# Synthesis of *β*-lactam-zidovudine pronucleosides as potential selective narrow-spectrum antibacterial agents

Miyanou Rosales-Hurtado, Fanny Faure, Filomena Sannio, Federica Verdirosa, Georges Feller, Elodie Carretero, Yen Vo-Hoang, Patricia Licznar-Fajardo, Suzanne Peyrottes, Jean-Denis Docquier, et al.

► To cite this version:

Miyanou Rosales-Hurtado, Fanny Faure, Filomena Sannio, Federica Verdirosa, Georges Feller, et al. Synthesis of *β*-lactam-zidovudine pronucleosides as potential selective narrow-spectrum antibacterial agents. Organic & Biomolecular Chemistry, 2024, 10.1039/D4OB01396D. hal-04828735

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Submitted on 10 Dec 2024

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<figure><img src="image_1.png" /></figure>
# Synthesis of β-lactam-zidovudine pronucleosides as potential selective narrow-spectrum antibacterial agents

Miyanou Rosales-Hurtado,ª Fanny Faure,ª Filomena Sannio,ᵇ Federica Verdirosa,ᵇ Georges Feller,ᶜ Elodie Carretero,ª Yen Vo-Hoang,ᵈ Patricia Licznar-Fajardo,ᵈ Suzanne Peyrottes,ª Jean-Denis Docquier,ᵇ,* Laurent Gavaraª,∗

**Table of contents:** Based on the Buchwald-Hartwig amination reaction, an original β-lactam pronucleosides series was synthesized and microbiologically evaluated.

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

**Abstract.** Since the discovery of penicillin, the forerunner of the most widely used class of antibiotics (i.e. B-lactams), natural compounds and their derivatives represented a major source of antibacterial therapeutic products whose availability enabled modern medical practices (invasive surgery, organ transplant, etc.). However, the relentless emergence of resistant bacteria is challenging the long-term efficacy of antibiotics, also decreasing their economic attractiveness for Big Pharma, leading to a significant decay in antibacterial development in the 21st century and an increased use of last-resort drugs such as carbapenems or colistin. Indeed, bacteria evolved an arsenal of resistance mechanisms, leading to the emergence of totally-drug resistant isolates, already sporadically isolated among Gram-negative bacterial species. To face this deadly peril, it is fundamental to explore new ground-breaking approaches. In view of the significance of both B-lactam antibiotics and the production of one or more B-lactamases as the major resistance mechanism (especially in Gram-negative bacteria), we implemented an original approach to selectively deliver antibacterial zidovudine (AZT) exploiting the B-lactamase-mediated hydrolysis of a B-lactam-conjugate prodrug. The synthesis of the targeted pronucleosides was performed in 5-7 steps and based on an original Pd-catalyzed cross-coupling reaction. Enzymatic and microbiological evaluations were performed to evaluate the synthesized pronucleosides, yielding new insights into molecular recognition of B-lactamase enzymes. This approach would potentially allow a targeted and selective eradication of antibiotic-resistant B-lactamase-producing (opportunistic) pathogens, as the inactive prodrug is unable to harm the commensal microbial flora.

**Keywords:** B-lactam, prodrug, bacterial resistance, Buchwald-Hartwig amination, zidovudine

<footnote>ª Institut des Biomolécules Max Mousseron, Univ Montpellier, CNRS, ENSCM, Montpellier, France</footnote>

<footnote>ᵇ Dipartimento di Biotecnologie Mediche, Università di Siena, I-53100 Siena, Italy</footnote>

<footnote>ᶜ Laboratoire de Biochimie, Centre d'Ingénierie des Protéines-InBioS, Université de Liège, B-4000 Liège, Belgium</footnote>

<footnote>ᵈ HSM, Univ Montpellier, CNRS, IRD, CHU Montpellier, France</footnote>
Introduction

Since the dawn of humanity, bacterial infections have been a major health threat and a main cause of death.¹ The discovery of vaccines and antibiotics for both prevention and therapy of infections, combined with improved hygienic conditions, led to a drastic decrease in the mortality associated with infectious diseases.² This great victory against microorganisms allowed numerous advances, notably in the field of invasive surgery and organ transplantation. Nevertheless, bacteria always find a way to adapt and after decades of evolution and dissemination, an increasing number of multidrug-resistant (MDR) pathogens have emerged both in the clinical setting and the community, significantly challenging our healthcare system. Today, hospital-acquired infections caused by MDR bacteria already account for >1.2 M deaths worldwide.³ To face this deadly threat and to help focus the antibiotic development pipeline on the most relevant issues, the World Health Organization (WHO) identified carbapenem-resistant Gram-negative organisms as critical priorities.⁴

The β-lactam antibiotics, after the discovery of penicillin G and thanks to the plethora of semi-synthetic
analogs, soon became and still represent a major therapeutic resource for the treatment of numerous
bacterial infections. All β-lactams, divided into four distinct subfamilies: penicillins, cephalosporins,
monobactams, and carbapenems, share a common four-membered β-lactam ring that is crucial for the
inactivation of their molecular targets, i.e. the Penicillin-Binding Proteins (PBPs), leading to bactericidal
activity.⁵ Under selective pressure, bacteria evolved resistance mechanisms, involving target
modification, decreasing the apparent permeability of biological membranes or the production of
enzymes able to hydrolyze the crucial β-lactam ring, the β-lactamases,⁶ the latter mechanism being
widespread and most relevant in Gram-negative bacteria. Today, β-lactamases count over 8,250
unique variants, divided into 4 different classes (A, B, C, and D) according to Ambler classification and
over 330 enzyme subfamilies.⁷ Among them, acquired carbapenemases, such as KPC-, NDM-, IMP-,
VIM- or OXA-type enzymes, also called the “big five”,⁸ represent a major issue in the clinical setting, by
conferring resistance to the life-saving carbapenems and representing a major evolution towards
extensively and pan-drug resistance phenotypes.

