PRODROGUE INTENDED FOR THE TREATMENT OF BACTERIAL INFECTIONS RESISTANT TO BETA-LACTAM ANTIBIOTICS

Abstract

The invention relates to the field of antibacterial treatments for combating antibiotic-resistant infections. More particularly, the invention concerns a prodrug for locally and specifically inhibiting bacterial infection caused by bacteria resistant to beta-lactam antibiotics. This prodrug comprises an antibacterial agent of a new therapeutic class, linked to a bait molecule including a functional group cleavable by a beta-lactamase as defined by the classifications of Ambler (1980) and Bush and Jacoby (2010). The antibacterial agent is masked and therefore inactive in the prodrug state and recovers its native structure and antibacterial properties once the prodrug is cleaved in situ by the pathogenic bacteria.

Description

Description Title of the invention: PRODROGUE INTENDED FOR TREATMENT OF BACTERIAL INFECTIONS RESISTANT TO ANTIBIOTICS OF THE FAMILY BETA-LACTAMS

[0001] = The invention relates to the field of antibacterial treatments intended to combat against antibiotic-resistant infections. More specifically, the invention concerns a prodrug that allows for the local and specific inhibition of a bacterial infection due to an antibiotic-resistant bacterium of the family beta-lactams. This prodrug includes a novel antibacterial agent. therapeutic class, and linked to a bait molecule comprising a group functional group cleavable by a beta-lactamase as defined according to the classifications d'Ambler (1980) and Bush and Jacoby (2010). The antibacterial principle is masked and therefore inactive in the pro-drug state and regains its native structure and anti- properties bacterial once the prodrug has been cleaved, in situ, by pathogenic bacteria. Scope of the invention

[0002] — The fight against bacterial infections is a public health issue of concern when it comes to dealing with multi-resistant bacteria. Indeed, many Bacteria have developed resistance mechanisms to conventional antibiotics. particularly those in the beta-lactam family. To treat these infections, anti- Second-generation biotics are administered, but these are responsible of side effects. Also, their systemic use (that is, not local and not specific) promotes the emergence of new resistances against these new molecules.

