylamide, *J Am Chem Soc*, 1975, **97**, 891-892.

76. K. R. Venrooij, L. de Bondt and K. M. Bonger, Mutually Orthogonal Bioorthogonal Reactions: Selective Chemistries for Labeling Multiple Biomolecules Simultaneously, *Top Curr Chem (Cham)*, 2024, **382**, 24.

77. D. Rifat, L. Chen, B. N. Kreiswirth and E. L. Nuermberger, Genome-Wide Essentiality Analysis of *Mycobacterium abscessus* by Saturated Transposon Mutagenesis and Deep Sequencing, *mBio*, 2021, **12**, e0104921.

78. M. Kanehisa, Y. Sato, M. Kawashima, M. Furumichi and M. Tanabe, KEGG as a reference resource for gene and protein annotation, *Nucleic Acids Res*, 2016, **44**, D457-462.

79. H. Ogata, S. Goto, K. Sato, W. Fujibuchi, H. Bono and M. Kanehisa, KEGG: Kyoto Encyclopedia of Genes and Genomes, *Nucleic Acids Res*, 1999, **27**, 29-34.

80. A. Kapopoulou, J. M. Lew and S. T. Cole, The MycoBrowser portal: a comprehensive and manually annotated resource for mycobacterial genomes, *Tuberculosis (Edinb)*, 2011, **91**, 8-13.

81. E. V. Koonin, Orthologs, Paralogs, and Evolutionary Genomics, *Annual Review of Genetics*, 2005, **39**, 309-338.

82. J. E. Griffin, J. D. Gawronski, M. A. DeJesus, T. R. Ioerger, B. J. Akerley and C. M. Sassetti, High-Resolution Phenotypic Profiling Defines Genes Essential for Mycobacterial Growth and Cholesterol Catabolism, *PLOS Pathogens*, 2011, **7**, e1002251.

83. C. M. Sassetti, D. H. Boyd and E. J. Rubin, Genes required for mycobacterial growth defined by high density mutagenesis, *Mol Microbiol*, 2003, **48**, 77-84.

84. K. Cotes, R. Dhouib, I. Douchet, H. Chahinian, A. de Caro, F. Carriere and S. Canaan, Characterization of an exported monoglyceride lipase from *Mycobacterium tuberculosis* possibly involved in the metabolism of host cell membrane lipids, *Biochem J*, 2007, **408**, 417-427.

85. A. Madani, I. Mallick, A. Guy, C. Crauste, T. Durand, P. Fourquet, S. Audebert, L. Camoin, S. Canaan and J. F. Cavalier, Dissecting the antibacterial activity of oxadiazolone-core derivatives against *Mycobacterium abscessus*, *PLoS One*, 2020, **15**, e0238178.

86. S. B. Cohen, B. H. Gern, J. L. Delahaye, K. N. Adams, C. R. Plumlee, J. K. Winkler, D. R. Sherman, M. Y. Gerner and K. B. Urdahl, Alveolar Macrophages Provide an Early *Mycobacterium tuberculosis* Niche and Initiate Dissemination, *Cell Host Microbe*, 2018, **24**, 439-446 e434.
87. L. Huang, E. V. Nazarova and D. G. Russell, *Mycobacterium tuberculosis*: Bacterial Fitness within the Host Macrophage, *Microbiol Spectr*, 2019, **7**, 7.2.04.
DOI: 10.1039/D5MD00102A

View Article Online

DOI: 10.1039/D5MD00102A

88. T. F. Byrd and C. R. Lyons, Preliminary characterization of a *Mycobacterium abscessus* mutant in human and murine models of infection, *Infect Immun*, 1999, **67**, 4700-4707.

89. T. Parish, In vitro drug discovery models for *Mycobacterium tuberculosis* relevant for host infection, *Expert Opin Drug Discov*, 2020, **15**, 349-358.

90. E. Le Run, M. Arthur and J. L. Mainardi, In Vitro and Intracellular Activity of Imipenem Combined with Tedizolid, Rifabutin, and Avibactam against *Mycobacterium abscessus*, *Antimicrob Agents Chemother*, 2019, **63**.

91. A. L. Lefebvre, V. Le Moigne, A. Bernut, C. Veckerle, F. Compain, J. L. Herrmann, L. Kremer, M. Arthur and J. L. Mainardi, Inhibition of the beta-Lactamase BlaMab by Avibactam Improves the In Vitro and In Vivo Efficacy of Imipenem against *Mycobacterium abscessus*, *Antimicrob Agents Chemother*, 2017, **61**.

92. B. C. VanderVen, R. J. Fahey, W. Lee, Y. Liu, R. B. Abramovitch, C. Memmott, A. M. Crowe, L. D. Eltis, E. Perola, D. D. Deininger, T. Wang, C. P. Locher and D. G. Russell, Novel inhibitors of cholesterol degradation in *Mycobacterium tuberculosis* reveal how the bacterium's metabolism is constrained by the intracellular environment, *PLoS Pathog*, 2015, **11**, e1004679.

93. P. Santucci, D. J. Greenwood, A. Fearns, K. Chen, H. Jiang and M. G. Gutierrez, Intracellular localisation of *Mycobacterium tuberculosis* affects efficacy of the antibiotic pyrazinamide, *Nat Commun*, 2021, **12**, 3816.
**data availability statement**

View Article Online

DOI: 10.1039/D5MD00102A

All data generated or analyzed during this study are included in this published article and its supplementary information files. The mass spectrometry proteomics data are available online through the ProteomeXchange Consortium (www.proteomexchange.org) with the dataset identifiers PXD057836.

<figure><img src="image_97.png" /></figure>