In our research group, we propose to hijack β-lactamase-mediated bacterial resistance by using β-
lactam-prodrug conjugates as a strategy to selectively target β-lactamase producers. Another potential
advantage to such selective delivery would be represented by limited damage of the gut microbiota
during antibiotic therapy, i.e. antibiotic-associated dysbiosis. Indeed, the resulting antibiotic-
associated dysbiosis may lead to several well-known side effects, such as secondary infections, that
represent a major cause of death in clinical settings.⁹ Furthermore, conventional antibacterial agents
are prone to select resistant strains, which could establish themselves in the microbiota of healthy
people, extending the human reservoir of antibiotic-resistant bacteria in healthy colonized
individuals.¹⁰ To break this vicious circle, the development of narrow-spectrum or more selective
antibacterial agents would be desirable. Our strategy would fill this gap by using the four-membered
ring of β-lactam agents as a molecular phishing for β-lactamase-producing bacteria, which upon β-
lactam hydrolysis, will release an antibacterial moiety through a spontaneous chemical rearrangement.
This reactivity has been well-known in the cephalosporin family since the discovery of the cephalothin,
the first marketed cephalosporin derivative (Scheme 1).¹¹ Based on this rearrangement, several
codrugs were developed and extensively studied such as Ro 23-9424, a combination between a PBPs
inhibitor (β-lactam moiety) and a DNA gyrase inhibitor (fluoroquinolone drug).¹² This strategy was
mainly developed to extend the antibacterial spectrum and enhance antibacterial activity. However,
due to the constantly increasing expression levels of β-lactamases, the β-lactam activity of such codrug on PBPs is seriously compromised. Recently, hybrid antibiotics and prodrugs have regained interest, mainly regarding the use of the cephalosporin core as a carrier.<sup>13-16</sup> Nevertheless, the constant use of clinically valuable β-lactam antibiotic scaffolds to design carrier-linked prodrugs is expected to maintain and even increase the selection of β-lactamase enzyme and corresponding β-lactamase producers, potentially compromising the antibacterial activity of crucial marketed cephalosporin β-lactam agents (e.g., Cefiderocol). To avoid any resistance contribution, a different design is required.

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

Ro 23-9424

<caption>**Scheme 1.** Mode of action of dual cephalosporin drugs and structure of Ro 23-9424.</caption>

## Prodrug design

We recently published an original prodrug strategy based on monobactam core as a carrier vector.<sup>17</sup> The advantage of this β-lactam structure is only shared by a few therapeutic agents. The first identified monobactam agents were the naturally occurring compounds Nocardicins (Scheme 2). These products displayed a good inhibition profile against several Gram-negative pathogens but none of them were clinically useful due to weak PBPs inhibition values and insufficient β-lactamases stability.<sup>18</sup> Currently, the only marketed monobactam drug is, with highly specific substituents such as the sulfonic acid function. Herein, we envisaged a repositioning strategy using zidovudine (AZT) as an antibacterial agent. Initially developed as an anti-cancer agent, it is still intensively employed in AIDS therapy. AZT also demonstrated potent and broad-spectrum antibacterial activities, especially against Gram-negative enterobacteria.<sup>19,20</sup> As previously stated, antibiotic therapy is always coupled with important and undesired effects on friendly bacteria, such as those constituting the gut microbiota, with dysbiosis and resistance selection phenomenon. To avoid all those deleterious contributions, it is necessary to design a specific delivery system to release antibiotics into pathogenic bacteria only. In Gram-negative organisms, the virulence is positively correlated to β-lactamase production, especially ESBL or carbapenemase enzymes.<sup>21,22</sup>

Monobactam-zidovudine prodrugs 1, such as the JMV8240, (Scheme 2) were formulated to be activated only by β-lactamase-producing strains.<sup>17</sup> The selective β-lactamase cleavage, with subsequent AZT release, was proven to occur both in *in vitro* enzyme assays, but also in the cellular context with a favorable ratio between pathogenic and commensal bacteria. Nevertheless, the affinity for β-lactamases remains moderate and the covalent bond between the AZT and the monobactam carrier introduces an uncontrolled stereogenic center. The corresponding epimers were difficult to separate and Isothermal Titration Calorimetry (ITC) experiments demonstrated that only one enantiomer was an effective β-lactamase substrate and thus biologically active. To bypass this limitation, we propose the introduction of a styrene linker to formulate corresponding conjugates 2 (Scheme 2). Besides abolishing the stereogenic issue, the double-bond feature is also known in penem
and cephem nuclei to facilitate the reactivity of the β-lactam carbonyl function towards β-lactamase enzymes.²³ The styrene linker has already demonstrated its self-immolative ability, especially on penem probes.²⁴,²⁵ The similar intermediate **3** is expected to release AZT after β-lactam hydrolysis mediated by serine- or metallo-β-lactamases. Based on these considerations, we envisaged a carrier-linker prodrug approach with a ternary conjugate constituted by a monobactam carrier, a styrene linker, and the antibacterial AZT. As previously described, the nature of the side-chain R is crucial to determine the β-lactamase affinity profile.¹⁷

<figure><img src="image_4.png" /><figcaption>**Scheme 2.** Described monobactam analogs and targeted conjugates **2**.</figcaption></figure>

## Results and discussion

Numerous synthetic pathways of the β-lactam ring are described in the literature.²⁶ Previously, we have already described the synthesis of β-lactam precursors **4** (Scheme 3) based on Miller's hydroxamate cyclization strategy with no substitution (**4a**), the penicillin V sidechain (**4b**) and Boc protected amine (**4c**).¹⁷ The next step consisted of the introduction of the carbon-carbon double bond. Even if numerous heterocycles share this feature (e.g., cephem, penem...), there are very few examples in the literature of acyclic derivatives. The main synthetic way involves an *N*-alkylation step by α-halogenated acetic reagents of the nitrogen atom of the β-lactam ring **4**. The resulting methylene group can be directly functionalized by halogenation reactions followed by a Wittig olefination²⁷ or by aldol condensation in strongly basic conditions.²⁸,²⁹ None of those approaches were successful or suitable in our case. Thus, we decided to focus on the Buchwald-Hartwig amination reaction to form the nitrogen-carbon bond between the β-lactam **4** and α-halogenated acrylate reagents **5** (Scheme 3). Cross-coupling reactions metal-mediated, e.g., palladium-based catalysts, emerged last decades as major tools for molecule synthesis.³⁰ Initially developed for carbon-carbon bond formation, the reaction scope gradually enlarged to heteroatom partners. In β-lactam analogs field, only intramolecular cross-coupling reactions are described.³¹ The Cu-catalyzed Ullmann-type reaction was initially investigated, but the required harsh conditions were demonstrated to be incompatible with the β-lactam stability.³² The development of copper-ligands permitted them to operate in milder conditions allowing the synthesis of some carbapenem analogs.³³ Finally, the introduction of Pd-
catalyzed cross-coupling reactions, especially Buchwald-Hartwig amination, led to the synthesis of
penem derivatives by intra-molecular cyclization.<sup>34,35</sup> We decided to implement this approach for a
bimolecular cross-coupling reaction (Scheme 3).