[0003] — Strategies for targeting antibacterial molecules to the site of infection of antibiotic-resistant bacteria have been proposed, notably by exploiting the A resistance mechanism implemented by the bacteria to target the treatment. This approach was described by Evans et al. (J. Med. Chem 2019, 62, 4411-4425); it consists of a prodrug that can be activated by beta-lactamases produced by bacteria resistant. Thus, a classic antibiotic molecule, ciprofloxacin, is conjugated chemically to a beta-lactam motif recognized by the bacterium and cleaved by it from so as to release the said antibiotic molecule. However, the specificity of this prodrug may be limited by the fact that the molecular structure of the antibiotic agent is under its "active" form within the prodrug. Thus, it is expected that an anti- biotic, even diminished, is exerted by the prodrug even before its cleavage by beta-lactamases. Thus, this limits the specificity of action of this prodrug on the Multidrug-resistant bacteria are a concern, but this aspect is crucial to avoid systemic exposure and prevent bacteria that are not already multidrug-resistant from developing resistance mechanisms. While this system provides localized antibacterial action at the site of infection, it does not solve the problem of antibiotic resistance. There is a real need for new therapeutic strategies to treat infections caused by antibiotic-resistant bacteria. Solutions to this need must incorporate (1) a new therapeutic target and (2) specific, local action to prevent systemic exposure and the development of resistance. Description of the invention The inventors propose a new therapeutic strategy in the fight against antibiotic-resistant bacteria. This approach is based on the antibacterial effect of small molecules targeting a novel molecular target whose mechanism of action is not countered by the classic defense or resistance systems developed by bacteria, such as certain molecules derived from essential oils. However, since these molecules are unstable and have low bioavailability, an innovative delivery system must be associated with them so that the therapeutic solution according to the invention takes the form of a prodrug. This approach consists of a prodrug that allows for the localized delivery of the active molecule only to the site of interest, the site of infection containing resistant bacteria, due to a specific enzymatic reaction of multi-resistant bacteria capable of cleaving the prodrug.This avoids any adverse systemic effects of the active ingredient. Thus, a first object of the invention relates to a prodrug intended for the treatment of bacterial infections resistant to beta-lactam antibiotics comprising an antibacterial principle linked to a bait molecule containing a functional group cleavable by a beta-lactamase, characterized in that it corresponds to the formula (D: [Chem.1] Low T4 505 ro Se =: SO Pa (D in which X is a spacer group whose presence is optional. R is a group chosen from among a methyl, a methylsulfonyleethyl, or a dimethylaminoethyl. R, is a group chosen from a methyl, a 2,2,2-trifluoroethyl, or a masked antibacterial principle corresponding to the molecular unit adjacent to the alcohol function of said antibacterial principle R; is a group chosen from a hydrogen atom, or a masked antibacterial principle corresponding to the molecular unit adjacent to the aldehyde function of said antibacterial principle provided that at least one of R, or R; is a masked antibacterial principle R is a beta-lactamase group. A second object of the invention relates to a pharmaceutical composition comprising a prodrug according to the invention and at least one pharmaceutically acceptable excipient. A third object of the invention relates to the use of a prodrug according to the invention in the treatment of bacterial infections resistant to beta-lactam antibiotics. Advantages of the invention The present invention differs from existing solutions by the choice of the antibacterial active ingredient and by the fact that this active ingredient is masked within the prodrug (and therefore rendered inactive in the prodrug form). Indeed, the inventors have developed an approach that makes it possible to exploit the antibacterial properties of small molecules whose instability and low bioavailability have hitherto been obstacles to their therapeutic use, as is the case for those derived from essential oils. These latter are known for their antibacterial activity and due to their novel mechanism of action, which is not likely to induce resistance in bacteria. Thus, the invention aims to provide a new class of effective and safe antibacterial (pro)drugs (without induction of new resistances) for treating infections caused by bacteria resistant to beta-lactams. The drug of the invention is in the form of a prodrug comprising an antibacterial principle derived from a small molecule bearing an aldehyde or alcohol functional group. These functional groups are subject to multiple rapid modifications by the patient's metabolism, resulting in low bioavailability of said small molecules. To ensure their delivery to the site of infection, they must be incorporated into molecular structures exhibiting suitable stability, water solubility, and bioavailability. The active principle is thus modified by conjugation to a