<figure><img src="image_5.png" /><figcaption>**Scheme 3.** Retrosynthetic analysis.</figcaption></figure>

Before the study of the coupling reaction, α-bromoacetate precursors **5** have to be prepared. The olefination reaction was mediated by a mixture of titanium (TiCl₄, 1M solution in DCM) and triethylamine on commercially available benzaldehydes to only give corresponding Z isomers. The benzaldehyde **6** was converted in good yield into corresponding α-bromoacetate **7** as described in the literature (Scheme 4).³⁶ The same methodology was successfully applied to the hydroxymethyl derivative **8** to give the olefin **9** with moderate yield. The benzylic halogenation was carried out by TMSBr reagent to provide the dibromo derivative **10**. Finally, AZT was introduced in basic media through a substitution reaction, leading to α-bromoacetate precursor **11**. As the nucleoside analog exhibits two reactive positions, the 5'-hydroxyl residue and the 3-nitrogen atom (Figure 1),³⁷ two types of derivative may be obtained. Both are already described for AZT prodrug strategy,³⁸ especially with a similar self-immolative linker.³⁹ Several experimental conditions were investigated with strong (NaH) or weak (DIEA and K₂CO₃) bases, polar (DMF), or apolar (THF) solvents and using a mesyl group instead of the bromine leaving atom. In all cases, only one isomer was obtained with a 10% overall yield over three steps, and the NMR study revealed it was the N-3 substituted derivative **11**. To improve the synthetic pathway, the commercially available bromo-aldehyde **12** was used as the starting material. The conversion into the corresponding α-bromoacetate **10** led to a complex mixture. However, the introduction of AZT, in the first step, provided a total conversion into the conjugate **13** without any purification. The olefination reaction was performed on the crude mixture from intermediates **9** or **13**, to provide the α-bromoacetate **11** in 33% over two steps.
<figure><img src="image_6.png" /><figcaption>**Scheme 4.** Synthesis of α-bromoacetate precursors. Reagents and conditions: (a) TiCl₄ (1.5 eq.), bromoacetate (2 eq.), TEA (2.5 eq), DCM; (b) TMSBr (2.5 eq.), CH₃CN; (c) AZT (1 eq), K₂CO₃ (3 eq.), DMF.</figcaption></figure>

The next step refers to the bond formation between the nitrogen atom of the β-lactam **4** and the previously synthesized α-bromoacetates **5** by the Buchwald-Hartwig amination reaction. Metal-catalyzed cross-coupling reactions depend on a lot of experimental conditions and parameters such as temperature, solvent, ligand, or nature of the precatalyst.⁴⁰,⁴¹ Initially, the reaction feasibility was evaluated on model substrates. The commercial 2-azetidinone **4a** reacted with bromo-derivative **7** in the presence of BINAP as ligand and Pd₂(dba)₃ as precatalyst in toluene at 80 °C to give the corresponding coupled compound **14a** with a moderate yield (Table 1, Entry 1). The same experimental conditions were assayed with acylated β-lactam **4b**, but no reaction occurred regardless of the nature of the precatalyst, Pd₂(dba)₃ or Pd(OAc)₂ (Table 1, Entries 2-3). To improve the reactivity, a common strategy consists of exploring more electron-rich and/or bulky ligands.⁴² No reaction occurred with the flexible DPEphos ligand regardless of palladium source (Table 1, Entries 4-5).⁴³ We decided to select two bulky ligands with slightly different behavior, the BrettPhos and the RuPhos.⁴⁴ The BrettPhos permitted the formation of a small amount of the desired compound and 20% of conversion with Pd(OAc)₂ and Pd₂(dba)₃ respectively (Table 1, Entries 6-7). Based on these results (Table 1, Entries 1 and 7), Pd₂(dba)₃ was set as the palladium source, and the combination with RuPhos ligand led to better conversion with 51% isolated yield of compound **14b**.
<table><caption>**Table 1.** Parameters optimization on model substrates.ª</caption><thead><tr><th>Entry</th><th>Cpd</th><th>Ligand</th><th>Precatalyst</th><th>Yield<sup>a</sup></th></tr></thead><tbody><tr><td>1</td><td><b>14a</b></td><td>BINAP</td><td>Pd<sub>2</sub>(dba)<sub>3</sub></td><td>46%</td></tr><tr><td>2</td><td><b>14b</b></td><td>BINAP</td><td>Pd<sub>2</sub>(dba)<sub>3</sub></td><td>-<sup>b</sup></td></tr><tr><td>3</td><td><b>14b</b></td><td>BINAP</td><td>Pd(OAc)<sub>2</sub></td><td>-<sup>b</sup></td></tr><tr><td>4</td><td><b>14b</b></td><td>DPEphos</td><td>Pd(OAc)<sub>2</sub></td><td>-<sup>b</sup></td></tr><tr><td>5</td><td><b>14b</b></td><td>DPEphos</td><td>Pd<sub>2</sub>(dba)<sub>3</sub></td><td>-<sup>b</sup></td></tr><tr><td>6</td><td><b>14b</b></td><td>BrettPhos</td><td>Pd(OAc)<sub>2</sub></td><td>trace<sup>c</sup></td></tr><tr><td>7</td><td><b>14b</b></td><td>BrettPhos</td><td>Pd<sub>2</sub>(dba)<sub>3</sub></td><td>(20%)<sup>d</sup></td></tr><tr><td>8</td><td><b>14b</b></td><td>RuPhos</td><td>Pd<sub>2</sub>(dba)<sub>3</sub></td><td>51%</td></tr></tbody></table>

ª Isolated yield; ᵇ No product detected; ᶜ Only detected by LCMS; ᵈ Conversion rate determined by LCMS.