bait molecule. Another advantage of this system is that it masks the activity of the active principle but ensures its release in the presence of a bacterium resistant to beta-lactam antibiotics. Once released, the molecule regains its active form and its antibacterial properties.The delivery of the active ingredient is therefore targeted to the site of infection, and avoids side effects and . risks of resistance development associated with systemic distribution of the antibacterial agent. The release of the active ingredient depends on the action of the resistant bacterium. The prodrug contains a group recognized by beta-lactamases secreted by the resistant pathogenic bacterium, which catalyze the cleavage of the prodrug's bait molecule, the beta-lactam unit. The breakdown of this unit releases the antibacterial agent and delivers an active molecule to the site of infection. The prodrug thus acts like a Trojan horse because the bacterium, by activating its defense system—the secretion of a beta-lactamase enzyme—induces the release of the antibacterial molecule that will destroy and / or inhibit it. In more detail, the prodrug comprises, between the beta group and the active ingredient (R3 group), an N / O-acetal-oxycarbonyl group linked to a spacer group R. This N / O-acetal-oxycarbonyl group ensures the incorporation and masking of the active ingredient, as well as its release following the collapse of the prodrug caused by the action of the target enzyme. This is an original element of the prodrug's structure, on which the innovative properties of the invention are based compared to similar prodrug molecules of the prior art: the release is efficient, but the prodrug is stable against spontaneous hydrolytic degradation in the absence of the target enzyme. This property also ensures that the prodrug cannot exert an intrinsic antibiotic action since the structure of the active ingredient is not recovered in the absence of cleavage.It is these two properties that make it possible to propose a clinical use of the prodrugs according to the invention. The system according to the invention is therefore based on a local, immediate, and specific action of an antibacterial molecule from a new therapeutic class that is not likely to induce new resistance. An example of such a molecule is trans-cinnamaldehyde, the active ingredient derived from cinnamon essential oil. DETAILED DESCRIPTION OF THE INVENTION A first object of the invention relates to a prodrug for the treatment of bacterial infections resistant to beta-lactam antibiotics comprising an antibacterial active principle linked to a bait molecule comprising a functional group cleavable by a beta-lactamase, characterized in that it corresponds to the formula (I): [Chem.1] L Ba N 9 Fe * Pr ' SF (D in which: X is a spacer group whose presence is optional. R is a group chosen from among a methyl, a methylsulfonyleethyl, or a dimethylaminoethyl. R; is a group chosen from a methyl group, a 2,2,2-trifluoroethyl group, or a masked antibacterial principle corresponding to the molecular unit adjacent to the alcohol function of said antibacterial principle R; is a group chosen from a hydrogen atom, or a masked antibacterial principle corresponding to the molecular unit adjacent to the aldehyde function of said antibacterial principle provided that at least one of R; or R3 is a masked antibacterial principle R, is a beta-lactam group. The term "prodrug" refers to a drug precursor, also called a "promedicine." It is an inactive or weakly active form of a molecule that is metabolized in vivo into an active metabolite. In the present invention, the prodrug consists of a masked antibacterial agent and a bait molecule; the metabolite is the antibacterial agent. The active agent is a small molecule (in the sense of a "small molecule drug") with antibacterial properties. It is masked in the prodrug, meaning it is in an inactive form. After cleavage and breakdown of the bait molecule, the alcohol or aldehyde chemical group is restored, and the active agent is returned to its active form.Such an active ingredient can be derived from different types of plants, including plants or lichens, and can in particular be derived from an essential oil, or from animals, especially marine animals. In a preferred embodiment of the invention, the antibacterial active ingredient is derived from an essential oil with known antibacterial properties. In a most preferred embodiment of the invention, the antibacterial active ingredient is selected from trans-cinnamaldehyde, eugenol, or carvacrol. Even more preferably, it is trans-cinnamaldehyde. The formulas of these 3 compounds are shown below: [Chem.2] 9 i cinnamaldehyde [Chem.3] EE A Friend eugenol [Chem.4] OH } carvacrol Within the prodrug, the active ingredient is rendered inactive by masking its aldehyde or alcohol group, as appropriate. This modification serves two purposes: it allows binding to the bait molecule and masks (temporarily suppresses) the antibacterial activity of the active ingredient while it is in the prodrug form. For the purposes of this invention, a "bait molecule" is defined as a group recognized by bacteria and capable of inducing the activation of bacterial defense mechanisms against beta-lactam antibiotics. These defense mechanisms consist of the secretion of an enzyme capable of