Once these experimental conditions were established, cross-coupling reaction with the AZT olefine **11** was investigated. The Buchwald-Hartwig amination with the unsubstituted β-lactam **4a** gave the corresponding conjugate **15a** with a moderate yield of 45% (Table 2, Entry 1). At the end of the reaction, uncompleted conversion was observed (starting material **11** was still present) and no more evolution was detected thereafter. To further improve the conversion rate, β-lactam equivalents and solvent parameters were investigated. Initially, a modest excess of β-lactam **4** was used (1.5 eq.) but a complete conversion was reached using 2 eq., leading to an improved yield of up to 85% (Table 2, Entry 2). Coupling-reactions can be carried out with a wide range of solvents, but the two most used in the Buchwald-Hartwig reaction are toluene and dioxane. When dioxane was used instead of toluene, a clean conversion was obtained, with a good yield of 73% (Table 2, Entry 3). As observed previously, acylated β-lactams **4b-c** showed slightly different reactivity. In toluene, with 1.5 or 2 eq. of β-lactam **4b**, only a limited conversion was detected (Table 2, Entries 4-5). To obtain a full conversion, the solvent had to be changed for dioxane leading to the desired conjugate **15b** with a 66% yield (Table 2, Entry 6). The Boc derivate **4c** gave similar results, with a better conversion-rate in dioxane than toluene (Table 2, Entries 7-8), and compound **15c** was isolated in 85% yield.
<table><caption>**Table 2.** Cross-coupling reaction on AZT conjugates.ª</caption><thead><tr><th>Entry</th><th>Cpd</th><th>Solvent</th><th>4 (eq.)</th><th>Yield<sup>a</sup></th></tr></thead><tbody><tr><td>1</td><td><b>15a</b></td><td>toluene</td><td>1.5</td><td>45%</td></tr><tr><td>2</td><td><b>15a</b></td><td>toluene</td><td>2</td><td>85%</td></tr><tr><td>3</td><td><b>15a</b></td><td>dioxane</td><td>2</td><td>73%</td></tr><tr><td>4</td><td><b>15b</b></td><td>toluene</td><td>1.5</td><td>24%</td></tr><tr><td>5</td><td><b>15b</b></td><td>toluene</td><td>2</td><td>(35%)<sup>b</sup></td></tr><tr><td>6</td><td><b>15b</b></td><td>dioxane</td><td>2</td><td>66%</td></tr><tr><td>7</td><td><b>15c</b></td><td>toluene</td><td>2</td><td>54%</td></tr><tr><td>8</td><td><b>15c</b></td><td>dioxane</td><td>2</td><td>85%</td></tr></tbody></table>

Thanks to the Pd-catalyzed amination reaction, key intermediates **15a-c** were successfully synthesized in 3 steps with 20-30% overall yield up to 1.5 mM scale. The last step of the synthetic pathway consists of the modulation of acyl substituent at position 3 of the β-lactam ring. Therefore, the derivative **15c** was treated in acidic conditions to give the free amino compound **16** with a 50% yield (Scheme 5). To decorate this position, several side chains, inspired by first generation β-lactam agents, were selected such as Penicillin V (b), G (d), Cefalotin (e), or Ampicillin (h). None of these substituents are described to significantly contribute to any β-lactamase resistance, which is a critical point for our approach. In addition, some specific biaryl substituents, demonstrating specific favorable interaction with β-lactamase enzymes, were also chosen based on literature data such as NDM-1 (p-Ph, f)<sup>45</sup> or AmpC (m-PhO, g).<sup>46</sup> The various envisioned sidechains were introduced by an amide coupling reaction between commercial carboxylic acid and compound **16** using a mixture of EDCI and HOBt as coupling agents. The corresponding amide compounds **17d-g** were obtained with a good yield (50-96% range). To avoid side reactions, the amine function of the ampicillin sidechain (h) had to be protected by a Boc group (17i). Removal of this protecting group was performed as previously described and gave the ampicillin analog **17h** with moderate yield (48%). The last step of the synthetic pathway consisted of methyl ester hydrolysis to provide the corresponding carboxylic acid function. This reaction was performed in basic-media using a water-THF solution of NaOH, providing a complete conversion. Finally, some final compounds **2a-h** were isolated as sodium salt to maximize aqueous solubility capability, and thus facilitate further microbiological evaluations (Scheme 5).

<footnote>ª Isolated yield; ᵇ Conversion rate determined by LCMS.</footnote>
<figure><img src="image_7.png" /><figcaption>**Scheme 5.** Deprotection and acylation reaction steps. Reagents and conditions: (a) TFA 50%, CH₂Cl₂, RT; (b) carboxylic acid (1.3 eq.), EDCI.HCl (1.1 eq.), HOBT (1.1 eq.), DIPEA (3.5 eq.), THF, 0°C; (c) NaOH [0.1 M], THF/H₂O (1:1), RT.</figcaption></figure>

Before the microbiological evaluation, the chemical stability of the newly synthesized prodrugs **2a-h** in an aqueous media has to be determined. As zidovudine (AZT) exhibits a very potent antibacterial activity, with MIC values well under 0.5 µg.mL⁻¹ on susceptible stains,²⁰ a chemical instability of the pronucleoside leading to subsequent AZT release may represent a significant interference, biasing the result of *in vitro* antibacterial testing. To monitor the chemical stability, the model compound **2b** was selected and evaluated by tandem mass spectrometry coupled to HPLC. A 0.9% NaCl physiological solution was used as the aqueous media, and the compound **2b** was incubated at 37 °C. Calibration curves were obtained for the compound and the AZT, and stability was monitored for 16 h. The prodrug **2b** proved to be completely stable, with no detectable AZT formation (See SI, Graph S1). Only compound **2h** exhibited weak chemical stability, even in organic solvents such as deuterated DMSO during the NMR characterization. To investigate the degradation pathway and elucidate the structure of the side product, a kinetic NMR study was conducted in DMSO-d₆ and in D₂O, followed by LC-MS analysis after 24 hours. While compound **2h** proved stable in water, the side product appeared immediately in DMSO to reach 80% ratio after 24h. The ¹H NMR signals of the acyl side chain showed the most significant changes compared to the starting compound **2h**. Mass analysis indicated the loss of a water molecule, associated with a less polar compound (see SI). The exact structure remains elusive, no specific signal of the free AZT was detected, and the chemical degradation is probably mediated by an intramolecular or bimolecular reaction between the amino group and the β-lactam ring. The presumable involvement of the NH₂ group in the decomposition process is also supported by the stability of the Boc derivate **2i**, with no associated degradation.