cleaving the beta-lactam ring present in this family of antibiotics. The bait molecule according to the invention comprises an R4 group recognized by beta-lactam-resistant bacteria. This R4 group is a beta-lactam group that mimics the presence of an antibiotic. As explained previously, recognition of this group acts as a trigger signal for the secretion of a beta-lactamase by the bacterium, which then acts in defense. The bait molecule also comprises the central group of formula (I) (N / O-acetal-oxycarbonyl group) and a spacer group X. One of the particularly innovative aspects of the invention lies in the structure of the cleavable functional group – which corresponds to the central N / O-acetal-oxycarbonyl group of formula (I) – which fulfills three functions: (i) the binding of the active ingredient to a beta-lactam group, (ii) the masking of the antibacterial activity of the active ingredient by its incorporation into the N / O-acetal-oxycarbonyl unit, and (iii) the activation of the antibacterial activity by cleavage of the beta-lactam group after secretion of a beta-lactamase by the bacteria, followed by the self-collapse of the structure, which results in the release of the active ingredient in a restored active form. which exhibits novel antibiotic properties. Indeed, it is remarkable to note that the cleavable group allows the active ingredient, which has been modified to synthesize the prodrug, to revert to its native form and antibacterial properties after release. In particular, the aldehyde or alcohol groups of the active ingredients reform in situ. This mechanism of action is illustrated in [Fig. 3]. The active ingredient may carry an aldehyde or an alcohol function. As can be seen from formula (I), when the active ingredient carries an aldehyde function, it is bound to the bait molecule at R3 and when it carries an alcohol function, it is bound to the bait molecule at R. In a particular embodiment of the invention, the prodrug may simultaneously comprise two different active ingredients, respectively on the R and Rs radicals. In a preferred embodiment of the invention, the beta-lactam group (R4 group) is derived from a cephalosporin, a penicillin, or a carbapenem. This group is cleavable by a beta-lactamase; the latter may be a carbapenemase. There are many R groups, described in the literature, and a person skilled in the art will be able to identify them. Cefalotin is a specific example. [Chem.5] QT, SA pe LX cefalotin The R4 group can be more generally represented by the following formula (11): [Chem.6] HIS oO FSPS g—w Û Ôo (qm in which Ry is chosen is chosen from among the alkyl, benzyl, aryl groups, substituted or not. Examples of specific R groupings are illustrated below: HN 8 HN. A x) % HN_S. onAa, Ra A RE NocHs, NOCHs, NO(CHA),CO2H, , The following are illustrated: HôN 2 + NA 7e la, NO(CHz), COZH, had 000$ CON RAA S. Ha; N ea 0HOH2SEHe H com HOgC” JL. TT La NOC(CHZ)2CO2H, NVC0H 77 der NEA OH 0 He N SN Léon “a LL “Q ki "CHF, HaG OH, Hg N° OH, ww è HO 7 ne " NH WT NH NH Ayhe he Ale CA eh, KA 0H, LA 0H, ON HO,C, Oo ne; ox OS. NH, OO; CH, dt, and) b 2N sH NOT NN HO— in ' NN Oo PO a; o N; Ha * CO ! Ÿ a VAT Hoocescn);— Ÿ FomeeE —p Q—CHe 4 CA * seen E The X spacer group allows the properties of the prodrug to be modulated according to the choice of the X group and the active ingredient associated with it. Its presence is optional. When the X group is absent, the R group is directly bonded to the N / O-acetal-oxycarbonyl group. In a preferred embodiment, X is a phenylene (-C6H4—), al-kynylene (-CH=CH-) or alkynylene (-C=C-) group. Examples of prodrugs according to the invention are the molecules OSTA-1, OSTAIb, OSTA-5, OSTA-6 and OSTA-7 shown below: [Chem.7] Sex, KS ÉyDE aged we 1 SRG 0 SO co 0 OSTA-1 [Chem.8] we. 310 k ve.30 GEO ES & X$ © Le A 04e ° = ET co” ô *s, Éd OSTA-1Ib [Chem.9] OSTA-5 cr Me, Ze 555, NE qu, a 0 1u5A 0e er” +4 E OSTA-5 be? [Chem.10] Me. $0 t To Mo. L0 PES, The _ &s to fe RE? These L ne” To, LG Héex 3 OSTA-6 [Chem.11] ï Me Î0 ÉTY taken; <é è LA. ia PR 8, 20008 Se CET # 8 x Ne = PP, Oo ko OSTA-7 A second object of the invention relates to a pharmaceutical composition comprising a prodrug as defined above and at least one acceptable excipient. A third object of the invention relates to the use of a prodrug as defined above in the treatment of bacterial infections resistant to beta-lactam antibiotics. Bacterial infections resistant to beta-lactam antibiotics are primarily caused by Gram-negative bacteria capable of producing beta-lactamases. However, some Gram-positive bacteria, such as MRSA, can also produce beta-lactamases. The prodrugs according to the invention are specifically targeted: - Gram-negative bacteria belonging to the following families: Moraxellaceae, Legionellaceae, Enterobacteriaceae, Pseudomonadaceae, Alcaligenaceae, Sphingomonadaceae, Pasteurellaceae, Neisseriaceae, Pasteurellaceae, Flavobacteriaceae, Bacteroidaceae, Fusobacteriaceae, Burkholderia cepacea, Cronobacter malonaticus, Cronobacter sakazakii, Helicobacter pylori, Micrococcus luteus, Prevotella, Proteus mirabilis, Proteus vulgaris G, Salmonella enteritidis G-, Salmonella enterica serovar typhi, Salmonella typhimuriu, Salmonella enterica serovar paratyphi A, Vibrio fluvialis (MDR), Vibrio parahaemolyticus, Acinetobacter baumannii, - Gram-negative bacteria