The antibacterial activity of the synthesized compounds was then evaluated using a selection of β-lactamase-producing isogenic E. coli laboratory strains (i.e. carrying a derivative of the pET-9a plasmid vector in which the gene encoding for the β-lactamase was cloned).⁴⁷ We chose four major β-lactamase enzymes highly prevalent in clinical isolates and significantly involved in β-lactam resistance: KPC-3, IMP-1, NDM-1, and AmpC. This representative panel is constituted of three different molecular classes (A, B and C) involving the two possible hydrolytic mechanisms (serine- and metallo-hydrolases). The
MIC values were determined using the broth microdilution method (Table 3) and confirmed the expectedly potent antibacterial activity of AZT (0.06-0.5 MIC range) on all tested strains, whereas the pronucleoside **JMV8240** demonstrated a strong selectivity for β-lactamase producers, although the nature of the β-lactamases produced appears to modulate the activity of the conjugate. However, none of the considered new pronucleosides **2a-h** displayed antibacterial activity toward the *E. coli* panel strains. This lack of activity can be associated with several factors, including an insufficient enzymatic recognition that may be investigated by ITC measurements or a lack of diffusion through the outer membrane. To determine the contribution of the lack of membrane permeability, the microbiological assays were reproduced in presence of a sub-inhibitory concentration of colistin (that did not affect bacterial growth on its own), a polymyxin antibiotic well known for its membrane permeabilizing properties. In these conditions, no significant modification of MIC values was observed with the model compound **2b**. These results indicate that passive diffusion may not be predominantly involved, even if there are several other bacterial barriers to limit drug periplasm accumulation, such as active efflux or requirements of specific outer membrane proteins.

<table><caption>**Table 3.** MIC determination by Broth micro-dilution method<sup>a</sup></caption><thead><tr><th rowspan="2">Entry</th><th rowspan="2">Bacterial strain</th><th colspan="10">MIC (mg.L<sup>-1</sup>)<sup>b</sup></th></tr><tr><th>AZT</th><th>JMV8240</th><th>2a</th><th>2b</th><th>2c</th><th>2d</th><th>2e</th><th>2f</th><th>2g</th><th>2h</th></tr></thead><tbody><tr><td>1<sup>c</sup></td><td><i>E. coli</i> BL21 (DE3)</td><td>0.06</td><td>16</td><td>&gt;128</td><td>&gt;128</td><td>&gt;128</td><td>&gt;128</td><td>&gt;128</td><td>&gt;128</td><td>&gt;128</td><td>&gt;128</td></tr><tr><td>2<sup>d</sup></td><td><i>E. coli</i> BL21 (DE3)<br>pET9a-KPC-3</td><td>0.5</td><td>0.25</td><td>&gt;128</td><td>&gt;128</td><td>&gt;128</td><td>&gt;128</td><td>&gt;128</td><td>&gt;128</td><td>&gt;128</td><td>&gt;128</td></tr><tr><td>3<sup>d</sup></td><td><i>E. coli</i> BL21 (DE3)<br>pET9a-IMP-1</td><td>0.03</td><td>2</td><td>&gt;128</td><td>&gt;128</td><td>&gt;128</td><td>&gt;128</td><td>&gt;128</td><td>&gt;128</td><td>&gt;128</td><td>&gt;128</td></tr><tr><td>4<sup>d</sup></td><td><i>E. coli</i> BL21 (DE3)<br>pET9a-NDM-1</td><td>0.06</td><td>4</td><td>&gt;128</td><td>&gt;128</td><td>&gt;128</td><td>&gt;128</td><td>&gt;128</td><td>&gt;128</td><td>&gt;128</td><td>&gt;128</td></tr><tr><td>5<sup>d</sup></td><td><i>E. coli</i> BL21 (DE3)<br>pET9a-AmpC</td><td>0.125</td><td>2</td><td>&gt;128</td><td>&gt;128</td><td>&gt;128</td><td>&gt;128</td><td>&gt;128</td><td>&gt;128</td><td>&gt;128</td><td>&gt;128</td></tr></tbody></table>

The interaction between β-lactamases and pronucleosides **2a-e** was investigated by Isothermal Titration Calorimetry (ITC), a thermodynamic technique based on enthalpy variation measurement (heat release of hydrolysis), already successfully used in our previous work, to demonstrate the hydrolysis of the β-lactam ring and allowing the measurement of kinetic parameters (such as the turnover rate, the Michaelis constant and the catalytic efficiency).<sup>48</sup> We selected two clinically relevant β-lactamases showing carbapenemase activity, KPC-2 and NDM-1, as representatives of the two mechanistically unrelated beta-lactamase families where beta-lactam hydrolysis relies on either acid-base catalysis assisted by a conserved catalytic serine residue (KPC-2) or metal-based catalysis assisted by zinc ions (NDM-1), as depicted in Scheme 2. As shown in Tables 4 and 5, the kinetic parameters of hydrolysis of several relevant conjugates were obtained with both tested enzymes. Data obtained with meropenem, here used as a control substrate of both carbapenemases, showed a very good agreement with those previously reported in the literature by different laboratories.<sup>49,50</sup>

With KPC-2, the previously described **JMV8240** pronucleoside showed a catalytic efficiency ($k_{cat}/K_M$) very similar to that measured with meropenem. As established in the literature, the N-acyl substitution on the azetidinone ring is a key factor in substrate recognition, leading to favorable interactions with