belonging to one of the following genera: Klebsiella, Serratia, Citrobacter, Salmonella, Shigella, Proteus, Haemophilus, Campylobacter, Legionella, Sphingomonas, Moraxella, Kingella, Pasteurella, Capnocytophaga, Neisseria, Bacteroides, Enterobacter, Pseudomonas, Fusobacterium, Achromobacter, Escherichia and Acinetobacter, - Gram-negative bacteria belonging to one of the following species: Klebsiella pneumoniae, Klebsiella oxytoca, Acinetobacter baumannii, Acinetobacter cal- coaceticuS, Serratia marcescens, Citrobacter freundii, Citrobacter koseri, Haemophilus influenzae, Haemophilus parainfluenza, Campylobacter jejuni, Campylobacter coli, Legionella preumophila, Legionella longbeachae, Legionella bozmanii, Legionella micdadei, Enterobacter cloacae, Escherichia coli, Escherichia hermannii, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas stutzeri, Achromobacter xylosoxidans, Achromobacter denitrificans, Sphingomonas pau- cimobilis, Moraxella catarrhalis, Kingella kingae, Pasteurella multicida, Capnocytophaga canimorsus, Neisseria gonorrhoeae, Neisseria actamica and Bacteroides fragilis - in particular the bacteria Pseudomonas aeruginosa and Acinetobacter baumannii (notably Acinetobacter baumannii 5377 or Acinetobacter baumannii mucoid). The following gram positive bacteria: Families : Staphylococcaceae, Micrococcaceae, Streptococcaceae, Enterococcaceae, Bacillaceae, Clostridiaceae, Listeraceae, Corynebacteriaceae Genre: Staphylococcus, Micrococcus, Streptococcus, Enterococcus, Bacillus, Clostridium, Listeria, Corynebacterium Species (examples): Staphylococcus aureus, Staphylococcus epidermitis, Staphylococcus saprophyticus, Micrococcus luteus, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus viridans, Enterococcus faecalis, Enterococcus faecium, Bacillus anthracis, Clostridium difficile, Listeria monocytogenes, Corynebacterium diphteriae D'autres non Gram-coloriables bacteria : Families: Mycobacteriaceae; Genre : Mycobacterium Species: Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium abscessus, Mycobacterium ulcerans These infections particularly affect the digestive tract, the urinary tract, the respiratory tract, or the skin and mucous membranes, and may affect a specific organ or be generalized. The prodrugs according to the invention can be used to treat all these forms of infection. Examples of infections (with or without comorbidities such as genetic factors, immunosuppression, etc.) for which the molecules according to the invention are particularly interesting are respiratory tract infections (with or without comorbidities): Pneumonia, PAH, VAP, tuberculosis, sinusitis, cystic fibrosis; blood or bloodborne infections: Bacteremia, Septicemia; Urinary tract infections: Cystitis, Pyelonephritis; skin and soft tissue infections: Abscesses, boils, impetigo, chronic wounds, necrotizing infections; surgical site infections; infections related to a prosthesis, a medical device (probe, catheter, etc.); gastrointestinal infections; sexually transmitted infections; infections of the central or peripheral nervous system, the heart, eyes, ears, mouth, bones and joints. The present invention will be better understood by reading the following examples, which are provided by way of illustration and should in no way be considered as limiting the scope of the present invention. DESCRIPTION OF THE FIGURES [Fig.1][Fig.1]: Representation of the synthesis process of the prodrug OSTA-1 [Fig.2][Fig.2]: UV absorbance spectrum at 285 nm of the OSTA-1 molecule in vitro. [Fig.3][Fig.3]: Activation mechanism of the active ingredient after collapse of the prodrug OSTA-1 EXAMPLES EXAMPLE 1: Synthesis of the OSTA-1 molecule The synthesis protocol for the prodrug molecule OSTI comprises 5 steps and two auxiliary steps. It is shown in [Fig.1]. EXAMPLE 2: Functional analysis of the OSTA-1 molecule A - In vitro cleavage of the OSTA-1 molecule The cleavage of the prodrug OSTA-1 was tested in vitro by contact with a beta-lactamase in an aqueous saline medium. Experimental conditions: The cleavage test is performed in physiological saline (Phosphate Buffer Saline, PBS) at pH 7.0. OSTA-1 is added at a concentration of 100 µM, along with commercial beta-lactamase at a concentration of 1 unit per mL. Incubation is carried out at 37°C. The appearance of free trans-cinnamaldehyde in the medium is then monitored by UV absorption spectroscopy at 285 nm. The results are presented in [Fig.2]. The release of the active ingredient, in this case trans-cinnamaldehyde, was monitored by UV absorption at 285 nm. The evolution of the release of the active ingredient (100 µM) from the prodrug molecule was observed in the presence and absence of beta-lactamase. It is clear that the active ingredient is released in a significant quantity after two hours of reaction in the presence of beta-lactamase. In the absence of beta-lactamase, no spontaneous release of the active ingredient was observed. Conclusion: The prodrug molecule OSTA-1 functions as expected: it is cleavable by beta-lactamase and allows the release of the active ingredient. B - Activation of the active ingredient after collapse of the prodrug The mode of activation of the active ingredient is illustrated in [Fig.3], which shows the mechanism of collapse of the prodrug OSTA-1 and the release of cinnamaldehyde in its active native form.