<footnote><sup>a</sup> Triplicate experiment; <sup>b</sup> Minimal Inhibitory Concentration; <sup>c</sup> Reference isolate; <sup>d</sup> Isogenic β-lactamase producer.</footnote>
the Ω-loop of KPC-2. Indeed, the unsubstituted derivative **2a** was expectedly unable to interact with that enzyme.⁵¹,⁵² The introduction of different acyl sidechains, as found in β-lactam antibiotics used in the clinical setting, greatly improved substrate recognition and, therefore, compound hydrolysis, the best result being obtained with the thiophene derivative **2e**.⁵³ Nevertheless, the hydrolysis observed with the newly synthesized compounds (**2a-e**) were deceivingly significantly slower than with **JMV8240**. Interestingly, this primarily relies on the turnover rates being up to ≈300-fold lower than that of **JMV8240**, while the Michaelis constants were largely unaffected, suggesting a lower reactivity of the substrate (i.e. leading to either a slower acylation, a slower deacylation, or both), rather than its recognition by the enzyme.⁵² This reduction of turnover rates obviously negatively impacted on the catalytic efficiency, with $k_{cat}/K_M$ values also decreased by up to ≈300 fold. Such data support the lack of antibacterial activity of these compounds on β-lactamase-producing strains. Mechanistically, and considering the catalytic mechanism of serine-β-lactamases, the $k_{cat}/K_M$ values is only affected by microscopic rate constants characterizing the acylation step.⁵⁴,⁵⁵ It may thus be speculated that the rate-limiting step in our system would be the formation of acyl-enzyme from the Michaelis complex, rather than the hydrolysis of the covalent adduct, equally leading to a significant drop in the turnover rate (indeed, $k_{cat} = (k_{acylation} \times k_{deacylation}) / [k_{acylation} + k_{deacylation}]$, which tends to reflect the rate-limiting step, i.e. either $k_{acylation}$ or $k_{deacylation}$). Although overall disappointing, the present data allow to identify critical structural requirements compatible with efficient hydrolysis and to establish further structure-activity relationship relevant for the design of future β-lactam-based conjugates relevant to our approach. When compared to compound **JMV8240**, the introduction of the styrene moiety appears to drastically reduce the reactivity of the compounds, although this reduction also depends on the substituent present on the azetidinone ring. Our data appears to confirm that a thiophene-containing sidechain (as in the first-generation cephalosporin cephalothin), as found in **2e**, is associated with a better hydrolysis than the substituents found in **2b** (as in penicillin V) or **2d** (as in penicillin G).

<table><caption>**Table 4.** Kinetic parameters of KPC-2 for the hydrolysis of pronucleosides **2a-e**, determined by ITC.</caption><thead><tr><th rowspan="2">Cpd</th><th colspan="3">KPC-2</th></tr><tr><th>k<sub>cat</sub> (s<sup>-1</sup>)<sup>a</sup></th><th>K<sub>M</sub> (mM)<sup>a</sup></th><th>k<sub>cat</sub>/K<sub>M</sub> (M<sup>-1</sup>S<sup>-1</sup>)</th></tr></thead><tbody><tr><td>Meropenem<sup>b</sup></td><td>5.8 ± 0.1<br>(4.1)</td><td>0.023 ±<br>0.002<br>(0.016)</td><td>2.5 x 10<sup>5</sup><br>(2.6 x 10<sup>5</sup>)</td></tr><tr><td>JMV8240</td><td>351.4 ±<br>8.8</td><td>1.5 ± 0.08</td><td>2.3 x 10<sup>5</sup></td></tr><tr><td>2a</td><td>ND<sup>c</sup></td><td>-</td><td>-</td></tr><tr><td>2b</td><td>7.4 ± 0.8</td><td>1.4 ± 0.1</td><td>5.3 x 10<sup>3</sup></td></tr><tr><td>2c</td><td>1.3 ± 0.1</td><td>1.7 ± 0.2</td><td>8.0 x 10<sup>2</sup></td></tr><tr><td>2d</td><td>6.4 ± 0.2</td><td>0.8 ± 0.1</td><td>8.0 x 10<sup>3</sup></td></tr><tr><td>2e</td><td>25.6 ± 2.9</td><td>1.9 ± 0.5</td><td>1.4 x 10<sup>4</sup></td></tr></tbody></table>

<footnote><sup>a</sup> Values are mean ± SD; <sup>b</sup> KPC-2 data in brackets are from Wang et al.⁴⁸; <sup>c</sup> Not detected, below the detection limit.</footnote>
Despite these disappointing results, a different set of conjugates (2b, 2e, and 2f) was nonetheless tested with NDM-1, with which only modest hydrolysis was obtained with the previously published series of pronucleosides.¹⁷ The inclusion of the biphenyl derivative 2f was dictated by our previous experience in the optimization of metallo-beta-lactamase inhibitors.⁴⁵ Such intuition appeared correct, as this compound exhibited the lowest $K_M$ value (in the micromolar range) with NDM-1, one order of magnitude lower than that observed with compound 2e (thiophene side chain). Strikingly, the measured turnover rates with NDM-1 (kcat, ≤0.2 s⁻¹) were even lower with that measured with KPC-2, while compound 2b (side chain as in penicillin V) did not appear to even bind to the enzyme or only by an unproductive binding way. The higher apparent affinity of compound 2f determined a two-fold higher catalytic efficiency, although these values were overall low ($k_{cat}/K_M$, ≤2 × 10³ M⁻¹ s⁻¹). Based on the different catalytic mechanism of metallo-β-lactamases, these data show that the rate-limiting step is not the binding of the substrate but its hydrolysis, possibly hindered by a perturbation of the coordination sphere of the hydroxide anion located the Zn ions, which represent the nucleophilic species able to attack the carbon carbonyl of the β-lactam ring. However, and taking into consideration the inherent very weak ability of metallo-β-lactamases to accommodate monobactam substrates,⁵⁶ those preliminary results are somewhat promising and surprising. In the case of aztreonam, the only marketed monobactam derivative, the sulfate group on the nitrogen atom of the monobactam is strongly involved in this hydrolytic stability profile.⁵⁷ The introduction of the regular carboxylic acid moiety, such as into the compounds 2e and 2f, allows to promote a recognition or hydrolysis rate up to three orders of magnitude greater than that of aztreonam, a notably bad substrate for metallo-β-lactamases (Table 5).⁵⁸ This favorable profile may be useful to selectively target metallo-β-lactamase producers, also commonly called superbugs,⁵⁹ highly involved in resistant bacterial infections.⁶⁰

<table><caption>**Table 5.** Kinetic parameters of NDM-1 for the hydrolysis of pronucleosides 2b, 2e and 2f, determined by ITC</caption><thead><tr><th rowspan="2">Cpd</th><th colspan="3">NDM-1</th></tr><tr><th>k<sub>cat</sub> (s<sup>-1</sup>)<sup>a</sup></th><th>K<sub>M</sub> (mM)<sup>a</sup></th><th>k<sub>cat</sub>/K<sub>M</sub><br>(M<sup>-1</sup>S<sup>-1</sup>)</th></tr></thead><tbody><tr><td>Meropenem<sup>b</sup></td><td>109 ± 1<br>(144.9)</td><td>0.06 ±<br>0.003 (0.06)</td><td>1.8 x 10<sup>6</sup><br>(2.4 x 10<sup>6</sup>)</td></tr><tr><td>Aztreonam<sup>c</sup></td><td>(0.014)</td><td>(9)</td><td>(1.5)</td></tr><tr><td>2b</td><td>ND<sup>d</sup></td><td>-</td><td>-</td></tr><tr><td>2e</td><td>0.187 ±<br>0.007</td><td>0.195 ±<br>0.032</td><td>9.6 x 10<sup>2</sup></td></tr><tr><td>2f</td><td>0.057 ±<br>0.004</td><td>0.027 ±<br>0.007</td><td>2.1 x 10<sup>3</sup></td></tr></tbody></table>

To further explain the lack of antibacterial activity, the compound stability was also investigated in the presence of CTX-M-15, KPC-2 and NDM-1 β-lactamases, using a LC-MS method. Rather surprisingly, the immediate disappearance of the studied compounds was observed (see SI for LC-MS chromatograms), although it was not associated with a significant release of AZT, and no prodrug metabolite was identified. As the prodrugs 2b-2e are stable in the absence of enzymes, their β-lactamase-specific enzymatic conversion may be followed by an unexpected chemical degradation.

<footnote><sup>a</sup> Values are mean ± SD; <sup>b</sup> NDM-1 data in brackets are from Ali et al.;<sup>50</sup> NDM-1 data in brackets are from Lohans et al.;<sup>58</sup> d Not detected, below the detection limit.</footnote>
Other experiments supported this observation and the absence of intact released AZT. Indeed, preincubating compound 2b with high concentrations of purified NDM-1 β-lactamases and dosing AZT using a sensitive microbiological assay (based on a disk diffusion assay using a susceptible *E. coli* laboratory strain [XL-1; AZT MIC value, 0.015 mg.mL⁻¹]) previously successfully used in our group,¹⁷ no significant bacterial growth inhibition could be observed, in agreement with a limited release of AZT or an unsuitable chemical rearrangement after the β-lactamase-mediated hydrolysis.

## Conclusion

A world without antibiotics is a grim perspective, although more realistic than ever! To successfully tackle the worrisome is of antibiotic resistance, the discovery of new antibiotics based on innovative strategies is crucial. The development of synthetic methodologies for the preparation of original β-lactam analogs is crucial to feed the antibacterial pipeline. In this context, we developed a new approach to synthesize monobactam derivatives by a selective and efficient Buchwald-Hartwig cross-coupling reaction in mild conditions. The scope of the reaction was determined, and several parameters were optimized, opening the door to new norcardicin analogs. This new synthetic methodology was effectively applied for the synthesis of original β-lactam-zidovudine conjugates leading to the obtention of 8 pronucleosides 2a-h, in 5 to 7 steps. As illustrated by the modest biological results obtained, the steric hindrance produced by the styrene linker seems to generate unfavorable interactions with the β-lactamases and interfere with the expected delivery of AZT. To improve the enzymatical recognition, the introduction of a smaller linker based on allyl group may represent a viable alternative to explore. Moreover, the addition of siderophore moieties to secure the bacterial accumulation have to be considered, especially on the acyl side chain of the β-lactam ring. Despite unsatisfactory antibacterial activity, ITC measurements provided interesting insights and SAR data relevant to our approach. Monobactams are described as unsuitable substrate for metallo-β-lactamases, but we demonstrated herein that it is possible to improve their affinity by adequate structural modifications. Taking advantage of these results, the design of new prodrug approaches will be developed in the near future.

**General methods** Nuclear Magnetic Resonance (NMR) spectra were recorded with a Bruker Avance 400 or 500 or 600 spectrometer (for ¹H and ¹³C NMR) and Bruker Avance 400 spectrometer (376.5 for ¹⁹F). Chemical shifts (δ) and coupling constants (J) are given in ppm and Hz, respectively, using residual solvent signals as reference for the ¹H and ¹³C. The following abbreviations are used: s = singlet, d = doublet, t = triplet, q = quartet, br s = broad signal, dd = double doublet, dt = double of triplet, m = multiplet. High-resolution mass spectra (HRMS) were registered on a JEOL JMS-SX-102A mass spectrometer. Reactions were monitored with Merck Kieselgel 60F₂₅₄ precoated aluminum silica gel plates (0.25 mm thickness) which were visualized under 254 nm light or by charring with a ninhydrin solution in butanol or with a vanillin solution in ethanol followed by heating or iodine stains. Purification experiments were carried out on Silica Gel premium Rf grade (40-63 µm) or were performed on a Biotage Isolera One using a 20-50 µm packed silica cartridge or using reverse phase preparative HPLC on Akta pure C18 column (Luna 21.2 x 100 mm, 5 µm, 100 Å). Eluent A: Water (10 mM AcONH₄ or 0.1% TFA or Neutral); B: Acetonitrile (Neutral or 0.1% TFA, respectively) with flow rate: 20 mL/min. LC-MS analyses were obtained on the Waters Alliance 2690 HPLC, coupled to a Waters Micromass Quattro micro API (electrospray ionization mode, ESI+). Conditions: RP C18 Chomolith High-Resolution column (25 x 4.6 mm) using a linear gradient (0-100%) of water and acetonitrile (each containing 0.1% of formic acid) over 3 min and at a flow rate of 3 mL/min, mono λ detection (214 nm).
Positive ion electrospray mass spectra were acquired at a solvent flow rate of 0.15 µL.min⁻¹. Nitrogen was used as both the nebulizing and drying gas. The data were obtained in a scan mode in 0.7 s intervals. UPLC analyses were obtained on the Waters Acquity H-Class UPLC, coupled to a Waters Zspray (electrospray ionization mode, ESI+). Conditions: Acquity UPLC® peptide HSS T3 column (2.1 x 50 mm, 1.8 µm, 100 Å) using a linear gradient (0-100%) of water and acetonitrile (each containing 0.1% of formic acid) over 5 min and at a flow rate of 0.8 mL.min⁻¹ flow rate, photodiode array detection (TUV - 214 nm). Positive ion electrospray mass spectra were acquired at a solvent flow rate of 0.8 µL.min⁻¹. Nitrogen was used as both the nebulizing and drying gas. The data were obtained in a scan mode in 0.2 s intervals. The purity of all synthetic compounds was determined by UPLC analysis and was greater than 95%. All detailed experimental procedures and associated spectrums are available in the supplementary data file.