Claims

Demands

1. Prodrug for the treatment of resistant bacterial infections to antibiotics of the beta-lactam family including a an antibacterial active ingredient linked to a bait molecule containing a functional group cleavable by a beta-lactamase, characterized in which corresponds to formula (I): = (60) Fe 0 N. where, hi Pi * YT oO Ms in which X is a spacer group whose presence is optional. R is a group chosen from among a methyl, a methylsulfony- ethyl or a dimethylaminoethyl R, is a group chosen from among a methyl, a 2,2,2-trifluoroethyl, or a masked antibacterial principle corresponding to the molecular unit adjacent to the alcohol function of said antibacterial principle R; is a group chosen from a hydrogen atom, or a masked antibacterial principle corresponding to the molecular unit adjacent to the aldehyde function of said antibacterial principle, provided that at least one of R; or R; is an anti- principle masked bacterial R4 is a beta-lactam group.

2. Prodrug according to claim 1 wherein R; and / or R3 are antibacterial principles derived from an essential oil.

3. Prodrug according to claim 2, wherein R; is the trans- cinnamaldehyde.

4. Prodrug according to claim 2, wherein R is chosen from eugenol or carvacrol.

5. Prodrug according to claim 2, wherein R; is the trans- cinnamaldehyde and R" is selected from eugenol or carvacrol.

6. Prodrug any one of the preceding claims, wherein said group R, is a derivative of cephalosporin, penicillin or car- Bapenem.

7. Prodrug one of the preceding claims, in which said group X is chosen from a hydrogen atom or a group phenylene, alkenylene or alkynylene.

8. Prodrug according to one of the preceding claims selected from the OSTA-1, OSTA-1b, OSTA-5, OSTA-6, or OSTA-7 molecules.

9. Pharmaceutical composition comprising a prodrug according to one of the claims | to 8 and at least one acceptable excipient.

10. | Prodrug according to one of the preceding claims for its use- lisation in the treatment of bacterial infections resistant to an- tibiotics of the beta-lactam family.