**Determination of kinetic parameters of hydrolysis by ITC.** Enzyme activity was recorded by ITC on a MicroCal ITC200 (GE-Malvern) equipped with a 200 µl Hastelloy sample cell and an automated 40 µl glass syringe rotating at 1000 rpm. Compounds were solubilized in 10 mM Hepes-NaOH, 0.15 M NaCl, pH 7.5, and both enzymes were diluted to the desired concentrations with the same buffer. For compounds 2a-h, the buffer also contained 1% DMSO and KPC-2 was brought to the same final DMSO concentration. The activity was recorded and calculated as described,⁴⁸ using a molar enthalpy value for β-lactam amide bond hydrolysis of 12 kcal/mol at 25°C. The reaction rate constant $k_{cat}$ (s⁻¹) is defined here as the number of amide bonds hydrolyzed per second and active site. For $K_m$ determination, activities were recorded at decreasing substrate concentrations, and the data points were fitted to the Michaelis-Menten equation.

**Enzyme Assays and Release of AZT from Prodrugs.** The β-lactam derivative 2b (2.5 µL) at 5 mg mL⁻¹ was preincubated with 8 µL of different enzymes at a concentration of 0.5 mg/mL (deposit A), 0.01 mg mL⁻¹ (deposit B), and no enzyme (deposit C) in 9.5 µL of 0.9% NaCl solution. After 30 min at room temperature, 2 µL was plated on the surface of Mueller-Hinton (MH) agar plate inoculated with *E. coli* XL1 strain (1.5 × 10⁸ CFU/mL) and incubated at 37 °C for 24 h.

**MICs determination.** The minimum inhibitory concentrations (MICs) of inhibitors were determined in triplicate using cation-supplemented Mueller-Hinton broth and a bacterial inoculum of 5 × 10⁴ CFU/well, as recommended by the CLSI and the European Committee on Antimicrobial Susceptibility Testing (EUCAST document "Terminology relating to methods for the determination of susceptibility of bacteria to antimicrobial agents", 2000). The MIC values of compounds were determined on *E. coli* BL21(DE3)-derivative laboratory strains hyperproducing a single β-lactamase.

**Stability assays by ultra-performance liquid chromatography separation and tandem mass spectrometry.** Identification and stability experiments were carried out by reverse phase liquid chromatography on a Waters Acquity Iclass UPLC system coupled to a high-resolution mass spectrometer (Xevo TQD Waters), equipped with an electrospray ionization source and controlled by MassLynx v.4.2 software for data acquisition. All the analyses were carried out using an Acquity UPLC BEH C18 2.1 x 50 mm column at 40°C. A flow rate of 0.8 mL.min⁻¹ and a gradient of (0-100%) B over 3.5 min were used: eluent A, water/0.1% HCO₂H; eluent B, acetonitrile/0.1% HCO₂H. The mass spectrometer operated in positive or negative ionization mode and parameters were set as follows: source block temperature, 150 °C, nebulizer nitrogen flow rate, 50 L/hour, capillary voltage, 2.0 or 2.5 kV; desolvation temperature, 550 or 600 °C; desolvation nitrogen gas flow, 900 or 1000 L/hour. The different investigated parameters were cone voltage (CV), collision energy (CE), ionization mode, and
MRM channels. Specific settings for each compound are provided in Supporting Information. All samples were prepared by dilution in a commercial physiological solution (0.9% NaCl). A calibration curve was determined for each compound as follows: samples used for the calibration were injected (1µl) in triplicate from the lowest concentration (0.0001 mg.ml⁻¹) to the highest one (0.01 mg.ml⁻¹) and the calibration curves were plotted with the Targetlynx tool from Masslynx. Then, samples used for stability experiments were injected (1µl) at 0.005 mg.mL⁻¹ at 37°C and monitored every hour over 24 hours. The stability curves were plotted with the Targetlynx tool from Masslynx. The MRM channel monitoring of AZT apparition and molecule degradation were plotted at the same time to check the release.

**Stability Assays by NMR.** Pronucleoside 2h were dissolved in 600 µL of solvent (DMSO-*d*₆ or *D*₂O) at a concentration of 10 mM. The ¹H NMR spectra were measured on a Bruker 600 MHz spectrometer at various time points at 310 K.

**Author contributions.** Miyanou Rosales-Hurtado: Synthesis and writing; Fanny Faure: Synthesis and microbiological assays; Filomena Sannio: Microbiological evaluation; Federica Verdirosa: Microbiological evaluation; Georges Feller: ITC evaluation; Elodie Carretero: Stability assay; Yen Vo-Hoang: Microbiological evaluation; Patricia Licznar-Fajardo: Microbiological evaluation; Suzanne Peyrottes: Supervision; Jean-Denis Docquier: Conceptualization and writing; Laurent Gavara: Conceptualization, writing, and project administration; All authors contribute to reviewing and editing.

**Data availability.** The data supporting this article have been included as part of the ESI.

**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.

**Acknowledgments.** This work was funded by the Agence Nationale de la Recherche (ANR-19-CE18-0003-01) and the LabUM chimie of the University of Montpellier through the "Investissements d'avenir" program of the ANR with reference number ANR-16-IDEX-0006. We also want to thank analytical and SynBio3 platforms and in particular Aurélien Lebrun, Luc Brunel, Dr. Pascal Verdié, and Pierre Sanchez for mass spectrometry analyses. J.D.D. was supported in part by the Italian MUR (Ministero dell'Università e Ricerca) in the frame of the PNRR PE-13 ("Piano Nazionale di Ripresa e Resilienza, Partenariato Esteso 13, Malattie infettive emergenti") INF-ACT project (One Health Basic and Translational Research Actions addressing Unmet Needs on Emerging Infectious Diseases).

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